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Graduate Theses and Dissertations Graduate School
2005
PACAP and VIP Modulation of Neuroexcitability in Rat PACAP and VIP Modulation of Neuroexcitability in Rat
Intracardiac Neurons Intracardiac Neurons
Wayne I. DeHaven University of South Florida
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PACAP and VIP Modulation of Neuroexcitability in Rat Intracardiac Neurons
by
Wayne I. DeHaven
A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy Department of Pharmacology and Therapeutics
College of Medicine University of South Florida
Major Professor: Javier Cuevas, Ph.D. David Morgan, Ph.D.
Paul E. Gottschall, Ph.D. Eric S. Bennett, Ph.D.
Keith R. Pennypacker, Ph.D.
Date of Approval: February 22, 2005
Keywords: Intracardiac ganglia, PACAP, VIP, VPAC2, PAC1, Calcium
© Copyright 2005, Wayne I. DeHaven
ACKNOWLEDGEMENTS
This dissertation would not have been completed without the love and
support from my family. I would like to give special thanks to my wife, Courtney,
for always supporting me in my scientific career, and reminding me of life outside
of the laboratory. I would like to thank my daughter, Eve, for always putting a
smile on my face. I would like to thank Mom and Dad for giving me the
opportunity to succeed. I would also like to thank my in-laws, Tom and Dennie
Masterson, for always lending a helping hand.
I would like to thank my mentor, Dr. Javier Cuevas, for giving me the
appropriate environment to grow as a scientist. I would also like to thank
Hongling Zhang for her scientific help and knowledge, and Crystal Reed and
Yolmari Cruz for maintaining the laboratory, and for putting up with sports radio
all of the time. I would like to thank my external reviewer, Dr. Rodney L.
Parsons, for taking the time to chair my dissertation committee. Finally, I would
like to thank the other members of my committee: Drs. David Morgan, Paul E.
Gottschall, Eric S. Bennett and Keith R. Pennypacker for their excellent scientific
advice.
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TABLE OF CONTENTS
List of Tables
……………………………………………………………………
v
List of Figures
………………………………………………………………….
vi
Abstract
…………………………………………………………………………..
xi
Chapter 1 Background and Significance ………………………………….. 1
Mammalian Intracardiac Ganglia ……………………………………… 1
PACAP and VIP effects on the Heart …………………………………. 4
PACAP and VIP receptors ……………………………………………... 6
PACAP and VIP relevance to cardiac function ……………………….
7
Chapter 2
Heterogeneity of PACAP and VIP receptors in rat intrinsic
cardiac neurons …………………………………………………. 12
Introduction ……………………………………………………………… 12
Methods ………………………………………………………………….. 15
Cell culture ………………………………………………………… 15
RT-PCR ……………………………………………………………. 16
Cytoplasm harvest of individual neurons ………………………. 17
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Results …………………………………………………………………… 18
PAC1 receptor mRNA isoforms detected in cardiac tissue …... 18
PAC1 mRNA expression in isolated rat intracardiac neurons … 19
VPAC1 and VPAC2 receptor transcripts detected in cardiac
tissue ……………………………………………………………….. 20
VPAC mRNA expression in isolated rat intracardiac neurons .. 21
Individual intracardiac neuron expression pattern of PAC1 and
VPAC mRNA ………………………………………………………. 22
Discussion ………………………………………………………………..
31
Chapter 3 VPAC receptor modulation of neuroexcitability in intracardiac
neurons: dependence on intracellular Ca2+ mobilization and
synergistic enhancement by PAC1 receptor activation ……… 33
Introduction ………………………………………………………………. 33
Methods ………………………………………………………………….. 37
Microfluorometric measurements ……………………………….. 37
Electrophysiology …………………………………………………. 38
Reagents and statistical analysis ……………………………….. 40
Results …………………………………………………………………… 41
PACAP and VIP directly increase free [Ca2+]i …………………... 41
Increases in free [Ca2+]i evoked by PACAP and VIP are
mediated by a VPAC receptor …………………………………... 42
PACAP and VIP mobilize Ca2+ from intracellular stores and
evoke Ca2+ entry through the plasma membrane ……………... 44
PACAP induces mobilization of [Ca2+]i from caffeine- and
ryanodine-sensitive stores ……………………………………….. 46
PACAP and VIP Induced Excitability in Rat Intracardiac
Neurons ……………………………………………………………. 49
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Increases in neuroexcitability evoked by PACAP and VIP are in
part mediated by a VPAC receptor ……………………………… 52
Effects of Ca2+-free extracellular conditions on PACAP-induced
intracardiac neuroexcitability …………………………………….. 54
Effects of Ryanodine on PACAP-induced intracardiac
neuroexcitability …………………………………………………… 56
Discussion ………………………………………………………………..
80
Chapter 4 Repetitive VPAC2 receptor-mediated calcium elevations in
intracardiac neurons is dependent on Ca2+, cADPR and store-
operated calcium (SOC) entry …………………………………. 87
Introduction ………………………………………………………………. 87
Methods ………………………………………………………………….. 92
Cell culture …………………………………………………………. 92
Microfluorometric measurements ……………………………….. 92
Reagents and statistical analysis ……………………………….. 93
Results …………………………………………………………………… 95
VPAC2 receptors mediate PACAP-induced elevations in free
[Ca2+]i. ……………………………………………………………….. 95
Forskolin does not increase [Ca2+]i in intracardiac neurons ….. 96
Role for cADP-ribose in the PACAP-elicited signal transduction
cascade …………………………………………………………….. 96
Effects of menthol and capsaicin on [Ca2+]i ……………………... 98
La3+ blocks VPAC2 mediated Ca2+ elevations and TRPC
channels in intracardiac neurons ………………………………... 99
PACAP evoked Ca2+ influx through store-operated channels
(SOC) ……………………………………………………………….
101
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The SOC channel component of the PACAP-induced Ca2+
response is blocked by 2-APB …………………………………... 103
2-APB blocks VPAC2 receptor mediated repetitive Ca2+
responses ………………………………………………………….. 104
Discussion ………………………………………………………………..
125
Chapter 5 Canonical transient receptor potential (TRPC) channels
regulate neuroexcitability in rat intracardiac neurons ……….. 132
Introduction ………………………………………………………………. 132
Methods ………………………………………………………………….. 135
Electrophysiology …………………………………………………. 135
Reagents and statistical analysis ……………………………….. 136
Results …………………………………………………………………… 138
2-APB decreases action potential (AP) firing in rat intracardiac
neurons …………………………………………………………….. 138
2-APB evoked changes in the single action potential waveform.. 139
Membrane currents underlying the 2-APB-elicited changes in
action potential properties ……………………………………….. 141
Discussion ………………………………………………………………..
151
Chapter 6 Summary …………………………………………………………. 155
Conclusion ………………………………………………………..
167
Works Cited
……………………………………………………………………... 170
About the author ………………………………………………………… End Page
v
LIST OF TABLES
Table 2.1 Sequences of oligonucleotide primers to detect PAC1
isoforms, as well as VPAC receptor transcripts in rat
intracardiac neurons ………………………………………...
25
Table 3.1 Effects of extracellular Ca2+ conditions and various
inhibitors on the PACAP-induced changes in
neuroexcitability in response to a 150 pA current pulse ... 79
vi
LIST OF FIGURES
Figure 1.1 Schematic representation of the connectivity among various
neurons involved in maintaining cardiac homeostasis ……...
9
Figure 1.2 Morphologically distinct intrinsic cardiac neurons identified in
vitro …………………………………………………………........
10
Figure 1.3 Schematic representation of the two neuropeptides, PACAP
and VIP, binding to their associated G-protein coupled PAC1,
VPAC1 and VPAC2 receptors ……………………………..…...
11
Figure 2.1 Expression of PAC1 receptor isoforms in cardiac tissue …….
26
Figure 2.2 Detection of PAC1 receptor isoforms in single intracardiac
neurons ……………………………………………………..…....
27
Figure 2.3 Expression of VPAC1 and VPAC2 receptor isoforms in cardiac
tissue ………………………………………………………….…..
28
Figure 2.4 Detection of VPAC receptor transcripts in single intracardiac
neurons ……………………………………………………..…....
29
Figure 2.5 Expression pattern of PAC1 isoforms and VPAC transcripts in
intracardiac neurons ………………………………………...….. 30
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Figure 3.1 PACAP and VIP, but not maxadilan, reversibly increase [Ca2+]i
in rat intracardiac neurons …………………………………......
59
Figure 3.2 PACAP-induced mobilization of [Ca2+]i in intracardiac neurons
is via VPAC receptor activation ………………………..……....
61
Figure 3.3 The transient, but not the sustained, component of the
PACAP-induced increase in [Ca2+]i is dependent on
extracellular Ca2+ …………………………………...…………...
63
Figure 3.4 PACAP evokes a rapid transient elevation of [Ca2+]i in
intracardiac neurons by releasing Ca2+ from
caffeine/ryanodine-sensitive internal stores ………...………..
65
Figure 3.5 The sustained, but not the transient, component of the
PACAP-induced increase in [Ca2+]i is blocked by 2-APB …...
67
Figure 3.6 PACAP increases neuroexcitability in neonatal rat intracardiac
neurons …………………………………………………………..
69
Figure 3.7 VIP-induced changes in neuroexcitability within neonatal rat
intracardiac neurons ………………………………………...…..
71
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Figure 3.8 PACAP-evoked changes in intracardiac neuroexcitability are
inhibited by the VPAC antagonist [N-Ac-Tyr1, D-Phe2]-GRF
(1-29) …………………………………………………………...
73
Figure 3.9 PACAP-induced changes in intracardiac neuroexcitability
are blocked by the removal of extracellular Ca2+ and
depletion of internal stores …………………………………...
75
Figure 3.10 PACAP-induced changes in intracardiac neuron excitability
are blocked by ryanodine ……………………………………..
77
Figure 4.1 PACAP-evoked mobilization of [Ca2+]i in rat intracardiac
neurons is via VPAC2 receptor activation …………………..
108
Figure 4.2 Forskolin does not increase free cytosolic calcium
concentrations in rat intracardiac neurons ………………….
110
Figure 4.3 The putative ryanodine receptor activator, cADP ribose, is
involved in the PACAP-elicited signal transduction cascade..
112
Figure 4.4 Neither menthol nor capsaicin increase free intracellular
Ca2+ concentrations in isolated rat intracardiac neurons ….
114
ix
Figure 4.5 La3+ blocks VPAC2 mediated Ca2+ elevations in intracardiac
neurons and the sustained component of the caffeine- and
muscarine-induced increase in [Ca2+]i ……………………....
116
Figure 4.6 The TRPC agonist, 1-oleoyl-2-acetyl-sn-glycerol (OAG),
does not increase [Ca2+]i in neonatal rat intracardiac
neurons ………………………………………………………....
118
Figure 4.7 Internal store depletion by PACAP or caffeine activates
store-operated Ca2+ (SOC) entry in intracardiac neurons ...
120
Figure 4.8 SOC channel activation by PACAP is inhibited by 2-APB ..
122
Figure 4.9 Repetitive elevations of [Ca2+]i mediated by VPAC2 receptor
activations are blocked by 2-APB …………………………...
124
Figure 5.1 2-APB effects on action potential firing in isolated intrinsic
cardiac neurons ………………………………………………..
144
Figure 5.2 The effects of 2-APB on the single action potential waveform
parameters ……………………………………………………..
146
Figure 5.3 Effects of the Ca2+-activated K+ inhibitors TEA, paxilline and
apamin, and the Ca2+ channel blocker Cd2+ on the action
potential and afterhyperpolarization …………………………
148
x
Figure 5.4 Inhibition of peak K+ channel currents in rat intracardiac
neurons by the TRP channel antagonist, 2-APB …………..
150
Figure 6.1 Schematic representation of the expression pattern of PAC1
receptor isoforms and VPAC transcripts in individual
intracardiac neurons …………………………………………..
164
Figure 6.2 Schematic diagram showing the effects of PACAP and VIP
on intracardiac neurons ………………………………………. 166
xi
PACAP and VIP Modulation of Neuroexcitability in Rat Intracardiac Neurons
Wayne I. DeHaven
ABSTRACT
Autonomic control of cardiac function depends on the coordinated activity
generated by neurons within the intracardiac ganglia, and intrinsic feedback
loops within the ganglia provide precise control of cardiac function. Both pituitary
adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal
polypeptide (VIP) are important regulators of cell-to-cell signaling within the
intracardiac ganglia, and PACAP and VIP action on these ganglia, mediated
through associated receptors, play an important role in the regulation of coronary
blood flow, cardiac contraction, relaxation, and heart rate. Results reported here
using PACAP and VIP provide direct evidence of some of the complex signaling
which occurs in neurons of the rat intracardiac ganglia.
The expression of PACAP and VIP receptors was investigated using
single-cell RT-PCR. Individual neurons were shown to express multiple isoforms
of the PACAP-selective receptor, PAC1, including the -short, -HOP1 and -HOP2
variants. These splice variants affect ligand binding, G-protein coupling and
xii
selectivity. Intracardiac neurons also express the non-selective receptors,
VPAC1 and VPAC2, with VPAC2 being found in greater proportion of cells. These
results demonstrate heterogeneity of PAC1 and VPAC receptors expressed in
intracardiac neurons, which has significant implications on the effects of PACAP
and VIP on cellular function.
Calcium imaging and electrophysiology were used to examine the
physiological effects of PACAP and VIP on isolated intracardiac neurons. Both
neuropeptides, through the activation of VPAC2 receptors, evoked rapid
increases in cytosolic calcium concentrations ([Ca2+]i) that exhibited both
transient and sustained components. The transient increases in [Ca2+]i were
mediated through the activation of ryanodine receptors, whereas the sustained
[Ca2+]i elevations were dependent on extracellular Ca2+ and pharmacologically
resembled canonical transient receptor potential (TRPC) channels.
PACAP and VIP also depolarized intracardiac neurons, and PACAP was
further shown to augment action potential firing in these cells. The depolarization
was dependent on activation of VPAC2 receptors and the concomitant increases
in [Ca2+]i, while PAC1 receptor stimulation potentiated the VPAC2 receptor-
induced depolarizations. Pharmacological evidence suggest TRPC channels
mediate this link between neuropeptide evoked changes in membrane properties
and [Ca2+]i. Thus, these results give insight into the complex PACAP and VIP
signaling which occurs in the intracardiac ganglia.
1
CHAPTER 1
BACKGROUND AND SIGNIFICANCE
Mammalian Intracardiac Ganglia
Parasympathetic influences on cardiac function are mediated through the
vagus nerve (CN X), and stimulation of this nerve results in multiple cardiac
effects, including bradycardia, atrioventricular (AV) block, and reduced
myocardial contractility. Intracardiac ganglia form the final pathway for
autonomic modulation of cardiac function, and in the classical view of
parasympathetic innervation of the heart, intracardiac neurons are considered
exclusively postganglionic efferent neurons. However, an increasing body of
data has supported the hypothesis that neurons within the intracardiac ganglia do
not serve as simple relays between the central nervous system (CNS) and heart
(Gray et al., 2004); but, instead, individual cardiac ganglia serve as complex
integrative centers within which processing of autonomic signals can occur.
These data have resulted in the proposal of the organization of the intrinsic
cardiac nervous system which includes parasympathetic efferent neurons,
sensory afferent neurons, intraganglionic and interganglionic neurons,
2
sympathetic neurons, as well as the terminals of cardiac neurons projecting from
higher centers (Gray et al., 2004; Ardell, 1994; Armour, 1994) (Fig 1.1).
Mammalian intracardiac ganglia are anatomically located in various fat
pads associated with the heart, and each distinct cluster of neurons
predominantly innervates different regions of the heart. For example, the
sinoatrial (SA) ganglion, located in a fat pad near the junction of the right atrium
and the superior vena cava, mediates a negative chronotropic effect (King and
Coakley, 1958; Gatti et al., 1995; Massari et al., 1996; Massari et al., 1994).
Similarly, the atrioventricular (AV) ganglion, found in a fat pad between the left
atrium and the inferior vena cava, potently reduces the AV conduction rate (Gatti
et al., 1995; Massari et al., 1995; Massari et al., 1996). The cranioventricular
(CV) ganglion, located at the cranial margin of the left ventricle, has been found
to contain neurons that selectively mediate negative inotropic effects on the left
ventricle, without influencing cardiac rate or AV conduction (Dickerson et al.,
1998; Gatti et al., 1997). In addition, there are numerous ganglia found
throughout the heart for which no functional roles have been described (Ardell,
1994; Armour, 1994; Calaresu et al, 1967; Johnson et al., 2004).
Morphologically, at least three distinct functional types of neurons exist
within these intrinsic cardiac ganglia. The first group of neurons is the
parasympathetic efferent postganglionic neurons (Jacobowitz et al, 1967, Xi-Moy
et al, 1993), innervating the coronary vasculature, cardiac myocytes, SA and AV
nodes. The second group of neurons is interneurons (Jacobowitz et al, 1967;
3
Armour and Hopkins, 1990; Randall and Wurster, 1994), which regulate the other
neurons of the ganglia. The third group of neurons is the small intensely
fluorescent cells (SIF cells), so named based on their high glyoxylate
fluorescence indicative of catecholamines (Seabrook et al., 1990). SIF cells do
not have processes; rather, SIF cells are small oval shaped cells which may have
paracrine function. Previous morphological studies describe principle
parasympathetic neurons as being monopolar or multipolar with long axons,
whereas interneurons display a dipolar or pseudodipolar arrangement (Edwards
et al, 1995). The axons of the interneurons appear to be shorter, with small
branches that terminate near other ganglion cells. Intracardiac neurons are also
morphologically differentiated on the basis of soma diameter with interneurons >
principle parasympathetic neurons >> SIF cells (Edwards et al., 1995; Allen and
Burnstock, 1990; Seabrook et al., 1990). In our isolated neuron preparation we
are able to identify various neuron types, including unipolar, bipolar and
multipolar neurons that correspond well to observations in intrinsic cardiac
neurons in situ (Edwards et al., 1995) (Fig 1.2)
Though some of the neuroanatomical and functional characteristics of
these intracardiac neurons are identified, little is known about how neural activity
is generated and coordinated within the intrinsic cardiac nervous system. Along
with the structural complexity of the intracardiac ganglia, a neurochemical
complexity exists which allows for a wide range of responses and ultimately
maintains cardiac homeostasis. Although acetylcholine is the primary
4
neurotransmitter in the mammalian heart, an expanded view of intracardiac
neurotransmission has evolved to include instances where substances other than
acetylcholine are localized and released and function as cotransmitters,
neuromodulators, or primary neurotransmitters themselves (Rubino et al, 1996).
Two such neurotransmitters with potent cardiovascular effects are pituitary
adenylate-cyclase activating polypeptide (PACAP) and vasoactive intestinal
polypeptide (VIP).
PACAP and VIP effects on the Heart
PACAP and VIP are pleiotropic neuropeptides that belong to the
secretin/glucagon/growth hormone-releasing factor family of peptides (Warren et
al, 1991; Ishizuka et al, 1992; Basler et al, 1995; Champion et al, 1996; Cardell et
al, 1997). These neuropeptides have a broad spectrum of effects in both the
central nervous system (CNS) and peripheral nervous system (PNS), including
the autonomic nervous system (Vaudry et al., 2000; Arimura, 1998). For
example, in the CNS, PACAP is involved in oxytocin and vasopressin release
(Murase et al., 1993; Seki et al., 1995), rhythmicity of melatonin production in the
pineal gland (Fukuhara et al., 1998), control of appetite and feeding behavior
(Christophe, 1998), inhibition of cell death and promotion of neurite outgrowth
during ontogenesis (Gonzalez et al., 1997; Cavallaro et al., 1996), synaptic
plasticity and learning in mice (Hashimoto et al., 2002) and mobilization of Ca2+
in astroglial cells (Tatsuno and Arimura, 1994). In the PNS, PACAP and VIP
5
appear to play a major role in the autonomic regulation of the cardiovascular
system, and the neuropeptides have been immunocytochemically identified in
nerve fibers and cell bodies within the heart, coronary vessels and cardiac
parasympathetic ganglia (Della et al., 1983; Weihe et al., 1984; Seabrook et al.,
1990; Gulbenkian et al., 1993; Braas et al., 1998; Horackova et al., 2000).
PACAP and VIP are extremely effective in dilating vascular beds,
including the coronary vessels that supply the heart (Fahrenkrug, 1993; Nillson,
1994). The vasodilatory effects of VIP on arteries are much greater than those
on veins because of the greater density of VIP receptors in arterial vessels (Luu
et al, 1993). Low concentrations of VIP can increase epicardial coronary artery
cross-sectional area by 27%, decrease coronary vascular resistance by 46%,
and increase coronary artery blood flow by 200% (Brum et al, 1986; Popma et al,
1990). PACAP has similar effects on coronary vasculature, except PACAP is
more efficacious than VIP as a vasodilator (Nillson, 1994). PACAP has also
been shown to modulate neuronal activity in canine intrinsic cardiac neurons in
situ and alter heart rate (Armour et al, 1993). These in situ experiments on
canine neurons suggest that not only do these neuropeptides enhance neuronal
activity, but also in some neurons, PACAP and VIP stimulation actually depress
neuronal activity. These neuropeptides have also been shown to increase sinus
rate and cardiac output (Karasawa et al, 1990; Poth et al, 1997; Sreedharan et
al, 1995), to initiate positive inotropic effects (Franco-Cereceda et al, 1987;
Henning et al, 2000), and to increase cardiac contractility (Henning, 1992). It is
6
our theory that this variability in response is the result of heterogeneity of
VIP/PACAP receptors in intracardiac ganglia.
PACAP and VIP receptors
The cellular effects of PACAP and VIP are mediated through G-protein
coupled receptors, and two main classes of PACAP and VIP receptors have
been categorized based on binding and in vitro functional data (Christophe,
1993; Shivers et al, 1991). The PAC1 receptors bind PACAP-27 and PACAP-38
with equal high affinity, but VIP with much lower affinity (Gourlet et al, 1995) (Fig
1.3). Thus, PAC1 receptors are known as PACAP-selective receptors. VIP
(VPAC1 and VPAC2) receptors recognize PACAP-27, PACAP-38 and VIP with
similar high affinity, and these are known as the non-selective VIP receptors.
At least seven isoforms of the PAC1 receptor exist due to alternative
splicing of the mRNA (PAC1-short, -very short, -HIP, -HOP1, -HOP2, -HIPHOP,
and TM4). Based on cloning experiments, the two major forms in mammals are
the normal (short) PACAP receptor and the -HOP1 PACAP receptor, which
contains a cassette insert in the third cytoplasmic loop (Spengler et al, 1993;
Svoboda, 1993). The original VPAC receptor was cloned from rat lung (Isihara et
al, 1992) and from human colon cells (Sreedharan et al, 1993). Presently known
as the VPAC1 receptor, this peptide may be considered the classical VIP
receptor described in lung, intestine, pancreas, and liver. A second VPAC
7
receptor was cloned from rat brain (Lutz et al, 1993) and was called the VPAC2
receptor. No splice variants of VPAC1 and VPAC2 are presently known to exist.
PACAP and VIP relevance to cardiac function
The intrinsic cardiac nerve plexus acts as much more than a simple relay
center for parasympathetic innervation to the heart. Rather, it functions as a
local integrative neural network capable of modulating extrinsic inputs to the
heart and in mediating local cardiac reflexes. The neuropeptides, PACAP and
VIP are important regulators of cell-to-cell signaling within the intracardial
ganglia, and PACAP and VIP action on these ganglia, mediated through the
associated receptors, play an important neuromodulatory role in the regulation of
coronary blood flow, cardiac contraction, relaxation, and heart rate. Even more
interesting are the possible roles these peptides play in different pathological
states. Despite many of the advances made in the research of PACAP and VIP
effects on the mammalian heart, the physiological and pathological roles of these
neuropeptides remain unclear and incomplete. The studies proposed here will
use novel approaches to give insight into the actions of PACAP and VIP in
intracardiac ganglia. Ultimately, this knowledge may contribute to the discovery
of new clinical tools used for diagnosing and treating diseased states such as
congestive heart failure (CHF), arrhythmias, hypertension and ischemic
conditions. The present study attempts to further the understanding of PACAP
and VIP regulation of intracardiac neurons. Considering this primary objective,
8
four specific aims were established: (1) Investigate whether individual rat
intracardiac ganglion neurons express the PAC1, VPAC1 and/or VPAC2 receptors
using the single-cell RT-PCR method. (2) Use whole-cell patch-clamp
techniques to determine both the passive and active membrane properties of
isolated intracardiac neurons in the absence and presence of PAC1, VPAC1
and/or VPAC2 stimulation. (3) Determine whether PACAP and/or VIP increase
[Ca2+]i in intracardiac neurons by using microfluorometric fura-2 Ca2+-imaging
techniques. (4) Characterize which Ca2+-activated ion channels are linked to
PACAP and/or VIP receptors.
