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).

References

Ahmad, S., Allescher, H.-D., & Daniel, E. E. (1988). [3H]Saxitoxin as a

marker for canine deep muscular plexus neurons. American Journal of

Physiology, 255, G462–G469.

Ahmad, S., Allescher, H. D., Jang, E., Rausa, J., & Daniel, E. E. (1991). a2-

adrenergic receptors on the circular smooth muscle of canine small intes-

tine. Canadian Journal of Physiology and Pharmacology, 69, 837–840.

Ahmad, S., Berezin, I., Vincent, J.-P., & Daniel, E. E. (1987). Neurotensin

receptors in canine intestinal smooth muscle: preparation of plasma

membranes and characterization of (Tyr3-I125)-labeled neurotensin bind-

ing. Biochimica et Biophysica Acta, 896, 224–238.

Ahmad, S., Rausa, J., Jang, E., & Daniel, E. E. (1989). Calcium channel

binding in nerves and muscle of canine small intestine. Biochemical and

Biophysical Research Communications, 159, 119–125.

Allescher, H. D., Ahmad, S., Kostka, P., Kwan, C. Y., & Daniel, E. E.

(1989). Distribution of opioid receptors in canine small intestine:

implications for function. American Journal of Physiology, 256,

G966–G974.

Allescher, H.-D., Daniel, E. E., Dent, J., Fox, J. E. T., & Kostolanska, F.

(1988). Extrinsic and intrinsic neural control of pyloric sphincter pres-

sure in the dog. Journal of Physiology (London), 401, 17–38.

Arunlakshana, O., & Schild, H. O. (1997). Some quantitative uses of

drug antagonists [originally published in 1958]. British Journal of

Pharmacology, 120 (4 Suppl.), 151–161; discussion 148–150.

Bauer, A. J., Sarr, M. G., & Szurszewski, J. H. (1991). Opioids inhibit

neuromuscular transmission in circular muscle of human and baboon

jejunum. Gastroenterology, 101, 970–976.

Bauer, A. J., & Szurszewski, J. H. (1991). Effect of opioid peptides on

circular muscle of canine duodenum. Journal of Physiology (London),

434, 409–422.

Becker, P. L., & Fay, F. S. (1987). Photobleaching of fura-2 and its effect

on determination of calcium concentrations. American Journal of

Physiology, 253, C613–C618.

Berezin, I., Huizinga, J. D., & Daniel, E. E. (1988). Interstitial cells of Cajal

in canine colon: a special communication network at the inner border of

the circular muscle. Journal of Comparative Neurology, 273, 42–51.

Berezin, I., Huizinga, J. D., & Daniel, E. E. (1990). Structural character-

ization and interconnections of interstitial cells of Cajal in myenteric

plexus and circular muscle of canine colon. Canadian Journal of

Physiology and Pharmacology, 68, 1419–1431.

Besse, J. C., & Furchgott, R. F. (1976). Dissociation constants and relative

efficacies of agonists acting on alpha adrenergic receptors in rabbit

aorta. Journal of Pharmacology and Experimental Therapeutics, 197,

66–78.

Bitar, K. N., & Makhlouf, G. M. (1985). Selective presence of opioid

receptors in circular muscle cells. Life Sciences, 37, 1545–1550.

Black, J. W., Gerskowitch, V. P., Leff, P., & Shankley, N. P. (1985a).

Pharmacological analysis of beta-adrenoceptor-mediated agonism in

the guinea-pig, isolated, right atrium. British Journal of Pharmacology,

84, 779–785.

Black, J. W., Leff, P., Shankley, N. P., & Wood, J. (1985b). An operational

model of pharmacological agonism: the effect of E/[A] curve shape on

agonist dissociation constant estimation. British Journal of Pharma-

cology, 84, 561–571.

Bolton, T. B. (1975). Effects of stimulating the acetylcholine receptor on the

current-voltage relationships of the smooth muscle membrane studied

by voltage clamp of potential recorded by micro-electrode. Journal of

Physiology (London), 250, 175–202.

Burgisser, E., Lefkowitz, R. J., & DeLean, A. (1981). Alternative explan-

ation for the apparent ‘‘two-step’’ binding kinetics of high-affinity rac-

emic antagonist radioligands. Molecular Pharmacology, 19, 509–512.

