Pharmacological techniques for the in vitro study of intestinal
smooth muscles
E.E. Daniel*, C.Y. Kwan, L. Janssen
Department of Medicine, Faculty of Health Sciences, McMaster University Hamilton, Ontario, Canada L8N 3Z5
1. Introduction
This review focuses on the use of intestinal (small and
large intestine) muscles for pharmacological studies, but
also uses data from other smooth muscles as appropriate.
Initially, it describes some aspects of the structure and
function and then proceeds to the variety of approaches
and techniques that can be used.
1.1. General attributes of intestinal tissues
1.1.1. Myogenic properties
In all species, the small and large intestines consist of four
or more muscle layers: outer longitudinal muscle, circular
muscle (which with the longitudinal muscle constitutes the
muscularis externa), and between the submucosa and the
mucosa, there are two layers of muscle (outer longitudinal
and inner circular) constituting the muscularis mucosa. In
small intestine, the circular muscle is divided by the deep
muscular plexus (see later) into an outer and an inner layer. As
described later, there is much species variation as to the
thickness of the inner circular muscle: in rodents and pigs, it is
only one or two cell layers thick, while in dogs and primates
including man it is several cell layers thick. Fig. 1 shows a
diagram of these layers in the small intestine.
The different muscle layers have structural differences:
only the outer circular muscle layer of small intestine has an
abundance of gap junctions identified by electron micros-
copy or by immunocytochemistry between cells. However,
the longitudinal muscle layers of small and large intestine
and the circular muscle of the muscularis externa of the
colon appear to have good electrical coupling and should be
considered as syncytial (i.e., single unit in behavior). In
contrast, the muscles of the muscularis mucosa are multiunit
in behavior.
Myogenic spontaneous activity of the small intestine has
been shown to be driven not by spontaneous variation in
muscle activity, but by special pacemaking networks, con-
sisting of arrays of special cells called interstitial cells of
Cajal (ICC). The main such network of small intestine is in
the myenteric plexus while that in large intestine is in the
submuscular plexus. ICC in the networks are interconnected
by gap junctions and it is assumed, but not proven, that they
transmit their pacing currents by gap junctions to the muscle
layers. However, these connections are rare, and no evidence
exists that pacemaking activity requires gap junctional con-
ductance. The pharmacology of the ICC cells (receptors,
second messengers, ion channels) themselves is poorly
known because they are difficult to study in isolation and
in tissues. Discerning effects on the pacing cannot be dis-
tinguished easily from effects on the driven smooth muscles.
1.1.2. Intrinsic innervation
The intrinsic innervation of the intestinal muscles is
mostly located in three plexuses: the myenteric (Auerbach’s)
plexus, between outer longitudinal and circular muscle, and
the submucosal (Meissner’s) plexus, between the circular
muscle and the muscularis mucosa, contain nerve cell bodies
and send nerve projections to muscle, to mucosa, and to one
another. The submucosal plexus can be subdivided into a
portion near the outer circular muscle and another portion
near the muscularis mucosa. The third plexus is called the
deep muscular plexus in the small intestine and as noted
above separates inner and outer circular muscle layers. In the
large intestine, it is located at the inner border of circular
muscle and called the submuscular plexus. Both of these
plexuses lack cell nerve cell bodies but have an abundance of
nerve endings as well as ICC.
The intestinal intrinsic nerve networks form complete
nerve circuits, which can drive peristalsis (see later) using
reflexes that originate in the mucosa in response to chemical
and physical (distention/distortion) stimuli and are transmit-
ted to the myenteric plexus. There, orally projecting nerves
1056-8719/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved.
PII: S1056 -8719 (01 )00131 -9
* Corresponding author.
Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158
mediate excitation (mainly by acetylcholine acting on mus-
carinic type 3 (sometimes also 2 or 1) receptors and
substance P (acting on neurokinin 1, 2, and in some species
neurokinin 3 receptors). Anally projecting nerves mediate
inhibition (mainly by releasing nitric oxide, vasoactive
intestinal polypeptide [VIP], or in some species ATP).
Inhibitory nerves have nNOS, VIP, and pituituary adenylate
cyclase activating peptide (PACAP) frequently colocalized
in their endings. NO acts usually on cytosolic guanylate
cyclase, VIP acts on VIP or PACAP-selective receptors.
PACAP is structurally closely related to VIP.
In addition to these main nerve mediators, there are
numerous others, notably 5-hydroxytryptamine (5-HT),
which acts on 5-HT3 or 5-HT4 receptors. Nearly every
mediator/modulator found in the central nervous system
also is found in the enteric nervous system. Detailing them
is beyond the scope of this chapter, but a useful reference is
The Handbook of Physiology (Wood, 1989), which contains
chapters related to all the matters discussed in this here.
The enteric (intrinsic) nervous system is connected to the
central nervous system by vagal and sacral parasympathetic
nerves and by sympathetic nerves. The upper gastrointestinal
tract, esophagus, stomach, and duodenum is under vagal
control in the sense that vagal stimulation activates postgan-
glionic fibers in the myenteric plexus and initiates motor
activity. The sacral parasympathetic nerves innervate part of
the colon and the rectum. Similarly, the sympathetic nerves
innervate enteric ganglia in both the myenteric and the
submucosal plexuses, and modulate (often inhibit) activity
of nerves. Guinea pigs are a commonly used model to study
peristalsis, the main mechanism controlling transit of food in
the small and large intestines. However, in many other
species that have a major contribution from the myogenic
pacemaking system from the ICC networks, peristalsic
activity interacts with and requires myogenic activity for
efficient transit.
Any study of the pharmacology of intestinal muscle must
be cognizant of the existence of themyogenic controls and the
numerous nerves reflexes of which the intestine is capable.
2. Studies with tissues
2.1. In vivo, ex vivo, or in vitro?
Pharmacological techniques can be applied in vivo, ex
vivo, and in vitro to intestinal smooth muscle. In contrast to
in vitro studies (when tissues are studied outside the body
and in an artificial environment), in vivo studies are carried
out while tissues remain in the living animal. Ex vivo
studies refer to those in which an organ is removed from
the animal for study, but not dissected. The goal is to
Fig. 1. Diagram of the layers of the small intestine (after Furness and Costa, 1980). The ICC networks are not illustrated, but they are located in the myenteric
plexus and the deep muscular plexus. In the colon, the primary ICC network is located in the submuscular plexus at the inner border of circular muscle. There is
no deep muscular plexus, but the submuscular plexus, like the deep muscular plexus, contains nerve endings but no nerve cell bodies.
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158142
maintain normal cellular relationships and, in the case of
intestine, the organ is perfused through the blood supply.
This review will focus on in vitro techniques that allow
greatermechanistic precision and depth of analysis. However,
it is important to remember that many tissues behave differ-
ently in vitro from in vivo or ex vivo (Fox et al., 1983). There
are many examples of this: for example, opioids acting at mand d receptors excite canine intestine (and that of other
species) in vivo or ex vivo, acting to decrease spontaneous
release of NO from nerves. This, in turn, increases release of
acetylcholine (Fox & Daniel, 1987a, 1987b; Fox-Threlkeld,
Woskowska, & Daniel, 1997; Fox-Threlkeld et al., 1994).
These opioids cause no excitation in vitro even though they
act by inhibiting release of inhibitory and other mediators
such as NO (Bauer & Szurszewski, 1991; Bauer, Sarr, &
Szurszewski, 1991). Opioid receptors are found on enteric
nerves in canine intestine (Allescher et al., 1989) and in most
other intestinal tissues including guinea pig intestine (Water-
field&Kosterlitz, 1975; Cowie et al., 1978;Kosterlitz, 1980),
where they act to modulate mediator release. However,
studies of isolated guinea pig intestinal muscle cells in vitro
(Bitar & Makhlouf, 1985; Kuemmerle & Makhlouf, 1992)
report that all opioid receptor classes (m, d, and k) are presentand opioids shorten cells.