The following chapters of this dissertation were written in manuscript form
and each chapter addresses a specific topic which relates to the aims of the
dissertation proposal.
9
FIGURE 1.1: Schematic representation of the connectivity among various
neurons involved in maintaining cardiac homeostasis. DRG, dorsal root ganglia;
MCG, middle cervical ganglia; SCG, superior cervical ganglia; ICG, intracardiac
ganglia; β1, adrenergic receptors; M1, muscarinic receptors (adapted from
Armour et al., 1998).
10
FIGURE 1.2: Morphologically distinct intrinsic cardiac neurons identified in vitro.
Unipolar (A), multipolar (B) and dipolar (C) neurons isolated from neonatal rat
intracardiac ganglia and cultured for 48 hr. Note that in addition to the difference
in the number of processes, the unipolar neuron (A) has a smaller soma than the
multipolar and dipolar neurons (B,C). (A) and (B) represent principle
parasympathetic neurons, whereas (C) is an interneuron. SIF cells, with an
average soma diameter of ~10 µm (Allen and Burnstock, 1987; Seabrook et al,
1990), are also observed in our preparation. Scale bars = 50 µm.
11
FIGURE 1.3: Schematic representation of the two neuropeptides, PACAP and
VIP, binding to their associated G-protein coupled PAC1, VPAC1 and VPAC2
receptors. While PACAP has similar affinities to both PAC1 and VPAC receptors,
VIP only binds to VPAC receptors with high affinity.
12
CHAPTER 2
HETEROGENEITY OF PACAP AND VIP RECEPTORS IN RAT INTRINSIC
CARDIAC NEURONS
INTRODUCTION
Mammalian intracardiac ganglia serve as integration centers for
parasympathetic, sympathetic and afferent signaling pathways in the heart
(Ardell, 1994). The structural complexity of the ganglia is complemented by a
neurochemical diversity that permits sophisticated processing of signals, and
ultimately allows proper control of cardiac function. While acetylcholine is the
primary parasympathetic neurotransmitter mediating intrinsic and extrinsic
innervation of the heart, other non-cholinergic, non-adrenergic neurotransmitters
have been found in cell bodies and nerve fibers within the ganglia (Weihe et al.,
1984; Rigel, 1998). These endogenous neurotransmitters often regulate
neuronal excitability, and thus facilitate the processing of information provided by
the diverse inputs. Two such neuromodulators are pituitary adenylate cyclase-
activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP), both
of which have been identified by immunocytochemistry in nerve fibers innervating
13
the heart, coronary vasculature and cardiac parasympathetic ganglia (Della et al,
1983; Braas et al., 1998; Seabrook et al., 1990; Weihe et al., 1984). PACAP
and VIP have been shown to modulate neuronal activity in canine intrinsic
cardiac neurons in situ and alter heart rate (Armour et al., 1993). These
neuropeptides have also been shown to increase sinus rate, cardiac contractility
and cardiac output (Henning, 1992; Rigel, 1998; Karasawa et al., 1990; Ross-
Ascuitto et al., 1993), and to induce vasodilation in coronary arteries (Feliciano
and Henning, 1998; Ross-Ascuitto et al., 1993).
VIP and PACAP can directly affect cardiac muscle and coronary blood
vessels (Hirose et al., 1997; Warren et al., 1984); however, there appears to be a
significant neural component to the cardiovascular effects of these neuropeptides
(Hirose et al., 1997). The cellular mechanisms by which PACAP and VIP
modulate cell-to-cell signaling in intracardiac ganglia and cardiac function are
poorly understood. Furthermore, conflicting data exists as to the subtype(s) of
PACAP and VIP receptors present in mammalian intracardiac neurons and
cardiac muscle. For example, in the adult guinea pig intracardiac neurons, only
the PAC1 receptor has been detected (Braas et al., 1998). The PAC1 receptor is
selective for PACAP and binds VIP with low affinity (Gourlet et al., 1995).
However, electrophysiological experiments on neonatal rat intrinsic cardiac
neurons suggest the presence of VPAC1 and/or VPAC2 receptors, both of which
bind PACAP and VIP with similar high affinities (Cuevas and Adams, 1996;
Gourlet et al., 1995). The PACAP/VIP receptors in intracardiac neurons from
14
these two species also appear to couple distinct signal transduction cascades
and modulate different effector targets. We have previously shown that PACAP
and VIP potentiate ACh-evoked currents in neonatal rat intracardiac neurons
(Cuevas and Adams, 1996; Liu et al., 2000); whereas in guinea pig intracardiac
neurons, PACAP has been shown to modulate neuronal excitability and
depolarize the cells (Braas et al., 1998).
Some of these discrepancies may reflect the complex nature of the
receptors of the PACAP/VIP family. For example, seven isoforms of the PAC1
receptor have been reported (PAC1-very short, -short, -HOP1, -HOP2, -HIPHOP,
-HIP, and TM4). These splice variations have been shown to affect ligand
binding, as well as G-protein coupling and selectivity. It has also been suggested
that mammalian intracardiac ganglia express multiple PAC1 isoforms (Braas et
al., 1998), but it remains to be determined if these receptors are exclusively on
neurons or on support cells and what PACAP/VIP receptor types are expressed
by individual neurons. Identification of the receptor types involved in PACAP and
VIP neurotransmission is the first step in elucidating the signal transduction
cascade that ultimately results in neuromodulation in intracardiac ganglia and
regulation of cardiac function.
15
METHODS
Cell Culture
Receptor transcripts were investigated in isolated parasympathetic
neurons of the rat intracardiac ganglia. As previously described (Fieber and
Adams, 1991), rats were killed by decapitation and the heart was carefully
removed and placed in a physiological saline solution (PSS) containing (mM):
140 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 7.7 glucose and 10 histidine; pH to 7.2
with HCl. Next, the atria was removed and incubated for 1 h at 37°C in saline
solution containing collagenase type 2 (1mg/ml; Worthington Biomedical Corp.,
Freehold, NJ, USA). After treatment with collagenase, clusters of ganglia were
dissected away from the remaining atrial tissue and transferred to a sterile
culture dish containing high-glucose culture media (Dulbecco’s Modified Eagle
Medium, DMEM), 10% (v/v) fetal calf serum, 100 U/ml penicillin and 0.1 mg/ml
streptomycin, and triturated using a fine-bore Pasteur pipette. The dissociated
neurons were finally plated onto poly-lysine (K) 18 mm glass coverslips and
incubated at 37°C for 36-72 h under a 95% air, 5% CO2 environment.
16
RT-PCR
The expression of PAC1, VPAC1, and VPAC2 receptors was studied in
cardiac tissues from neonatal rats (3-7 days old). Total RNA was isolated from
intracardiac ganglia and associated tissue, atria, ventricles and pituitary gland
(positive control) (RNeasy, Qiagen, Hilden, Germany). The RNA was reverse
transcribed and the resultant cDNA amplified using PCR techniques. PCR
products were resolved using agarose gel (1.5%) electrophoresis with ethidium
bromide as the label, and visualized using UV illumination. In all PCR
experiments from tissue extracts, RT-negative controls were conducted to
screen for contaminants. RNA was reverse-transcribed in a 20 µl reaction
volume using the SuperScript First-Strand Synthesis System for RT-PCR
(Invitrogen Co., San Diego, CA, USA). As a negative control, a PCR reaction
with only water was conducted to eliminate the possibility of false positives due
to contaminating cDNA. Primer pairs specific for PAC1, VPAC1 and VPAC2
receptor transcripts (Table 2.1) were designed to span an intron in order to
discriminate between genomic DNA and cDNA. PCR reactions were conducted
using the SuperScript System with Platinum Taq DNA polymerase (Invitrogen
Co.). The cycling parameters were one cycle of 94°C for 2 min; 30 cycles of
94°C for 30 s, 61°C for 45 s, and 72°C for 1 min; and one cycle of 72°C for 5
min.
17
Cytoplasm harvest of individual neurons
For single cell RT-PCR experiments, intracardiac neurons were
dissociated, and cytoplasm extracted from isolated neurons as previously
described (Poth et al., 1997). The intracellular contents of individual intracardiac
neurons were harvested by applying suction on a patch pipette in the dialyzing
whole-cell recording configuration. The pipettes were filled with 3 µL of 1X
Superscript One-Step RT-PCR Reaction Mix (Invitrogen Co.) containing 1 U/µL
RNAsin (Promega Co., Madison, WI, USA). After cytoplasm extraction, the
contents of the pipette were expelled into a microfuge tube and frozen on dry ice.
Immediately following extractions, single cell RT-PCR experiments were
conducted using Superscript One-Step RT-PCR with Platinum Taq (Invitrogen
Co.). Negative controls were obtained by suctioning extracellular solution near
the location of the harvested neuron into a separate boricillate pipette. This
control was carried out to exclude contamination with cytoplasm from nearby
cells or by contamination with VIP/PACAP receptor containing plasmids routinely
used in the laboratory. The cycling parameters were one cycle of 50°C for 30
min and 95°C for 2 min; 40 cycles of 94°C for 30 s, 61°C for 45 s, and 72°C for 1
min; and one cycle of 72°C for 5 min.
RT-PCR products were gel purified using a QIAEX II Gel Purification kit
(QIAGEN) and sequenced by the Molecular Biology Core Facility at the H. Lee
Moffitt Cancer Center and Research Institute.
18
RESULTS
PAC1 receptor mRNA isoforms detected in cardiac tissue
Experiments were conducted to determine the distribution of PAC1
receptor transcripts in cardiac tissue of neonatal rats. The tissues examined
were intracardiac ganglia and associated tissue (e.g. cardiac myocytes, Schwann
cells, vascular smooth muscle, endothelial cells, and fibroblasts), the auricles of
the atria, and the apex of the ventricles. Whole tissue RNA extracts were probed
for PAC1 receptor isoforms using the PAC1-1 and PAC1-2 oligonucleotide primers
(table 2.1), designed to differentiate between splice variations in the third
cytoplasmic loop, which is essential for G-protein interaction and influences G-
protein specificity, and the amino terminus of the PAC1 receptor (Fig 2.1A). RT-
PCR amplification of the extracts using PAC1-1 primers demonstrated that
multiple PAC1 receptor isoforms are expressed in intracardiac neurons and
associated tissues, atria and ventricles of neonatal rats (Fig 2.1B). Products of
the predicted sizes for PAC1-short and/or -very short, -HIP and/or -HOP, and -
HIPHOP were detected in all extracts.
The oligonucleotide primers (PAC1-1) designed to distinguish between
splice variations in the third cytoplasmic loop of the PAC1 receptor do not
19
discriminate between the PAC1-short or PAC1-very short variations. This splicing
occurs within the domains encoding the amino terminus of the receptor, and is
significant in ligand binding affinities. Therefore, PAC1-2 primers were developed
to amplify PAC1 cDNA associated with the extracellular, N-terminus (Fig 2.1A).
The resultant bands following RT-PCR suggest that neonatal rat cardiac tissues
only express the short sequence variant of the PAC1 receptor (Fig 2.1C).
PAC1 mRNA expression in isolated rat intracardiac neurons
The presence of PAC1 receptor transcripts in non-neuronal tissues of the
heart necessitated the use of single-cell PCR to determine if individual neurons
express PAC1 receptors and the specific isoform(s) present. Figure 2.2A shows
RT-PCR reaction results for five isolated neurons using the PAC1-1 primers.
Similar to the whole tissue samples, individual rat intracardiac neurons express
one or more splice variations in the region encoding the third cytoplasmic loop of
the PAC1 receptor. The amino terminus region was also investigated at the
single-neuron level to distinguish between short and very short transcript
presence within such neurons (Fig 2.2B). RT-PCR reaction results, following two
rounds of PCR, for two isolated neurons using the PAC1-2 oligonucleotide
primers indicate only the presence of the -short variant.
PCR products from both sets of primers were gel purified and cloned
using the pGEM-T Easy Vector System (Promega). As expected, sequences and
translation of the 493 bp size RT-PCR product (PAC1-2) from two individual
20
neurons aligned perfectly with the sequence of the PAC1-short (PAC1-s; Z23279).
None aligned with the PAC1-very short sequence (PAC1-vs). Sequences and
translation of the 303 bp size RT-PCR product (PAC1-1) from two individual
neurons also aligned with the sequence of the PAC1-short receptor, further
proving the presence of only PAC1-short splicing within the N-terminus of
neonatal rat intracardiac neurons. Sequences and translation of the 384/387 bp
size RT-PCR products (PAC1-1) from five individual neurons aligned with the
sequences for PAC1-short, PAC1-HOP1 (Z23274) and PAC1-HOP2 (Z23275).
Although experiments seemed to indicate the presence of -HIPHOP transcripts
(468/471 bp products) within these neurons and cardiac tissue, no PAC1-
HIPHOP sequences were elucidated. Thus, rat intracardiac neurons express
PAC1 transcripts lacking a cassette insert in the third cytoplasmic loop and
transcripts containing either the -HOP1 or -HOP2 cassette. No transcripts
containing the -HIP or -HIPHOP insert were detected. Single-cell RT-PCR
studies confirm that neonate rat intracardiac neurons only express PAC1-short
isoform.
VPAC1 and VPAC2 receptor transcripts detected in cardiac tissue
Related to the PAC1 receptor, the VIP (VPAC) receptor is also a member
of the secretin/glucagon receptor family. Two subtypes of the VIP receptors
(VPAC1 and VPAC2) have been cloned and sequenced, and there is fifty percent
homology between VPAC1 and VPAC2 (Rubino et al., 1996). Unlike the PAC1
21
receptor, VPAC1 and VPAC2 bind both PACAP and VIP with similar affinity
(Gourlet et al., 1995). Also, no splice variations of VPAC1 and VPAC2 are known
to exist. To investigate the presence of VPAC1 and VPAC2 receptor transcripts in
the intracardiac neurons of neonatal rats, specific oligonucleotide primers were
developed to amplify non-homologous regions of the receptor transcripts (Fig
2.3A,C). Whole tissue RNA extracts from the indicated tissues were reverse
transcribed and amplified via PCR using these primers. Both VPAC1 and VPAC2
transcripts were detected in cardiac associated tissues (Fig 2.3A,C). Among
these tissues, it was obvious that VPAC2 was the predominant messenger RNA
transcribed. Restriction digests of the resultant bands for both VPAC1 (Nde I)
and VPAC2 (Van91I) support the data that these receptor transcripts are present
in cardiac associated tissues (Fig 2.3B,D).
VPAC mRNA expression in isolated rat intracardiac neurons
To complete the investigation of VPAC1 and VPAC2 receptor transcripts,
neonatal rat intracardiac neurons were studied using single-cell RT-PCR. Figure
2.4A shows the reaction results for five isolated neurons using the VPAC1 F4R4
primers. RT-PCR reaction results for five isolated neurons using the VPAC2
F4R4 primers are shown in figure 2.4B. The two single-cell RT-PCR gels clearly
demonstrate that VPAC2 is the most abundant VIP receptor transcript within
these single neurons. Sequences of RT-PCR products from two individual
neurons identically matched with the sequence of the VPAC2 receptor (Z25885).
22
Unlike in the adult guinea pig, neonatal rat intracardiac neurons and/or
associated tissue do express the PACAP- and VIP- associated receptors VPAC1
and VPAC2, with VPAC2 being the predominant species. This supports previous
electrophysiological findings suggesting the presence of a receptor that binds
with high affinity to the ligand VIP (Liu et al., 2000).
Individual intracardiac neuron expression pattern of PAC1 and VPAC
mRNA
Splice variation of the neonatal rat PAC1 receptor was further scrutinized
by relating the data discovered in the two previous figures (Fig 2.5A,B,C). The
first bar graph depicts the percent of intracardiac neurons expressing the
indicated combinations of PAC1 receptor variants (Fig 2.5A). Neurons exhibit
three distinct expression patterns of PAC1 isoforms: 1)PAC1-short, 2)PAC1-HOP,
and 3)PAC1-short and PAC1-HOP. No PAC1-very short, PAC1-HIP, or PAC1-
HIPHOP transcripts were detected. The second bar graph indicates the percent
of neurons expressing either the PAC1-short or the PAC1-HOP (alone or with
other isoforms), and -HOP1 versus -HOP2 transcripts (cloned sequences) (Fig
2.5B). Of all of the neurons studied, the majority expressed the -HOP variant
and nearly half of the neurons expressed the short variant. Of the PAC1-HOP
isoforms, the HOP1 was expressed predominantly. The last bar graph depicts
the optical density of PAC1-HOP1 and PAC1-short receptor transcripts in cells
expressing both sequence variants (Fig 2.5C). Optical density of individual
23
bands was determined using a GS 700 Imaging Densitometer (BIO-RAD) and
Multi-Analyst (BIO-RAD) software. Figure 2.5C shows that in neurons which
expressed both PAC1-HOP and PAC1-short transcripts, PAC1-HOP1 was the
predominant species. Thus, three distinct expression patterns of PAC1 receptor
isoforms were observed in isolated intracardiac neurons. The PAC1-HOP1
receptor isoform is the most common sequence variant present in rat intracardiac
neurons and is expressed at higher levels than the PAC1-short variant.
24
TABLE 2.1: Table showing the sequence of oligonucleotide primers used in this study and the predicted size for
the individual products. a All PAC1 splice variants containing a cassette insert in the third cytoplasmic loop (PAC1-
HIP, -HOP, and –HIPHOP) have the short insert in the amino terminus region, and thus their RT-PCR products will
be indistinguishable from the PAC1-short when the PAC1-2 primers are used.
25
Receptor Primer Sequence (5’ to 3’) Variant Size (bp)
Very short 303
Short 303
HIP 387
FWD: CTTGTACAGAAGCTGCAGTCCCCAGACATG
HOP1 387
HOP2 384
HIPHOP1 471
PAC1-1
REV: CCCGTGCTTGAAGTCCATAGTGAAGTAACGGTTCACCTT
HIPHOP2 468
FWD: TGTAAGCTGCCCTGAGGTCT Very short 430
PAC1
PAC1-2 REV: CACCACGCAGTAGTGGAAGA Shorta 493
FWD: TCTTCAACAGCGGGGAGATAGACCACTGC VPAC1 VPAC1-1
REV: GAAACCCTGGAAAGAGCCCACGACAAGTTC
__ 554
FWD: ATCCTTCCTCCCAGCAGGTGTTTCC VPAC2 VPAC2-1
REV: GTATCTGTAGGGCGCTTTCTGAGCCATTCC
__ 565
26
FIGURE 2.1: Expression of PAC1 receptor isoforms in cardiac tissue. (A)
Schematic representation of the PAC1 receptor indicating the regions amplified
by the PAC1-1 and PAC1-2 primer pairs. The locations of the cassette inserts
that contribute to the different PAC1 isoforms are also shown. RT-PCR results
for RNA extracts from the indicated tissues using the primers PAC1-1 (B) and
PAC1-2 (C). STDS, 100 bp ladder standards; PIT, pituitary gland; IC, intracardiac
ganglia; VENT, ventricles. Arrows indicate predicted sizes for different splice
variants of the PAC1 receptor. *See note in Table 2.1.
27
FIGURE 2.2: Detection of PAC1 receptor isoforms in single intracardiac neurons.
(A) RT-PCR results obtained from individual intracardiac neurons using the
PAC1-1 (A) and PAC1-2 (B) primers. Arrows indicate the predicted sizes for
different splice variants of the PAC1 receptor (see Table 2.1); STDS, 100 bp
ladder standards.
28
FIGURE 2.3: Expression of VPAC1 and VPAC2 receptor isoforms in cardiac
tissue. Schematic representations of the VPAC1 and VPAC2 receptors indicating
the regions amplified by the VPAC1 F4R4 primers (A) and VPAC2 F4R4 primers
(C), as well as RT-PCR results for RNA extracts from the indicated tissues using
these oligonucleotide primer sets, respectively. Restriction digests of the
resultant bands for VPAC1 (Nde I) (B) and VPAC2 (Van91I) (D), supporting the
data that these receptor transcripts are present in cardiac associated tissues.
Arrows indicate the predicted sizes for VPAC1 and VPAC2 (see Table 2.1).
29
FIGURE 2.4: Detection of VPAC receptor transcripts in single intracardiac
neurons. RT-PCR results obtained from individual intracardiac neurons using the
VPAC1 F4R4 (A) and VPAC2 F4R4 (B) oligonucleotide primers. Arrows indicate
the predicted sizes for VPAC1 and VPAC2 (see Table 2.1). (C) Sequences of the
565 bp size RT-PCR product from two individual neurons aligned with the
sequence of the VPAC2 receptor (GI: 414188). PCR products were gel purified
(Qiagen) and cloned using the pGEM-T Easy Vector System (Promega).
30
FIGURE 2.5: Expression pattern of PAC1 isoforms and VPAC transcripts in
intrinsic cardiac neurons. (A) Comparison of the number of cells expressing
either PAC1-short, -HOP1 or –HOP2 transcripts (n = 34), VPAC1 (n = 32) or
VPAC2 (n = 30). (B) Percentage of intracardiac neurons expressing the indicated
combinations of PAC1 receptor variants (n = 28). (C) Optical density of the
PAC1-short and PAC1-HOP1 receptor transcripts in cells expressing both
sequence variants (n = 10). Asterisk denotes significant difference (p < 0.05).
31
DISCUSSION
PACAP and VIP peptides have a wide range of neuromodulatory roles in
the peripheral nervous system, potentially through the receptors PAC1, VPAC1,
or VPAC2. Expression of all three receptors within intracardial neurons was
investigated using RT-PCR. Initially, whole tissue RNA extracts of cardiac
associated tissue were reverse transcribed, and the resultant cDNA was
amplified using specific primers for each of the three receptors. But because the
primary interest was intracardiac neurons, further examination needed to be
completed to prove that these receptor transcripts are present in these neurons.
Therefore, single-cell RT-PCR was initiated to verify PAC1, VPAC1, and VPAC2
transcripts within the intracardiac neurons.
We conclude that over 90% of rat intracardiac ganglion neurons express
transcripts encoding the PACAP selective receptor PAC1. This finding is
consistent with electrophysiological experiments previously conducted.
Individual neurons express one or more isoforms of the PAC1 receptor. The
sequence variations occur in the domain encoding the third cytoplasmic loop,
which is responsible for G-protein regulation, selectivity and coupling. Neurons
exhibit three distinct expression patterns of PAC1 isoforms: 1)PAC1-short,
32
2)PAC1-HOP, and 3)PAC1-short and PAC1-HOP. No PAC1-very short, PAC1-
HIP, or PAC1-HIPHOP transcripts were detected. Rat intracardiac neurons
primarily express the PAC1-HOP1 receptor isoform, in contrast to guinea pig
intracardiac neurons where the PAC1-very short is the most abundant sequence
variant. In addition to PAC1, intracardiac neurons express VPAC1 and VPAC2
transcripts, with the latter being found in greater proportion of the neurons. This
observation is consistent with the cellular effects of VIP in intracardiac neurons.
The relationship between these receptors, signal transduction and down stream
effector targets remain to be determined.