Burks, T. F. (1973). Mediation by 5-hydroxytryptamine of morphine stimu-

lant actions in dog intestine. Journal of Pharmacology and Experimen-

tal Therapeutics, 185, 530–539.

Burks, T. F., & Long, J. P. (1967a). Responses of isolated dog small intes-

tine to analgesic agents. Journal of Pharmacology and Experimental

Therapeutics, 158, 264–271.

Burks, T. F., & Long, J. P. (1967b). Release of 5-hydroxytryptamine from

isolated dog intestine by nicotine. British Journal of Pharmacology, 30,

229–239.

Bylund, D. B. (1986). Graphic presentation and analysis of inhibition data

from ligand-binding experiments. Analytical Biochemistry, 159, 50–57.

Cayabyab, F. S., de Bruin, H., Jimenez, M., & Daniel, E. E. (1996). Ca2 +

role in myogenic and neurogenic activities of canine ileum circular

muscle. American Journal of Physiology, 271, G1053–G1066.

Cayabyab, F. S., Jimenez, M., Vergara, P., de Bruin, H., & Daniel, E. E.

(1997). Influence of nitric oxide and vasoactive intestinal peptide on the

spontaneous and triggered electrical and mechanical activities of the

canine ileum. Canadian Journal of Physiology and Pharmacology,

75, 383–397.

Chadwick, C. C., Saito, A., & Fleischer, S. (1990). Isolation and character-

ization of the inositol trisphosphate receptor from smooth muscle. Pro-

ceedings of the National Academy of Sciences of the United States of

America, 87, 2132–2136.

Chanda, B., Tiwari, J. K., Varshney, A., & Mathew, M. K. (1999). Trans-

planting the N-terminus from Kv1.4 to Kv1.1 generates an inwardly

rectifying K+ channel. Neuroreport 10, 237–241.

Chen, C., McDonald, T. J., & Daniel, E. E. (1994a). Characterization of

galanin receptor in canine small intestinal circular muscle synapto-

somes. American Journal of Physiology, 266, G106–G112.

Chen, C. K., McDonald, T. J., & Daniel, E. E. (1994b). Galanin receptor in

plasma membrane of canine small intestinal circular muscle. American

Journal of Physiology, 266, G113–G117.

Cimprich, P., & Slavik, J. (1996). Artifacts in fluorescence ratio imaging.

In J. Slavik (Ed.), Fluourescence microscopy and fluorescent probes

( pp. 119–123). New York: Plenum Press.

Cobbold, P. H., & Rink, T. J. (1987). Fluorescence and bioluminescence

measurement of cytoplasmic free calcium. Biochemical Journal, 248,

313–328.

Cornelissen, W., De Laet, A., Kroese, A. B., Van Bogaert, P. P., Scheuer-

mann, D. W., & Timmermans, J. P. (2000). Electrophysiological fea-

tures of morphological Dogiel type II neurons in the myenteric plexus

of pig small intestine. Journal of Neurophysiology, 84, 102–111.

Costa, M., & Furness, J. B. (1976). The peristaltic reflex: an analysis of

the nerve pathways and their pharmacology. Naunyn Schmiedebergs

Archives of Pharmacology, 294, 47–60.

Cowie, A. L., Kosterlitz, H. W., & Waterfield, A. A. (1978). Factors influ-

encing the release of acetylcholine from the myenteric plexus of the

E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158154

ileum of the guinea-pig and rabbit. British Journal of Pharmacology,

64, 565–580.

Crema, A., Frigo, G. M., & Lecchini, S. (1970). A pharmacological anal-

ysis of the peristaltic reflex in the isolated colon of the guinea-pig or cat.

British Journal of Pharmacology, 39, 334–345.

Daniel, E. E., Brown, R. D., Wang, Y. F., Low, A. M., Lu-Chao, H., &

Kwan, C. Y. (1999). a-adrenoceptors in canine mesenteric artery are

predominantly 1A subtype: pharmacological and immunochemical evi-

dence. Journal of Pharmacology and Experimental Therapeutics, 291,

671–679.

Daniel, E. E., Costa, M., Furness, J. B., & Keast, J. R. (1985). Peptide

neurons in the canine small intestine. Journal of Comparative Neurol-

ogy, 237, 227–238.