Many other examples exist, for example, motilin, which
acts on intestinal nerves of canine (and other) species in
vivo or ex vivo (Fox et al., 1984; Hirning & Burks, 1986;
Fox & Daniel, 1987b; Fox-Threlkeld et al., 1991b), but may
act directly on gastrointestinal smooth muscle in vitro
(Depoortere et al., 1991, 1993; Lu et al., 1998). Thus it is
advisable not to extrapolate in vitro findings to the in vivo
state without a direct check using techniques like close intra-
arterial perfusion in vivo (Fox & Daniel, 1986; Fox et al.,
1983, 1984; Daniel & Kostolanska, 1989) or ex vivo (Burks
& Long, 1967a, 1967b; Manaka et al., 1989; Daniel and
Kostolanska, 1989; Fox-Threlkeld et al., 1991a, 1991b,
1991c, 1993) while recording mechanical response to ago-
nists, antagonists, or nerve stimulation. This will determine
any qualitative differences that exist.
The ex vivo infused segment of intestine was introduced
by Burks and used by him to identify sites and mechanisms
of action of several agents (Burks & Long, 1967a, 1967b;
Burks, 1973; Hirning & Burks, 1986; Northway & Burks,
1979; Stewart & Burks, 1977; 1980). It was modified by our
laboratory (Manaka et al., 1989; Fox-Threlkeld et al., 1991a,
1991b, 1991c, 1993, 1994, 1997, 1999; Vergara et al., 1995,
1996) and used to identify sites and mechanisms of action of
additional agents. It has several advantages over the use of
close intra-arterial perfusion in vivo, even though prepara-
tion of intra-arterial perfusion is easier and does not require
the same use of a perfusion pump. The perfused region is
uncertain and variable when intra-arterial perfusion of intest-
ine is used in vivo, depending on whether an end artery to the
intestine or a larger artery is chosen for perfusion, on the
volume and force of the infusion, and on whether any agent
in the perfusate affects the distribution of vascular flow.
Also, with this technique, the site is still perfused by blood
when no infusion is made, allowing endogenous agents to
affect outcomes and allowing recirculation of a potent
compound to have secondary actions. Moreover, if the neural
connections are intact, extrinsic reflexes may release local
mediators to complicate interpretations.
In contrast, the ex vivo preparation (in which a segment of
intestine is totally isolated from the body, perfused through its
artery, instrumented to record mechanical activity, if desired
to stimulate nerves, and the venous outflow collected) avoids
most of these problems (Manaka et al., 1989). Outcomes
qualitatively resemble those obtained by other in vivo prep-
arations, but are more reproducible. The collection of the
venous outflow allows measurement over time of the release
of endocrine, neural, and other endogenous mediators/mod-
ulators/products. Burks and colleagues (cited above) used it to
measure 5-HT release and we used it to measure VIP,
substance P, and prostanoid release. The disadvantages of
this preparation include the need for additional equipment to
heat solution and perfuse the segment, the difficulty of
cannulating the veins for measurement of outflow (arteries
are easy), and that it has a limited lifetime, depending on the
nature of the perfused fluid. Eventually edema interferes with
the flow and distribution of the perfusate. Taking account of
changes due to deterioration of the preparation requires
checking myogenic responses (infusions of acetylcholine or
KCl) before and after experimental variables are introduced
(Fox-Threlkeld et al., 1991a, b, c, 1994, 1997, 1999; Daniel
et al., 1994). If neural responses to electrical field stimulation
are being assessed, then they, too, should be evaluated at the
beginning and at the end of the experiment. Since in vivo
preparations require anesthesia and fatigue over time, similar
controls are required.
Although quantitative pharmacological study is possible
from in vivo or ex vivo experiments, it is more difficult than
from in vitro experiments. In practice, it is not feasible to
carry out multiple concentration effect curves to an agonist
with increasing concentrations of antagonist because of the
inability to maintain the prerequisite equilibrium conditions
for the agonists and antagonists in vivo as the perfusion site
is blood-perfused except when an agent is administered. In
ex vivo experiments, the limitation of being unable to
perform multiple concentration-effect curves to agonists in
the presence of increasing antagonist concentration exists
because of the limited survival time of the preparation.
Obviously, too, it is not feasible to study multiple prepara-
tions simultaneously in vivo or ex vivo; thus time controls
of changes unrelated to the experiment are impossible. Such
preparations are best used to identify sites of drug action
and to provide qualitative information of receptor identity
and of site and mechanism of drug action.
2.2. Ex vivo studies of peristalsis
A special ex vivo preparation is the isolated intestine
perfused at the oral end to induce peristaltic activity,
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158 143
recorded as expulsion of fluid at the anal end or as
contractions of circular and/or longitudinal muscle or all
of these (see Costa & Furness, 1976 and references therein).
This can be done in vascularly perfused or bath-perfused
conditions. The preparation has been widely used, primarily
in the guinea pig intestine, to define the neural mechanisms
(Costa & Furness, 1976; Crema et al., 1970; Foxx-Orenstein
& Grider, 1996) and the roles of 5-HT, released in response
to distention or mechanical distortion in initiation of peri-
staltic reflexes by activating sensory nerves in the mucosa
(Grider et al., 1996; Gershon et al., 1990; Gershon, 1991;
Kadowaki et al., 1996; Jin et al., 1999; Yuan et al., 1994)
and transmitting activity at myenteric synapses (Johnson
et al., 1980). Once activated, sensory nerves and interneur-
ones transmit the information to myenteric neurons that
project orally to cause excitation of both muscle layers
(Costa & Furness, 1976; Yokoyama & North, 1983) and
anally to cause inhibition (Furness et al., 1982; Daniel &
Kostolanska, 1989; Smith et al., 1991; Shuttleworth &
Sanders, 1996; Iversen et al., 1997; Kunze et al., 2000;
Cornelissen et al., 2000; Furness, 2000).
Many specialized modifications of this technique have
been used to identify nerves firing during peristalsis, their
projections, their mediators, and the transduction mecha-
nisms used by their mediators (Furness, 2000). This vast
subject area is beyond the scope of this review. However,
the fact that the guinea pig has poor myogenic control over
intestinal function (see Introduction) means that findings
from its intestine cannot be extrapolated automatically to
other species.
2.3. Quantitative pharmacology in vitro
Intestinal smooth muscles can be used for qualitative and
quantitative pharmacological studies in vitro. They can be
used as strips of the wall, prepared either in the circular or
longitudinal muscle axis to measure contractions, semi-
isometrically (since no smooth muscle undergoes pure
isometric contractions). Alternatively, if feasible without
significant damage, each muscle layer can be dissected free
and studied, avoiding any mechanical interactions between
layers. Strips obtained by dissecting tissues to remove one
or the other muscle layer must be checked to ensure that
tissue damage by dissection has not affected contractile
responses of interest.
First, we focus on contractile responses, later on relaxa-
tion responses. The simplest preparation to make is the strip
containing the whole external musculature, after opening
the intestine and removing the mucosa and submucosa.
Strips can be cut in either the longitudinal or the circular
direction and strung up in a muscle bath connected to a
strain gauge or other tension recorder. If the strips are small
in width, the contractions recorded in the long axis of
muscle cells will be virtually unaffected by concomitant
contractions of the cross-sectioned cells of the other layer.
Optimal length and tension is determined by stretching the
strip in increments, waiting for passive relaxation to be
complete, and successively checking the response to a
standard contractile agent. For the latter, 60 mM KCl works
well and can be washed out and repeated as many times as
needed. Muscarinic agonists, carbamoyl choline or meth-
acholine (10� 7 to 10� 5 M), are good alternatives. It is
better to use a concentration yielding at least 50 to 80% of
the maximal response.
After finding the optimal length or tension (Schild, 1969,
1975, 1997; Arunlakshana & Schild, 1997; Kenakin, 1985)
for contractile responses, agonists can be applied in increas-
ing concentrations to obtain a complete (including a plateau
response) concentration-effect curve. This can be followed
by a supramaximal (30–100� the Kd) concentration of
putative, selective antagonists to provide qualitative
information about the nature of receptors present and
responding to applied agonists, an approach also applicable
in vivo or ex vivo.