33
CHAPTER 3
VPAC RECEPTOR MODULATION OF NEUROEXCITABILITY IN
INTRACARDIAC NEURONS: DEPENDENCE ON INTRACELLULAR CALCIUM
MOBILIZATION AND SYNERGISTIC ENHANCEMENT BY PAC1 RECEPTOR
ACTIVATION
INTRODUCTION
Pituitary adenylate-cyclase activating polypeptide (PACAP) and
vasoactive intestinal polypeptide (VIP) are pleiotropic neuropeptides that belong
to the glucagon/secretin/growth hormone-releasing factor family of peptides
(Warren et al., 1991; Ishizuka et al., 1992; Basler et al., 1995; Cardell et al.,
1997). These neuropeptides have pronounced effects on the central nervous
system, as well as on neurons and effector targets of the autonomic nervous
system. For example, in the central nervous system, PACAP has been shown to
be involved in hippocampal synaptic plasticity and associative learning in mice
(Hashimoto et al., 2002), while VIP has been shown to regulate intrathalamic
rhythm activity and may modulate information transfer through the
thalamocortical circuit (Lee and Cox, 2003). In the peripheral nervous system,
PACAP and VIP appear to play a major role in autonomic regulation of the
cardiovascular system. VIP has been shown to cause positive chronotropic and
34
inotropic effects (Rigel et al., 1989; Karasawa et al., 1990; Yonezawa et al.,
1996). In contrast, PACAP has been shown to cause a transient increase in
sinus rate and atrial contractility that is followed by negative chronotropic and
inotropic responses in isolated canine atria (Hirose et al., 1997). Also, both
PACAP and VIP are potent dilators of coronary arteries (Huang et al., 1993;
Kawasaki et al., 1997). While these neuropeptides can directly influence cardiac
tissues, the effects of PACAP and VIP on the heart are in part due to
neuroregulatory effects on parasympathetic intracardiac ganglia (Yonezawa et
al., 1996; Hirose et al., 1997; Seebeck et al., 1996; Armour et al., 1993).
Mammalian intracardiac ganglia play a major role in neuronal control of
the heart and are believed to be capable of independently monitoring and
influencing cardiac function. A variety of neurotransmitters and neuromodulators,
including PACAP and VIP, appear to be involved in the relaying of information
within intracardiac ganglia and onto effector targets such as cardiac valves,
coronary arteries and cardiomyocytes. PACAP and VIP have been detected by
immunocytochemistry in nerve fibers in the heart, coronary vasculature and
cardiac parasympathetic ganglia (Della et al., 1983; Weihe et al., 1984; Seabrook
et al., 1990; Gulbenkian et al., 1993; Braas et al., 1998; Horackova et al., 2000).
Although extrinsic fibers may be one source of these neuropeptides in the heart
(Calupca et al., 2000), PACAP and VIP are also found in cell bodies and nerve
fibers of intrinsic cardiac neurons (Della et al., 1983; Weihe et al., 1984;
Seabrook et al., 1990; Gulbenkian et al., 1993; Braas et al., 1998; Horackova et
35
al., 2000). Despite the fact that data clearly demonstrate that these endogenous
neuropeptides are potent regulators of the cardiovascular system, the cellular
mechanisms mediating their effects remain poorly understood. There are also
conflicting reports in the literature concerning the mechanisms by which PACAP
and VIP regulate the function of intrinsic cardiac neurons. For example, it has
been suggested that PACAP and VIP modulate neurotransmission in rat cardiac
ganglia by potentiating nicotinic acetylcholine receptors (Cuevas and Adams,
1996; Liu et al., 2000), whereas studies on guinea pig intracardiac neurons
indicate that PACAP, but not VIP, regulates the excitability of intrinsic cardiac
neurons, possibly by influencing the K+ conductance, IA (Miura, A. et al., 2001;
Braas et al., 1998).
PACAP and VIP evoke their effects via the activation of specific G-protein
coupled receptors (Christophe, 1993; Shivers et al., 1991). The PAC1, or
PACAP-selective receptors, bind both active forms of PACAP, PACAP-27 and
PACAP-38, with equally high affinities, but VIP with much lower affinity (Gourlet,
et al., 1995); whereas, non-selective VPAC (VPAC1 and VPAC2) receptors
recognize PACAP-27, PACAP-38 and VIP with similar high affinities (Gottschall
et al., 1990; Gottschall et al, 1991; Cauvin et al., 1990; Cauvin et al, 1991; Lam
et al., 1990; Robberecht et al., 1992; Suda et al., 1992). At least seven isoforms
of the PAC1 receptor exist due to alternative splicing of the mRNA, while no
splice variants of VPAC1 or VPAC2 are presently known to exist. These different
PAC1 isoforms and VPAC receptor types are known to couple to distinct second
36
messenger pathways (Spengler et al., 1993; Pantaloni et al., 1996; Chatterjee et
al., 1996). Rat intrinsic cardiac neurons express several isoforms of the PAC1
(PAC1-short, PAC1-HOP1, PAC1-HOP2), as well as both VPAC receptors
(DeHaven and Cuevas, 2002).
The functional consequences of the PAC1/VPAC receptor heterogeneity, which is
also observed in other neuron types, such as mammalian cortical neurons,
remain to be fully elucidated. The present study examined the ability of PACAP
and VIP to regulate the electrical properties of intrinsic cardiac neurons from
neonatal rats, and to modulate [Ca2+]i in these cells. Both PACAP and VIP
depolarize intracardiac neurons and increase [Ca2+]i by evoking Ca2+ release
from ryanodine-sensitive intracellular stores and promoting Ca2+ entry through
the plasma membrane. PACAP, but not VIP, also increased the number of
action potentials fired by neurons in response to depolarizing current pulses.
The changes in neuroexcitability evoked by PACAP and VIP were blocked by
inhibiting the [Ca2+]i elevations produced by the neuropeptides.
37
METHODS
Membrane currents and [Ca2+]i were investigated in isolated intracardiac
ganglion neurons of neonatal rats (4-7 days old). The preparation and culture of
isolated intrinsic cardiac neurons has been previously described (Fieber and
Adams, 1991). Dissociated neurons were plated onto poly-L-lysine coated glass
coverslips and incubated at 37°C for 36-72 h under a 95% air, 5% CO2
environment prior to the experiments.
Microfluorometric measurements
The calcium sensitive dye fura-2 acetoxymethylester (fura-2-AM) was
used for measuring intracellular free calcium concentrations ([Ca2+]i) in
intracardiac neurons at room temperature, as previously described (Smith and
Adams, 1999). Cells plated on coverslips were then incubated for 1 hour at room
temperature in physiological saline solution (PSS) consisting of (in mM): 140
NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 7.7 glucose and 10 HEPES (pH to 7.2 with
NaOH), which also contained 1 µM fura-2-AM and 0.1 % dimethyl sulfoxide
(DMSO). The coverslips were then washed in PSS (fura-2-AM free) prior to the
38
experiments being carried out. All solutions were applied via a rapid application
system identical to that previously described (Cuevas and Berg, 1998).
A Sutter DG-4 high-speed wavelength switcher (Sutter Instruments Co.,
Novato, CA) was used to apply alternating excitation with 340 nM and 380 nM
ultraviolet light. Fluorescent emission at 510 nm was captured using a Cooke
Sensicam digital CCD camera (Cooke Corporation, Auburn Hills, MI) and
recorded with Slidebook 3.0 software (Intelligent Imaging Innovations, Denver,
CO) on a Pentium IV computer. Changes in [Ca2+]i were calculated using the
Slidebook software (Intelligent Imaging Innovations, Denver, CO) from the
intensity of the emitted fluorescence following excitation with 340 and 380 nm
light, respectively, using the equation:
[Ca2+]i = Kd Q (R-Rmin)/(Rmax-R)
where R represents the fluorescence intensity ratio (F340/F380) as determined
during experiments, Q is the ratio of Fmin to Fmax at 380 nm, and Kd is the Ca2+
dissociation constant for fura-2. Calibration of the system was performed using
the Molecular Probes (Eugene, OR) Fura-2 Calcium Imaging Calibration Kit and
values were determined to be: Fmin/Fmax (23.04), Rmin (0.31), Rmax (8.87).
Electrophysiology
Coverslips plated with the dissociated neurons were transferred to a 0.5
ml recording chamber mounted on a phase-contrast microscope (400X). The
extracellular recording solution was identical to the Ca2+ imaging PSS. The Ca2+-
39
free PSS contained no CaCl2 and included 1 mM ethylene glycol-bis(2-
aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). All drugs were bath applied
in PSS. In some electrophysiological and Ca2+ imaging experiments the order of
drug administration was reversed, that is PACAP was given in the presence of
other drugs (or in Ca2+-free media) prior to being given alone as a control. This
alteration in the protocol was done to ensure that any change in response was
not due to rundown. Currents were amplified and filtered (5 kHz) using an
Axoclamp-200B Amplifier, digitized with a 1322A DigiData digitizer (20kHz), and
collected on a Pentium IV computer using the Clampex 8 program (Axon
Instruments, Inc., Foster City, CA, USA). Data analysis was conducted using the
pClamp 8 program, Clampfit.
The whole-cell perforated-patch variation of the patch-clamp recording
technique was used as previously described (Rae et al., 1991; Xu and Adams,
1992). This configuration preserves intracellular integrity, preventing the loss of
cytoplasmic components and subsequent alteration of functional responses of
these neurons (Cuevas and Adams, 1996; Cuevas et al., 1997). The pipette
solution contained (mM): 75 K2SO4, 55 KCl, 5 MgSO4, 10 HEPES, 198 µg/ml
amphotericin B, and 0.4% DMSO. All electrophysiological recordings were
performed at room temperature.
Membrane potential responses to hyperpolarizing and depolarizing current
pulses (-100 pA to +200 pA) were determined in the absence and presence of
VIP/PACAP receptor ligands. The mean resting membrane potential, latency for
40
action potential firing, action potential duration, amplitude of
afterhyperpolarization and the number of action potentials elicited by depolarizing
current pulses were determined in the absence and presence of PAC1 and VPAC
receptor ligands. Membrane input resistance was also determined as described
previously (Xu and Adams, 1992).
Reagents and statistical analysis
All chemicals used in this investigation were of analytic grade. The
following drugs were used: amphotericin B, DMSO, mecamylamine, caffeine, 2-
aminoethoxydiphenyl borate (2-APB) (Sigma-Aldrich, St. Louis, MO); PACAP-27,
PACAP-38, L-8-K (VIP receptor binding inhibitor), [N-Ac-Tyr1, D-Phe2]-GRF (1-
29) and VIP (American Peptide Co., Sunnyvale, CA); tetrodotoxin (TTX),
ryanodine, thapsigargin and forskolin (Alomone Labs, Jerusalem, Israel); barium
chloride (Fisher Scientific, Hampton, NH2), and fura-2-AM (Molecular Probes,
Eugene, OR). Recombinant maxadilan and M65 were generous gifts from E. A.
Lerner (Massachusetts General Hospital, Harvard Medical School). All data are
presented as the mean ± SEM of the number of observations indicated.
Statistical analysis was conducted using SigmaPlot 8 (SPSS, Chicago, IL) and
paired and unpaired t-tests were used for within group and between group
comparisons, respectively.
41
RESULTS
PACAP and VIP directly increase free [Ca2+]i
The most abundant PAC1 receptor isoform expressed in neonatal rat
intrinsic cardiac neurons, PAC1-HOP, has been shown to elicit elevations in
intracellular calcium in cortical neurons (Grimaldi and Cavallaro, 2000).
However, the relationship between PACAP and intracellular calcium has not
been investigated in the autonomic neurons. The effect of PACAP on Ca2+
handling in intracardiac neurons was studied using fura-2 Ca2+-imaging
techniques. Figure 3.1A shows a representative trace of change in [Ca2+]i
(∆[Ca2+]i) as a function of time recorded from a neuron before, during and
following 2 minute application of 100 nM PACAP-27 (black trace). Intracellular
Ca2+ concentrations increased by >400 nM when PACAP-27 was applied, and
returned to near control levels upon washout of drug. In similar experiments
PACAP-27 significantly increased intracellular calcium by ~400%, from a
baseline of 65.3 ± 5.5 nM to a peak value of 335.1 ± 65.4 nM (n=23) (Fig 3.1B).
In all cells responding to PACAP, the elevations in [Ca2+]i were reversed when
PACAP was removed from the bath, and subsequent applications of PACAP to
the same cell produced [Ca2+]i increases of similar magnitude.
42
In a series of experiments, the PAC1 receptor-selective agonist,
maxadilan, and VPAC receptor-selective agonist, VIP, were used to determine if
the PACAP-induced elevations in [Ca2+]i are mediated exclusively by activation
of PAC1 receptors, or if VPAC receptors contribute to this phenomenon. Figure
3.1A also shows representative traces of change in [Ca2+]i recorded from two
neurons as a function of time before, during and following 2 minute application of
maxadilan (10 nM, dotted trace) and VIP (100 nM, gray trace), respectively.
Maxadilan, at a concentration (10 nM) specific for PAC1 (Moro and Lerner, 1997),
failed to elicit a change in [Ca2+]i. In contrast, VIP, at a concentration (100 nM)
that preferentially activates VPAC receptors (Gourlet et al., 1995), reversibly
increased [Ca2+]i by > 225 nM in this neuron. In 14 similar experiments 100 nM
VIP significantly increased mean peak [Ca2+]i by more than 225%, from 73.2 ±
12.1 nM to 239.1 ± 27.4 nM, which is an increase comparable to that observed
when PACAP-27 was used as the agonist (Fig 3.1B). Maxadilan (10 nM-100
nM), however, did not elicit any detectable changes in [Ca2+]i in any of the cells
tested (Fig 3.1B; n = 7), which suggests that the PAC1 receptor is not responsible
for the PACAP-induced increases in [Ca2+]i observed in these cells.
Increases in free [Ca2+]i evoked by PACAP and VIP are mediated by a VPAC
receptor
To confirm that activation of VPAC receptors is responsible for the
PACAP-induced elevations in [Ca2+]i, the PAC1 receptor-specific antagonist M65,
43
and VPAC receptor-specific antagonists L-8-K and [N-Ac-Tyr1, D-Phe2]-GRF (1-
29), were used. Figure 3.2 shows representative traces of change in [Ca2+]i as a
function of time during application of 100 nM PACAP-27 alone (black traces Fig
3.2A-C) or when the neuropeptide was co-applied with 100 nM M65 (Fig 3.2A,
gray trace), 5 µM L-8-K (Fig 3.2B, gray trace) or 100 nM [N-Ac-Tyr1, D-Phe2]-
GRF (1-29) (YF-GRF) (Fig 3.2C, gray trace). Only the VPAC receptor
antagonists were shown to block the effects of PACAP on [Ca2+]i. A bar graph of
results obtained in similar experiments is shown in Figure 3.2D, and reveals that
application of either L-8-K (n = 9) or [N-Ac-Tyr1, D-Phe2]-GRF (1-29) (YF-GRF, n
= 5) significantly attenuated PACAP-evoked elevations in [Ca2+]i in isolated
intracardiac neurons, while M65 (n = 5) had no effects on the neuropeptide-
induced changes in [Ca2+]i. These data further support the conclusion that the
PACAP and VIP evoked increases in [Ca2+]i are mediated by VPAC receptors
and not PAC1 receptors in these cells.
PACAP and VIP are known to modulate nicotinic acetylcholine receptors
(nAChRs) in intrinsic cardiac neurons (Liu et al., 2000). This raises the possibility
that the observed PACAP- and VIP-induced elevations in [Ca2+]i may be due to
regulation of presynaptic or postsynaptic nAChRs by the neuropeptides. Thus, to
verify that the effect of exogenously applied PACAP and VIP on [Ca2+]i are not
indirectly caused by nAChR modulation, the Na+ channel blocker TTX (400 nM)
and ganglionic nAChR blocker mecamylamine (25 µM) and were used to block
nicotinic neurotransmission pre- and post-synaptically, respectively. Neither the
44
application of TTX (n=4) nor mecamylamine (n=3) prevented the PACAP-induced
rise in [Ca2+]i in rat intracardiac neurons (data not shown). The application of
PACAP-27 (100 nM) in the presence of TTX significantly (p<0.01) elevated
[Ca2+]i from 107.1 ± 13.8 nM to 286.8 ± 25.1 nM, values comparable to the
calcium elevations seen in the absence of TTX. Similarly, PACAP-27 increase in
[Ca2+]i was unaffected by the application of mecamylamine (65.4 ± 9.5 nM to
236.6 ± 31.2 nM ), a 265% increase in intracellular calcium (data not shown).
These results suggest that the observed PACAP-induced rise in [Ca2+]i is not
dependent on neurotransmission.
PACAP and VIP mobilize Ca2+ from intracellular stores and evoke Ca2+ entry
through the plasma membrane.
PAC1 and VPAC receptors have been linked to both Ca2+ entry through
the plasma membrane via the activation of L-type calcium channels (Chatterjee
et al., 1996; Tanaka et al., 1998) and Ca2+ release from intracellular stores
(MacKenzie et al., 2001). Experiments were conducted to determine the source
of Ca2+ responsible for the VPAC mediated increase in [Ca2+]i. Application of
PACAP-27 produced an increase in [Ca2+]i that exhibited both a transient and a
sustained component (Fig 3.3A, black trace). Similar results were observed
when VIP was used as the agonist (data not shown). However, when PACAP
was applied in Ca2+-free PSS, the initial transient increase in [Ca2+]i was
observed, but the sustained component was abolished (Fig 3.3A, gray trace).
45
Figure 3.3B shows a bar graph of mean peak and sustained [Ca2+]i recorded
under normal and Ca2+-free conditions from 12 isolated intracardiac neurons.
Even though the magnitude of the transient peak [Ca2+]i remained unchanged
(normal PSS: 179.2 ± 17.6 nM; Ca2+-free PSS: 173.4 ± 17.8 nM), the sustained
component significantly decreased from 103.6 ± 11.5 nM to 45.7 ± 6.7 nM
following removal of extracellular Ca2+. This latter [Ca2+]i value was equivalent to
control baseline [Ca2+]i. Thus, the sustained component is dependent on
extracellular Ca2+ and is likely due to Ca2+ flux through the plasmalemma.
The presence of a PACAP-induced transient elevation in [Ca2+]i in the
absence of extracellular calcium suggested that activation of VPAC receptors
mobilized Ca2+ from intracellular stores in these neurons. In order to test this
hypothesis, we used the endoplasmic reticulum (ER) Ca2+-ATPase inhibitor,
thapsigargin, which specifically blocks Ca2+ reuptake leading to depletion of the
ER Ca2+ stores (Lytton et al., 1991). Prior to the experiments, dissociated
intracardiac neurons were incubated for 1 hr in 20 µM thapsigargin (in PSS, 37
°C). Experiments were then conducted to determine if PACAP increased [Ca2+]i
under these conditions. Untreated neurons collected from either the same
neonatal rat or animals from the same litter were used as a positive control to
determine if the neurons responded to PACAP. Figure 3.3C shows traces of
change in [Ca2+]i recorded from two neurons preincubated in 20 µM thapsigargin
and exposed to either 100 nM PACAP-27 or 500 µM acetylcholine (ACh).
PACAP failed to elicit a change in [Ca2+]i in these neurons, but ACh, which
46
promotes Ca2+ entry via plasma membrane nicotinic acetylcholine receptors in
intracardiac neurons (Beker et al., 2003), produced a pronounced transient
elevation in [Ca2+]i. In similar experiments, thapsigargin-pretreatment
significantly decreased the peak PACAP response from 370.3 ± 40.9 nM to 105.0
± 8.3 nM (n = 6; Fig 3.3D), with the latter value being indistinguishable from the
baseline [Ca2+]i measured in this group of cells (104.4 ± 16.5 nM). In contrast,
thapsigargin preincubation had no effect on the peak [Ca2+]i elevations produced
by rapid focal application of 500 µM ACh (Fig 3.3D). Thapsigargin also
eliminated the rise in [Ca2+]i elicited by bath application of 5 mM caffeine (Fig
3.3D), which has been shown to evoke release of Ca2+ from intracellular stores in
these cells (Beker et al., 2003). Therefore, depleting intracellular Ca2+ stores via
thapsigargin pretreatment abolished both the transient and sustained PACAP-
induced changes in [Ca2+]i.
PACAP induces mobilization of [Ca2+]i from caffeine- and ryanodine-
sensitive stores
Previous studies have demonstrated that rat intrinsic cardiac neurons
have two distinct ER Ca2+ stores, one that is sensitive to inositol 1,4,5-
trisphosphate (IP3) and a second that is sensitive to ryanodine and caffeine
(Smith and Adams, 1999; Beker et al., 2003). The PACAP/VIP receptors have
been shown to mobilize Ca2+ from both IP3- and ryanodine/caffeine-sensitive
stores in other cell types (Grimaldi and Cavallaro, 2000; Tanaka et al., 1998). To
47
determine if PACAP evokes release of Ca2+ from ryanodine/caffeine-sensitive
stores we used two approaches: 1) depleting the ryanodine/caffeine-sensitive
stores of Ca2+ by bath applying 5 mM caffeine and 2) blocking the release of Ca2+
from these stores by bath applying 10 µM ryanodine. Figure 3.4 shows
representative traces of change in [Ca2+]i in response to 5 mM caffeine (Fig 3.4A)
or 10 µM ryanodine (Fig 3.4C) recorded from two neurons (gray traces), and to
PACAP-27 (100 nM) in the absence (black traces) and presence (dotted traces)
of these drugs (Figs 3.4A and 3.4C, respectively). Both the transient and
sustained PACAP-evoked elevations in [Ca2+]i were suppressed by pretreatment
with caffeine or ryanodine. In 6 identical experiments, caffeine significantly
decreased peak ∆[Ca2+]i increase from 139.3 ± 32.5 nM to 21.3 ± 4.6 nM (Fig
3.4B). Similarly, ryanodine depressed the peak elevation in ∆[Ca2+]i from 162.2 ±
27.0 nM to 32.7 ± 10.9 nM (Fig 3.4D). The initial application of caffeine produced
an increase in [Ca2+]i (236.8 ± 34.1 nM) similar to that elicited by PACAP, but,
unlike PACAP, was often associated with Ca2+ oscillations (see Fig 3.4A).
Ryanodine, however, did not promote calcium release in any of the cells tested,
which is consistent with the drug acting as an inhibitor of the ryanodine receptor
at this concentration (Chu et al., 1990).
Our experiments with ryanodine, however, cannot rule out the possibility
that neuropeptide-stimulated IP3 production evokes small, local changes in
[Ca2+]i that initiate the release of Ca2+ from the caffeine/ryanodine stores. The
IP3-receptor antagonist 2-APB (Maruyama et al., 1997) was used to determine if
48
IP3-mediated Ca2+-induced-Ca2+ release is responsible for the transient elevation
in [Ca2+]i observed following VPAC receptor stimulation. Figure 3.5A shows
[Ca2+]i traces elicited by focal application of PACAP in intracardiac neurons in the
absence and presence of 2-APB (50 µM). 2-APB does not block the transient
elevation in [Ca2+]i produced by either PACAP or caffeine (Fig 3.5B). In contrast,
2-APB attenuated the increase in [Ca2+]i evoked by bath application of muscarine
(5 µM, Fig 3.5C), which is primarily due to release of Ca2+ from IP3-sensitive
stores (Beker et al., 2003). Figure 3.5D shows a bar graph of the mean peak
increase in [Ca2+]i evoked by PACAP, caffeine and muscarine in the absence and
presence of 2-APB. Whereas 2-APB significantly depressed the peak increases
in [Ca2+]i evoked by muscarine from 107 ± 25 nM to 31 ± 8 nM, it had no effect
on those elicited by PACAP or caffeine.
Whereas the transient component of the PACAP-induced elevation in
[Ca2+]i was unaffected by 2-APB, the sustained component was depressed in the
presence of the drug (Fig 3.5A). In 6 identical experiments the PACAP-induced
sustained increase in [Ca2+]i decreased from 66 ± 7 nM to 30 ± 6 nM (Fig 3.5E).
Similarly, the sustained elevations in [Ca2+]i observed following application of
caffeine or muscarine were also attenuated by 2-ABP (Figs 3.5E). At the
concentration used here (50 µM), 2-APB also directly blocks several subtypes of
transient receptor potential (TRP) ion channels (Clapham et al., 2001).
Therefore, these data suggest that capacitative Ca2+ entry through transient
49
receptor potential channels contributes to the sustained elevation in [Ca2+]i
evoked by VPAC receptor activation.
