186

PACAP Modulates Rat Sympathetic Neuron Depolarization Through IP

3

VICTOR MAY,

a

MATTHEW M. BEAUDET, RODNEY L. PARSONS, AND KAREN M. BRAAS

Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, Vermont 05405, USA

INTRODUCTION

During the last several years, our laboratories among many others, have demon-strated the physiological relevance of PACAP and the PACAP-selective PAC

1

recep-tor as the noncholinergic neuromodulatory system in sympathetic autonomicphysiology. We have shown that PACAP and the PAC

1

receptors are distributed inthe appropriate tissues to participate in sympathetic function, that PACAP peptideselicit sympathetic neuronal responses at physiologically relevant concentrations, andgiven the well established neurotrophic functions of the VIP/PACAP family of pep-tides, that PACAP and PAC

1

receptors have potential roles in sympathetic neuronaldevelopment and regeneration following injury. The initial studies demonstrated thepotent effects of PACAP27 and PACAP38 on superior cervical ganglion (SCG)sympathetic neuron neurotransmitter and neuropeptide Y (NPY) production andsecretion.

1,2

Subsequently, we have employed molecular, cellular, and biochemicalapproaches to demonstrate the expression of PACAP mRNA in spinal cord sympa-thetic preganglionic projection neurons to the SCG,

3

to identify the preferentialexpression of one specific PAC

1

(

short

)HOP1 receptor variant in the majority of SCGpostganglionic neurons, and to establish, using selective inhibitors to particular sec-ond messenger pathways, the ability of that SCG receptor variant to activate potentlymultiple intracellular signaling cascades.

1,4–6

These results were in agreement withthe elegant work of a number of other laboratories, demonstrating that the high den-sity of PACAP-immunoreactive fibers in the SCG is diminished on transection of thecervical sympathetic trunk,

7–9

the SCG PAC

1

receptor mRNA is preferentiallyexpressed in postganglionic neurons by

in situ

hybridization,

4,10

and that PACAPpeptides may have proliferative roles in sympathetic neuroblast development.

6,11

The expression of PACAP is augmented considerably following neuronal injury, duein part to alterations in posttranscriptional mechanisms suggesting that PACAP pep-tides may be important mediators of the regeneration response.

12–14

The multitudeof different intracellular signaling pathways coupled to the PAC

1

receptor, the diver-sity of neuronal responses to PACAP and the unique regulatory mechanisms under-lying the expression of PACAP peptides all suggest that the PACAP peptidergic

a

Address for correspondence: Victor May, Ph.D., Department of Anatomy and Neuro-biology, University of Vermont College of Medicine, Given Health Science BuildingC421, Burlington, Vermont 05405. Voice: 802-656-0398; fax: 802-656-8704.

vmay@zoo.uvm.edu

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system must have extraordinary neuroregulatory roles in nervous system develop-ment and function.

PACAP POTENTLY DEPOLARIZES SYMPATHETIC NEURONS

More recently, we have expanded our studies to better understand, not only themore rapid neurotransmitter roles of PACAP in modulating neuronal membranecharacteristics, but also the ionic mechanisms that are regulated by PACAP and thelong term consequences of these responses following altered neuronal transcription-al activity. Intracellular microelectrodes were used to measure the electrophysiolog-ical responses in cultured primary SCG neurons. Treatment of SCG neurons withPACAP peptides elicited membrane depolarizations in more than 90

%

of the neu-rons examined, which correlated well with previous studies demonstrating the prev-alence of PAC

1

receptor expression in a majority of adult and neonatal SCG, and

FIGURE 1. PACAP27 induces membrane depolarizations and inward currents insympathetic neurons. A. Phase contrast micrograph of the postganglionic sympathetic neu-rons in vitro used for current and voltage recordings with intracellular microelectrodes.B. Representative SCG neuron action potential with overshooting peak and after hyperpo-larization. C. PACAP-induced depolariztion of a sympathetic neuron in response to 1 secpressure ejection of 50 µM PACAP27 (arrow). D. In the presence of 300 nM TTX, pressureapplication of 50 µM PACAP27 to sympathetic neurons voltage clamped to −50 mVrevealed the current underlying the PACAP-induced depolarizations.