The experimenter often has to decide what to do about
spontaneous activity. Spontaneous activity is frequently
present during in vitro as well as during in vivo or ex vivo
experiments. Blocking nerve activity with TTX often pro-
vokes spontaneous activity by turning off spontaneous
release of nitric oxide from nerves. Extensive dissections,
removing the ICC pacemaking regions described above, will
usually abrogate spontaneous activity. Cooling to 30 or
25�C will also markedly diminish spontaneous activity. As
described later, stable spontaneous tonic or phasic activity is
desirable when inhibitory effects are to be studied.
If no spontaneous activity is present, contractile
responses to agonists can be measured as the maximum
height of response or the area under the curve (several
computer programs to this end exist). The latter is more
pertinent when contractions have major phasic as well as
tonic components. These components can be measured
separately if they appear to be affected differentially (see
Allescher et al., 1988). Spontaneous activity, if present, has
to be measured and subtracted from each data point to
determine agonist responses. This is problematic if multiple
responses are to be measured noncumulatively because each
wash-out will result in different spontaneous activity levels.
Waiting for recovery to the original level of spontaneous
activity may be impractical. Cumulative concentration-
response curves may avoid (or hide) this dilemma.
Contractile data can be presented in international units
of force. However, for pharmacological analysis, it is
often useful to normalize them as a percentage of the
maximum response to a full agonist (defined below). This
provides information about the ability of various agonists
to attain maximal responses. Another alternative fre-
quently used is normalization to the response to a non-
specific agonist such as 60 mM KCl. We use repeated
exposure to KCl to evaluate that tissues are viable and
yield consistent responses.
Tissue strips will have different cross-sectional areas of
smooth muscle contributing to contraction. Sometimes it is
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158144
important to normalize responses to this area to make them
comparable. The most accurate way to do this is by
preparing tissue cross sections and examining them by
histochemistry to find the area of smooth muscle. A simpler
alternative is to measure tissue weight (wet or dry weight)
and normalize to that. If there are tissues present other than
the smooth muscle contributing to contraction, this simpler
method may lead to significant error. Normalization to
cross-sectional area of muscle is not necessary for most
pharmacological analysis but is important if treatments or
experimental conditions that affect ability of muscle to
contract have been applied and the effects need to be
compared to controls.
In gastrointestinal tissue strips with numerous intrinsic
nerves, agonists may act in part by releasing mediator, either
enhancing or inhibiting the response of interest. Also, tonic
nerve activity may contribute hidden modulation of
responses. If such possibilities exists, nerve axonal transmis-
sion can be blocked with tetrodotoxin (210–6 M) alone.
Also w conotoxin GVIA (210–7 M) can be added to block
transmitter release from nerve endings by inhibiting N Ca
channels. NOS inhibitors (e.g., L-NOARG or L-NAME at
10–4M) can be used if tonicNO release is affecting responses.
Quantitative information about affinity of the antagonist
and the nature of its interaction with the receptor can be
derived from Schild plots (Schild, 1969, 1975, 1997;
Arunlakshana & Schild, 1997), in which concentration-
effect curves are repeated after increasing concentrations
of the antagonist to determine the rightward shift of the
concentration-effect curves to the agonist. The reactions are
assumed to be represented by:
Agonist ðAÞ þ Receptor ðRÞUAR ! Effect
Antagonist ðAnÞ þ RUAnR
Fig. 2 illustrates this on the assumption that one agonist
and one antagonist compete for the same receptor site. If
care is taken to avoid or account for any time decay of the
receptor-mediated response, checked by using strips as time
controls in which the concentration-effect curves are
repeated throughout the experiment without any antagonist;
a Schild plot of log of the dose ratios minus 1 (log [Dose
Ratio � 1]) against the log of the antagonist concentrations
provides much important information. If the plot is a straight
line with a slope of 1, its intercept with the zero axis (at log
[2 � 1]) provides a valid measure of the log KD value for
the antagonist (Fig. 3). This is a fundamental measure of
antagonist affinity for the receptor and usually will corre-
spond to the value from ligand binding (see below).
A slope of the Schild plot different from 1 implies a more
complex interaction between antagonist, agonist, and recep-
tor, including antagonism of agonist action at more than one
receptor, stoichiometry different from one antagonist and
one agonist molecule competing for one receptor, a receptor
with promiscuous interactions with second messenger sys-
tems, and others, which have been summarized in various
reviews (Kenakin, 1985, 1988, 1990; Scaramellini & Leff,
1998). Sorting out explanations for a slope other than 1 may
not be easy and may be impossible using tissue techniques
alone. An example is the difficulty in identifying the nature
of the a1 adrenergic receptor in urinary tract smooth
muscles. It appears to be a1A by some criteria, a1L by other
criteria (low affinity for prazosin), or a promiscuous recep-
tor by other ones (see references in Daniel et al., 1999).
Not all antagonists are competitive and reversible. Some
act at sites other than the binding site for agonists and
prevent the agonist from competing with the antagonist at
high concentrations, yielding concentration-effect curves
Fig. 2. Consequences for agonist concentration response of adding
increasing concentrations of a competitive antagonist, competing 1:1 with
an agonist for a receptor.
Fig. 3. A Schild plot of the logarithm of various (dose ratios � 1) of agonist
required to reach 50% maximum response as concentrations of antagonist
are increased. These values are plotted against the logarithm of the
antagonist concentration. The pA2 value is the negative logarithm of the
antagonist concentration at which the value of the logarithm (dose ratio �- 1) is zero (i.e., when the DR is 2). This characterizes the interaction of the
antagonist with the receptor, provided the slope of the curve in not different
from 1.
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158 145
(Fig. 4) in which the maximum response is no longer
attained. Some antagonists such as phenoxybenzamine, an
adrenergic receptor selective antagonist, are competitive but
irreversible antagonists. Its irreversible antagonism is pre-
vented competitively by the presence of another agonist or
competitive antagonist, but once it interacts with the recep-
tor, its antagonism is irreversible (Nickerson, 1967).
Moreover, not all agonists are full agonists (i.e., some
cannot at any concentration reach the maximum response of
which the tissue or cell is capable) (Fig. 5a); they are partial
agonists. Many full agonists are so efficacious that they
initiate a full tissue response with only partial occupation of
receptors (Fig. 5b). Increasing concentrations of an irrevers-
ible antagonist such as phenoxybenzamine will then shift
the concentration-effect curve to the right until occupation
of the residual receptors will no longer produce a maximum
response (Fig. 6). Further increased concentrations of ant-
agonist shift the concentration-response curve further right
and further decrease the maximum response.
An agonist can produce a maximum response after some
of its receptors are irreversibly inactivated because the
percentage occupancy of receptors is not equivalent to the
percentage response achieved. This occurs because there is
frequently amplification of the signal produced by receptor
occupancy before the response is initiated. The various ways
in which signal amplification can be achieved are beyond the
scope of this review. However, the distinction between extent
of receptor occupancy and degrees of response (Fig. 5b)
Fig. 4. Plots comparing effects of noncompetitive to competitive
antagonists on agonist concentration-response curves. Note that the
noncompetitive antagonist not only shifts the concentration-response curve
to the right, it also reduces the maximum response. See Fig. 6.
Fig. 5. (a) Plots of responses to different agonists acting at the same receptor: at left, a potent full agonist reaches the maximum tissue response; at right, another
full agonist, but a less potent one, also reaches the maximum tissue response; in the middle, another agonist of intermediate potency (can be more or less potent
than any full agonist) fails to produce the tissue maximum response at any concentration and is a partial agonist. This illustrates that potency and efficacy are
not determined by the same agonist properties. (b) Distinction between potency and efficacy. Potency is determined as the EC50 (effective concentration for
50% of maximum response). This has no necessary connection to receptor occupancy and may be at a much lower percentage.