PACAP and VIP Induced Excitability in Rat Intracardiac Neurons
Earlier studies in intracardiac neurons from adult guinea pigs suggest that
PACAP, but not VIP, modulates neuroexcitability in these cells (Braas et al.,
1998). However, no such changes have been reported in neonatal rat
intracardiac neurons (Cuevas and Adams, 1996; Liu et al., 2000). Data suggest
that intracardiac neurons of these two species express different PACAP and VIP
receptor subtypes (Braas et al., 1998; DeHaven and Cuevas, 2002), and thus the
neuropeptides may have distinct cellular effects. For example, in contrast to
observations reported here, application of PACAP has not been shown to effect
[Ca2+]i in guinea pig intracardiac neurons. Thus, it seemed prudent to further
investigate the effect of PACAP and VIP on the electrical properties of rat
intrinsic cardiac neurons. Figure 3.6A shows a family of representative voltage
responses elicited by 500 ms, 150 pA depolarizing current pulses from an
isolated intracardiac neuron held under current-clamp mode using the whole-cell
perforated patch method. Membrane responses were recorded in the absence
(Control) and presence of bath applied PACAP-27 (100 nM), and following
washout of the neuropeptide (Wash). Application of PACAP depolarized the
neuron and increased the number of action potentials elicited by the current
pulse. The PACAP evoked membrane depolarization was observed in a majority
50
of the cells tested (54 of 73 cells) (Fig 3.6B). The resting membrane potential
(RMP) measured in intracardiac neurons was -52.0 ± 0.6 mV under control
conditions and depolarized to -47.7 ± 0.7 mV in the presence of PACAP-27. The
effect of PACAP on the RMP of these neurons was reversible upon washout of
drug (-51.3 ± 0.6). This difference was statistically significant (p < 0.01).
In most neurons tested the depolarizing current pulses evoked short,
adapting trains of action potentials (46 of 73 neurons), whereas 15 neurons fired
single action potentials and 12 neurons exhibited tonic firing when challenged
with similar current pulses. In a population of neurons (42 of 73), the number of
action potentials elicited was increased by 115%, from 3.2 ± 0.3 to 6.9 ± 0.5, by
100 nM PACAP-27 (Fig 3.6C). Following a 15 minute washout of the
neuropeptide, the number of action potentials induced by the current pulses
returned to near control levels (3.9 ± 0.5). The increase in action potentials
observed in the presence of PACAP was statistically significant when compared
to either control or washout. The population of neurons exhibiting augmented
action potential firing included phasic, adapting and tonic neurons. Thus, the
effect of PACAP was not associated with a cell type with distinct active
membrane properties. Examining the action potential characteristics of neurons
that showed increased firing revealed that PACAP significantly decreased the
action potential latency period from 6.0 ± 0.7 ms to 4.7 ± 0.5 ms (n = 7; p < 0.01)
and increased the afterhyperpolarization from -13.3 ± 6.1 mV to -21.4 ± 6 mV (n
= 7; p < 0.05). However, other parameters including action potential duration and
51
overshoot were not altered (data not shown). It is important to note that the
membrane depolarization and increase in action potential firing were not
correlated, and only 30 of 73 cells showed both a depolarization and increased
action potential firing. This observation suggests that two distinct mechanisms
are mediating these membrane responses.
The effects of PACAP were in part mimicked by the related neuropeptide,
VIP (Fig 3.7). Bath application of VIP depolarized a population of neurons (8 of
13) from -47.3 ± 1.7 to -44.5 ± 1.5 (Fig 3.7B). This depolarization was of lesser
magnitude than the PACAP-induced depolarization, but was statistically
significant, and reversed upon washout of the peptide (-45.9 ± 1.6). VIP affected
a population of neurons similar to PACAP (~60%). However, unlike PACAP, VIP
had no effects on action potential firing in these cells (Fig 3.7C). The number of
action potentials fired in response to 150 pA depolarizing current pulses before,
during and after washout of 100 nM VIP were 2.8 ± 0.5, 3.2 ± 0.6 and 3.4 ± 0.6,
respectively.
Membrane input resistance was investigated to determine whether the
neuropeptide-induced changes in excitability were associated with opening
and/or closing of ion channel(s). Current pulses (-100 pA) were applied to elicit
hyperpolarizations before, during and after PACAP or VIP (100 nM) application.
The amplitude of the sustained hyperpolarization was used to determine input
resistances. Neither PACAP nor VIP significantly altered the input resistance.
This observation suggests that the neuropeptides are likely exerting their effects
52
by simultaneously opening and closing multiple membrane channels resulting in
no net change in input resistance. Likewise, there was no correlation between
changes in input resistance and PACAP-induced modulation of neuroexcitability
in guinea pig intrinsic cardiac neurons (Braas et al., 1998).
Increases in neuroexcitability evoked by PACAP and VIP are in part
mediated by a VPAC receptor
In order to substantiate that VPAC receptors contribute to the PACAP and
VIP induced changes in intrinsic cardiac neuron excitability, the VPAC receptor
antagonist [N-Ac-Tyr1, D-Phe2]-GRF (1-29) was used. Figure 3.8A shows
representative action potentials evoked from a single neuron in response to a
depolarizing current pulse (300 ms, 150 pA) in the absence and presence of 100
nM PACAP-27 and/or 100 nM [N-Ac-Tyr1, D-Phe2]-GRF (1-29). Whereas,
PACAP depolarized the cell by 3.6 mV and increased action potential firing in the
cell when administered alone, PACAP failed to change the resting membrane
potential when co-applied with [N-Ac-Tyr1, D-Phe2]-GRF (1-29) (PACAP-27 + YF-
GRF). Bath application of [N-Ac-Tyr1, D-Phe2]-GRF (1-29) alone (YF-GRF) had
no direct effects on the active or passive membrane properties of the cell. In
similar experiments, co-application of [N-Ac-Tyr1, D-Phe2]-GRF (1-29) blocked
PACAP-induced depolarizations in cells that were shown to significantly respond
to the neuropeptide (Fig 3.8B). Figure 3.8C shows a bar graph of the number of
action potentials elicited by identical depolarizing current pulses in cells exposed
53
to PACAP prior to or following incubation in [N-Ac-Tyr1, D-Phe2]-GRF (1-29).
While PACAP increased firing from 2.7 ± 0.3 to 6.3 ± 1.5 action potentials under
control conditions, the neuropeptide had no effect on action potential firing
following application of [N-Ac-Tyr1, D-Phe2]-GRF (1-29).
Although VIP in part mimicked the depolarizing effects of PACAP, the
magnitude of the VIP-induced depolarization was only ~60% of that elicited by
PACAP. Also, VIP had no effects on action potential firing in these cells. This
observation suggests that PAC1 receptors may also contribute to the PACAP-
induced changes in the electrical properties of intracardiac neurons. To examine
this hypothesis the PAC1-selective antagonist, M65, was used in a series of
experiments to determine if inhibition of PAC1 receptors alters the PACAP-
evoked increase in excitability. Co-application of M65 (10 nM) depressed both
the PACAP-induced depolarization and increase in action potential firing in
neurons shown to respond to 100 nM PACAP-27 (Table 3.1). Surprisingly, bath
application of maxadilan (10-100 nM) failed to produce depolarizations in these
neurons (Table 3.1). Taken together these data suggest that stimulation of
VPAC receptors increases neuroexcitability in intrinsic cardiac neurons, and that
this increase in neuroexcitability is enhanced by concurrent activation of PAC1
receptors.
54
Effects of Ca2+-free extracellular conditions on PACAP-induced intracardiac
neuroexcitability
Our observation that stimulation of VPAC receptors evokes elevations in
[Ca2+]i raises the possibility that this change in [Ca2+]i may contribute to the
observed enhancement in neuroexcitability evoked by PACAP. This hypothesis
is further supported by the noted association of increases in the amplitude of the
[Ca2+]i-dependent action potential afterhyperpolarization with augmented action
potential firing in the cells. To determine the role of PACAP-induced Ca2+
mobilization in intracardiac neuroexcitability, whole-cell perforated patch
experiments were conducted in current-clamp mode under Ca2+-free extracellular
conditions. In rat intracardiac neurons, removal of extracellular Ca2+ over a short
period of time (minutes) not only prevents Ca2+ conductance through the plasma
membrane, but also results in depletion of Ca2+ from internal stores (unpublished
observation). Thus, PACAP-induced [Ca2+]i mobilization is blocked by washing
the intracardiac neurons with Ca2+-free PSS. Figure 3.9A shows representative
action potentials evoked from a neuron by depolarizing current pulses (300 ms,
150 pA) in the absence and presence of PACAP-27 (100 nM) under control
conditions (2.5 mM Ca2+) and in Ca2+-free conditions (0 Ca2+, 1 mM EGTA). In
the presence of external Ca2+, PACAP depolarized the neuron from -51.5 mV to -
47.8 mV and increased the number of action potentials fired in response to the
current pulse. However, PACAP failed to produce either of these changes when
extracellular Ca2+ was removed. In similar experiments, Ca2+-free conditions
55
inhibited the ability of PACAP to alter the resting membrane potential (Fig 3.9B)
and the action potential firing properties (Fig 3.9C) of neurons shown to respond
to the neuropeptide under control conditions. In normal extracellular Ca2+ (2.5
mM), PACAP-27 (100 nM) depolarized the cells from -51.1 ± 1.6 mV to -46.8 ±
1.9 mV, whereas in Ca2+-free PSS the resting membrane potential was -50.9 ±
1.0 mV and -51.9 ± 1.8 mV in the absence and presence of PACAP, respectively
(n=6). Similarly, in 5 neurons that exhibited increased action potential firing when
PACAP was bath applied in PSS containing normal Ca2+ (2.5 mM), removal of
extracellular Ca2+ abolished the neuropeptide-mediated effects (Fig 3.9C). Thus,
PACAP-evoked changes in excitability are dependent on the neuropeptide-
elicited elevation in [Ca2+]i.
In order to confirm that in the absence of extracellular Ca2+ intracardiac
neurons were capable of firing multiple action potentials, 1 mM Ba2+ was bath
applied in Ca2+-free PSS. Barium (1 mM) has previously been shown to block
membrane K+ channels and to increase neuroexcitability in intracardiac neurons
(Xu, Z.J. and Adams, D.J., 1992). Under these Ca-free conditions barium
significantly depolarized (p<0.01) the intracardiac neurons from -51.1 ± 1.9 mV to
-43.8 ± 1.5 mV and increased action potential firing (p<0.01) from a mean of 1.8
± 0.3 to 5.8 ± 0.5 action potentials (n = 5) (data not shown). These effects were
reversible upon washout of Ba2+. Thus, removal of extracellular Ca2+ does not
prevent intracardiac neurons from exhibiting increased neuroexcitability.
56
Effects of Ryanodine on PACAP-induced intracardiac neuroexcitability
Electrophysiological experiments conducted under Ca2+-free conditions
suggested that PACAP-induced changes in excitability were at least in part due
to the effect of the neuropeptide on [Ca2+]i. Further experiments were conducted
to determine if PACAP-induced mobilization of Ca2+ from ryanodine-sensitive
intracellular stores contributed to the increased neuroexcitability observed in the
presence of the neuropeptide. Figure 3.10A shows representative action
potentials evoked in response to a depolarizing current pulse (300 ms, 150 pA)
during bath application of 100 nM PACAP-27 before and after pretreatment with
ryanodine (10 µM). Whereas, PACAP increased action potential firing in this cell
under control conditions, preincubation in ryanodine eliminated the PACAP-
induced changes in firing characteristics. Moreover, preincubation in ryanodine
also prevented PACAP from depolarizing the cell, since PACAP depolarized the
cell by 3.7 mV under control conditions but failed to alter the resting membrane
potential in the presence of ryanodine. In similar experiments, preincubation in
ryanodine blocked PACAP-induced depolarizations in cells that were shown to
depolarize in the presence of the neuropeptide (Fig 3.10B). Figure 3.10C shows
a bar graph of the number of action potentials (± SEM) elicited by identical
depolarizing current pulses in cells exposed to PACAP prior to and following
incubation in ryanodine. Although PACAP increased firing from 1.7 ± 0.3 to 5.3 ±
0.9 action potentials under control conditions, PACAP had no effects on action
potential firing following application of ryanodine. This observation further
57
establishes elevations in [Ca2+]i as a obligatory component in the PACAP/VIP
enhancement of excitability in these cells.
58
FIGURE 3.1: PACAP and VIP, but not maxadilan, reversibly increase [Ca2+]i in
neonatal rat intracardiac neurons. (A) Graph of change in [Ca2+]i (∆[Ca2+]i),
defined as peak [Ca2+]i minus baseline [Ca2+]i, as a function of time for single
intracardiac neurons recorded before, during and following 2 minute bath
application of 100 nM PACAP-27 (black trace), 100 nM VIP (gray trace) and the
PAC1-selective agonist maxadilan (10 nM) (dotted trace). The solid line above
the traces indicates peptide application times. Individual images were captured
at 0.5 Hz. (B) Bar graph of mean peak [Ca2+]i (± SEM) recorded before
(baseline), during (drug) and following (wash) PACAP-27 (n=23), VIP (n=14) and
maxadilan (n=7) application in different preparations. Asterisks denote significant
difference (p<0.01) from respective baseline [Ca2+]i values. No significant
difference exists between the PACAP-27 and VIP induced responses.
59
60
FIGURE 3.2: PACAP-induced mobilization of [Ca2+]i in rat intracardiac neurons is via VPAC receptor activation.
Representative traces of ∆[Ca2+]i as a function of time recorded from 3 neurons (A-C, respectively) in response to 100 nM
PACAP-27 application in the absence (black traces) and presence (gray traces) of the PAC1 receptor antagonist, M65 (A,
100 nM), or the VPAC receptor antagonists L-8-K (B, 5 µM) or [N-Ac-Tyr1, D-Phe2]-GRF (1-29) (C, YF-GRF, 100 nM).
The solid line above the traces indicates PACAP-27 bath application times. All antagonists were pre-applied for 5 min
prior to and during the administration of PACAP. (D) Bar graph of mean peak [Ca2+]i (± SEM) before (Baseline), during
(Drug) and following washout (Wash) of PACAP or PACAP and the indicated PAC1/VPAC antagonists. Asterisk denotes
significant difference (p<0.01) from the baseline [Ca2+]i recorded for each experimental condition; PACAP-27 (n = 19);
M65 + PACAP-27 (n = 5), L-8-K + PACAP-27 (n=9), YF-GRF + PACAP-27 (n=5).
61
62
FIGURE 3.3: The sustained, but not the transient, component of the PACAP-induced increase in [Ca2+]i is dependent on
extracellular Ca2+. (A) Typical traces of ∆[Ca2+]i recorded from a single neuron in response to 2 min bath application of
PACAP-27 (100 nM) in normal physiological extracellular Ca2+ (Control, black trace) and Ca2+-free conditions (0 Ca2+,
gray trace). Cells were exposed to the Ca2+-free PSS only during the PACAP application to prevent depletion of Ca2+
from intracellular stores. (B) Bar graph of mean peak [Ca2+]i (± SEM) before (Baseline), during (Drug) and following
washout (Wash) of PACAP recorded in normal and Ca-free conditions (n=12). Asterisks denote significant difference
(p<0.01) from baseline [Ca2+]i, and # denotes significant difference from sustained [Ca2+]i evoked by PACAP in normal
extracellular Ca2+. (C) Representative ∆[Ca2+]i traces recorded in response to 1.5 second application (arrow) of 500 µM
acetylcholine (ACh, gray trace) and 100 nM PACAP (black trace) from two neurons preincubated in thapsigargin (20 µM,
1 hr, 37° C). (D), Bar graph of mean baseline and mean peak [Ca2+]i (±SEM) following addition (1.5 s) of acetylcholine
(500 µM), PACAP-27 (100 nM) and caffeine (5 mM) in control neurons and neurons pretreated for 1 hr in thapsigargin.
Asterisks denote significant difference (p<0.01) from baseline [Ca2+]i. The # symbols denote significant difference
(p<0.01) from control PACAP and caffeine responses.
63
64
FIGURE 3.4: PACAP evokes a rapid transient elevation of [Ca2+]i in intracardiac neurons by releasing Ca2+ from
caffeine/ryanodine-sensitive internal stores. (A) Typical time courses of ∆[Ca2+]i recorded in response to PACAP-27 (100
nM) application before (black trace) and after (dotted trace) preincubation in 5 mM caffeine (gray trace). Caffeine was
applied until [Ca2+]i baseline stabilized (~10 min) and continued through the PACAP application (PACAP-27 + Caffeine) .
(B) Bar graph of mean ∆[Ca2+]i (± SEM) evoked by 100 nM PACAP, 5 mM caffeine or 100 nM PACAP following 5 mM
caffeine preincubation (n=6). Asterisk denotes significant difference (p<0.01) from the PACAP control. (C)
Representative ∆[Ca2+]i trace recorded in response to application of PACAP-27 (100 nM), ryanodine (10 µM) or PACAP
following ryanodine preincubation. PACAP was bath applied for 2 min, while ryanodine was applied for ~10 min. (D) Bar
graph of mean ∆[Ca2+]i (± SEM) recorded under the same conditions as (C); n = 9 for each condition. Asterisk denotes
significant difference (p<0.01) from PACAP control peak ∆[Ca2+]i.
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FIGURE 3.5: The sustained, but not the transient, component of the PACAP-induced increase in [Ca2+]i is blocked by 2-
APB. Shown are typical time courses of ∆[Ca2+]i recorded in response to 100 nM PACAP-27 (A), 5 mM caffeine (B), or 5
µM muscarine (C) before (Control, black trace) and after preincubation in 50 µM 2-APB (+2-APB, gray trace). Each panel
represents recordings obtained from a single cell. 2-APB was applied for 10 min and continued through the application of
drug (PACAP, caffeine, or muscarine). Shown are bar graphs of mean peak ∆[Ca2+]i (± S.E.M.) (D) and mean sustained
∆[Ca2+]i (± S.E.M) (E) evoked by 100 nM PACAP-27 (n = 6), 5 mM caffeine (n = 7), or 5 µM muscarine (n = 6) in the
absence (Control) and presence of 50 µM 2-APB (+2-APB). The asterisks denote significant difference (p < 0.01) from the
corresponding control.
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FIGURE 3.6: PACAP increases neuroexcitability in neonatal rat intracardiac neurons. (A) Action potentials elicited from a
single current-clamped neuron in response to 150 pA depolarizing current pulses in the absence (Control) and presence
of 100 nM PACAP-27 (PACAP-27), and following washout of drug (Wash). (B) Scatter plot of mean resting membrane
potential (± SEM) recorded under identical conditions as in (A) from 54 neurons. (C) Bar graph of the mean number of
action potentials (± SEM) elicited by depolarizing current pulse (500 ms, 150 pA) from neurons (n = 42) in the absence
(Control) and presence of 100 nM PACAP (PACAP) and following washout of the neuropeptide (Wash). Asterisks denote
significant difference (p<0.01).
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FIGURE 3.7: VIP-induced changes in neuroexcitability within neonatal rat intracardiac neurons. (A) Action potentials
elicited form a single current-clamped neuron in response to 150 pA depolarizing current pulses in the absence (Control)
and presence of 100 nM VIP (VIP), and following washout of drug (Wash). (B) Scatter plot of mean resting membrane
potential (± SEM) recorded under identical conditions as in (A) from 8 neurons. (C) Bar graph of the mean number of
action potentials elicited by depolarizing current pulse (300 ms, 150 pA) from neurons (n = 8) in the absence (Control) and
presence of 100 nM VIP (VIP) and following washout of the neuropeptide (Wash). Asterisk denotes significant difference
(p<0.01).
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FIGURE 3.8: PACAP-induced changes in intracardiac neuroexcitability are inhibited by the VPAC antagonist [N-Ac-Tyr1,
D-Phe2]-GRF (1-29). (A) Action potentials evoked from a single intracardiac neuron by depolarizing current pulse (300
ms, 150 pA) in the absence (Control) and presence of 100 nM PACAP-27 (PACAP), 100 nM [N-Ac-Tyr1, D-Phe2]-GRF (1-
29) (YF-GRF), or 100 nM [N-Ac-Tyr1, D-Phe2]-GRF (1-29) and 100 nM PACAP-27 (PACAP-27 + YF-GRF). (B) Scatter
plot of mean resting membrane potential (RMP) (± SEM) and (C) bar graph of the average number of action potentials (±
SEM) elicited by depolarizing current pulse (300 ms, 150 pA) for neurons (n = 7) studied under the same conditions as
described above in (A), and following washout of the peptides (Wash). Asterisks denote significant difference from control
(p<0.01).
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FIGURE 3.9: PACAP-induced changes in intracardiac neuroexcitability are blocked by removal of extracellular Ca2+ and
depletion of internal Ca2+ stores. (A) Action potentials evoked in response to a depolarizing current pulse (300 ms, 150
pA) from a single intracardiac neuron in the absence and presence of 100 nM PACAP in normal PSS (Control; PACAP
100 nM) or in Ca2+-free PSS (Ca2+ 0 mM, EGTA 1 mM; Ca2+ 0 mM, EGTA 1 mM, PACAP), respectively. Cells were
exposed to Ca2+-free PSS for ~10 min prior to the Ca2+-free experiments. (B) Scatter plot of mean resting membrane
potential (RMP) (± SEM) recorded from 6 neurons under control and Ca2+-free conditions (0 Ca2+) in the absence
(Control, Wash) and presence of bath applied PACAP-27 (100 nM; PACAP). (C) Bar graph of the average number of
action potentials (± SEM) elicited by depolarizing current pulse (300 ms, 150 pA) in intracardiac neurons studied under the
same conditions as described for panel (B). Asterisks denote significant difference from control (p<0.01).
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FIGURE 3.10: PACAP induced changes in intracardiac neuron excitability are blocked by ryanodine. (A) Action
potentials recorded from a single intracardiac neurons in response to a depolarizing current pulse (300 ms, 150 pA) in the
absence (Control) and presence of 100 nM PACAP-27 (PACAP 100 nM) or following pretreated with ryanodine (10 µM)
for 10 min in the absence (Ryanodine) and presence (Ryanodine and PACAP) of bath applied PACAP-27 (100 nM), and
following washout of drugs (Wash). (B) Scatter plot of mean resting membrane potential (RMP) (± SEM) from 5 neurons
using the same conditions as described in panel (A), with the addition of the value obtained following washout of drugs
(Wash). (C) Bar graph of the mean number of action potentials (± SEM) elicited by depolarizing current pulse (300 ms,
150 pA) on single intracardiac neurons (n = 3) using identical conditions as in panel (B). Asterisks denote significant
difference from control (p<0.01).
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TABLE 3.1: Effects of extracellular Ca2+ conditions and various inhibitors on the PACAP-induced changes in membrane
potential and action potential firing in response to a 150 pA current pulse. All neurons recorded under Ca2+-free,
ryanodine, [N-Ac-Tyr1, D-Phe2]-GRF (1-29) (YF-GRF), and M65 conditions were first shown to respond to PACAP-27
alone (data not shown), and control conditions shown indicate steady-state recordings after pretreating the cells under
each condition, respectively. Error Bars represent ± SEM for the number of cells shown in parentheses. Asterisks denote
significant difference (p<0.01).
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Treatment Membrane Potential (mV) Action Potential Firing
PACAP Control Drug (n) Control Drug (n)
2.5 mM Ca2+ -52.0 ± 0.6 -47.7 ± 0.7* 54 3.2 ± 0.3 6.9 ± 0.5* 42
Ca2+-free -50.9 ± 1.0 -51.9 ± 1.8 6 3.6 ± 0.6 3.7 ± 1.1 4
Ryanodine -51.4 ± 1.3 -52.3 ± 1.3 5 2.0 ± 0.6 2.3 ± 0.9 3
M65 -52.2 ± 2.7 -52.8 ± 2.3 7 2.0 ± 0.6 1.7 ± 0.3 3
YF-GRF -50.8 ± 1.7 -51.0 ± 1.5 3 3.0 ± 0.6 3.3 ± 0.3 3
VIP -47.3 ± 1.7 -44.5 ± 1.5* 8 2.8 ± 0.5 3.2 ± 0.6 8
Maxadilan -50.9 ± 1.9 -51.9 ± 1.7 4 3.5 ± 1.2 3.8 ± 1.1 3
Caffeine -55.3 ± 2.1 -55.9 ± 2.0 4 3.5 ± 1.0 3.5 ± 1.2 4
80
DISCUSSION
The major finding reported here is that activation of VPAC receptors
increases neuroexcitability in intrinsic cardiac neurons, and that concurrent
activation of VPAC and PAC1 receptors results in a synergistic augmentation of
neuroexcitability. Furthermore, the PACAP and VIP mediated increase in
excitability is dependent on VPAC receptor induced Ca2+ release from caffeine-
and ryanodine-sensitive intracellular stores.