188 ANNALS NEW YORK ACADEMY OF SCIENCES

cultured sympathetic neurons.

4,5,8–10

Superfusion of 100 nM PACAP27 for 30 sec-onds or one second pressure application of 50

µ

M PACAP27 depolarized sympathet-ic neurons 10

±

0.5 mV and 12

±

0.6 mV, respectively (see

F

IGURE

1). Inward currentsunderlying the PACAP-induced depolarizations were measured in tetrodotoxin(TTX)-treated preparations using the single electrode voltage-clamp recording tech-nique. Pressure application of 50

µ

M PACAP27 onto SCG neurons voltage-clampedto

−

50 mV produced a mean inward current of

−

145

±

9.0 pA (

F

IG

.

1).

Similar to the peptide concentration–dependence curves for the potent activationof second messengers by PACAP, superfusion of cultured sympathetic neurons cur-rent clamped to

−

50 mV with either PACAP27 and PACAP38 produced nearly iden-tical dose response profiles. PACAP27 or PACAP38 produced maximaldepolarizations of 10–15 mV at 32–100 nM peptide, with an EC

50

of approximately5 nM. VIP, which shares 68

%

amino acid homology with PACAP27, failed to pro-duce any responses at concentrations up to 1

µ

M peptide; application of the PACAPantagonist PACAP(6–38) for 15 min prior to PACAP27 superfusion diminished thedepolarization response by approximately 80

%

.

SCG neurons in culture are frequently associated with processes and to ensurethat the PACAP-induced membrane depolarizations represented direct neuronalresponses to PACAP peptides and were not secondary to PACAP-induced release ofother neuroregulators, the neuronal cultures were treated with either TTX or Cd

2+

prior to peptide application to block stimulated neurotransmitter/neuromodulatorsecretion and interactions from adjacent terminals. In these experiments, superfu-sion of 100 nM PACAP27 in the presence of 300 nM TTX or 200

µ

M Cd

2+

, depo-larized SCG neurons 13

±

0.9 mV or 9

±

0.8 mV, respectively, values not statisticallydifferent from the observed PACAP-induced depolarizations in the absence of chan-nel inhibitors. These neuropharmacological studies provided direct evidence thatPACAP peptides elicit primary responses in sympathetic postganglionic neurons fol-lowing PACAP-selective PAC

1

receptor activation.

PACAP MEDIATED DEPOLARIZATIONS IN SCG NEURONS RESULT FROM INHIBITION OF A POTASSIUM CONDUCTANCE AND

ACTIVATION OF A NONSELECTIVE CATIONIC CONDUCTANCE

The slow depolarizations elicited by many neurotransmitters and neuropeptidesare frequently a consequence of inhibition of a membrane potassium conductanceand/or activation of a nonselective cationic conductance (NSCC).

15–18

Accordingly,several approaches were undertaken to examine whether similar ionic mechanismsunderlie the observed PACAP-induced depolarization. In sympathetic neurons, heldat

−

50 mV and maintained in a HEPES buffered physiological solution containing300 nM TTX, pressure application of 50 µM PACAP27 (1 sec) produced an inwardcurrent of

−

129

±

12 pA. To examine the possibility that the observed PACAP-induced current was mediated in part by inhibition of a potassium conductance, KClwas added to the bathing medium to raise the extracellular potassium concentrationfrom 5.9 mM to 20 mM and shift the potassium equilibrium from

−

90 mV to approx-imately

−

50 mV.