Fig. 6. Effects of a competitive, irreversible antagonist on responses to an
agonist of high efficacy leaving spare receptors when a maximum response
is reached. When the agonist has high efficacy and there are spare receptors,
the antagonist may initially shift the curve to the right without reducing the
response. Once spare receptors are occupied, further increases in antagonist
concentration will reduce the maximum response.
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158146
makes determination of agonist affinities for receptors from
concentration-response curves less straightforward than
determination of antagonist affinities. Agonist KD is not
equal to EC50. This means that potency per se is not a
measure of agonist affinity. Another variable has to be
considered: the ability of a given agonist to activate the
signal amplification system leading to response, sometimes
called intrinsic activity or efficacy.
From tissue bath studies, Furchgott and coworkers
(Furchgott, 1967, 1978; Besse & Furchgott, 1976) proposed
a way to estimate agonist affinities and relative efficacies. It
depended on successive inactivations of increasing numbers
of receptors by an irreversible antagonist until the maximum
responses were successively reduced (Kenakin & Beek,
1984). Other more sophisticated techniques have also been
proposed (DeLean et al., 1978; Black et al., 1985b; Kena-
kin, 1985, 1995; Leff et al., 1990b) because concentration-
response curves are not always hyperbolic (needed for
accurate use of the Furchgott null method) or because of
evidence that the measurement of affinity and efficacy were
not necessarily independent using this method and errors in
achieving maximum agonist responses introduced incorrect
outcomes (Black et al., 1985a, 1985b; Leff et al., 1990a,
1990b; Leff & Giles, 1992; Van der Graaf & Danhof, 1997;
Van der Graaf & Stam, 1999).
Study of relaxation responses requires a different
approach in part. In gastrointestinal sphincters, including
in some species the ileo-colic sphincter, active tension
develops spontaneously when they are passively stretched.
Thus attempts to study length-tension relation are difficult,
requiring specialized apparatus and approaches. They will
not be covered here. However, passive stretch sufficient to
induce high levels of active tension can be determined
empirically. Nerve stimulations and agonists that cause
relaxation of active tension can be studied quantitatively if
tension is stable and recovers after removal of the stimulus.
The percentage relaxation of active tension (zero active
tension determined at the end of the experiment by remov-
ing all external Ca2 + and adding 1 mM [EGTA] Etylene
Glycol-bis-( b-aminoethylether)-N, N, N0, N0-tetracetic acid)
can be used in dose-effect determinations. The most com-
mon complication is the introduction by experimental inter-
ventions (such as a receptor antagonist) of changes in the
active tension from which relaxation is being estimated. If
these changes cannot be eliminated by blocking nerve
function, or if the relaxation is nerve-mediated, a choice
must be made. The relaxation estimated thereafter will differ
depending on whether it is calculated based on the ampli-
tude of relaxation or the level of relaxation achieved. There
is no theoretically valid solution to the dilemma as to which
is more accurate. For relaxations from spontaneous active
tension (tone), we prefer the use of the percentage of
relaxation achieved, the nadir, estimated in terms of the
original tension as 100%. In other words, if the control
relaxation was from 10 g to 2 g, it would reach a nadir of
20% of control tension. If a subsequent experimental
variable reduced or increased control tension, but the
relaxation achieved was still to 2 g, we would conclude
that the relaxation was unchanged to 20% of the initial
control tension. If the relaxation was now to 5 g, we would
conclude that it was reduced to a nadir of 50% of initial
control tension.
For study of relaxations from active tension induced by
an agonist, it is necessary to choose an agonist that produces
contractions stable over the time needed to carry out
relaxation-response curves. If this is impossible, as it often
is, the only option is to repeatedly wash and readd agonist,
checking that a constant contractile response repeatedly
reoccurs. The difficulties with quantitation of relaxation
responses have resulted in fewer strip studies of receptor
mechanisms mediating relaxation responses compared to
studies of contracting agents.
3. Methods dependent on subcellular
membrane methods
3.1. Membrane receptor ligand binding
The alternative way to determine agonist (or antagonist)
affinity for a receptor is to measure binding of a radioactive
ligand to the receptor. This can now be done in intact tissues
using autoradiography (Young & Kuhar, 1979; Rainbow
et al., 1982a, 1982b, 1984; Power et al., 1988), but with
more accuracy in tissue homogenates and most accuracy in
isolated, purified, and characterized membranes (Ahmad
et al., 1987). One necessary precondition for valid data is
that the receptors to which binding is carried out come only
from the cells responding in vitro. Techniques have been
worked out and applied to intestine to separate synapto-
somes of embedded nerves in each plexus of intestinal
smooth muscle from membranes of the muscle layer and
to characterize each (Ahmad et al., 1987, 1988, 1989, 1991;
Allescher et al., 1989; Chen et al., 1994a, 1994b; Grover
et al., 1980a, 1984; Kostka et al., 1987, 1992, 1989a, 1989b;
Mao et al., 1991, 1992, 1995, 1996a, 1996b, 1997). When
applied appropriately after careful separation of muscle
layers and characterization of membrane fractions, this
technique allows the locations of receptors as well as
binding affinities to be determined.
Another precondition for valid data, if the ligand is an
agonist, is that binding does not alter the state of the receptor
(Burgisser et al., 1981; Leung et al., 1991; Rodbard et al.,
1986). The limitation of ligand binding studies of agonists is
that although they may provide affinity for the receptor, they
do not register the amplification that occurs in a particular
tissue between agonist binding and contractile response (i.e.,
they do not yield intrinsic activity or efficacy).
For ligand binding, it is important to start with a tissue
uncontaminated with several cell types, which may contain
the similar or related binding sites. The intestine is com-
posed of multiple layers of muscle (longitudinal, circular in
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158 147
the muscularis externae, and two such layers in the muscu-
laris mucosae). The circular muscle of the muscularis
externae of small intestine is also subdivided into an outer
(with gap junctions and intramuscular ICC) and an inner
portion (without gap junctions or ICC). They are separated
by the deep muscular plexus, composed of bundles of nerve
axons and varicosities and a network of ICC, but without
nerve cell soma. In some species like the dog and human the
inner circular muscle is many cell layers thick, while in
rodents it is one or two cells layers thick (Ahmad et al.,
1988; Duchon et al., 1974; Daniel et al., 1985; Komuro,
1999; Toma et al., 1999; Wang et al., 1999; Rumessen et al.,
1982, 1992). The other network of ICC is in the myenteric
plexus, and this is the dominant pacemaker in canine small
intestine (Cayabyab et al., 1997; Daniel et al., 1998;
Jimenez et al., 1996) as well as mouse intestine (Sanders,
1996; Thuneberg, 1999; Lee et al., 1999).
In the large intestine of most species, the circular muscle
has a layer of ICC and a plexus of nerve endings at the inner
border, now called the submuscular plexus (Berezin et al.,
1988). Damage to the ICC network of the submuscular
plexus prevents the usual spontaneous contractions driven
by slow waves (Durdle et al., 1983). In the colon, the ICC
network in the myenteric plexus is a secondary pacemaking
system (Berezin et al., 1990; Sanders, 1996).
One obvious problem for ligand binding studies is how
to separate these layers, which usually have different recep-
tors and functions. Longitudinal and circular muscles of the
muscularis externae can be separated reasonably well by
dissection (Furness et al., 1982; Furness, 2000; Daniel et al.,
1985; Allescher et al., 1989; Ahmad et al., 1989), which
usually removes the longitudinal muscle and myenteric
plexus along with a very thin layer of circular muscle,
leaving the circular muscle with its deep muscular plexus.