Our studies show that bath application of VIP or PACAP evokes an
elevation in intracellular free-Ca2+ that is completely blocked by the VPAC
receptor antagonists L-8-K and [N-Ac-Tyr1, D-Phe2]-GRF (1-29). Furthermore,
the effects of PACAP and VIP were not mimicked by the PAC1 selective agonist
maxadilan. Taken together these data suggest that the effects of PACAP and
VIP on [Ca2+]i are mediated by activation of VPAC receptors in these cells.
Previous studies have shown that rat intracardiac neurons express three PAC1
receptor isoforms (PAC1-short, PAC1-HOP1, and PAC1-HOP2), as well as VPAC1
and VPAC2 receptors (DeHaven and Cuevas, 2002). The predominant PAC1
receptor, PAC1-HOP, has been shown to evoke release of calcium from
intracellular stores via both cAMP and IP3 pathways in various cell types
including neuroepithelial cells, chromaffin cells, astrocytes, and cortical neurons
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(Grimaldi and Cavallaro, 2000; Zhou et al., 2001; Payet et al., 2003). However,
our data indicate that stimulation of PAC1 has no direct effect on [Ca2+]i in
neonatal rat intracardiac neurons. Earlier studies using single-cell RT-PCR
detected VPAC1 receptor transcripts in < 20% of neonatal rat intracardiac
neurons, whereas VPAC2 receptor transcripts were expressed in > 90% the cells
(DeHaven and Cuevas, 2002). In light of the fact that PACAP increase [Ca2+]i in
over > 95% of the cells tested, VPAC2 receptors are likely to be mediating these
increases.
In contrast to PAC1 receptors, there is limited information concerning
VPAC receptor mediated regulation of [Ca2+]i, and to date, there have been no
reports of VPAC receptors evoking elevations of [Ca2+]i in neurons. Stimulation
of VPAC receptors with low concentrations of VIP (1 nM) has been shown to
evoke calcium oscillations in pancreatic acinar cells (Kase et al., 1991). In
contrast to our observations, however, higher concentrations of VIP (100 nM)
failed to elicit elevations in [Ca2+]i and had an inhibitory effect on ACh-evoked
[Ca2+]i responses in acinar cells (Kase et al., 1991). VPAC-mediated increases
in [Ca2+]i have also been reported in the lymphoblastic MOLT4 and SUPT1 cells
(Anton et al., 1993; Xia et al., 1996). Exogenously expressed VPAC1 and VPAC2
receptors have also been shown to stimulate [Ca2+]i in COS7 cells and chinese
hamster ovary (CHO) cells (MacKenzie et al., 2001; Langer et al., 2001).
The VPAC evoked increases in [Ca2+]i exhibited both a transient and a
sustained component. The transient component was observed in the absence of
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extracellular Ca2+, but was blocked by ryanodine (10 µM) or depletion of the Ca2+
stores by caffeine or thapsigargin. These results suggest that the transient
component is due to VPAC mobilization of intracellular Ca2+, and more
specifically, due to Ca2+ release from ryanodine-sensitive Ca2+ stores. In
pancreatic and submandibular gland cells, VPAC receptor activation promotes
Ca2+ oscillations via a phospholipase C (PLC)/IP3 cascade (Luo et al., 1999).
Thus, in those secretory cells, VIP is likely increasing [Ca2+]i by evoking Ca2+
release from IP3-sensitive Ca2+ stores. Although rat intracardiac neurons also
contain IP3-sensitive stores (Beker et al., 2003), our results with ryanodine and 2-
APB suggest that a PLC-IP3-IP3-receptor pathway is not involved in the VPAC
receptor-induced increases in [Ca2+]i seen in these cells.
Unlike the transient component, the VPAC-mediated sustained elevations
in [Ca2+]i were abolished by removal of extracellular Ca2+, suggesting that they
are due to calcium entry through the plasma membrane. In various cell types,
including αT3-1 gonadotrophs (Rawlings et al., 1995) and HIT-T15 insulinoma
cells (Leech et al., 1995), VPAC2 receptor activation has been reported to
increase [Ca2+]i by enhancing Ca2+ entry through plasma membrane voltage-
gated Ca2+ channels. However, previous studies have shown that VIP does not
alter the biophysical properties of high-voltage-activated calcium channels in
intracardiac neurons (Cuevas and Adams, 1996). Furthermore, VPAC-mediated
modulation of voltage-gated Ca2+ channels appears to involve a cAMP/PKA
dependent mechanism (Rawlings et al., 1995; Leech et al., 1995), but in intrinsic
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cardiac neurons the activator of adenylate cyclase, forskolin (100 µM), failed to
mimic the effects of PACAP and VIP on [Ca2+]i. Thus, the influx of Ca2+ observed
following VPAC receptor stimulation is unlikely to be mediated by Ca2+ entry
through voltage-gated Ca2+ channels. Given that the sustained component is
only observed following Ca2+ release from the ryanodine-sensitive stores, and
that it may also be produced by depletion of these stores by caffeine (data not
shown), it is likely mediated by store-operated channels. This conclusion is
supported by our observation that the sustained elevation in [Ca2+]i produced by
PACAP is blocked by the TRP channel antagonist 2-APB. Depletion of
caffeine/ryanodine-sensitive Ca2+ stores has previously been shown to evoke
capacitative Ca2+ entry in both excitable and non-excitable cells (Weigl et al.,
2003; Xue et al., 2000).
We have now shown that, in addition to increasing [Ca2+]i, VIP and
PACAP enhance neuronal excitability in intrinsic cardiac neurons. However, the
effects on excitability produced by these neuropeptides are not identical. These
differences may shed light into possible distinct roles for the two neuropeptides in
the regulation of intracardiac neurons, and thus of the cardiovascular system.
Bath application of PACAP or VIP results in a depolarization of intrinsic cardiac
neurons. While PACAP has previously been shown to depolarize guinea pig
intracardiac neurons, VIP had no effect on the excitability of those cells (Braas et
al., 1998). The observation that VIP depolarizes intracardiac neurons and that
VPAC-specific antagonists block PACAP-induced depolarizations suggests that
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VPAC receptors directly influence neuroexcitability in rat intracardiac neurons.
VPAC receptor activation has been shown to depolarize thalamic relay and
medial pontine reticular formation neurons by 2-3 mV (Lee and Cox, 2003;
Kohlmeier and Reiner, 1999), which is similar to the VIP-induced depolarizations
reported here (2.4 ± 0.4 mV).
The observation that maxadilan, a PAC1 receptor specific agonist (Moro
and Lerner, 1997), does not evoke a depolarization of intracardiac neurons or
increase action potential firing suggests that PAC1 receptors do not have a direct
effect on neuroexcitability in these cells. PAC1 receptor activation, however,
potentiates VPAC receptor-induced depolarizations. Evidence for this conclusion
is provided by two observations reported here: 1) PACAP depolarizes neurons to
a greater extent than VIP, and 2) the PAC1 receptor antagonist, M65, depressed
PACAP induced depolarizations. Furthermore, increases in action potential firing
in response to depolarizing current pulses were only observed under conditions
in which both PAC1 and VPAC receptors were stimulated, that is, when PACAP
was used as the agonist and neither PAC1 nor VPAC receptor-specific
antagonists were applied. Thus, simultaneous PAC1 and VPAC receptor
stimulation elicits a synergistic enhancement of neuroexcitability and produces
changes in the active membrane properties that are not seen with stimulation of
either receptor alone.
The VPAC-mediated increases in [Ca2+]i appear to be critical for the
enhanced neuroexcitability of intracardiac neurons by PACAP. PACAP failed to
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depolarize intracardiac neurons and to increase action potential firing when
applied in the presence of ryanodine, when the intracellular stores were depleted
with caffeine, or in the absence of extracellular Ca2+. It is interesting to note that
caffeine had no direct effects on neuroexcitability (Table 3.1), and thus Ca2+
release from intracellular stores is necessary, but not sufficient to promote
enhanced neuroexcitability. Muscarinic receptor activation has been shown to
depolarize intracardiac neurons and to augment action potential firing in these
cells (Xu and Adams, 1992). Recent studies have shown that muscarinic
receptor activation mobilizes Ca2+ from intracellular stores in intracardiac neurons
(Beker et al., 2003). Even though inhibition of the K+ conductance, IM, appears to
be a major factor contributing to muscarinic-receptor induced neuroexcitability in
these neurons (Xu and Adams, 1992), it would be of interest to determine if
muscarine-induced increases in [Ca2+]i also play a role in the enhanced
neuroexcitability, and thus if mobilization of intracellular [Ca2+]i is a mechanism by
which various excitatory neurotransmitters regulate the electrical properties of
intrinsic cardiac neurons.
In conclusion, the present study demonstrates the first evidence of VPAC
receptor-mediated regulation of intracellular Ca2+ in neurons, and shows that by
increasing [Ca2+]i, VPAC receptors enhance neuroexcitability. Furthermore, our
results demonstrate that the heterogeneity in PAC1 and VPAC receptors
observed in intracardiac neurons has significant implications for the effects of the
neuropeptides on cellular function. The ability of PACAP to depolarize the
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neurons and increase action potential firing is dependent on PACAP stimulation
of both PAC1 and VPAC receptors. VIP, by acting exclusively on VPAC
receptors, evokes a depolarization of lesser magnitude than PACAP and fails to
increase action potential firing. The ability of PACAP and VIP to differentially
influence the electrical activity of intracardiac neurons may also help explain why
these related neuropeptides have distinct effects on the cardiovascular system.
While both PACAP and VIP have been shown to dilate coronary arteries and to
produce positive chronotropic effects (Karasawa et al., 1990; Hirose et al., 1997;
Champion et al., 1996), PACAP, but not VIP, evokes pronounced negative
chronotropic and negative inotropic effects that are mediated by activation of
intrinsic cardiac neurons (Hirose et al., 1997).
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CHAPTER 4
REPETITIVE VPAC2 RECEPTOR-MEDIATED CALCIUM ELEVATIONS IN
INTRACARDIAC NEURONS IS DEPENDENT ON Ca2+, cADPR AND STORE-
OPERATED CALCIUM (SOC) ENTRY
INTRODUCTION
The intracardiac ganglion is capable of independently regulating the
mammalian heart, and the pituitary adenylate cyclase activating polypeptide
(PACAP) and vasoactive intestinal polypeptide (VIP) have significant effects on
the cardiovascular system. Experiments in our laboratory and by Parsons and
colleagues (Braas et al., 1998; Merriam et al., 2004; Braas et al., 2004) have
given insight into how these neuropeptides may be regulating cardiac function via
the modulation of intrinsic cardiac neurons. Our studies have recently shown
that activation of the PACAP (PAC1) and VIP (VPAC) receptors in neonatal rat
intracardiac neurons increases neuroexcitability in these cells, and that
simultaneous activation of VPAC and PAC1 receptors results in a synergistic
enhancement of excitability (DeHaven and Cuevas, 2004). Importantly, it seems
the neuropeptide mediated increase in excitability is dependent on VPAC
receptor induced elevations in intracellular calcium ([Ca2+]i) that arise from both
88
release of Ca2+ from ryanodine- and caffeine-sensitive stores, and calcium entry
through the plasma membrane. While our studies showed that VPAC receptors
mediate this effect, neither the specific VPAC receptor involved nor the signal
transduction system coupling the VPAC receptor to the ryanodine receptor have
been identified. Furthermore, the ion channel facilitating Ca2+ through the
membrane has not been unequivocally determined.
Similar to intracardiac neurons, activation of PACAP receptors in bovine
adrenal medullary cells has been shown to release Ca2+ from ryanodine- and
caffeine-sensitive intracellular stores independent of IP3 receptor activation
(Tanaka et al., 1998). Interestingly, the PACAP-evoked release from the internal
stores was found to be mediated by a Na2+-influx-dependent membrane
depolarization (Tanaka et al., 1996). Furthermore, the depolarizing effects of
PACAP were shown to be independent of Ca2+. However, the PACAP-induced
changes in neuroexcitability in intracardiac neurons appear dependent on the
release of Ca2+ from these intracellular stores (DeHaven and Cuevas, 2004).
Thus, it seems unlikely that PACAP is regulating the ryanodine receptors in
intracardiac neurons the same way as the peptide does in bovine adrenal
medullary cells. Ryanodine receptors are regulated by numerous factors,
including the physiological agents Ca2+, ATP, Mg2+ and cyclic ADP-ribose
(cADPR), as well as various cellular processes, including phosphorylation (Fill
and Copello, 2002). Both Ca2+ and cADPR have been suggested as the
endogenous ligands of neuronal ryanodine receptors (Sitsapesan et al., 1995;
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Thomas et al., 2001; Li et al., 2001), and the data presented here suggest that
Ca2+ and cADPR are both crucial elements in the regulation of ryanodine
receptors in isolated rat intracardiac neurons by VPAC2 receptors.
Pharmacological studies with 2-APB suggest the plasma membrane
component of the PACAP and VIP elicited Ca2+ response is through transient
receptor potential (TRP) channels. TRP channels are a member of a large family
of non-selective cation channels expressed in neurons, skeletal and smooth
muscle, and in non-excitable cells (Clapham, 2003). In excitable cells such as
neurons, the opening of TRP channels has two significant effects, depolarization
of the cell, and sustained elevation of [Ca2+]i. The TRP family of ion channels is
divided into several branches, including the TRPC (canonical), TRPM
(melastatin) and TRPV (vanilloid) subfamilies, as well as the distant TRP
channels, TRPP, TRPA and TRPML. Some members of the TRPC channel
subtype appear to be activated directly by diacylglycerol (Clapham, 2003).
However, significant controversy exists as to the mechanisms regulating TRPC
activity. For example, TRPC3 has been shown to directly couple to IP3 receptors
in order to activate the channel (Zhang et al., 2001). Conversely, in IP3 knockout
cells, muscarinic receptor activation of PLC and DAG signaling activated TRPC3,
independent of IP3 receptors (Venkatachalam et al., 2001). Furthermore, some
members of the TRPC channel family are believed to be activated as a result of
elevations in intracellular free Ca2+ or by depletion of intracellular Ca2+ stores
(Clapham, 2003).
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Earlier studies in intracardiac neurons suggested that a TRP-like channel
is activated following VPAC receptor-induced Ca2+ release from ryanodine/
caffeine-sensitive stores. This channel was not activated when VPAC receptors
were stimulated but Ca2+ mobilization was inhibited (DeHaven and Cuevas,
2004). Thus, TRP channels in intracardiac neurons appear to be regulated by
either elevations of [Ca2+]i and/or by depletion of the ryanodine/caffeine-sensitive
stores. In many non-neuronal and neuronal cell types, the depletion of
intracellular Ca2+ stores causes activation of plasma membrane store-operated
(SOC) channels (Putney, 1986; Berridge, 1995; Parekh and Penner, 1997). The
physiological functions of the store-operated Ca2+ channels include the
maintenance of proper Ca2+ levels in the intracellular stores, and the generation
of prolonged Ca2+ signals beyond those provided by the intracellular messengers
inositol (1,4,5)-trisphosphate (IP3) and cyclic ADP ribose (cADPR) (Putney,
2004). The most widely studied current involved in this capacitative Ca2+ entry is
the Ca2+-release-activated Ca2+ current (ICRAC) (Hoth and Penner, 1992).
However, unlike TRP channels, ICRAC is a divalent cation-selective channel that is
characteristically found in hematopoietic cells and not neurons. Furthermore, the
currents activated by Ca2+-store depletion in other cell types maintain different
electrophysiological properties compared to the ICRAC.
Data presented here show PACAP elevates intracellular Ca2+ in these
intracardiac neurons through the activation of VPAC2 receptors, and that
mobilization of intracellular calcium from ryanodine- and caffeine- sensitive
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internal stores by VPAC2 is dependent on Ca2+ and cADPR, and not adenylyl
cyclase activity. The sustained component of the PACAP-evoked elevations in
[Ca2+]i are, indeed, through SOC channels, and further pharmacological evidence
suggests TRPC channels are the underlying channels mediating the capacitative
Ca2+ entry. Finally, 2-APB inhibited these neurons from repetitive VPAC2
mediated increases in [Ca2+]i, suggesting a role for TRP channels in VPAC2
receptor-elicited changes in neuroexcitability seen in rat intracardiac neurons.
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METHODS
Cell culture
The preparation and culture of isolated intrinsic cardiac neurons has been
previously described (Fieber and Adams, 1991). Dissociated neurons were
plated onto poly-L-lysine coated glass coverslips and incubated at 37°C for 36-72
h under a 95% air, 5% CO2 environment prior to the experiments.
Microfluorometric measurements
The calcium sensitive dye fura-2 acetoxymethylester (fura-2-AM) was
used for measuring intracellular free calcium concentrations ([Ca2+]i) in
intracardiac neurons at room temperature, as previously described (Smith and
Adams, 1999). Briefly, a 1 mM fura-2-AM stock was made using DMSO as the
solvent. Cells plated on coverslips were then incubated for 1 hour at room
temperature in physiological saline solution (PSS) consisting of (in mM): 140
NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 7.7 glucose and 10 HEPES (pH to 7.2 with
NaOH), which also contained 1 µM fura-2-AM and 0.1 % dimethyl sulfoxide
(DMSO). The coverslips were then washed in PSS (fura-2-AM free) prior to the
experiments being carried out. All solutions were applied via a rapid application
system identical to that previously described (Cuevas and Berg, 1998).
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A Sutter DG-4 high-speed wavelength switcher (Sutter Instruments Co.,
Novato, CA) was used to apply alternating excitation with 340 nM and 380 nM
ultraviolet light via a liquid light guide. Fluorescent emission at 510 nm was
captured using a Cooke Sensicam digital CCD camera (Cooke Corporation,
Auburn Hills, MI) and recorded with Slidebook 3.0 software (Intelligent Imaging
Innovations, Denver, CO) on a Pentium IV computer. Changes in [Ca2+]i were
calculated using the Slidebook software (Intelligent Imaging Innovations, Denver,
CO) from the intensity of the emitted fluorescence following excitation with 340
and 380 nm light, respectively, using the equation:
[Ca2+]i = Kd Q (R-Rmin)/(Rmax-R)
where R represents the fluorescence intensity ratio (F340/F380) as determined
during experiments, Q is the ratio of Fmin to Fmax at 380 nm, and Kd is the Ca2+
dissociation constant for fura-2. Calibration of the system was performed using
the Molecular Probes (Eugene, OR) Fura-2 Calcium Imaging Calibration Kit and
values were determined to be: Fmin/Fmax (23.04), Rmin (0.31), Rmax (8.87).
Reagents and statistical analysis
All chemicals used in this investigation were of analytic grade. The following
drugs were used: Dimethyl sulfoxide (DMSO), 2-aminoethoxydiphenylborate (2-
APB), 8-bromo-cyclic adenosine diphosphate ribose (8-Br-cADPR), lanthanum
chloride, 1-oleoyl-2-acetyl-sn-glycerol (OAG), menthol, capsaicin and caffeine
(Sigma-Aldrich, St. Louis, MO); PACAP-27, PACAP(6-38) (American Peptide
94
Co., Sunnyvale, CA); forskolin (Alomone Labs, Jerusalem, Israel); and fura-2-AM
(Molecular Probes, Eugene, OR). All data are presented as the mean ± SEM of
the number of observations indicated. Statistical analysis was conducted using
SigmaPlot 8 (SPSS, Chicago, IL) and paired t-tests were used for within group
comparisons.
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RESULTS
VPAC2 receptors mediate PACAP-induced elevations in free [Ca2+]i
Our laboratory recently discovered that in neonatal rat intracardiac
neurons, PACAP and VIP significantly elevates intracellular calcium levels, and
furthermore, these increases in free [Ca2+]i are mediated through VPAC
receptors and not PAC1 (DeHaven and Cuevas, 2004). Earlier studies using
single-cell RT-PCR detected both VPAC1 and VPAC2 receptor transcripts in rat
intracardiac neurons (DeHaven and Cuevas, 2000). To determine the specific
VPAC receptor subtype mediating these effects, PACAP(6-38), a truncated form
of PACAP-38 which selectively inhibits VPAC2 at the concentrations used here
(100 nM) was applied simultaneously with PACAP-27. Changes in [Ca2+]i were
then measured using fura-2 mediated Ca2+-imaging. Figure 4.1A shows
representative traces of change in [Ca2+]i (∆[Ca2+]i) as a function of time for a
single intracardiac neuron in response to 2 minute PACAP-27 (100 nM) in the
absence (black trace) and presence (gray trace) of 100 nM PACAP(6-38).
PACAP(6-38) pretreatment in this cell reduced the PACAP-mediated elevations
in [Ca2+]i. In six similar experiments, PACAP(6-38) significantly decreased
elevations in [Ca2+]i evoked by PACAP from 410.9 ± 72.9 nM to 219.4 ± 23.0 nM,
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a 44.4% peak decrease (Fig 4.1B). Thus, VPAC2 receptors mediate the PACAP
and VIP induced elevations in [Ca2+]i in rat intracardiac neurons.
Forskolin does not increase [Ca2+]i in intracardiac neurons
As the name implies, the pituitary adenylyl cyclase-activating polypeptide
(PACAP) was originally discovered to increase the activity of adenylyl cyclase in
the pituitary gland, and thus elevate cAMP levels (Miyata et al., 1989). Thus,
involvement of the AC-cAMP-PKA cascade in the VPAC2 receptor-mediated
[Ca2+]i increases in intracardiac neurons was tested by directly activating adenylyl
cyclase with forskolin (10 µM). Figure 4.2A depicts typical traces of ∆[Ca2+]i as a
function of time for a single intracardiac neuron in response to 6 minute caffeine
(5 mM) (black trace) or forskolin (10 µM) (gray trace) applications. In all
intracardiac neurons tested (n = 15), forskolin failed to elevate [Ca2+]i, yet these
same neurons did respond to caffeine (positive control) (Fig 4.2B). Forskolin
application times were varied (2 min to 16 min) to ensure enough time to activate
the AC-cAMP-PKA cascade. These data suggest that intracardiac neuron
elevations in [Ca2+]i through caffeine- and ryanodine-sensitive stores are
independent of the AC-cAMP-PKA cascade.
Role for cADP-ribose in the PACAP-elicited signal transduction cascade
In addition to cAMP, the intracellular second messenger, cADPR, has
been shown to mobilize Ca2+ from ryanodine sensitive stores. To date, neither
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PAC1 nor VPAC receptors have been linked to the production of cADPR.
However, it seemed prudent to determine if this Ca2+-releasing signal metabolite
is involved in the VPAC2-ryanodine receptor pathway. For these experiments,
the cell permeable cADPR antagonist, 8-Br-cADPR (1 µM), was used. Figures
4.3A and 4.3C show representative traces of change in [Ca2+]i as a function of
time recorded from different isolated intracardiac neurons in response to 100 nM
PACAP-27 (A) or 5 mM caffeine (C) applications in the absence and presence of
8-Br-cADPR (1 µM). While 8-Br-cADPR alone had no effect on [Ca2+]i, it did
inhibit the PACAP-induced mobilization of [Ca2+]i in this neuron (Figure 4.3A).
However, on a different intracardiac neuron, 1 µM 8-Br-cADPR had no effect on
a caffeine mediated increase in [Ca2+]i (Figure 4.3C), as predicted from results in
other studies (Prakash et al., 2000). In 7 similar experiments, 8-Br-cADPR
significantly inhibited the PACAP-mediated mean peak increases in [Ca2+]i, from
260.7 ± 31.6 nM to 144.1 ± 14.8 nM, a 45% decrease in peak Ca2+ influx (Figure
4.3B). Nonetheless, figure 4.3D shows no effect of the cell permeable 8-Br-
cADPR on caffeine-induced increases in [Ca2+]i (caffeine alone: 336.9 ± 34.4 nM;
+ 8-Br-cADPR: 371.2 ± 34.7 nM) (n=9). These data suggest that in intracardiac
neurons VPAC2 receptors couple to ryanodine receptors via a cADPR dependent
pathway. However, the binding sites for cADPR and caffeine on the neuronal
ryanodine receptors appear to be different. These data support previous findings
which suggest a different binding site for caffeine and cADPR on the ryanodine
receptor (Walseth and Lee, 1993; Prakash et al., 2000).