19

When sympathetic neurons are voltage clamped to

−

50 mV underthese recording conditions, a reduction in the amplitude PACAP-induced current

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would be anticipated if inhibition of a potassium conductance participated in thepeptide response. Elevation of the external potassium concentration to 20 mMdiminished the PACAP-induced inward current by approximately 35

%

comparedto values recorded in the HEPES-buffered solution containing 5.9 mM KCl, support-ing the view that inhibition of a potassium conductance contributed in part tothe PACAP-induced depolarization. The fact that a significant component of thePACAP-induced current remained in 20 mM KCl implied that activation of otherionic conductances represent key mechanisms contributing to the PACAP-inducedcurrent.

To investigate whether activation of a NSCC in sympathetic neurons wasinvolved, the sodium ion concentration in the HEPES-buffered bathing solution wasdecreased. In neurons voltage clamped at

−

50 mV, sodium ion influx contributes asignificant portion of the inward current through NSCC channels; hence, reducingthe sodium ion concentration by replacing all of the external NaCl with the mem-brane impermeant N-methyl-

D

-glucamine (NMG; 121 mM) (sodium deficient medi-um) should reduce the size of the PACAP current. As anticipated, the PACAP-induced inward current was reduced 70

%

from

−

129 pA in sodium containingHEPES-buffered medium, to

−

40

±

10 pA under sodium deficient conditions. Toestablish whether most of the remaining PACAP-induced current recorded in sodiumdeficient solution could be attributed to potassium conductance inhibition, both thesodium and potassium concentrations in the HEPES physiological bathing solutionwere modified simultaneously. As previously, external NaCl was replaced withNMG and the potassium concentration was elevated to 20 mM; under these condi-tions, the PACAP-induced current was reduced more than 90

%

to approximately

−

12 pA. In sum, both activation of a NSCC and inhibition of a potassium conduc-tance appeared to generate the observed PACAP-induced inward current.

Potassium M-Current (I

M

) Inhibition Represents a Minor Component of the PACAP-Induced Inward Current at

−

50 mV

Rat sympathetic neurons possess a well characterized voltage dependent, non-inactivating barium-sensitive outward potassium conductance, the M-current(

I

M

).

20,21

If

I

M

inhibition represents one mechanism generating the PACAP-inducedcurrents, then inhibition of

I

M

prior to peptide application should reduce the size ofthe inward current. Accordingly, to test that possibility, the effect of sympatheticneuron pretreatment with 1 mM Ba

2+

on PACAP-induced currents was evaluated.For sympathetic neurons voltage-clamped at

−

50 mV and bathed in Krebs solutioncontaining 300 nM TTX and 1 mM Ba

2+

, application of 50

µ

M PACAP initiated aninward current of

−

138

±

17 pA. Thus, the PACAP-induced current recorded in thepresence of 1 mM barium was only reduced by 5

%

compared to control inward cur-rents of

−

145

±

9 pA. In light of these results, and given the voltage-dependence of

I

M

(i.e., that only a small portion of M-current conductance is activated at

−

50 mV),inhibition of

I

M

under these recording conditions was not a prominent contributor tothe PACAP-induced current.

Residual PACAP-Induced Current is Not Mediated by Chloride Ions

Although the previous experimental perturbations, examining the contributionsof increased NSCC and decreased potassium conductance, abrogated nearly all of

190 ANNALS NEW YORK ACADEMY OF SCIENCES

the PACAP-induced ionic currents, a residual PACAP-induced current of approxi-mately

−

12 pA remained. In some neurons, activation of a chloride current can con-tribute to peptide-induced inward currents;

22

accordingly, one possibility consideredwas that a chloride conductance may have represented that residual current. To testthis possibility, the chloride ion concentration in the Krebs bathing solution wasreduced by replacing extracellular sodium chloride with sodium propionate. In thision replacement paradigm, the chloride ion equilibrium potential should shift to amore positive value and any chloride component of the PACAP-induced currentshould be increased in magnitude, thus increasing the total PACAP-induced current.Contrary to expectations, the SCG neuronal PACAP-induced inward current wasdiminished rather than increased in the chloride-reduced medium (

−

104

±

22 pA).Thus, PACAP-induced activation of a chloride conductance did not contribute to thePACAP-induced current in sympathetic neurons.