The synaptosomes of the myenteric plexus and deep mus-
cular plexus can be separated after homogenization by
differential centrifugation since they spin down with centri-
fugal force like that required to bring down mitochondria,
while plasma membrane spins down with much higher force
(Ahmad et al., 1987, 1988, 1989, 1991; Allescher et al.,
1989; Chen et al., 1994a, 1994b; Grover et al., 1980a, 1984;
Kostka et al., 1987, 1992, 1989a, 1989b; Mao et al., 1991,
1992, 1995, 1996a, 1996b, 1997, 1998). Further purification
can be obtained with sucrose (or other) density gradient
techniques, either continuous or discontinuous (Kostka et
al., 1992, 1989a, 1989b; Mao et al., 1991, 1992, 1995,
1996a, 1996b, 1997). Markers for nerves and muscle and for
the various membrane fractions to estimate their purities
have been described, for example, 50 nucleotidase for
smooth muscle membranes (Matlib et al., 1979; Kwan et
al., 1983; Ahmad et al., 1987), [3H] saxitoxin binding for
synaptosomal membranes (Ahmad et al., 1988), and various
markers for sarcoplasmic reticulum and mitochondrial mem-
branes (Matlib et al., 1979; Kwan et al., 1983). Of course,
specific binding to plasma membrane receptors provides an
additional marker when the receptor is uniquely located on a
given cell type. For large intestine (colon), the approach to
separation of layers is similar. However, the main ICC
network is at the inner border of circular muscle, the
submuscular plexus (Berezin et al., 1988, 1990), where it
can easily be damaged during removal of the submucosa.
After application of the above approaches, several uncer-
tainties remain: the locus of membranes from interstitial
cells and membranes of nerve cell soma. Since these
contribute a small fraction of the total membranes derived
from the intestine, they are usually ignored. As noted below,
receptors on these cell types can be identified by use of
immunohistochemistry, when antibodies to given receptors
exist. Presently, the main limitation to ligand binding
approaches is that relatively large quantities of membranes
are needed. This limits use of membrane isolation with
animals such as the mouse, the major source of animals with
genetic manipulation of proteins including receptors.
If ligand binding is carried out to simple homogenates of
the whole muscularis externae, the locus and unique nature
of any receptors studied will be unclear. Alternately, purified
and characterized membrane fractions, plasma membrane of
a muscle layer, or synaptosomes from a given plexus in
mitochondrial fractions can be used. The advantages of using
purified membranes include higher receptor density and less
background contamination, as well as better insight as to the
locus of receptors. This is also an advantage when receptors
or other proteins are to be identified by Western blotting.
Ligands to receptors are usually made radioactive (some-
times fluorescent), most commonly labeled with tritium (3H)
or 125I. Of course, any radioisotope that is a stable atom of
the ligand can be used, such as 14C, 35S, 32P. Radioisotopes
with higher specific activity (e.g., 125I) are necessary for
accurate binding of very high affinity ligands, which must be
used in very low concentrations to cover the full concentra-
tion range. However, 125I is a large molecule, usually not
present endogenously in ligands. If an iodine label is added
near or as part of the ligand moiety involved in binding, it
may inhibit or preclude binding (see Grover et al., 1984a).
Computer programs are available to analyze binding data,
whether from a primary ligand binding curve or a competi-
tion curve (Rodbard et al., 1986; Bylund, 1986).
3.2. Binding and transport of Ca2+
Major advances in the studies of smooth muscle contract-
ile function have come from the study of the role of Ca2+ in
the excitation-contraction coupling in smooth muscles. The
use of subcellular membranes in smooth muscle research can
be dated back about three decades (Matlib et al., 1979,
1982). Earlier experimental approaches to the direct study of
the Ca2+ mobilization and the subcellular distribution of
Ca2+ with the use of radiotracer technique, such as 45Ca2+ ,
in intact smooth muscle tissues (by wash-out techniques and
curve-peeling analysis, or by autoradiographic techniques at
the electron microscopic level) were seriously hampered by
the high background signals due to the high level of
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158148
extracellular relative to intracellular Ca2+ and the compart-
mentalization of intracellular Ca2+ , as well as by the
structural complexity of the smooth muscle tissues (see
review by Kwan et al., 1983). Despite the rapid development
of more advanced biophysical techniques for measuring
cytosolic Ca2+ content and mobilization in smooth muscle
cells with the use of sensitive fluorescent Ca2+ indicator dyes
or the study of Ca2+ or Ca2+ -dependent currents across the
cellular membranes using sophisticated electrophysiological
methods (to be discussed below), the subcellular membrane
approach continues to have an important role in smooth
muscle research because it still represents a relatively inex-
pensive and quite versatile approach, which allows molecu-
lar characterization of receptors affecting Ca2+ handling at
the membrane recognition site (as previously described),
interaction of the second messengers with the effector sites,
and the dynamic of regulation of Ca2+ binding and transport
at specific subcellular membrane sites. Furthermore, it also
allows better understanding of the interactions of Ca2+ with
some membrane-bound enzymes, thus facilitating the char-
acterization of fractionated membranes as previously
described and providing alternative investigatory tools to
study membrane transduction and ion transport mechanisms.
Using membrane fraction techniques, two distinctly
different Ca2+ -ATPase pumps, one in the sarcoplasmic
reticulum (SR)-enriched membrane fraction and the other
in plasmalemmal-enriched fractions, have been identified
and characterized first in intestinal smooth muscles (Wuy-
tack et al., 1985). These Ca2+ pumps were believed to be
physiologically more important in the regulation of cyto-
solic Ca2+ concentrations than the Na + -Ca2+ exchanger
located in the smooth muscle plasmalemma, based on their
Km values for the ATP-dependent transport (Grover et al.,
1981). In using this methodological approach, the purity of
the membranes, the leakiness and the sidedness of the
membrane vesicles intrinsic to the fractionation procedures
must be taken into consideration in the interpretation of the
data (Grover et al., 1980b). It is likely that much of the ATP-
dependent Ca2+ -transport activities observed in the plasma-
lemma-enriched fraction of smooth muscle tissues may be
attributed to the presence of the of plasmalemmal micro-
domain, such as caveolae (the surface membrane vesicles).
This hypothesis is suggested by recent evidence that smooth
muscle caveolae may represent a potentially important site
for signal transduction events, including Ca2+ -signaling
(Darby et al., 2001).
Studies of Ca2+ channels in smooth muscle tissues have
used the subcellular membrane approach. These Ca2+
channels include the dihydropyridine binding sites in the
plasmalemma-enriched membranes (Grover et al., 1984b),
Ca2+ -induced Ca2+ -release channels evaluated as the rya-
nodine binding sites (Zhang et al., 1993a, 1993b) as well
as IP3-induced Ca2+ -release channels (Chadwick et al.,
1990) in the SR-enriched membrane fractions. Although
the subcellular distribution, density, binding affinities, and
the regulatory factors of these Ca2+ channels can be
characterized using the subcellular membrane technique,
this approach cannot demonstrate the full function of these
channels in isolated membranes vesicles (e.g., the demon-
stration of Ca2+ release by these Ca2+ -channel modulators
under physiological conditions). For example, for the study
of the release of Ca2+ from 45Ca2+ -preloaded membrane
vesicles by dihydropyridine Ca2+ agonists, right-side-out
plasmalemmal vesicles are required, but not easily
obtained. Radioactive [45Ca2+ ] cannot be actively loaded
in the presence of ATP even if right-side-out vesicles are
available; the pump would be operating from inside-
outward. Thus, Ca2+ -loading would have to be carried out
passively at high Ca2+ concentrations. This protocol requires
the subsequent use of high concentrations of EGTA to
remove the high extravesicular Ca2+ (Moore & Abercrom-
bie, 1996). The effect of high EGTA concentration on the
binding of dihydropyridines (or ryanodine) and membrane
leakiness may mask the physiological event of Ca2+ release.
In a study of ryanodine-induced Ca2+ release from SR-
enriched fractions isolated from rat vas deferens and act-
ively loaded with Ca2+ in the presence of ATP and oxalate
(Zhang et al., 1996), ryanodine at concentrations near its Km
value in binding studies (nM range) caused little Ca2+
release. Instead, it caused significant inhibition of the
ATP-dependent Ca2+ transport at high concentrations
( > 30 uM). This finding offers an alternative interpretation
of the findings in contractile functional studies, that the
inhibition by ryanodine of the transient smooth muscle
contraction in Ca2+ -free medium due to Ca2+ release from
the SR occurs at high concentration of ryanodine. Thus,
ryanodine may inhibit the SR-Ca2+ pump at the concen-
trations frequently used.