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Effects of Menthol and Capsaicin on [Ca2+]i
As previously mentioned, several subfamilies of TRP channels exist,
including TRPC, TRPM and TRPV. While TRPC channels are the most widely
implicated TRP channels in store-operated Ca2+ entry, TRPV and TRPM
activation may elevate [Ca2+]i in intracardiac neurons. Ca2+ imaging experiments
were performed using menthol and capsaicin in order to determine if the
activation of certain TRPV or TRPM receptors increase [Ca2+]i in rat intracardiac
neurons. Menthol has been shown to activate the TRPM8 channels (Hu et al,
2004; Tsuzuki et al., 2004), and capsaicin has been shown to activate TRPV1
channels (Hu et al, 2004; Krause et al., 2005). Figure 4.4A shows changes in
[Ca2+]i traces elicited by 6 minute applications of menthol (10 µM) or capsaicin (1
µM) onto different isolated intracardiac neurons. Neither menthol nor capsaicin
elevated [Ca2+]i in intracardiac neurons (Fig 4.4B), suggesting that TRPM8 and
TRPV1 do not play a role in the sustained phase of the Ca2+ response in these
cells. Furthermore, 2-APB is a common activator of TRPV1, TRPV2 and TRPV3
(Hu et al, 2004), and activation of these receptors by 2-APB elevates [Ca2+]i.
However, 2-APB alone had no effects on the cytosolic Ca2+ concentration in
neonatal rat intracardiac neurons. Taken together, these data suggest that
TRPV1, TRPV2, TRPV3 and TRPM8 channels do not play a role in the sustained
phase of the VPAC2 receptor-evoked Ca2+ response in neonatal rat intracardiac
neurons. Since a considerable number of studies have implicated each of the
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TRPC channels as store-operated channels, and because 2-APB, menthol, and
capsaicin elicited no changes in cytosolic free Ca2+ levels, we propose that TRPC
channels are the likely candidate for the sustained component of the [Ca2+]i
elevations seen in neonatal rat intracardiac neurons.
La3+ blocks VPAC2 mediated Ca2+ responses and TRPC channels in
intracardiac neurons.
Lanthanum was used to investigate both the ryanodine receptors and the
TRPC channels in rat intracardiac neurons. The regulation of ryanodine
receptors directly by Ca2+ has been widely established in the literature (Fill and
Copello, 2002). However, in isolated intracardiac neurons, VPAC2 receptor
activation induces an initial, transient mobilization of [Ca2+]i from caffeine- and
ryanodine-sensitive stores which appears to be regulated by cADPR, and not
Ca2+. Nonetheless, experiments were performed with lanthanum in order to
determine the intracardiac neuronal regulation of ryanodine receptors by Ca2+.
Furthermore, lanthanum (La3+), a trivalent cation that inhibits all membrane Ca2+
channels, including voltage-gated, store-operated Ca2+ and TRPC channels
(Aussel et al., 1996; Halaszovich et al., 2000; Beedle et al., 2002) will provide
insight to the sustained elevation of [Ca2+]i which appears to be mediated by
activation of TRPC channels. The concentration of La3+ used (100 µM) in these
experiments was selected because this is the concentration shown previously to
maximally inhibit TRPC channels (Takai et al., 2004). Figures 4.5A, B and C
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show changes in [Ca2+]i traces elicited by 2 minute application of PACAP-27 (100
nM), caffeine (5 mM) or muscarine (5 µM) onto different isolated intracardiac
neurons in the absence and presence of La3+ (100 µM). Lanthanum alone
produced no significant measurable changes in [Ca2+]i, and did not block the
transient elevation in [Ca2+]i produced by caffeine (Fig 4.5C) or muscarine (Fig
4.5B). However, 100 µM La3+ significantly reduced the peak change in [Ca2+]i
(∆[Ca2+]i) mediated by PACAP receptor activation in this neuron, from 139.5 nM
to 16.2 nM (Fig 4.5A). Figure 4.5D shows a bar graph of the mean peak
increase in [Ca2+]i evoked by PACAP, caffeine and muscarine in the absence and
presence of La3+. While La3+ significantly inhibited the peak Ca2+ response to
PACAP (n = 7; 137.7 ± 17.9 nM to 24.5 ± 8.0 nM), La3+ had no effect on the peak
increases in [Ca2+]i evoked by caffeine (n = 11) or muscarine (n = 4) (caffeine:
330.1 ± 60.9 nM to 285.9 ± 38.6 nM; muscarine: 88.7 ± 23.2 nM to 95.7 ± 22.4
nM). Thus, it seems likely that while IP3 receptor-mediated elevations in Ca2+ in
intracardiac neurons are not directly dependent on Ca2+, ryanodine receptor-
evoked Ca2+ elevations are dependent on Ca2+ regulation. Furthermore,
caffeine-mediated increases in Ca2+, by directly acting on the ryanodine
receptors and sensitizing them to Ca2+, do not directly depend on Ca2+ influx
through La3+-sensitive plasma membrane channels. Also, because La3+ had no
effect on the peak increases in [Ca2+]i evoked by caffeine, La3+ does not enter the
cell and alter ryanodine receptors directly.
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Because the transient component of the PACAP-induced elevation in
[Ca2+]i was blocked by La3+, the sustained component was also significantly
abolished (Figure 4.5A,E). Therefore, La3+ would give us little insight to the
sustained component of the Ca2+ responses in neonatal rat intracardiac neurons.
However, the transient component of the caffeine-and muscarine-evoked
elevation in [Ca2+]i was unaffected by La3+, and therefore would allow us to
determine whether the sustained component of Ca2+ responses in these cells are
mediated through La3+ (and 2-APB) sensitive TRPC channels. Figures 4.5B and
C show that unlike the peak Ca2+ responses, the sustained Ca2+ increases
mediated by caffeine and muscarine in these cells are largely reduced by
application of 100 µM La3+. While the caffeine-mediated sustained increase in
[Ca2+]i decreased from 92.6 ± 11.8 nM to 15.7 ± 5.4 nM, the muscarine-evoked
sustained Ca2+ response decreased from 31.1 ± 1.8 nM to 3.2 ± 4.8 nM (Fig
4.5E). These data further support the observation that TRPC channels contribute
to the sustained elevation in [Ca2+]i evoked by VPAC2 receptor activation.
PACAP evoked Ca2+ influx through store-operated channels (SOC)
While TRPC receptors are implicated as the channels underlying store-
operated Ca2+ entry, the link between receptor activation and channel gating is
undetermined. Many of the TRPC channels that have been implicated in
capacitative Ca2+ entry have also been shown to be activated by the intracellular
messenger sn-1,2-diacylglycerol (DAG) (Hu et al, 2004; Venkatachalam et al.,
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2003). Thus, we wanted to see the effects of the cell permeable analog of DAG,
1-oleoyl-2-acetyl-sn-glycerol (OAG), on intracellular Ca2+ levels in isolated
intracardiac neurons. Figure 4.6A shows the change in [Ca2+]i elicited by a 6
minute bath application of 100 µM OAG. In all neurons tested (n=23), OAG
failed to elevate [Ca2+]i (Fig 4.6B), suggesting that TRPC channels in neonatal rat
intracardiac neurons are not regulated by DAG.
The paradigm that we used to demonstrate that the TRPC channels
mediating the sustained component of the Ca2+ response are store-operated
Ca2+ (SOC) channels is based on a depletion of internal stores with drug
(PACAP-27 or caffeine) under Ca2+ free conditions (+1 mM EGTA). After store
depletion, application of extracellular Ca2+ results in Ca2+ influx through the
plasma membrane store-operated channels. Figure 4.7A shows typical traces of
change in [Ca2+]i (∆[Ca2+]i) as a function of time for 2 minute applications of 100
nM PACAP-27 (PACAP), 5 mM caffeine (Caffeine) and no drug (PSS) under
Ca2+ free conditions (solid line above traces), followed by the introduction of 2.5
mM Ca2+ (dashed line above traces). Both PACAP and caffeine released Ca2+
from intracellular stores under Ca2+ free conditions (+ 1 mM EGTA), and the
introduction of 2.5 mM Ca2+ in the external medium elevated [Ca2+]i above control
levels (PSS alone) under both conditions. In six similar experiments, PACAP-
and caffeine-mediated release of Ca2+ from intracellular stores under Ca2+ free
conditions caused significant increases in Ca2+ influx through the plasma
membrane after Ca2+ was introduced to the medium, when compared to PSS
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alone (Figure 4.7B). Under 0 Ca2+ (+ 1 mM EGTA), 100 nM PACAP increased
[Ca2+]i from 68.3 ± 4.1 nM to 210.2 ± 46.4 nM, a 210% increase. The application
of 2.5 mM Ca2+ to the extracellular medium of neurons treated with PACAP
increased the [Ca2+]i to 122.5 ± 12.3 nM. Thus, the peak store-operated
component of the VPAC2 elicited Ca2+ elevations was 58% of the peak elevations
in [Ca2+]i caused by internal store release. Similar percentages were seen for
caffeine. Figure 4.7C shows a bar graph of change in [Ca2+]i (∆[Ca2+]i) measured
for PACAP, caffeine and PSS alone, under calcium free (0 Ca2+) or 2.5 mM Ca2+
conditions. Thus, the TRPC component of VPAC2 receptor-evoked elevations in
[Ca2+]i is dependent on extracellular Ca2+ and is activated by the internal store
depletion in intracardiac neurons.
The SOC channel component of the PACAP-induced Ca2+ response is
blocked by 2-APB
We previously showed that the sustained component of the Ca2+ response
in these isolated intracardiac neurons is completely abolished by the TRPC
channel antagonists, 2-APB and La3+. Furthermore, figure 4.7 showed the
depletion of internal stores by PACAP under Ca2+-free conditions activates SOC
channels located in the plasma membrane. However, the question remains
whether the SOC channels are the same as the 2-APB-sensitive TRPC
channels. Figure 4.8 shows that the PACAP-mediated Ca2+ response through
SOC channels is significantly inhibited by the pretreatment with 50 µM 2-APB.
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PACAP-27 (100 nM) was applied to isolated intracardiac neurons under Ca2+
free conditions (1 mM EGTA) for ~5 min to ensure complete store depletion from
PACAP–sensitive pools, and the return to baseline Ca2+ levels. The
reintroduction of Ca2+ (2.5 mM) to the extracellular solution evoked a Ca2+ influx
through SOC channels, similar to what was shown in figure 4.7. However, in the
presence of 50 µM 2-APB, the influx of Ca2+ through these SOC channels was
significantly blocked. Figures 4.8A and B depict typical traces of changes in
[Ca2+]i as a function of time for 100 nM PACAP- (Fig 4.8A) and 5 mM caffeine-
(Fig 4.8B) mediated activation of SOC channels, before (black traces) and after
(gray traces) application of 2-APB. In 6 similar recordings, pretreatment with 2-
APB significantly reduced the VPAC2 receptor-mediated influx of Ca2+ through
SOC channels by more than 80%, from 102.4 ± 12.2 nM to 18.9 ± 1.4 nM (Fig
4.8C). Similarly, caffeine-mediated SOC channel activation was also significantly
blocked by the application of 2-APB (~75% decrease) (n = 7). Thus, the store-
operated component is the same as the 2-APB-sensitive component of the Ca2+
response in neonatal rat intracardiac neurons.
2-APB blocks VPAC2 receptor mediated repetitive Ca2+ responses
Store-operated calcium entry (SOC), also known as capacitative calcium
entry, plays an essential role in calcium signaling, where it can generate signals
directly or serve to replenish the internal stores in order to generate repetitive
calcium increases. Several models have been established to describe how
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internal stores regulate SOC through the plasma membrane, including the
existence of diffusible messengers, or through a direct protein-to-protein
interaction (Putney et al.,2001; Berridge, 1995). We wanted to investigate the
role of SOC in generating repetitive Ca2+ increases in our isolated intracardiac
neurons. Figure 4.9 depicts repetitive elevations of [Ca2+]i mediated by VPAC2
receptor activations or caffeine, blocked by the TRP channel antagonist, 2-APB.
This figure shows representative traces of change in [Ca2+]i (∆[Ca2+]i) as a
function of time recorded from single neurons in response to 100 nM PACAP-27
(Fig 4.9Ai) or 5 mM caffeine (Figure 4.9Aii) application, in the absence and
presence of 2-APB (50 µM). Figure 4.9B shows a bar graph of mean peak
[Ca2+]i for the first and second bath applications of caffeine or PACAP, before
(Control) or during (+ 2-APB) 2-APB application. Importantly, under the presence
of 2-APB, neither PACAP nor caffeine were capable of eliciting a Ca2+ response
after the initial peak response occurred. While the initial peak response to
PACAP was 107.2 ± 20.3 nM under 2-APB conditions, the repetitive 2nd
application of PACAP only increased [Ca2+]i to 22.2 ± 5.5 nM, a 80% decrease in
the peak [Ca2+]i. Identical to what was previously published (DeHaven and
Cuevas, 2004), figure 4.9C depicts a bar graph of mean sustained [Ca2+]i
detailing that in these intracardiac neurons, 2-APB significantly inhibits the
sustained component of the VPAC2 receptor mediated elevations in intracellular
calcium. Thus, the SOC is essential in replenishing the internal Ca2+ stores of
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intracardiac neurons, and as a result, regulates repetitive elevations of [Ca2+]i in
mammalian intracardiac neurons.
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FIGURE 4.1: PACAP-induced mobilization of [Ca2+]i in rat intracardiac neurons
is via VPAC2 receptor activation. (A) Representative traces of change in [Ca2+]i
(∆[Ca2+]i), defined as peak [Ca2+]i minus baseline [Ca2+]i, as a function of time
recorded from a single neuron in response to 100 nM PACAP-27 application in
the absence (black trace) and presence (gray trace) of the PAC1 and VPAC2
receptor antagonist, PACAP(6-38) (100 nM). The solid line above the traces
indicates PACAP-27 bath application times. All antagonists were pre-applied for
5 min prior to and during the administration of PACAP. (B) Bar graph of mean
peak [Ca2+]i (± SEM) before (Baseline), during (Drug) and following washout
(Wash) of PACAP or PACAP and PACAP(6-38) (n = 7). Asterisks denote
significant difference (p<0.01) from the baseline [Ca2+]i recorded for each
experimental condition; # symbol denotes significant difference (p<0.01) from
control PACAP responses.
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FIGURE 4.2: Forskolin does not increase free cytosolic calcium concentrations
in isolated rat intracardiac neurons. (A) Representative traces of change in
[Ca2+]i (∆[Ca2+]i) as a function of time recorded from a single neuron in response
to 5 mM caffeine (black trace) application and 10 µM forskolin (gray trace). The
solid line above the traces indicates caffeine and forskolin bath application times
for this cell (6 min). In other similar experiments, forskolin was bath applied in a
time range from 2 to 16 min, and similar results were seen under all conditions.
(B) Bar graph of mean peak [Ca2+]i (± SEM) before (Baseline), during (Drug) and
following washout (Wash) of forskolin (white) or caffeine (black) (n = 15).
Asterisk denotes significant difference (p<0.01) from the baseline [Ca2+]i
recorded.
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FIGURE 4.3: The putative ryanodine receptor activator, cADP ribose, is involved in the PACAP-elicited signal
transduction cascade. (A) Typical time courses of )[Ca2+]i recorded in response to PACAP-27 (100 nM) application
before (black trace) and after (gray trace) preincubation in 1 µM 8 Br-cADP ribose. 8 Br-cADP ribose was applied for ~10
min prior to and continued through the PACAP application (8 Br-cADP ribose + PACAP). (B) Bar graph of peak [Ca2+]i (±
SEM) evoked by 100 nM PACAP or 100 nM PACAP following 1 µM 8 Br-cADP ribose preincubation (n=7). Asterisks
denote significant differences (p<0.01) from baseline, and # sign denotes significant difference from PACAP control peak
[Ca2+]i. (C) Representative )[Ca2+]i trace recorded in response to application of caffeine (5 mM) or caffeine following 1 µM
8 Br-cADP ribose preincubation. Caffeine was applied for 1 min, while 8 Br-cADP ribose was applied for ~10 min. (D)
Bar graph of peak [Ca2+]i (± SEM) recorded under the same conditions as (C); n = 9 for each condition. Asterisks denote
significant differences (p<0.01) from respective baselines.
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FIGURE 4.4: Neither capsaicin nor menthol elicit any changes in [Ca2+]i in
neonatal rat intracardiac neurons. (A) Representative traces of change in [Ca2+]i
(∆[Ca2+]i) as a function of time recorded from two different neurons in response to
either 1 µM capsaicin (black trace) or 10 µM menthol (gray trace). The solid line
above the traces indicates bath application times. (B) Bar graph of mean peak
[Ca2+]i (± SEM) before (Baseline), during (Drug) and following washout (Wash) of
capsaicin (n=16) or menthol (n = 16).
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FIGURE 4.5: La3+ blocks VPAC2 receptor mediated Ca2+ responses and the sustained component of the Ca2+ responses
in neonatal rat intracardiac neurons. Typical time courses of ∆[Ca2+]i recorded in response to 100 nM PACAP-27 (A), 5
mM caffeine (B), or 5 µM muscarine (C) before (Control, black trace) and after preincubation in 100 µM La3+ (+La3+, gray
trace). Each panel represents recordings obtained from a single cell. La3+ was applied for 10 min and continued through
the application of drug (PACAP, caffeine or muscarine). Bar graph of mean peak ∆[Ca2+]i (± SEM) (D) or mean sustained
∆[Ca2+]i (± SEM) (E) evoked by 100 nM PACAP-27 (n = 7), 5 mM caffeine (n = 11), or 5 µM muscarine (n = 4) in the
absence (Control) and presence of 100 µM La3+ (+ La3+). Asterisks denote significant difference (p<0.01) from the
corresponding control.
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FIGURE 4.6: The TRPC agonist, 1-oleoyl-2-acetyl-sn-glycerol (OAG), does not
increase [Ca2+]i in neonatal rat intracardiac neurons. (A) Representative traces
of change in [Ca2+]i (∆[Ca2+]i) as a function of time recorded from two different
neurons in response to 100 µM OAG (black trace). The solid line above the trace
indicates bath application times. (B) Bar graph of mean peak [Ca2+]i (± SEM)
before (Baseline), during (Drug) and following washout (Wash) of OAG (n=23).
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FIGURE 4.7: Internal store depletion by PACAP or caffeine activates SOC
channels. (A) Representative traces of change in [Ca2+]i (∆[Ca2+]i) as a function
of time recorded from a single neuron in response to 2 minute bath application of
100 nM PACAP-27 (black trace), 5 mM caffeine (gray trace) or nothing (dotted
trace), in the absence (+1 mM EGTA) and presence of 2.5 mM Ca2+. The solid
line above the traces indicates Ca2+ free conditions, while the dashed line
indicates introduction of 2.5 mM Ca2+ in the bath solution. (B) Bar graph of mean
peak [Ca2+]i (± SEM) under control (Baseline), 0 Ca2+ (0 Ca2+ Peak), and
physiological Ca2+ (2.5 mM Ca2+ Peak) conditions, in the presence of 100 nM
PACAP(black), 5 mM caffeine (gray) or no drug (PSS) (n = 6). Asterisks denote
significant difference (p<0.01) from the baseline [Ca2+]i recorded for each
experimental condition; # symbol denotes significant difference (p<0.01) from the
PSS response. (C) Bar graph (± SEM) of peak change in [Ca2+]i (∆[Ca2+]i) for
application of PACAP, caffeine and no drug (PSS) under no extracellular Ca2+ (0
Ca2+) and physiological extracellular Ca2+ (2.5 mM Ca2+) conditions (n = 6). Data
was collected from the same cells as shown in (B), and # denotes significant
difference from PSS alone condition.
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FIGURE 4.8: SOC channel activation by PACAP is inhibited by 2-APB. (A) Representative traces of change in [Ca2+]i
(∆[Ca2+]i) as a function of time recorded from a single neuron in response to bath application of 2.5 mM Ca2+ before (black
trace) and after (gray trace) the treatment with 50 µM 2-APB. PACAP-sensitive internal Ca2+ stores were depleted by 100
nM PACAP-27 application under Ca2+ free conditions (+1 mM EGTA) (~5 min). Recordings started (time = 0 min) after
cytosolic free Ca2+ levels returned to baseline following PACAP or caffeine application. The solid line above the traces
indicates PACAP application under Ca2+ free conditions, while the dashed line indicates introduction of 2.5 mM Ca2+ in the
bath solution. (B) Same as (A), except 5 mM caffeine in the place of PACAP in order to deplete caffeine-sensitive internal
Ca2+ stores. (C) Bar graph of mean peak change in [Ca2+]i (Peak ∆[Ca2+]i), before (black) and after (gray) 2-APB
treatment, evoked by the application of 2.5 mM Ca2+ after the internal store depletion by PACAP (n = 6) and caffeine (n =
7). Asterisks denote significant difference (p<0.01) from control responses recorded for each experimental condition.
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FIGURE 4.9: Repetitive elevations of [Ca2+]i mediated by VPAC2 receptor activations are blocked by 2-APB.
Representative trace of change in [Ca2+]i (∆[Ca2+]i) as a function of time recorded from a single neuron in response to 5
mM caffeine (i) or 100 nM PACAP-27 (ii) applications in the absence and presence of 2-APB (50 µM). The solid lines
above the trace indicate 2 minute caffeine or PACAP bath application times, and the dashed line indicates 2-APB
application times. (B) Bar graph of mean peak [Ca2+]i (± SEM) for the first and second bath applications of caffeine or
PACAP, before (Control) or during (+ 2-APB) 2-APB application. Asterisks denote significant difference (p<0.01) from the
first application and # symbol denotes significant difference (p<0.01) from control responses. (C) Bar graph of mean
sustained [Ca2+]i (± SEM) for the first and second bath applications of caffeine or PACAP, before (Control) or during (+ 2-
APB) 2-APB application. Asterisks denote significant difference (p<0.01) from the first application. (caffeine: n=5; PACAP:
n=6).
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DISCUSSION
The major finding reported here is that PACAP-induced elevations in
[Ca2+]i in intrinsic cardiac neurons are mediated by the activation of VPAC2
receptors that couple to ryanodine-sensitive Ca2+ stores via a signal transduction
cascade involving Ca2+ and cADPR. Furthermore, we show pharmacological
evidence that VPAC2 receptor activation results in the opening of TRP channels,
most probably TRPC. These channels open in response to depletion of Ca2+
evoked mobilization of Ca2+ from intracellular stores, and are thus functioning as
store-operated channels that mediate capacitative calcium entry. In contrast to
previous reports on some native TRPC receptors (Venkatachalam et al, 2003;
Grimaldi et al., 2003; Hu et al., 2004), TRPC channels mediating the sustained
Ca2+ response in intracardiac neurons are not activated by DAG.
Our laboratory has previously shown that rat intracardiac neurons express
three PAC1 receptor isoforms (PAC1-short, PAC1-HOP1, and PAC1-HOP2), as
well as VPAC1 and VPAC2 receptors (DeHaven and Cuevas, 2002), and
activation of VPAC receptors mediates PACAP- and VIP-elicited elevations in
[Ca2+]i (DeHaven and Cuevas, 2004), as well as influences the electrical
properties of the cell. These data are the first to show that VPAC2 receptors
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mediate the PACAP- and VIP-induced increases in cytosolic Ca2+ concentrations
in neonatal rat intracardiac neurons. Our results are supported by the fact that
VPAC1 receptor transcripts were detected in less than 20% of neonatal rat
intracardiac neurons, whereas VPAC2 receptor transcripts were expressed in >
90% the cells (DeHaven and Cuevas, 2002). While VPAC1 receptors can couple
to AC (Ishihara, T. et al., 1992), VPAC2 receptors can additionally activate the
PLC -IP3-PKC cascade (Lutz et al., 1993; Inagaki et al., 1994). Moreover,
PACAP-mediated increases in [Ca2+]i have been linked to both AC and PLC
activation (Payet et al., 2003; Tanaka et al., 1996; Grimaldi and Cavallaro, 2000);
however, neither of these pathways appears to be critical in the VPAC2 receptor
mediated increases in [Ca2+]i in neonatal rat intracardiac neurons.
The activation of VPAC2 receptors and subsequent increase in [Ca2+]i in
rat intracardiac neurons is not directly associated with increased levels of cAMP.