Daniel, E. E., Haugh, C., Woskowska, Z., Cipris, S., Jury, J., & Fox-

Threlkeld, J. E. T. (1994). Role of NO-related inhibition in intestinal

function: relation to vasoactive intestinal polypeptide (VIP). American

Journal of Physiology, 266, G31–G39.

Daniel, E. E., Honour, A. J., & Bogoch, A. (1960). Electrical activity of the

longitudinal muscle of the dog small intestine studied in vivo using

microelectrodes. American Journal of Physiology, 198, 113–118.

Daniel, E. E., & Kostolanska, F. (1989). Functional studies of nerve projec-

tions in the canine intestine. Canadian Journal of Physiology and Phar-

macology, 67, 1074–1085.

Daniel, E. E.,&Wang,Y. F. (2000).Gap junctions in intestinal smoothmuscle

and interstitial cells of Cajal. Microscopy Research and Technique, 47,

309–320.

Daniel, E. E., Wang, Y. F., & Cayabyab, F. (1998). Role of gap junctions in

structural arrangements of interstitial cells of Cajal and canine ileal

smooth muscle. American Journal of Physiology, 274, G1125–G1141.

Darby, P. J., & Daniel, E. E. (2000). Caveolae from canine airway smooth

muscle contain the necessary components for a role in CA2+ handling.

American Journal of Physiology (Lung Cellular & Molecular Physiol-

ogy), 279, L1226–L1235.

DeLean, A., Munson, P. J., & Rodbard, D. (1978). Simultaneous analysis

of families of sigmoidal curves: application to bioassay, radioligand

assay, and physiological dose-response curves. American Journal of

Physiology, 235, E97–E102.

Depoortere, I., Peeters, T. L., & Vantrappen, G. (1991). Evidence for motilin

receptor subtypes in the rabbit. Journal of Gastrointestinal Motility, 3,

179–184.

Depoortere, I., Peeters, T. L., & Vantrappen, G. (1993). Distribution and

characterization of motilin receptors in the cat. Peptides, 14, 1153–1157.

Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M.,

Cohen, S. L., Chait, B. T., & MacKinnon, R. (1998). The structure of

the potassium channel: molecular basis of K+ conduction and selectiv-

ity. Science, 280, 69–77.

Drummond, R. M., Mix, T. C., Tuft, R. A., Walsh, J. V. Jr., & Fay, F. S.

(2000). Mitochondrial Ca2+ homeostasis during Ca2+ influx and Ca2+

release in gastric myocytes from Bufo marinus. Journal of Physiology

(London), 522, 375–390.

Duchon, G., Henderson, R., & Daniel, E. E. (1974). Circular muscle layers

in the small intestine. In E. E. Daniel, Proceedings of the Fourth Inter-

national Symposium on GI Motility ( pp. 635–646). Vancouver: Mitch-

ell Press.

Durdle, N. G., Kingma, Y. J., Bowes, K. L., & Chambers, M. M. (1983).

Origin of slow waves in the canine colon. Gastroenterology, 84,

375–382.

Forsythe, I. D. (1994). Direct patch recording from identified presynaptic

terminals mediating glutamatergic EPSCs in the rat CNS, in vitro.

Journal of Physiology (London), 479, 381–387.

Fox, J. E. T., & Daniel, E. E. (1986). Substance P: a potent inhibitor of the

canine small intestine in vivo. American Journal of Physiology, 250,

G21–G27.

Fox, J. E. T., & Daniel, E. E. (1987a). Exogenous opiates: their local

mechanisms of action in the canine small intestine and stomach.

American Journal of Physiology, 253, G179–G188.

Fox, J. E. T., & Daniel, E. E. (1987b). Activation of endogenous excitatory

opiate pathways in the canine small intestine by field stimulation and

motilin. American Journal of Physiology, 253, G189–G194.

Fox, J. E. T., Daniel, E. E., Jury, J., Fox, A. E., & Collins, S. M. (1983).

Sites and mechanisms of action of neuropeptides on canine gastric

motility differ in vivo and in vitro. Life Sciences, 33, 817–825.

Fox, J. E. T., Daniel, E. E., Jury, J., & Robotham, H. (1984). The mech-

anisms of motilin excitation of the canine small intestine. Life Sciences,

34, 1001–1006.

Fox-Threlkeld, J. E. T., Daniel, E. E., Christinck, F., Woskowska, Z.,

Cipris, S., & McDonald, T. J. (1993). Peptide YY stimulates circular

muscle contractions of the isolated perfused canine ileum by inhibiting

nitric oxide release and enhancing acetylcholine release. Peptides, 14,

1171–1178.