Dysfunction of smooth muscles, including gastrointesti-
nal smooth muscles, noted in various diseases may manifest
itself in the form of membranes defects in Ca2+ transport
(see review by Kwan, 1992, 1999; Sakai & Kwan, 1993).
Data from studies of this kind should be interpreted cau-
tiously. When the observed Ca2+ uptake activities are less in
the membranes isolated from the tissues of diseased animal
compared to those from the control healthy animals, it is
tempting to attribute the changes as a manifestation of the
pathophysiological state or disease mechanisms. However,
it may be an artifact due to differential responses to the same
methodological regimen of the tissue in health and in
pathological state. For example, highly hypertrophied
smooth muscle tissues or smooth muscle tissue containing
excessive amounts of connective tissues and calcified tis-
sues subjected to suboptimal initial tissue homogenization
may yield different losses and/or distributions of membrane
materials. Thus differential purification and quantitative
yield of the resultant membranes will affect the comparison
of membrane composition or function to control prepara-
tions. This may result in misleading, quantitative changes of
the observed parameters (e.g., receptor binding sites,
amount of Ca2+ binding and transport, membrane-bound
enzymatic activities, etc.). This methodological pitfall may
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158 149
be avoided by considering the total percentage yield of the
activities in question in all the membrane fractions, the
relative enrichment of the membranes in question over the
initial crude homogenates, or normalization of the activities
of interest by a parameter, which is associated with the same
membrane but known to be unaltered.
In general, the subcellular membrane methods, whether
applied to receptor or Ca2+ handling studies, suffer from the
major disadvantage that they offer very little information on
the interaction and cross talk of the signaling events that occur
in intact cells and tissues under physiological conditions. This
aspect will be further discussed in a later section.
4. Electrophysiological methods
4.1. Intracellular microelectrodes
Intracellular electrophysiology had its origins in the
pioneering work of Hodgkin and Huxley (1939) and Hodg-
kin and Katz (1949) and their studies of axon potential
propagation in nerves; they used the squid axon because its
relatively large size (500 mm in diameter) facilitated the
insertion of bulky electrodes intracellularly. Since then, the
technology behind fabricating microelectrodes has been
refined to such an extent that even intact smooth muscle
cells (with diameters generally two orders of magnitude
smaller than the squid axon) can now be successfully and
routinely impaled. However, this requires microelectrodes
with tips sufficiently small and sharp as to penetrate the cell
membrane cleanly without tearing it, yet strong enough to
be forced through connective tissue and extracellular matrix
without breaking. At the same time, the body of the
electrode needs to be insulated (further increasing its dia-
meter), with only the tips bared to the cytosol, to properly
quantify the potential difference across the membrane.
Finally, the electrical resistance of the entire electrode needs
to be as low as possible. Generally, these demands are
adequately met by glass microelectrodes filled with 3 M
KCl (to minimize electrical resistance) having a tip diameter
of less than 100 nm and tip resistance of 30–100 MV.
These are then carefully advanced into the target tissue
using micromanipulators (with positional control as fine as a
few microns) and antivibration tables (to prolong the dura-
tion of impalement and minimize vibration-related arte-
facts), and membrane potential recordings are made using
a voltage amplifier, Faraday cage (to ground out electrical
‘‘noise’’ coming from lights, computers, etc.), oscilloscope
(to monitor the recordings during the experiment), and some
form of data storage.
In this way, many laboratories have been able to study
membrane potential changes in a variety of smooth muscle
tissues under many different conditions (Bolton, 1975; Cor-
nelissen et al., 2000; Cayabyab et al., 1996, 1997; Jimenez et
al., 1996). In addition to making passive recordings from the
cells, sophisticated electronics have been added in order to
allow injection of electrical current into the cell and thereby
alter/control the membrane potential, evoke electrical
responses, assess changes in membrane resistance, etc.
However, there are several drawbacks and limitations to
using this technique. Spontaneous mechanical activity and/
or contractile responses evoked by excitatory stimuli often
lead to ejection of the microelectrode, thereby limiting the
duration of recordings; the latter can be prolonged using
agents that fully relax the muscle or by attempting to
aggressively immobilize the tissues (e.g., using dissection
pins) or by using ‘‘floating microelectrodes,’’ which move
with the tissue (e.g., Daniel et al., 1960; Koch et al., 1988,
1991). Also, the relatively small size of the electrodes does
not allow one to introduce agents into the cell (e.g., to alter
the intracellular concentration of Ca2+ , or Cl � , or some
other single ion), and the high resistance of the electrodes
produces voltage errors and polarization artefacts, particu-
larly during injection of large currents into the cells, and
limits the ability to ‘‘clamp’’ the membrane potential. Patch-
clamp electrophysiology has provided the necessary break-
through to circumvent these limitations.
Additional pharmacological data about smoothmuscle can
be obtained by use of microelectrodes to penetrate single
muscle or nerve cells, recording membrane potential changes
on nerve stimulation or agonist administration, hyperpolari-
zation associated with inhibition of contractile activity, or
depolarization usually associated with activation of contract-
ile activity. Electrophysiological responses can be correlated
with tissue contractile responses by immobilization of the
sector of tissue impaled by the microelectrode or by using
microelectrodes that move with the tissue (see above). Since
contraction or relaxation may occur due to altered sensitivity
of the contractile apparatus to [Ca2+ ]i with varied or no
membrane potential change, additional data for identification
of action mechanisms of drugs or neurotransmitters can be
obtained by showing that the membrane potential changes
correspond to those of agonists known to act on a specific
receptor or on the postjunctional receptor to a neuromediator.
Further, the ability of selective antagonists or ion channel
blockers to impede both the depolarization-hyperpolarization
and the contraction-relaxation would support but not prove a
putative receptor identification or ionic activation mech-
anism. Further supportive evidence could be derived by
showing that the same second messengers were involved by
blocking their activation or actions.
5. Studies with isolated cells
5.1. General
Techniques are now available to isolate single smooth
muscle cells from any tissue (Janssen and Sims, 1992, 1993;
Salapatek et al., 1998). If the tissue has multiple layers of
muscle, as do gastrointestinal tissues (longitudinal, circular
muscles, and muscularis mucosae), it is essential to ensure
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158150
that the cells derive from the layer of interest. Cells in
different intestinal layers have different morphology and
properties [e.g., circular muscle cells are densely connected
by gap junctions while longitudinal muscles cells have no
detectable gap junctions (Daniel & Wang, 2000) and lon-
gitudinal muscle has differently configured slow waves
compared to circular muscle, as well as a different mem-
brane potential (Hara et al., 1986; Jimenez et al., 1996).
There are various criteria for ensuring that the cells
isolated have not been damaged seriously by the isolation
process: (1) cell length should approximate that in situ; (2)
cell ultrastructure should reveal that intracellular structures
are intact, except that the basement membrane and gap
junctions will be missing; (3) intracellular [Ca2+ ] should not
be seriously elevated over 100–200 nM; (4) the cells should
respond to contractile agonists by shortening, which is
reversible on washing and repeatable.
Many studies have been carried out with cells which are
shorter than in situ, shorten only 20–25% with potent
contractile agonists, and never relax after washing (e.g.,
Bitar & Makhlouf, 1985; Kuemmerle & Makhlouf, 1992;
Murthy et al., 2000). The results from such cells may be
difficult to relate to in vivo findings (e.g., they show
responses never seen in vivo or even in tissues in vitro,
such as contracting to opioid agonists to all three receptor
subtypes (Bitar & Makhlouf, 1985; Kuemmerle & Makh-
louf, 1992)].
If cells are to be used for patch-clamp studies, additional
criteria should be applied: (1) the membrane potential
should approximate that observed when cells were studied
with microelectrodes in situ if the pipette solution resembles
that of the cytosol; (2) the cell should retain its shape on
patching; (3) cell capacitance and access resistance should
be appropriate for smooth muscle (see below). When cells
are to be used after short-term culture, retention of the
contractile phenotype is crucial for studies of ion channels
by patch clamp, of shortening or [Ca2+ ]i changes in
response to agonists, and of receptor binding. For patch-
clamp studies, the following brief summary provides the
major points of methodology.