Current–Voltage (I–V) Data also Support Involvement of Both an Increased NSCC and Decreased Potassium Conductance

The voltage-dependence of the PACAP-induced current was assessed fromcurrent–voltage (

I

–

V

) relationships measured prior to and during PACAP stimula-tion.

I

–

V

curves were generated by applying voltage ramps over the range -120 to -30 mV at a rate of 25 mV/sec; the current component related to PACAP was deter-mined as the difference in the current recorded before PACAP application from thetotal current measured after peptide treatment. Control

I-V

curves were generatedwith neurons bathed in Krebs physiological solution with the voltage ramp initiatedfrom a holding potential of

−

50 mV. Subsequently, 50

µ

M PACAP27 was applied tothe same neuron by pressure ejection (1 sec) and a second

I-V

curve was generatedas soon as the PACAP-induced current reached maximum. In all neurons, applica-tion of PACAP shifted the zero current value to a more positive potential. Further-more, for cells bathed in Krebs solution, the PACAP-induced current appeared to beessentially voltage-independent, an observation consistent with both an increase inNSCC and a decrease in potassium conductance contributing to the generation of thePACAP-induced current.

The same experimental design was used to generate

I-V

curves with the recordingsolution modified to eliminate involvement of the NSCC. In cells maintained inthe sodium deficient, NMG-substituted HEPES-buffered recording medium, thePACAP-induced current increased at potentials greater than

−

50 mV and decreasedat potentials less that than

−

50 mV. Even though the PACAP-induced currents didnot reverse at the potassium equilibrium potential, the results were consistent withthe involvement of the inhibition of a potassium conductance contributing to the gen-eration of the PACAP-induced depolarization.

SPECIFIC INTRACELLULAR SIGNALING PATHWAYS ARE INVOLVED IN THE PACAP-INDUCED DEPOLARIZATIONS

The PAC

1

(

short

)HOP1 receptor variant expressed in SCG neurons is coupled toboth the adenylyl cyclase and phospholipase C (PLC) signaling cascades. To inves-tigate whether one of these pathways mediated the PACAP-mediated depolarizations

191MAY et al.: RAT SYMPATHETIC NEURON DEPOLARIZATION

in sympathetic neurons, the primary neuronal cultures were pretreated with selectiveinhibitors to each component of the pathways to assess their ability to attenuate thePACAP response. Treatment of the cultures with cholera toxin to downregulate Gαs,or pertussis toxin or N-ethylmaleimide to selectively target Gαi/o, had no effects onSCG PAC1 receptor-induced depolarizations. Furthermore, direct activation of sym-pathetic neurons with forskolin, dibutyryl cAMP or 8-bromo-cAMP, or inhibition ofprotein kinase A with H-89 did not simulate or blunt, respectively, the PACAPresponse. In sum, these results indicated that the PACAP-mediated depolarizationswere not generated following cAMP production or protein kinase A activation (seeTABLE 1).

However, the PLC inhibitor U73122 reduced the PACAP-induced depolarizationsin SCG neurons more than 90%, suggesting that PLC activation was a critical step ingenerating the PACAP-induced response. Since PLC hydrolysis of phosphatidyl-inositol bisphosphate results in the generation of two second messengers, diacylgly-erol (DAG) to activate protein kinase C, and inositol triphosphate (IP3) to bindendoplasmic reticulum IP3 receptors and stimulate intracellular calcium release, otherinhibitors and activators were employed to test roles of these downstream pathways.Activation of protein kinase C by acute stimulation with phorbol myristate acetate(PMA) did not elicit depolarizations in any of the neurons tested. Furthermore, nei-ther inhibition of protein kinase C with bisindoylmaleimide I (BimI) nor downregu-lation of protein kinase C upon chronic PMA treatment had any effect on PACAP-induced depolarizations, thereby suggesting that IP3 may represent the pertinent sig-naling pathway. Recently, several cell permeant xestospongin compounds have beendescribed to inhibit IP3 receptors23 and among these reagents, xestospongin C (XeC)