Fox-Threlkeld, J. E. T., Daniel, E. E., Christinck, F., Hruby, V. J., Cipris, S.,

& Woskowska, Z. (1994). Identification of mechanisms and sites of

actions of Mu and Delta opioid receptor activation in the canine intes-

tine. Journal of Pharmacology and Experimental Therapeutics, 268,

689–700.

Fox-Threlkeld, J. E. T., Manaka, H., Manaka, Y., Cipris, S., & Daniel, E. E.

(1991a). Stimulation of circular muscle motility of the isolated perfused

canine ileum: relationship to VIP output. Peptides, 12, 1039–1045.

Fox-Threlkeld, J. E. T., Manaka, H., Manaka, Y., Cripis, S., Woskowska, Z.,

& Daniel, E. E. (1991b). Mechanism of noncholinergic excitation of

canine ileal circular muscle by motilin. Peptides, 12, 1047–1050.

Fox-Threlkeld, J. E. T., McDonald, T. J., Cipris, S., Woskowska, Z., &

Daniel, E. E. (1991c). Galanin inhibition of VIP release and circular

muscle motility in the isolated perfused canine ileum. Gastroenterology,

101, 1471–1476.

Fox-Threlkeld, J. E. T., McDonald, T. J., Woskowska, Z., & Daniel, E. E.

(1999). Pituitary adenylate cyclase-activating peptide as a neurotrans-

mitter in the canine ileal circular muscle. Journal of Pharmacology and

Experimental Therapeutics, 290, 66–75.

Fox-Threlkeld, J. E. T.,Woskowska, Z., &Daniel, E. E. (1997). Sites of nitric

oxide (NO) actions in control of circular muscle motility of the perfused

isolated canine ileum. Canadian Journal of Physiology and Pharmacol-

ogy, 75, 1340–1349.

Foxx-Orenstein, A. E., & Grider, J. R. (1996). Regulation of colonic pro-

pulsion by enteric excitatory and inhibitory neurotransmitters. American

Journal of Physiology, 271, G433–G437.

Furchgott, R. F. (1967). The pharmacological differentiation of adrener-

gic receptors. Annals of the New York Academy of Sciences, 139,

553–570.

Furchgott, R. F. (1978). Pharmacological characterization of receptors: its

relation to radioligand-binding studies. Federation Proceedings, 37,

115–120.

Furness, J. B. (2000). Types of neurons in the enteric nervous system.

Journal of the Autonomic Nervous System, 81, 87–96.

Furness, J. B., & Costa, M. (1980). Types of nerves in the enteric nervous

system. Edinburgh, UK: Churchill Livingstone.

Furness, J. B., Costa, M., Murphy, R., Beardsley, A. M., Oliver, J. R.,

Llewellyn-Smith, I. J., Eskay, R. L., Shulkes, A. A., Moody, T. W.,

& Meyer, D. K. (1982). Detection and characterisation of neurotrans-

mitters, particularly peptides, in the gastrointestinal tract. Scandinavian

Journal of Gastroenterology, 71 (Suppl.), 61–70.

Gershon, M. D. (1991). Serotonin: its role and receptors in enteric neuro-

transmission. Advances in Experimental Medicine and Biology, 294,

221–230.

Gershon, M. D., Wade, P. R., Kirchgessner, A. L., & Tamir, H. (1990).

5-HT receptor subtypes outside the central nervous system. Roles in

the physiology of the gut. Neuropsychopharmacology, 3, 385–395.

Gordienko, D. V., Greenwood, I. A., & Bolton, T. B. (2001). Direct visual-

ization of sarcoplasmic reticulum regions discharging Ca(2+)sparks in

vascular myocytes. Cell Calcium, 29, 13–28.

Grider, J. R., Kuemmerle, J. F., & Jin, J. G. (1996). 5-HT released by

mucosal stimuli initiates peristalsis by activating 5–HT4/5–HT1p re-

ceptors on sensory CGRP neurons. American Journal of Physiology,

270, G778–G782.

E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158 155

Grover, A. K., Crankshaw, J., Garfield, R. E., & Daniel, E. E. (1980a).