5.2. Patch-clamp electrophysiology
One of the main features that distinguishes this technique
from the intracellular microelectrode technique described
above is the electrode itself. In particular, the tip is much
blunter and wider (diameter of approximately 1 mm; elec-
trical resistance of 3–5 MV) and is ‘‘fire-polished’’ to
remove any residues or particles and to round off the sharp
edges, thereby allowing the tip to form an electrically tight
seal with the cell membrane (‘‘leak’’ resistance on the order
of 109 V). As a result, usually less than a handful of ion
channels are isolated within the patch of membrane at the tip
of the electrode, and ionic currents flowing through those
can be recorded with high resolution while the patch is still
attached to the cell (‘‘cell-attached patch’’) or following
excision of the patch from the cell (‘‘excised patch’’)
(Hamill et al., 1981).
On the other hand, the current through all of the channels
throughout the cell can be measured by gaining access to the
cell interior either by rupturing the patch at the tip of the
pipette (‘‘whole-cell’’) or perforating it with pore-forming
agents included in the electrode solution (‘‘perforated-
patch’’) (Horn & Marty, 1988). The relatively low access
resistance of the tip (relative to the intracellular microelectr-
odes described above) decreases the overall series resistance
of the electrode, thereby reducing voltage errors to previ-
ously unimaginably low levels, improving spatial and tem-
poral resolution, and allowing exquisitely fine control of the
membrane potential. Furthermore, the wide mouth of the
pipette allows one to dialyze the cytosol and/or introduce
agents into the cell (e.g., enzymes, antibodies, antagonists,
etc.) to manipulate the intracellular environment and signal-
ing pathways (Janssen & Sims, 1992, 1993). It is even
possible, after characterizing the ionic currents in the cell
(along with other physiological responses such as [Ca2+ ]iusing fura-2, or changes in cell length), to siphon out the
contents of that cell, particularly its nucleic acid content, and
perform single-cell RT-PCR (Tsumura et al., 1998; Huizinga
et al., 2000; Robinson et al., 2000).
The resolution of this relatively novel technique was
already astounding one or two decades ago, providing
exquisitely sensitive measurements on the order of picoam-
peres on a very fast time scale (microsecond intervals). But it
has been further enhanced by the development of compli-
cated mathematical algorithms that allow one to unmask
ionic currents several orders of magnitude smaller (on the
order of femptoamperes) than the inherent background noise
of the recordings (‘‘noise analysis’’), or to devise detailed and
sophisticated molecular models of the various activation/
inactivation states/substates of the ion channels. These algo-
rithms are beyond the scope of this review, but can be found
elsewhere (Klein et al., 1997; Steffan & Heinemann, 1997).
Finally, another development that has further expanded
the scope and abilities of this technique has been the recent
identification of pharmacological agonists and antagonists,
particularly many toxins, which are fairly selective for
different types of ion channels. This list includes charybdo-
toxin and iberiotoxin (large conductance Ca2+ -dependent
K+ channels), dendrotoxin (voltage-dependent K+ chan-
nels), tetrodotoxin and saxitoxin (voltage-dependent Na+
channels), maitotoxin (nonselective cation channels), sev-
eral conotoxins (various voltage-dependent Ca2+ channels),
and xestospongins (IP3-gated Ca2+ -release channels), to
name a few. One can also use electrode solutions composed
of various permeant and impermeant ions to isolate a
particular ionic current of interest: for example, Cs+ and
tetraethylammonium are routinely used to block K+ currents
nonselectively and thereby unmask voltage-dependent Ca2+
currents, while inorganic cations (Cd2+ , Ni2+ , Mg2+ , Mn2+ ,
etc.) and large anions (niflumic acid, 4-acetomido-40-iso-
thiocyanato-stilbene-2,20disulphonic acid [SITS], 9-anthra-
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158 151
cene, etc.) are used to block Ca2+ and Cl � currents,
respectively. In addition to these pharmacological criteria,
ionic currents are also distinguished on the basis of their
whole-cell or macroscopic electrophysiological properties,
that is, the voltage-dependence and time-dependence of
their activation and inactivation. For example, voltage-
dependent Ca2+ -currents have generally been subclassified
into L-, T-, and N-subtypes depending on whether they
require very negative (T-) or more positive (L- and N-)
potentials to become activated, and whether they inactivate
slowly (L-) or very quickly (T- and N-), for example. At the
single channel level, similar information is obtained by
measuring parameters such as opening probabilities and
durations of channel opening and closure at different vol-
tages. The ion channels that have been studied in detail
range from the very small conductance (20 femtosiemens)
Ca2+ -selective channels that contribute to refilling of the
sarcoplasmic reticulum (‘‘calcium-release-activated cur-
rent’’ [CRAC]) (Parekh & Penner, 1997) to the very large
conductance (several hundred picosiemens) Ca2+ -activated
K+ channels (Prasad et al., 1999).
Using these relatively new electrophysiological, math-
ematical, and pharmacological tools, it is now possible to
identify not only the major functional parts of the channel
(such as the transmembrane pore region), but to also
identify the individual amino acid(s) responsible for its
ionic selectivity (Doyle et al., 1998; Liao & Torre, 1999),
or its voltage sensor, which triggers channel opening (‘‘gat-
ing charge’’; Islas & Sigworth, 1999), or a cluster of amino
acids, which acts as a ‘‘ball and chain’’ that plugs up the
channel pore and inactivates it after somewhat of a delay
(Chanda et al., 1999).
The practical limits of this technique continue to be
extended in other ways. For example, the majority of
whole-cell recordings in the past had to be done in single
isolated cells (dissociated enzymatically from their multi-
cellular environment) for reasons related to spatial spread of
the voltage commands into adjacent cells. Now, however,
many groups are studying gap junction coupling between
pairs of cells (Miyoshi et al., 1996), or ion currents in clumps
of cells (the tissue being only partially digested to minimize
enzyme-induced damage to the preparation; Quinn & Beech,
1998), or currents in whole-tissue slices (Forsythe, 1994).
One fascinating development has seen a patch of membrane
removed from one cell and ‘‘crammed’’ into another cell,
using the Ca2+ -dependent or cyclic nucleotide-dependent
channels of the former as bioprobes for second messenger
levels in the latter (Trevidi & Kramer, 1998).
6. Fluorescent molecular probe methods in
Ca2+ measurement
Although 45Ca2+ tracer techniques have been widely
applied to intact tissues, isolated/cultured cells, and fractio-
nated membrane vesicles from smooth muscle tissues, and
provided important new insights in the understanding of
development of methods in the measurement of cytosolic
Ca2+ (Thomas, 1982), they do suffer from many disadvan-
tages as described earlier. The development of optical
molecular probes for Ca2+ , especially the membrane-per-
meable, Ca2+ -selective metallochromic fluorescent probes
(reviewed by Tsien, 1989), has opened new opportunities in
noninvasively studying sensitive changes of cytosolic Ca2+
in living tissues and cells, including smooth muscle.
This method is based on the principle of spectral changes
of the fluorescent dye upon binding with Ca2+ in the
cytosol, where the uncharged ester form of the dye is
hydrolyzed by the abundant cytosolic esterases and the
resulting anion form of the dye becomes trapped in the
cytosol. Following the development of the first two gen-
erations of the Ca2+ -fluorescent dyes, such as quin-2, fura-
2, and indo-1, several brighter and even organelle-selective
and Ca2+ -selective dyes have been made commercially
available (Haugland, 1996). Some of these dyes (e.g.,
fura-2 and indo-1 and their derivatives) offer advantages
because the estimates are ratiometric (i.e., the Ca2+ -bound
and the unbound forms elicit distinctly different fluo-
rescence spectra with an isosbetic point; for example, the
excitation spectrum of Ca2+ -bound fura-2 elicits a peak
near 340 nm, whereas that of the unbound fura-2 elicits a
peak near 380 nm with an isosbetic point, which is
independent of Ca2+ concentration). Since the increase of
fluorescence at 340 nm and the decrease of fluorescence at
380 nm occur simultaneously with increasing free Ca2+
concentration, the signal will be greatly enhanced by
expression as the ratio of the fluorescence, F340 nm/F380
nm. This allows measurement of Ca2+ to be done with less
dye-loading (therefore, less production of cytotoxic materi-
als as a result of esterase actions, less Ca2+ buffering effect,
and less cost) and not critically dependent on the dye
concentration present in the cytosol (Herman, 1996; Cim-
prich & Slavik, 1996).