The mechanisms by which PACAP and VIP increase [Ca2+]i appears to differ
from cell-type to cell-type. For example, in five main cell-types of the rat anterior
pituitary, PACAP raises [Ca2+]i three different ways: via cAMP, phospholipase C
(PLC) and a third unknown novel mechanism (Alarcon and Garcia-Sancho,
2000). In fetal human chromaffin cells, PACAP elevates [Ca2+]i through caffeine-
and ryanodine-sensitive internal stores, and forskolin causes an identical
elevation in [Ca2+]i to PACAP (Payet et al., 2003). Pharmacological evidence
with specific PKA antagonists showed that the PACAP-induced release of Ca2+
from caffeine- and ryanodine-sensitive stores occurs through the AC-cAMP-PKA
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cascade in fetal human chromaffin cells. However, in neonatal rat intracardiac
neurons, the AC-cAMP-PKA cascade does not play a direct role in the VPAC2
receptor-mediated elevations in [Ca2+]i. Although the AC-cAMP-PKA intracellular
signal pathway does not directly increase [Ca2+]i in these intracardiac neurons,
our experiments do not rule out the possibility that cAMP may have a different
role in PACAP modulation of neuroexcitability seen in these cells. PAC1
activation in isolated guinea pig intracardiac neurons has been shown to
enhance the cAMP-regulated, hyperpolarization-activated nonselective cationic
conductance, Ih (Merriam et al., 2004). Ih has previously been demonstrated in
neonatal rat intracardiac neurons by electrophysiology techniques (Cuevas et al.,
1997).
Cyclic ADP-ribose (cADPR) was first shown to elevate [Ca2+]i in sea-urchin
eggs (Clapper et al., 1987), and it has since been shown to mobilize Ca2+ from
intracellular stores in a wide variety of cell types (Lee, 2001), including canine
and murine autonomic neurons (Smyth et al., 2004). Convincing
pharmacological evidence suggests that cADPR may be an endogenous
modulator of the ryanodine receptor (RYR) (Perez et al, 1998; Locuta et al,
1998). For instance, the elevations of intracellular Ca2+ mediated by cADPR
have been shown to be blocked by specific RYR inhibitors and enhanced by
caffeine (Galione et al, 1991; Lee, 1993; Lee et al, 1995). Both the synthesis
(ADP-ribosyl cyclase activity) and the degradation (cADPR-hydrolase activity) of
cADPR are mediated by the orphan receptor, CD38 (Takasawa et al., 1993;
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Zocchi et al., 1993). However, a paradox exists because of the extracellular
production of cADPR by CD38, and few data are available to explain the
transport of extracellular cADPR into the cell (Franco et al., 1998). This paradox
can partially be solved by the fact that experimental evidence has shown CD38-
like activity away from the plasma membrane and associated with several
intracellular organelles, including the mitochondria (Liang et al., 1999) and
endoplasmic reticulum (Bacher et al., 2004). Our data shown here suggests that
in isolated intracardiac neurons, VPAC2 receptor mobilization of Ca2+ through
caffeine- and ryanodine-sensitive internal stores is dependent on the production
of cADPR. However, caffeine was unaffected by the bath application of the
antagonist, 8-Br-cADPR. These data support previous findings which suggest a
different binding site for caffeine and cADPR on the ryanodine receptor (Walseth
and Lee, 1993; Prakash et al., 2000).
Lanthanum experiments performed here show that the VPAC2 receptor
mobilization of Ca2+ through caffeine- and ryanodine-sensitive internal stores is
dependent on a small Ca2+ influx through the plasma membrane. However, the
channel underlying these effects is yet to be determined. Furthermore, the
effects of La3+ on membrane Ca2+ channels appears to be faster than the
exchange of Ca2+ in bathing solution, since experiments performed under Ca2+
free conditions failed to block the VPAC2 receptor-evoked release of Ca2+ from
ryanodine-sensitive internal stores (see Fig 3.3). All three known ryanodine
receptor types (RYR1, RYR2 and RYR3) have been shown to be regulated by
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Ca2+, both on the cytosolic and luminal surfaces of the receptors (Meissner, G.,
2004). While RYRs are activated by high-affinity, Ca2+ specific binding sites,
these receptors are also inhibited by the binding of Ca2+ to low affinity, less
specific sites, giving rise to the characteristic bimodal Ca2+ dependence of
channel activity (Meissner, 2004). However, micromolar concentrations of Ca2+
are required to fully activate the RYR; therefore, it seems unlikely to be the only
essential regulator of RYRs in neurons. More likely, several key players, such as
Ca2+, cADPR, ATP and calmodulin are required to activate RYRs. Experiments
shown here with La3+ and 8-Br-cADPR support this hypothesis. Our earlier
studies failed to show any intracellular Ca2+ events that preceded the release of
[Ca2+] from the ryanodine/caffeine-sensitive stores. Thus, it seems likely that
La3+ is not inhibiting a PACAP-induced membrane influx, but rather blocking a
Ca2+ leak channel that maintains [Ca2+]i at levels which permit cADPR activation
of the RYR. Given that 2-APB did not block PACAP-induced activation of the
RYR, this leak channel is a distinct molecular entity from the TRPC channel.
However, we cannot exclude the possibility that PACAP is promoting a Ca2+
influx that is below our threshold for detection. Further, we can not exclude the
possibility that 100 µM La3+ directly blocks PACAP binding to the VPAC2
receptors, leading to the inhibition of VPAC2 receptor-mediated Ca2+ responses
under the presence of La3+.
Capacitative calcium entry through store-operated Ca2+ channels plays an
essential role in Ca2+ signaling. We show here that the PACAP activation of
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VPAC2 receptors in rat intracardiac neurons stimulates Ca2+ influx through TRP
channel antagonist-sensitive store-operated channels; however, the mechanisms
underlying the link between the intracellular stores and the TRP channels
remains to be determined. A number of models have been proposed to explain
how internal stores can regulate SOC channels in the plasma membrane. Some
models suggest the existence of diffusible messengers, whereas others consider
that information may be transferred more directly through a protein-protein
interaction (Putney et al.,2001; Berridge, 1995). Many of the TRPC channels
that have been implicated in capacitative Ca2+ entry have also been shown to be
activated by the intracellular messenger sn-1,2-diacylglycerol (DAG) (Hu et al,
2004; Venkatachalam et al., 2003). However, application of 100 µM 1-oleoyl-2-
acetyl-sn-glycerol (OAG) failed to increase [Ca2+]i in these intracardiac neurons,
suggesting that in intrinsic cardiac neurons, these ion channels primarily function
as store-operated channels.
While the mechanisms linking store-operated calcium channels to
intracellular Ca2+ stores remain elusive, the physiological roles for capacitative
Ca2+ entry is more clearly defined. Store-operated Ca2+ channels maintain
proper Ca2+ levels in the endoplasmic reticulum and generate prolonged Ca2+
responses (Putney, 2004). However, the majority of literature on physiological
functions of the SOC channels comes from studies on muscle and non-excitable
cells, not neurons. These data show that the SOC channels present in these rat
intracardiac neurons, indeed, function to replenish the intracellular Ca2+ stores,
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and that activation of these channels is necessary to preserve functional
responses to PACAP. Given that TRPC channels are also activated upon Ca2+
release from intracellular stores elicited by muscarinic receptor activation, TRPC
channels are likely to be important in preserving responses to other
neurotransmitters as well.
In conclusion, the present study demonstrates the first evidence of VPAC2
receptor mediated regulation of intracellular Ca2+ in neurons, and shows that
Ca2+ and cADPR play a part in the signal transduction cascade mediating these
effects. Furthermore, the store-operated Ca2+ channels present in these neurons
pharmacologically resemble TRPC channels, and these channels are crucial in
the ability of the cells to replenish intracellular Ca2+ stores.
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CHAPTER 5
CANONICAL TRANSIENT RECEPTOR POTENTIAL (TRPC) CHANNELS
REGULATE NEUROEXCITABILITY IN RAT INTRACARDIAC NEURONS
INTRODUCTION
The modulation of excitability of intracardiac neurons by PACAP and VIP
is in part dependent on elevations of intracellular calcium (Ca2+). These
elevations in calcium result from both mobilization of Ca2+ from ryanodine- and
caffeine-sensitive intracellular stores and capacitative Ca2+ entry through the
plasma membrane. While inhibition of Ca2+ release from intracellular stores has
been shown to abolish the enhancement of neuroexcitability induced by these
neuropeptides, less is known about the role of the capacitative Ca2+ entry on the
electrical properties of these neurons. Furthermore, the store-operated Ca2+
(SOC) channels mediating this phenomenon have not been fully characterized.
Given the importance of intrinsic cardiac neurons in the regulation of the heart,
understanding mechanisms regulating the excitability of these cells is of
significant interest. Moreover, the relationship between SOC channels and
neuroexcitability in peripheral and central neurons remans poorly understood,
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and thus insight into how these ion channels influence passive and active
membrane properties of intracardiac neurons may have broad implications.
Previously, our laboratory showed that the SOC channels present in
intracardiac neurons pharmacologically resemble short transient receptor
potential (TRPC) channels, inhibited by both 2-aminoethoxydiphenylborate (2-
APB) and lanthanum (La3+). Interestingly, inhibition of these TRPC channels
block the ability of the intracardiac neurons to sequentially elevate free
intracellular calcium concentrations ([Ca2+]i) mediated by VPAC2 receptor
activation. VPAC2 receptor regulation of TRPC channels may underlie the
changes in the resting membrane potential (RMP) and neuroexcitability seen in
the presence of PACAP in these cells.
TRP channels are a large family of non-selective cation channels
expressed in neurons and other non-excitable cells. Originally discovered as the
Drosophila photoreceptor channels (Montell and Rubin, 1989; Wong et al, 1989;
Phillips et al., 1992; Xu et al., 2000), TRP channels are composed of tetrameric
assemblies of six transmembrane spanning units. Three main subfamilies exist
based on sequence homology: canonical TRP (TRPC), melastatin TRP (TRPM)
and vanilloid TRP (TRPV) channels, and heteromers can be formed by members
of each same subfamily. While the Ca2+ permeability makes them transducers
leading to elevations in [Ca2+]i, the non-selective cationic nature of TRP channels
makes them depolarizing channels.
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The ionic mechanisms underlying the PACAP- and VIP-evoked changes
in the resting membrane potential (RMP) and neuroexcitability remain unclear.
TRPC channels, thought to be the store-operated component of the Ca2+
response in intracardiac neurons, may in part explain the increased
neuroexcitability and depolarizations seen in the presence of PACAP. The
present study examined the effects of the TRPC channel antagonist, 2- APB, on
neuroexcitability of neonatal rat intracardiac neurons. These data show that 2-
APB decreases the number of action potentials elicited by small depolarizing
current pulses. The reduction of action potentials occurs because of a 2-APB-
mediated hyperpolarization of the resting membrane potential (RMP) and
significant decrease in the action potential afterhyperpolarization (AHP) in
intracardiac neurons. While the effects of 2-APB on the RMP suggests a direct
role for TRPC channels on intracardiac neuroexcitability, the effects of 2-APB on
the AHP suggests TRPC regulation of Ca2+ activated K+ channels in intracardiac
neurons. Outward membrane currents underlying these effects produced by 2-
APB indeed support the indirect inhibition of such a Ca2+ activated K+
conductance. These data support our hypothesis that the store-operated phase
of the PACAP-evoked increase in [Ca2+]i, through TRPC channels, in part leads
to the changes in neuroexcitability seen with PACAP administration in neonatal
rat intracardiac neurons.
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METHODS
Electrophysiology
The 2-APB effects on neuroexcitability were investigated in isolated
intracardiac ganglion neurons of neonatal rats (4-7 day old), as previously
described (Fieber and Adams, 1991). Coverslips containing the dissociated
neurons were transferred to a 0.5 mL recording chamber mounted on a phase-
contrast microscope (400X). The extracellular recording solution was
physiological saline solution (PSS) consisting of (in mM): 140 NaCl, 3 KCl, 2.5
CaCl2, 1.2 MgCl2, 7.7 glucose and 10 HEPES (pH to 7.2 with NaOH). All drugs
were bath applied in PSS. Ca2+-sensitive and -insensitive K+ channel currents
were isolated by adding tetrodotoxin (TTX, 400 nM) to the PSS to inhibit voltage
activated Na+ channel currents. Action potentials and currents were amplified
and filtered (5 kHz) using an Axoclamp-200B Amplifier, digitized with a 1322A
DigiData digitizer (20kHz), and collected on a Pentium IV computer using the
Clampex 8 program (Axon Instruments, Inc., Foster City, CA, USA). Data
analysis was conducted using the pClamp 8 program, Clampfit.
The whole-cell perforated-patch variation of the patch-clamp recording
technique was used, as previously described (Horn and Marty, 1988; Rae et al.,
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1991; Xu and Adams, 1992). This configuration preserves intracellular integrity,
preventing the loss of cytoplasmic components and subsequent alteration of
functional responses of these neurons (Cuevas and Adams, 1996; Cuevas et al.,
1997). The pipette solution contained (mM): 75 K2SO4, 55 KCl, 5 MgSO4, 10
HEPES, 198 µg/ml amphotericin B, and 0.4% DMSO. Final patch pipette
resistance was 1.0 to 1.3 MΩ to permit maximal electrical access under the
present recording configuration.
Membrane potential responses to a depolarizing current pulse (-150 pA) were
determined in the absence and presence of various drugs. The mean resting
membrane potential, peak amplitude (overshoot), rise and decay slope, and
amplitude of afterhyperpolarization (AHP) were determined in the absence and
presence of 2-APB. Membrane currents were elicited by step depolarization
from -60 mV to +120 mV, or an applied voltage ramp from -120 mV to +50 mV.
Reagents and statistical analysis
All chemicals used in this investigation were of analytic grade. The following
drugs were used: Dimethyl sulfoxide (DMSO), tetraethylammonium salt (TEA),
tetrodotoxin (TTX), 2-aminoethoxydiphenylborate (2-APB), lanthanum chloride,
and cadmium chloride (Sigma-Aldrich, St. Louis, MO); and paxilline, apamin,
charybdotoxin, iberiotoxin, slotoxin (Alomone Labs, Jerusalem, Israel). All data
are presented as the mean ± SEM of the number of observations indicated.
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Statistical analysis was conducted using SigmaPlot 8 (SPSS, Chicago, IL) and
paired t-tests were used for within group comparisons.
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RESULTS
2-APB decreases action potential (AP) firing in rat intracardiac neurons
Earlier experiments (see Figure 3.5) showed that 2-APB effectively
blocked the store-operated channel, TRPC, activated by VPAC2 receptors in
intracardiac neurons. Therefore, this drug was used to determine the role of
TRPC in the active and passive membrane properties of intracardiac neurons.
The effects of 2-APB on action potential firing in rat intracardiac neurons were
investigated using the whole-cell perforated patch technique under current clamp
mode. Figure 5.1A shows a family of voltage responses elicited by 800 msec
depolarizing current pulses (150 pA) from an isolated intracardiac neuron.
Voltages were recorded in the absence (Control) and presence of 50 µM 2-APB
(2-APB), and following washout of the drug (Wash). Application of 2-APB
reduced the number of action potentials fired in this neuron from 19 under control
conditions to 8 during bath application of 2-APB, and this effect was completely
reversible. In 9 neurons studied, the depolarizing current pulses evoked
accommodating trains of action potentials in 4 cells (adapting neurons),
sustained firing of action potentials in 3 cells (tonic neurons), and a single action
potential in 2 cells. These data coincide with previously published reports on the
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firing properties of neonatal rat intracardiac neurons (DeHaven and Cuevas,
2004). While 2-APB did not inhibit firing in neurons firing single action potentials,
this drug reduced the number of action potentials elicited by depolarizing current
pulses in both accommodating (adapting) and tonic neurons. In neurons firing
multiple action potentials (adapting and tonic), 2-APB significantly reduced the
number of action potentials by ~50%, from 12.4 ± 2.4 to 6.1 ± 1.0 action
potentials fired (Fig 5.1B).
2-APB-evoked changes in the single action potential waveform
To gain insight into the underlying cause of the reduction in action
potential firing evoked by bath application of 2-APB, current clamp experiments
were performed to look at the single action potential waveform parameters.
Figure 5.2A shows single action potentials elicited from brief depolarizing pulses
(100 pA) before (Control), during (2-APB) and after (Wash) bath application of 50
µM 2-APB. 2-APB hyperpolarized the intracardiac neuron from -55.9 mV to -61.1
mV, and reduced the afterhyperpolarization (AHP) by ~10 mV, from -17.9 mV to -
4.6 mV. Along with the significant resting membrane potential (RMP) and AHP
changes evoked by 2-APB, the TRP channel antagonist was also shown to alter
the rise and decay slope of the single action potential, with the latter being a
significant change (-14.2 ± 3.3 mV/sec to -12.8 ± 3.5 mV/sec). The effects of 2-
APB on the action potential waveform configuration are summarized in Figure
5.2B. Similar findings on RMP, AHP and rise and decay slope were made with
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100 µM La3+ (Data not shown), which inhibits voltage gated ion channels and
TRP channels (Aussel et al., 1996; Halaszovich et al., 2000; Beedle et al., 2002).
The effects of 2-APB on the action potential AHP suggests a role for TRP
channel regulation of Ca2+ activated K+ channels in rat intracardiac neurons
(Franciolini et al., 2001). The effects of various direct acting (charybdotoxin,
paxilline, iberiotoxin, slotoxin, apamin, TEA) and indirect acting (Ca2+ free PSS,
La3+, Cd2+) Ca2+ activated K+ channel blockers on action potential waveform and
AHP were investigated in isolated rat intracardiac neurons. Using the perforated-
patch whole-cell recording technique, neurons were held at -50 mV under current
clamp conditions, and single action potentials were evoked by 100 pA
depolarizing current pulses for 100 msec. Interestingly, neither of the specific BK
channel toxins, charybdotoxin (100 nM, n=5) and paxilline (200 nM, n=5), or the
SK channel toxin, apamin (100 nM, n=4) elicited any changes in rat intracardiac
neuron waveform parameters, including the relative AHP (Fig 5.3C,D,E).
Iberiotoxin (10 nM) and slotoxin (10 nM) also had no effects on relative AHP
(Data not shown). Conversely, TEA (500 µM), which has been shown to inhibit
BK channels in rat intracardiac neurons (Franciolini, F. et al., 2001), increased
the action potential duration by decreasing the decay slope; however, no
significant change in the relative AHP was seen (Fig 5.3A,E). Furthermore, the
indirect acting Ca2+ activated K+ channel blockers Cd2+ (100 µM) and La3+ (10-
100 µM), and the replacement of extracellular Ca2+ with Mg2+ (Ca2+-free PSS),
significantly reduced the relative AHP in intracardiac neurons by 30%, 58% and
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39%, respectively (Fig 5.3B,E). Thus, these data show 2-APB significantly
reduces the decay slope and AHP in rat intracardiac neurons, as well as
hyperpolarizes these cells. These effects are in part mimicked by La3+, Ca2+-free
PSS, TEA and Cd2+; yet, none of the specific toxins for Ca2+ activated K+
channels alter the action potential waveform parameters. Therefore, the identity
of the channel mediating the AHP remains to be determined.
Membrane currents underlying the 2-APB-elicited changes in action
potential properties
To analyze the effects of 2-APB on intracardiac neuron membrane K+
currents, outward currents evoked by 20 mV steps from -60 mV to +120 mV were
recorded before (Control), during (2-APB) and after (not shown) bath application
of 50 µM 2-APB (Fig 5.4A). The net 2-APB sensitive current (2-APB Sensitive)
was determined by subtracting the current remaining after 2-APB application
from that observed under control conditions (Control) (Fig 5.4A, bottom). The 2-
APB-sensitive current, and thus the TRPC channel regulated K+ current in these
neurons, exhibits pronounced time-dependent inactivation at positive potentials,
but such inactivation was not observed at negative potentials. Figure 5.4B
shows the current-voltage relationship of both the mean peak 2-APB-sensitive
current () and the mean sustained 2-APB-sensitive current (), determined at
the location of the arrows shown in figure 5.4A, for 4 neurons. The 2-APB-
sensitive current activated at potentials positive to -60 mV, and maximal
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activation occurs near +100 mV. The 2-APB-sensitive peak and sustained
current densities at +120 mV were 106.4 ± 37.2 mV and 20.2 ± 12.9 mV,
respectively.
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FIGURE 5.1: 2-APB induced changes in the number of action potentials fired in neonatal rat intracardiac neurons. (A)
Action potentials elicited from a single current-clamped neuron in response to 150 pA depolarizing current pulses (800
msec) in the absence (Control) and presence of 50 µM 2-APB (2-APB), and following washout of drug (Wash). (B) Bar
graph of the mean number of action potentials ± SEM recorded from 9 neurons under identical conditions as in (A).
Dashed lines indicate 0 mV. The asterisk denotes significant difference (p<0.05)
144
145
FIGURE 5.2: 2-APB-evoked changes in the single action potential waveform.
(A) Single action potentials elicited by brief depolarizing current pulses (100 ms,
150 pA) on a single intracardiac neuron before (Control, black trace), during (2-
APB, red trace) and following (Wash, gray trace) bath applied 2-APB (50 µM).
Dashed line indicates 0 mV. (B) Effects of 2-APB on the resting membrane
potential (RMP) and action potential waveform parameters. Action potentials
were evoked by 100 pA current injections for 100 msec. 2-APB was bath applied
and the data are shown as the mean ± S.E.M. for 4 cells. Asterisks denote
significant difference from control (p<0.05).
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CONTROL 2-APB (50 µM) WASH
RMP (mV) -52.9 ± 2.4 -56.1 ± 2.9* -52.5 ±0.8
PEAK AMPLITUDE (mV) 20.3 ± 1.5 20.2 ± 3.3 19.8 ± 0.7
RISE SLOPE (mV/sec) 33.4 ± 9.1 40.4 ± 12.1 38.5 ± 9.2
DECAY SLOPE (mV/sec) -14.2 ± 3.3 -12.8 ± 3.5* -16.1 ± 3.3
AHP (mV) -17.4± 2.7 -8.2 ± 3.5* -20.1 ± 1.9
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FIGURE 5.3: Effects of various direct- and indirect-acting Ca2+-activated K+
inhibitors on the action potential waveform and afterhyperpolarization (AHP) in rat
intracardiac neurons. Resting membrane potential (RMP) was held at -50 mV
under current clamp conditions and action potentials were elicited by brief
depolarizing current pulses (150 pA, 100 msec). Action potentials were obtained
in the absence (Control) and presence of bath applied (A) 500 µM TEA, (B) 100
µM Cd2+, (C) 200 nM paxilline, or (D) 100 nM apamin. Dashed lines indicate 0
mV. (E) Bar graph depicting the relative afterhyperpolarzations (AHP), defined
as the AHP under the presence of drug divided by the control AHP, of
intracardiac neurons under the presence of the direct acting (charybdotoxin,
paxilline, apamin, TEA) and indirect acting (Ca2+ free, La3+, Cd2+) Ca2+ activated
K+ channel blockers (± SEM). Asterisks denote a significant decrease in AHP
(p<0.05).
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149
FIGURE 5.4: Inhibition of outward currents in rat intracardiac neurons by the TRP channel antagonist, 2-APB. (A)
Membrane currents evoked by depolarizing steps between -60 mV to +120 mV from a holding potential of -60 mV in
normal physiological saline solution (PSS) before (Control) and during (2-APB) bath applied 2-APB (50 µM). The net 2-
APB sensitive current (2-APB Sensitive) was determined by subtracting the current remaining after 2-APB application
from that observed under control conditions (Control). (B) Current-voltage relationship of both the peak 2-APB-sensitive
current (--) and the sustained 2-APB-sensitive current (-±-), determined at the location of the arrows shown in (A)
bottom (n=4). Error bars indicate ± S.E.M.
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DISCUSSION
The major finding reported here is that TRP channels, most probably
TRPC, in intrinsic cardiac neurons regulate the active and passive membrane
properties of these cells. Inhibition of TRPC channels with 2-APB or La3+
hyperpolarized intracardiac neurons, depressed repetitive firing and eliminated
the action potential afterhyperpolarization (AHP) in these cells. The effects of 2-
APB and La3+ on AHP were in part mimicked by Cd2+ and Ca2+-free conditions,
suggesting that the AHP is mediated by a Ca2+-activated current that can be
regulated by TRPC. Furthermore, currents mediated by TRPC channels were
recorded at voltages near the resting membrane potential of these cells (circ. -50
mV), and exhibited little or no time-dependent inactivation at negative potentials.
However, at positive potentials the TRPC channels showed pronounced time-
dependent inactivation. These data further support that 2-APB mediates its
affects through: (1) the inhibition of non-selective TRPC channels, and (2) either
the direct inhibition of Ca2+-activated K+ channels, or more probable, the indirect
inhibition of Ca2+-activated K+ channels due to TRPC inhibition.