Smooth muscle membrane vesicle orientation: a study on intactness and

sidedness of rat myometrium plasma membrane vesicles. Canadian

Journal of Physiology and Pharmacology, 58, 1202–1211.

Grover, A. K., Kwan, C. Y., Garfield, R. E., McLean, J., Fox, J. E. T., &

Daniel, E. E. (1980b). Fractionation and Ca uptake studies on mem-

branes of rabbit longitudinal and circular intestinal smooth muscle.

Canadian Journal of Physiology and Pharmacology, 58, 1102–1113.

Grover, A. K., Kwan, C. Y., & Daniel, E. E., (1981). Na-Ca exchange in rat

myometrium membrane vessicles highly enriched in plasma mem-

branes. American Journal of Physiology, 240, C175–C182.

Grover, A. K., Kwan, C. Y., Kostka, P., Sheppard, S. M., & Daniel, E. E.

(1984a). Binding and degradation studies on angiotensin II. Canadian

Journal of Physiology and Pharmacology, 62, 1203–1208.

Grover, A. K., Kwan, C. Y., Luchowski, E., Daniel, E. E., & Triggle, D. J.,

(1984b). Subcellular distribution of [3H]nitrendipine binding in smooth

muscle. Journal of Biological Chemistry, 259, 2223–2226.

Hamill, O. P., Marty, A., Neher, E., Sakmann, B., & Sigworth, F. J. (1981).

Improved patch clamp techniques for high-resolution current recording

from cells and cell-free membrane patches. Pflugers Archives, 391,

85–100.

Hara, Y., Kubota, M., & Szurszewski, J. H. (1986). Electrophysiology of

smooth muscle of the small intestine of some mammals. Journal of

Physiology (London), 372, 501–520.

Haugland, R. P. (1996). Indicators for Ca2+ , Mg2+ , Zn2+ and other met-

als. In Handbook of fluorescent probes and research hemicals, 6th ed.

(pp. 484–502). Eugene, OR: Molecular Probes.

Herman, B. (1996). Fluorescence microscopy: state of the art. In J. Slavik,

Fluourescence microscopy and fluorescent probes ( pp. 1–14). New

York: Plenum Press.

Hirning, L. D., & Burks, T. F. (1986). Neurogenic mechanism of action of

motilin in the canine isolated small intestine ex vivo. European Journal

of Pharmacology, 128, 241–248.

Hodgkin, A. L., & Huxley, A. F. (1939). Action potentials recorded from

inside a nerve fiber. Journal of Physiology (London), 144, 710–717.

Hodgkin, A. L., & Katz, B. (1949). The effect of sodium ions on the

electrical activity of the giant axons from Sepia. Journal of Physiology

(London), 119, 513–528.

Horn, R., & Marty, A. (1988). Muscarinic activation of ionic currents

measured by a new whole-cell recording method. Journal of General

Physiology, 92, 145–159.

Hotta, K., Yamamoto, Y., & Oba, T. (1985). Transient intracellular calcium

movement coupled with mechanical activity of smooth muscle. Japa-

nese Journal of Physiology, 35, 1079–1083.

Huizinga, J. D., Robinson, T. L., & Thomsen, L. (2000). The search for the

origin of rhythmicity in intestinal contraction; from tissue to single

cells. Neurogastroenterology and Motility, 12, 3–9.

Islas, L. D., & Sigworth, F. J. (1999). Voltage sensitivity and gating charge

in Shaker and Shab family potassium channels. Journal of General

Physiology, 114, 723–742.

Iversen, H. H., Celsing, F., Leone, A. M., Gustafsson, L. E., & Wiklund, N.

P. (1997). Nerve-induced release of nitric oxide in the rabbit gastro-

intestinal tract as measured by in vivo microdialysis. British Journal of

Pharmacology, 120, 702–706.

Jaggar, J. H., Porter, V. A., Lederer, W. J., & Nelson, M. T. (2000).

Calcium sparks in smooth muscle. American Journal of Physiology,

278, C235–C256.

Janssen, L. J., & Sims, S. M. (1992). ACh activates nonselective cation

and chloride conductances in canine and guinea-pig tracheal myocytes.

Journal of Physiology (London), 453, 197–218.

Janssen, L. J., & Sims, S. M. (1993). Histamine activates Cl – and K+

currents in guinea-pig tracheal myocytes: convergence with muscarinic

signalling pathway. Journal of Physiology (London), 465, 661–677.