Some dyes (e.g., fluo-3) that lack the ratiometric advan-
tage offer the technical advantage due to their very bright
fluorescence. Also, the excitation wavelength for fluo-3 falls
outside the range of that for the fluorescence of NADH and
related chemicals that are present in the cell as abundant
endogenous fluorophores, which decreases problems arising
from the autofluorescence of the cells or tissues.
Most of these fluorescent dyes are subject to photo-
bleaching effect, resulting in progressively decreasing fluo-
rescence intensity with longer period of exposure to the light
source (Becker & Fay, 1987). This, however, can be mini-
mized or prevented by reducing the oxygen tension and
limiting the exposure of cells or tissues to the continual
illumination of light. Although the fluorescence dyes are
trapped as anions in the cytosol after hydrolysis by
esterases, in some cells the fluorescence intensity drops
steadily due to the fluorescent dye leaking out (largely
extruded via the anion transporter in the plasma mem-
branes). Such leaks are largely inhibited by the anion
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158152
exchanger, probenecid, usually at millimolar range (Munsch
& Deitmer, 1995). Any dye loss to the extracellular space is
accompanied by an increase of the total fluorescence signal
due to binding of leaked material to extracellular Ca2+ ,
creating both a false-positive result and reducing the signal-
to-noise ratio. This difficulty may be reduced by shortening
the measurement time and by washing out the extruded
dyes, by inclusion of probenecid inhibiting the anion
exchanger, and by introduction of the dye in the form of
dextran-conjugated dye through microinjection to minimize
the loss of dye.
Whole-tissue or isolated cells can be used to study
changes in [Ca2+ ]i, although different approaches have to
be taken (see below). For isolated cells, the procedures and
precautions for loading the cells with fura-2 or other
fluorescent molecules have been described earlier in great
detail (Cobbold & Rink, 1987). In essence, the loading
conditions, such as dye concentration (usually 1–5 mM),
loading period (15–60 min), and temperature (25–37�C)are highly empirical and require experimentation. The same
precautions also apply to the loading of intact smooth
muscle tissues (Hotta et al., 1985; Sato et al., 1988; Mitsui
& Karaki, 1990; Nagasaki et al., 1991; Mo & Kwan, 1998).
However, for intact tissue strips, higher concentration of
dyes (>10 uM) and longer incubation time (>1 h) are usually
necessary because of the many diffusion barriers of the
smooth muscle tissues. However, whether overloading of
dyes actually occurs has rarely been tested. In case of
overloading of the fluorescent dye, incomplete hydrolysis
of the fluorescence dye molecules may introduce significant
error in the estimation of the cytosolic Ca2+ concentrations,
as unhydrolyzed and partially hydrolyzed dyes do not
demonstrate the same Ca2+ -dependent spectra and tend to
be compartmentalized. The advantage of using fluorescence
dye-loaded smooth muscle tissues is that the contractile
events can be monitored simultaneously in real time with
the change of fluorescence ratio (an indication of cytosolic
Ca2+ concentration) such that the changes of cytosolic Ca2+
concentration can be related to the contractile events.
However, one must be cautious in data interpretation as
the baseline of the fluorescence signal tends to decrease
gradually with time and under certain conditions or in
certain tissues, especially thick tissue strips, and generation
of optical artifacts associated with tissue contraction is not
uncommon (Sato et al., 1988; Mo & Kwan, 1998).
There is a wealth of information on the use of the
fluorescent dye method in the measurement of cytosolic
Ca2+ in smooth muscle cells, especially in vascular smooth
muscle cells. The advantage of using single cells compared
to tissues for Ca2+ measurement is that the interference and
diffusion difficulties associated with the presence of extra-
cellular matrix can be avoided. Also, given the proper
equipment setup and manual skills, simultaneous measure-
ment of ionic currents and the mobilization of Ca2+ can be
made (Ohta and Nakazato, 1994; Romero et al., 1998;
ZhuGe et al., 1999).
On the other hand, use of isolated cells has a number of
technical disadvantages. As mentioned earlier, cultured
smooth muscle cells may lose their native receptor charac-
teristics or change their sensitivity to receptor activation.
Enzymatically digested cells may require time for recovery
from the digestive processes. Since they are usually isolated
in very low Ca2+ medium to maintain them in a relaxed
state, it is imperative to check their tolerance to physio-
logical concentrations of Ca2+ introduced extracellularly. It
should also be kept in mind that, when cultured or enzy-
matically dispersed smooth muscle cells are studied at the
single cell level, individual cells may respond in a highly
variable manner differing in the magnitude of the signals
and the onset time to generate the signals. In tissues the
same cells are often connected by gap junctions and behave
as a syncytium.
Commercial instrumentation such as dynamic fluo-
rescence digital imaging and laser confocal fluorescent
microscopy have become available for the temporal and
spatial mapping of Ca2+ mobilization in different regions of
one single smooth muscle cell (Oh et al., 1997; Low et al.,
1997; Kirber et al., 2000; Drummond et al., 2000). The
calibration of the fluorescence signals can be transformed
into pseudocolor for easy mapping and visualization of the
Ca2+ concentration gradient across the entire area of the cell
or in a focal area of interest. Since the resolving power of
optical microscope is about 0.2 mm, superimposition of
signals within this plan of focus obscures spatial details that
might otherwise be resolved. Furthermore, for specimens
thicker than this depth of field, light from out of focus
planes creates diffuse halos around the object of study. This
difficulty is diminished with the optical sectioning power of
the confocal microscope. However, at this juncture of
technological development, the use of digital fura-2 fluo-
rescence imaging has the advantage over the laser confocal
fluorescence microscope for its lower cost and the ratio-
imaging capability, if the focal interest rests only on the
general mapping of the intracellular Ca2+ . However, extra
caution is required to avoid imaging artifacts arising from
background defocusing while focusing on the subject of
interest, from the change of the refraction index of the oil
due to change of temperature (if temperature-controlled
platform and oil immersion objective are used), from photo-
bleaching, and from cell movement. The laser confocal
fluorescence imaging, on the other hand, can provide higher
temporal resolution power by sacrificing the two-dimen-
sional spatial information. A single line across a region of
interest in the cell can be repeatedly scanned by the laser at
intervals between 10 and 100 msec. In fact, the line-
scanning component of the confocal microscope could be
deactivated to allow repetitive measurement of the same
spot of the cell to gain even higher temporal resolution. The
temporal component of the above fluoromicroscopic meas-
urement of Ca2+ is of particular importance for the detection
of Ca2+ sparks (Jaggar et al., 2000), which presumably
initiate the Ca2+ waves (Stevens et al., 2000), while the
E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158 153
spatial component determines the region (e.g., carveolae) or
organelles (superficial SR) that are involved in the origin
and maintenance of the Ca2+ dynamics (Lohn et al., 2000;
Murthy et al., 2000; Stevens et al., 2000; ZhuGe et al., 2000;
Gordienko et al., 2001).
7. Summary
The variety of techniques for pharmacological analysis
that can be applied to intestinal, as to most, smooth muscle
is great and growing. These techniques allow determination
of pharmacological properties at all levels of organization,
including molecular (not fully covered here). Users would
do well to keep in mind that smooth muscles may have
different properties at different levels of organization (i.e.,
they are not the sum of their properties as studied by
reductionist techniques).
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