Our studies show that bath application of 50 µM 2-APB hyperpolarizes
intracardiac neurons by ~3 mV, and in neurons which fire tonic or
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accommodating (adapting) action potentials in response to depolarizing current
pulses, 2-APB reduces the number of action potentials fired significantly. The
hyperpolarizing affects of 2-APB are mediated through the inhibition of the
depolarizing, non-selective TRPC channels in these intracardiac neurons.
Furthermore, the effects of 2-APB on the single action potential waveform
properties of intracardiac neurons showed a significant decrease in the peak
afterhyperpolarization (AHP) when compared to control conditions, as well as a
change in the decay slope, meaning the action potential duration increased. The
effects of 2-APB on AHP suggest a role for TRPC channel regulation of Ca2+-
activated K+ channels. Thus, TRPC channels directly, and TRPC regulation of
Ca2+ activated K+ channels, play a role in the regulation of neuroexcitability in
intracardiac neurons. Importantly, 2-APB does not block Ca2+ activated Cl-
channels in Xenopus oocytes (Chorna-Ornan et al., 2001). However, 2-APB may
block other membrane chloride channels undetermined to date. Interestingly, the
PACAP-evoked increase in action potential firing in these cells was associated
with an increase in AHP (DeHaven and Cuevas, 2004).
Ca2+ activated K+ channels are known to alter excitability in autonomic
neurons (Franciolini et al., 2001), and it previously has been shown that both IP3
(Hoesch et al., 2004) and ryanodine (Moore et al., 1998; Jobling et al., 1993;
Kawai and Watanabe, 1989, 1991) receptor-evoked Ca2+ release activates a K+
conductance which alters the AHP properties of neurons. However, these
studies focused directly on the IP3 and ryanodine receptor intracellular Ca2+
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stores activating the Ca2+ activated K+ channels, and failed to mention the
common denominator between these two stores, the store-operated Ca2+
channels.
These studies show that while charybdotoxin, paxilline, iberiotoxin,
slotoxin and apamin have no effects on the AHP of rat intracardiac neurons, the
replacement of extracellular Ca2+ with Mg2+, or the addition of the voltage-gated
Ca2+ channel blocker, Cd2+, significantly reduced the relative AHP. These data
suggest the role of toxin insensitive, Ca2+ activated conductances in the
regulation of the AHP in these cells. Furthermore, La3+, which inhibits voltage
gated and store-operated Ca2+ channels, as well as TRP channels (Aussel et al.,
1996; Halaszovich et al., 2000; Beedle et al., 2002), was shown to have nearly
identical effects to 2-APB on the action potential properties, including the RMP
and AHP. This finding is important because 2-APB is known to inhibit IP3
receptors along with plasma membrane SOC channels. The fact that La3+
showed similar effects on the intracardiac neuroexcitability and action potential
waveform properties suggests the 2-APB effects on action potential firing are not
mediated by the inhibition of IP3 receptors, but instead inhibition of TRP channels
in the plasma membrane. To our knowledge, these are the first data showing the
regulation of plasma membrane ion channels important in the production of the
AHP by the TRP channel antagonists, 2-APB and La3+.
Finally, we find a reversible inhibition of an outward current elicited by
voltage steps from -60 mV to +120 mV before and after 2-APB bath application.
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Two phases of the outward currents were investigated, the initial peak K+ current
and the sustained K+ current. While the peak K+ current is generally thought to
be mediated by BK and IA currents, the sustained conductance is through
delayed rectifier K+ channels (Wang et al., 2002; Sun et al., 2003). BK channels
are present in these neurons (Franciolini et al., 2001), but IA has yet to be
identified. These data show that the majority of the 2-APB sensitive outward
current in rat intracardiac neurons is the peak, transient current; however, a very
small inhibition of the sustained current was noted. Thus, 2-APB blocks a peak
transient outward current in rat intracardiac neurons which resembles BK or IA.
However, these effects are probably also due to actions on the TRPC channels
themselves. To date, very little is known about the regulation of excitability by
TRPC channels. Thus, further experiments must be performed in order to
determine (1) the direct effects of SOC channels (TRPC) on membrane currents
and (2) the regulation of other Ca2+ activated ion channels, such as K+ channels,
by these SOC channels in rat intracardiac neurons. These studies will give
further insight to the Ca2+ dependent regulation of neuroexcitability seen with the
neuropeptides, PACAP and VIP, in rat intracardiac neurons.
In conclusion, the Ca2+ dependent regulation of neuroexcitability in rat
intracardiac neurons by PACAP and VIP can in part be explained by the
activation of 2-APB- and La3+-sensitive SOC channels in the plasma membrane,
indirectly associated with the regulation of other Ca2+ activated ion channels,
most notably the Ca2+ activated K+ channels.
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CHAPTER 6
SUMMARY
Results reported here using PACAP and VIP provide direct evidence of
some of the complex signaling which occurs in neurons of the mammalian
intracardiac ganglia (Fig 6.2), and this study provides the first description of
modulation of intracardiac neuron excitability through the VPAC2 receptor-
mediated regulation of intracellular Ca2+. Furthermore, these data describe, in
general, a novel method in which neurons can increase excitability through G
protein regulated changes in Ca2+ handling, and these results can be related to
various mammalian cells.
The expression of PACAP and VIP receptors were investigated in isolated
intracardiac neurons of neonatal rat intracardiac ganglia using single-cell reverse
transcription-polymerase chain reaction (RT-PCR). Individual intracardiac
neurons were shown to express various isoforms of the PAC1 receptor, including
the PAC1-short, -HOP1 and -HOP2 variants, which differ in the region encoding
the G protein-binding domain. Neither the PAC1-very short, -HIP, nor –HIPHOP
variants were detected. The PAC1-HOP1 was expressed at higher levels and in
a greater number of cells than other PAC1 variants. VPAC1 and VPAC2
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transcripts were also detected in intracardiac neurons, with VPAC2 being found in
nearly all of the neurons (Fig 6.1). These data support previous cloning
experiments which suggest the two major forms in mammals are the PAC1-short
and -HOP1 receptors (Spengler et al, 1993; Svoboda, 1993). In contrast, in
isolated adult guinea pig intracardiac neurons, the expression of PAC1-very short
and PAC1-HOP2 isoforms predominate (Braas et al., 1998), and no VPAC
receptor isoforms have been reported. The present findings underlie the
complex effects of PACAP and VIP on neuroexcitability and Ca2+ handling in the
intracardiac ganglia.
The effects of PACAP and VIP on neonatal rat intracardiac
neuroexcitability were investigated using the whole cell perforated patch
technique under current clamp mode. PACAP and VIP have been shown to
modulate the activity of various neuronal populations. For instance, in the CNS,
administration of PACAP in the hypothalamus increased the firing rate activity
and caused membrane depolarization (Uchimura et al., 1996; Shibuya, et al.,
1998). Likewise, in the PNS, application of PACAP on isolated adult guinea pig
intracardiac neurons also depolarized the cells and changed the action potential
firing frequencies (Braas et al., 1998; Parsons et al., 2000). The present results
show both PACAP and VIP enhanced neuronal excitability in isolated neurons of
rat intracardiac ganglia. Interestingly, the effects on excitability produced by
these neuropeptides were not identical. While bath application of PACAP or VIP
resulted in a depolarization of intracardiac neurons, PACAP depolarized the
157
neurons to a greater extent than VIP. However, maxadilan, a PAC1 receptor
specific agonist, did not evoke a depolarization of intracardiac neurons or
increase action potential firing, suggesting that PAC1 receptors do not have a
direct effect on neuroexcitability in these cells. Nonetheless, the PAC1 receptor
antagonist, M65, depressed PACAP induced depolarizations. Furthermore,
increases in action potential firing in response to depolarizing current pulses
were only observed under conditions in which both PAC1 and VPAC receptors
were stimulated, that is, when PACAP was used as the agonist and neither PAC1
nor VPAC receptor-specific antagonists were applied. Thus, simultaneous PAC1
and VPAC receptor stimulation elicits a synergistic enhancement of
neuroexcitability and produces changes in the active membrane properties that
are not seen with stimulation of either receptor alone.
The effects of PACAP and VIP on Ca2+ handling in intracardiac neurons
was investigated using fura-2 Ca2+ imaging techniques. While PACAP and VIP
increased [Ca2+]i in isolated intracardiac neurons, maxadilan, a PAC1-selective
agonist, failed to elicit a response. Furthermore, the VPAC-selective antagonists,
L-8-K and [N-Ac-Tyr1, D-Phe2]-GRF (1-29), significantly blocked the
neuropeptide-evoked Ca2+ elevations, while M65, the PAC1-selective antagonist
failed to inhibit the response. Lastly, application of PACAP(6-38) significantly
inhibited the PACAP-induced elevations in [Ca2+]i. Taken with the fact that
neonatal rat intracardiac neurons predominantly express the VPAC2 receptor
158
(>90%), these results show that activation of VPAC2 receptors mediates the Ca2+
responses in these cells.
PACAP and VIP are known to modulate nicotinic acetylcholine receptors
(nAChRs) in isolated rat intracardiac neurons (Liu et al., 2000). However,
blocking neurotransmission with the Na2+ channel blocker, tetrototoxin (TTX), and
the ganglionic nAChR blocker, mecamylamine, had no effect on the VPAC2
receptor-elicited changes in [Ca2+]i in rat intracardiac neurons. Thus, the
observed rise in [Ca2+]i is not dependent on neurotransmission.
VPAC2 receptor-elicited changes in [Ca2+]i in rat intracardiac neurons
exhibited both a transient and sustained component. VPAC receptors have been
linked to both Ca2+ entry through the plasma membrane via the activation of L-
type Ca2+ channels (Chatterjee et al., 1996; Tanaka et al., 1998) and Ca2+
release from intracellular stores. Two distinct ER intracellular Ca2+ stores exist in
intracardiac neurons, one that is sensitive to inositol 1,4,5-trisphosphate (IP3) and
another sensitive to ryanodine and caffeine. Under Ca2+-free conditions, PACAP
and VIP still elicited a Ca2+ response in these neurons. However, the sustained,
plateau component of the Ca2+ response was lost. Conversely, VPAC2 receptor-
induced increases in [Ca2+]i were completely blocked by the application of
ryanodine, or depletion of the intracellular Ca2+ stores by caffeine or thapsigargin.
Furthermore, 2-APB, an IP3 receptor and TRP channel antagonist, only blocked
the sustained component of the PACAP-elicited elevations in [Ca2+]i. These data
suggest VPAC2 receptor activation in intracardiac neurons leads to an elevation
159
in [Ca2+]i which initially comes from ryanodine- and caffeine-sensitive intracellular
pools, followed by the influx of Ca2+ through the plasma membrane. Moreover,
the plasma membrane component appears to be through some unknown, 2-APB
sensitive TRP channel.
While our studies showed that VPAC2 receptors mediate a Ca2+ response
in isolated intracardiac neurons initially through the release of Ca2+ from caffeine-
and ryanodine-sensitive intracellular pools, followed by the activation of 2-APB
sensitive plasma membrane channels, the signal transduction system coupling
the VPAC2 receptor to the ryanodine receptor had not been identified.
Furthermore, the 2-APB sensitive channel facilitating Ca2+ through the plasma
membrane had not been determined. Fura-2 mediated Ca2+ imaging
experiments were performed in order to elucidate some of the factors in the
VPAC2 receptor regulation of ryanodine receptors and plasma membrane TRP
channels in isolated rat intracardiac neurons. The activation of VPAC2 receptors
and subsequent increase in [Ca2+]i in rat intracardiac neurons was not directly
associated with increased levels of cAMP. Forskolin, an adenylyl cyclase
activator, failed to elevate [Ca2+]i in these isolated neonatal rat intracardiac
neurons. However, 8-Br cADPR significantly reduced the VPAC2-evoked Ca2+
elevations, suggesting that ryanodine receptor activation is dependent on the
production of cADPR in neonatal rat intracardiac neurons. Furthermore, the
binding site of caffeine and cADPR to the ryanodine receptor is different,
because caffeine was unaffected by the bath application of 8-Br cADPR.
160
Lanthanum experiments performed here showed that the VPAC2 receptor
mobilization of Ca2+ through caffeine- and ryanodine-sensitive internal stores is
dependent on a small Ca2+ influx through the plasma membrane. However, the
channel underlying these effects is yet to be determined. Interestingly, the
inhibition of membrane channels by La3+ appears to be faster than the exchange
of Ca2+ in the bathing solution, since experiments performed under Ca2+ free
conditions failed to block the VPAC2-evoked release of Ca2+ from ryanodine-
sensitive internal stores. Conversely, this paradoxical effect mediated by La3+
might be explained by the direct inhibition of PACAP binding to the VPAC2
receptor. Nonetheless, several key factors, including cADPR and Ca2+ are
required to activate ryanodine receptors in neonatal rat intracardiac neurons.
The inhibition of the VPAC2 receptor-mediated influx of Ca2+ through the
plasma membrane by 2-APB suggested TRP channels mediates this effect. The
inhibition of the sustained component of the Ca2+ response by La3+ in these
neurons further supported that TRP channels underlie the response. However,
several subfamilies of TRP channels exist, including TRPC, TRPM and TRPV.
While TRPC channels are the most widely implicated in store-operated Ca2+
entry, TRPV and TRPM needed to be ruled out. Fura-2 mediated Ca2+ imaging
experiments showed that neither menthol, nor capsaicin increased [Ca2+]i in
isolated neonatal rat intracardiac neurons. While menthol has been shown to
activate TRPM8 channels (Hu et al., 2004; Tsuzuki et al., 2004), capsaicin has
been shown to activate TRPV1 channels (Hu et al., 2004; Krause et al., 2005).
161
Thus, these data suggest TRPM8 and TRPV1 do not play a role in the sustained
phase of the Ca2+ response in these neurons. Furthermore, 2-APB is a common
activator of TRPV1, TRPV2 and TRPV3 (Hu et al., 2004). However, 2-APB
alone had no effects on the [Ca2+]i in intracardiac neurons. Taken together,
these data suggest TRPV and TRPM do not play a role in the sustained phase of
the VPAC2 receptor-evoked Ca2+ response in neonatal rat intracardiac neurons.
Thus, TRPC channels are the likely candidate for this effect.
TRPC channels have been implicated in store-operated Ca2+ entry, as well
been shown to be activated by the intracellular messenger DAG (Hu et al., 2004;
Venkatachalam et al., 2003). However, application of OAG failed to increase
[Ca2+]i in these intracardiac neurons, suggesting that these ion channels primarily
function as store-operated channels in these cells. This was supported by the
fact that store depletion by PACAP under Ca2+ free conditions evoked a store
dependent influx of Ca2+ across the plasma membrane, and that influx was
significantly blocked by bath application of 2-APB. Thus, TRPC channels are
regulated by store depletion in neonatal rat intracardiac neurons.
Store-operated Ca2+ channels maintain proper intracellular store Ca2+
levels, as well as generate prolonged Ca2+ responses (Putney, 2004). These
data showed that 2-APB significantly inhibited the ability of the neuron to
repetitively increase [Ca2+]i. Thus, TRPC channels regulated by store depletion
function to replenish the intracellular Ca2+ stores, and activation of these
channels is essential to preserve functional responses to PACAP.
162
Experiments using electrophysiological techniques showed that the
modulation of excitability of intracardiac neurons by PACAP and VIP is
dependent on elevations of [Ca2+]i. Bath application of ryanodine, and the
removal of extracellular Ca2+ significantly reduced the depolarization and
increases in action potential firing induced by application of PACAP. Thus, the
inhibition of the Ca2+ release from ryanodine-sensitive has been shown to abolish
the enhancement of neuroexcitability induced by PACAP in these intracardiac
neurons. However, little is known about the role of store-operated Ca2+ entry on
the electrical properties of these neurons. Store-operated TRPC channels may
in part underlie the changes in neuroexcitability seen in the presence of PACAP
and VIP in intracardiac neurons. While the Ca2+ permeability of TRPC channels
makes them candidates for store-operated Ca2+ channels, the non-selective
cationic nature of TRPC channels makes them depolarizing agents. Thus,
electrophysiological experiments were performed to investigate the ionic
mechanisms underlying the PACAP and VIP evoked changes in resting
membrane potential (RMP) and neuroexcitability seen in isolated neonatal rat
intracardiac neurons. Data presented here showed that 2-APB decreased the
number of action potentials elicited by small depolarizing current pulses. The
reduction of action potentials occurred because of a 2-APB mediated
hyperpolarization of the resting membrane potential (RMP) and significant
decrease in the action potential afterhyperpolarization (AHP) in these intracardiac
neurons. The effects of 2-APB on the RMP suggests a direct role for TRP
163
channels on the regulation of intracardiac neuron passive and active membrane
properties. However, the effects of 2-APB on the AHP suggests TRPC channels
regulate Ca2+ activated K+ channels in intracardiac neurons. These data were
supported by the fact that 2-APB inhibited transient outward currents that
resembled Ca2+ activated K+ currents.
While these results discussed give insight to the Ca2+-dependent changes
in neuroexcitability evoked by VPAC2 receptor activation in isolated intracardiac
neurons, the PAC1 receptor mediated effects have yet to be determined.
Although the AC-cAMP-PKA intracellular signal pathway does not directly
increase [Ca2+]i in these cells, our experiments do not rule out the possibility that
cAMP may have a different role in PACAP modulation of neuroexcitability seen in
intracardiac neurons. PAC1 activation in guinea pig intracardiac neurons has
been shown to enhance the cAMP-regulated, hyperpolarization-activated
nonselective cation conductance, Ih (Merriam et al., 2004). Ih has previously
been demonstrated in neonatal rat intracardiac neurons by electrophysiological
techniques (Cuevas et al., 1997).
164
FIGURE 6.1: Schematic representation of the expression pattern of PAC1 and
VPAC2 receptors in individual intracardiac neurons. The percentages (%)
located inside the cells indicate the predicted percent of individual neonatal rat
intracardiac neurons that express the various PAC1 receptor isoforms, as well as
VPAC2 receptors. Data are interpreted from experiments performed using single-
cell RT-PCR (Chapter 2). In order to avoid confusion, VPAC1 receptors were left
out of the diagram. However, VPAC1 receptor transcripts were detected in a
small proportion of isolated neonatal rat intracardiac neurons (>20%).
165
FIGURE 6.2: Schematic representation of the pathways mediating the PACAP- and VIP-induced elevations in [Ca2+]i and
enhancement of neuroexcitability seen in isolated rat intracardiac neurons. PACAP and VIP increase [Ca2+]i in
intracardiac neurons through the activation of G-protein coupled VPAC2 receptors, and this mobilization of Ca2+ exhibits
both a transient and sustained component. The transient component is in part due to mobilization of Ca2+ from
caffeine/ryanodine-sensitive stores, and the signal transduction is Ca2+ and cADPR-dependent. Ca2+ release from these
stores regulates the entry of Ca2+ through the plasma membrane, and pharmacological evidence suggests TRPC
channels mediate this effect. TRPC is critical in these intracardiac neurons abilities to repetitively elevate intracellular
Ca2+ levels and replenish internal Ca2+ stores. PACAP-induced changes in Ca2+ handling by intracardiac neurons is
linked to the neuroexcitability produced by the neuropeptide in these cells, and simultaneous activation of VPAC2 and
PAC1 receptors results in a synergistic amplification of excitability. Pharmacological evidence suggests inhibition of TRPC
decreases action potential firing in intracardiac neurons through changes in resting membrane potential (RMP),
suggesting a direct role for TRPC in the regulation of neuroexcitability. Furthermore, 2-APB effects on the action potential
afterhyperpolarizations (AHP) and outward currents suggest TRPC channels regulate Ca2+ activated K+ channels.
However, PAC1 receptor mediated effects have yet to be elucidated.
166
167
CONCLUSION
Autonomic control of cardiac function depends on the coordinated activity
generated by neurons within the intracardiac ganglia, and intrinsic feedback
loops within the ganglia provide precise control of cardiac function. Interestingly,
the progressive development of heart disease is associated with a remodeling of
this intrinsic cardiac ganglion, and an adaptation of the control mechanisms
which regulate these intrinsic cardiac neurons. Such a remodeling process likely
involves both afferent and efferent neurons, hormones, receptors, and signal
transduction pathways within the intracardiac neurons. Although recent studies
have identified some of the anatomical and functional characteristics of the
intracardiac ganglia, we are just beginning to understand how interactions among
the network of neurons act to regulate cardiac function. Understanding the
intracardiac ganglia in normal physiological conditions, as well as the remodeling
that occurs in diseased states, will lead to novel approaches to the development
of therapy to treat heart disease.
Results reported here using PACAP and VIP provide direct evidence of
some of the complex signaling which occurs in neurons of the rat intracardiac
ganglia. The physiological effects of VPAC2 receptor activation on intracardiac
168
neurons have clinical implications. Interestingly, VIP has been shown by both in
vivo and in vitro experiments to act as a positive inotropic (Unverferth et al.,
1986; Bell and McDermott, 1994) and chronotropic agent (Christophe et al.,
1984; Smitherman et al., 1989; Rigel and Lanthrop, 1990). Moreover, VIP has
been shown to be elevated in patients suffering from early heart failure, while
decreased concentrations of VIP are related to the progressive worsening of the
disease (Lucia et al., 2003). Furthermore, it has been suggested that some of
the beneficial effects seen with angiotensin converting enzyme (ACE) inhibitor
therapy in heart failure patients may be caused by VIP (Duggan and Ye, 1998).
ACE inhibitors result in an increase in cardiac output without an increase in heart
rate, suggesting a positive inotropic effect. However, this cannot be explained by
a reduction in angiotensin II and bradykinin concentrations. It has been
suggested that the angiotensin converting enzyme may metabolize VIP (Duggan
and Ye, 1998), and the increased concentrations of VIP that would result from
ACE inhibitor therapy would explain the positive inotropic effects seen. However,
several studies have now shown that the angiotensin converting enzyme does
not metabolize VIP (Farmer and Togo, 1990; Duggan and Ye, 1998).
Nonetheless, VIP concentrations do increase during ACE inhibitor therapy, and
these changes probably contribute to the improvement in cardiac function
following therapy with these agents (Duggan and Ye, 1998).
In conclusion, the mammalian intracardiac ganglion exerts intrinsic regulation
over cardiac performance, and is thus a potential pharmacological target for
169
treating disease. Experiments performed here give insight into some of the
complex signaling which occurs in neurons of the mammalian intracardiac
ganglia, and these results support the hypothesis that the intrinsic cardiac nerve
plexus acts as much more than a simple relay center for parasympathetic
innervation to the heart. PACAP and VIP are important regulators of neuronal
signaling within the intracardiac ganglia, and VPAC2 and PAC1 receptor
activation plays an important neuromodulatory role in the regulation of cardiac
homeostasis. Even more interesting is the possible roles these peptides play in
various diseases associated with the heart. Despite the progress made in
intracardiac ganglion research, much of the physiological and pathological roles
of these ganglia remain incomplete. Further investigations of the intracardiac
ganglia will shed light on the intrinsic beat-to-beat regulation of the mammalian
heart. For instance, the molecular identities of many of the membrane proteins
associated with intracardiac neuroexcitability have yet to be determined.
Furthermore, the electrophysiological properties of intrinsic cardiac neurons have
not been completely established (Adams and Cuevas, 2004). To my knowledge,
data shown here are the first to indicate the importance of TRPC activity on the
resting membrane potential (RMP) in intracardiac neurons, as well as the
regulation of Ca2+ activated K+ channels. Nonetheless, progress is being made
in the understanding of intracardiac neuronal control of the mammalian heart.
170
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ABOUT THE AUTHOR
Wayne Isley DeHaven was born in St. Petersburg, Florida on April 8,
1976, to Eugene W. and Kathy I. DeHaven. He received his elementary
education at Bear Creek and Azalea Middle Schools. In 1994, Wayne graduated
Boca Ciega High School in Gulfport, Florida. Wayne earned his bachelor’s
degree in Entomology from the University of Florida in 1998. Two years later,
Wayne earned his Master’s degree in Biomedical Sciences from Barry University
in Miami, Florida. In 2000, Wayne entered the Ph.D. program at the University of
South Florida College of Medicine to pursue further education and training in
science. Wayne completed his studies at the University of South Florida
Pharmacology and Therapeutics Department in the Spring of 2005.