TABLE 1. Effects of second messenger activators or inhibitors on PACAP-inducedneuron depolarizations

NOTE: SCG neurons in vitro were incubated in defined medium containing the indicateddrugs as described in the Methods. The effects of these agents on PACAP27-induced depolariza-tion was examined in the presence of 300 nM TTX. The data represent the mean membranepotential or depolarization ± SEM, and the numbers of cells tested under each paradigm areshown in parentheses.

aSignificantly different from vehicle control (p < 0.05). The resting membrane potential ofneurons in Krebs solution in the absence of TTX was −55 ± 1 mV (n = 35); PACAP27-induceddepolarization in these cells was 12.6 ± 0.7 mV.

Treatment Membrane Potential (mV)

PACAP-Induced Depolarization (mV)

Vehicle, DMSO −54 ± 10 15.6 ± 1.3 (10)

Forskolin (10 µM) −59 ± 3 11.5 ± 1.2 (4)

H-89 (25 µM) −56 ± 5 12.4 ± 1.9 (5)

U73122 (5.6 µM) −46 ± 1 1.1 ± 0.5 (7)a

BimI (2.5 µM) −58 ± 3 13.3 ± 1.5 (6)

PMA (500 nM) −52 ± 8 11.0 ± 0.6 (3)

XeD (20 µM) −52 ± 5 7.6 ± 1.2 (10)a

XeC (10 µM) −47 ± 3 7.7 ± 1.8 (3)a

192 ANNALS NEW YORK ACADEMY OF SCIENCES

and xestospongin D (XeD) decreased the PACAP-elicited depolarizations by morethan 50% (TABLE 1). These observations imply that PACAP activation of SCG PAC1receptors and subsequent generation of IP3 may result in IP3 receptor-mediatedrelease of intracellular calcium to modulate a store operated nonselective cationicchannel. To investigate whether intracellular calcium release elicited the peptide-mediated depolarizations, SCG neurons were pretreated with either thapsigargin orcyclopiazonic acid to allow the progressive depletion of internal calcium stores, orwith the calcium chelator BAPTA to buffer transient elevations in intracellular calci-um. Under all of these circumstances, however, the PACAP-induced depolarizationswere not affected.

Although these results appear to be conflicting, the data were consistent with cur-rent studies describing the regulation and activation of nonselective cationic Trp(transient receptor potential) channels first described by Drosophila genetics.24

There are multiple Trp, Trp-like, and Trp-related channels in a variety of tissuesacross many species, and although many Trp channels are store operated, some havebeen shown to be dependent directly on IP3 receptor interactions, in a xestosponginsensitive manner, to maintain the channels in an active state.25 Other Trp channelsappear to be modulated directly by DAG or DAG metabolites;26,27 whether these Trpchannel subtypes participate in the PACAP-mediated sympathetic response remainsto be investigated. Among the many mammalian Trp channels described to date, wehave shown by reverse transcription polymerase chain reaction that at least fourTrp mRNAs appear to be expressed in sympathetic neurons. The functional associa-tions between SCG PAC1 receptors and Trp channels are still under study, but thecurrent observations appear novel for peptidergic receptors and may reveal newinsights into the mechanisms through which PACAP peptides regulate autonomicneuronal function.

ACKNOWLEDGMENTS

This work was supported by Grants HD-27468 (V.M. and K.M.B.), NS-01636(V.M.), and NS-23978 (R.L.P.) from the National Institutes of Health and Grant975043N (R.L.P. and K.M.B.) from the American Heart Association.

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