Jimenez, M., Cayabyab, F. S., Vergara, P., & Daniel, E. E. (1996). Hetero-

geneity in electrical activity of the canine ileal circular muscle: inter-

action of two pacemakers. Neurogastroenterology and Motility, 8,

339–350.

Jin, J. G., Foxx-Orenstein, A. E., & Grider, J. R. (1999). Propulsion in

guinea pig colon induced by 5-hydroxytryptamine (HT) via 5-HT4 and

5-HT3 receptors. Journal of Pharmacology and Experimental Thera-

peutics, 288, 93–97.

Johnson, S. M., Katayama, Y., & North, R. A. (1980). Multiple actions of 5-

hydroxytryptamine on myenteric neurones of the guinea-pig ileum.

Journal of Physiology (London), 304, 459–470.

Kadowaki, M., Wade, P. R., & Gershon, M. D. (1996). Participation of 5–

HT3, 5–HT4, and nicotinic receptors in the peristaltic reflex of guinea

pig distal colon. American Journal of Physiology, 271, G849–G857.

Kenakin, T. P. (1985). The quantification of relative efficacy of agonists.

Journal of Pharmacological Methods, 13, 281–308.

Kenakin, T. (1988). Are receptors promiscuous? Intrinsic efficacy as a

transduction phenomenon. Life Sciences, 43, 1095–1101.

Kenakin, T. (1990). Drugs and receptors. An overview of the current state

of knowledge. Drugs, 40, 66–87.

Kenakin, T. (1995). Agonist-receptor efficacy. I: Mechanisms of efficacy

and receptor promiscuity. Trends in Pharmacological Sciences, 16,

188–192.

Kenakin, T. P., & Beek, D. (1984). Relative efficacy of prenalterol and

pirbuterol for beta-1 adrenoceptors: measurement of agonist affinity

by alteration of receptor number. Journal of Pharmacology and Exper-

imental Therapeutics, 229, 340–345.

Kirber, M. T., Guerrero-Hernandez, A., Bowman, D. S., Fogarty, K. E., Tuft,

R. A., Singer, J. J., & Fay, F. S. (2000). Multiple pathways responsible

for the stretch-induced increase in Ca2+ concentration in toad stomach

smooth muscle cells. Journal of Physiology (London), 524, 3–17.

Klein, S., Timmer, J., & Honerkamp, J. (1997). Analysis of multichannel

patch clamp recordings by hidden Markov models. Biometrics, 53,

870–884.

Koch, T. R., Carney, J. A., Go, V. L., & Szurszewski, J. H. (1988). Sponta-

neous contractions and some electrophysiologic properties of circular

muscle from normal sigmoid colon and ulcerative colitis. Gastroenter-

ology, 95, 77–84.

Koch, T. R., Carney, J. A., Go, V. L., & Szurszewski, J. H. (1991). Inhib-

itory neuropeptides and intrinsic inhibitory innervation of descending

human colon. Digestive Diseases and Sciences, 36, 712–718.

Komuro, T. (1999). Comparative morphology of interstitial cells of Cajal:

ultrastructural characterization. Microscopy Research and Technique,

47, 267–285.

Kosterlitz, H. W. (1980). Opioid peptides and their receptors. Progress in

Biochemical Pharmacology, 16, 3–10.

Kostka, P., Ahmad, S., Berezin, I., Kwan, C. Y., Allescher, H.-D., &

Daniel, E. E. (1987). Subcellular fractionation of the longitudinal

smooth muscle/myenteric plexus of dog ileum: dissociation of the dis-

tribution of two plasma membrane marker enzymes. Journal of Neuro-

chemistry, 49, 1124–1132.

Kostka, P., Ahmad, S., Kwan, C. Y., Daniel, E. E., Gordon, R. K., &

Chiang, P. K. (1992). Prejunctional muscarinic receptors in the deep

muscular plexus of canine ileum: comparison with smooth muscle

receptors. Journal of Pharmacology and Experimental Therapeutics,

263, 226–231.

Kostka, P., Kwan, C. Y., & Daniel, E. E. (1989a). Presynaptic and postjunc-

tional muscarinic receptors in dog ileum: binding studies. European

Journal of Pharmacology, 173, 35–42.

Kostka, P., Sipos, S. N., Kwan, C. Y., Niles, L. P., Daniel, E. E. (1989b)

Identification and characterization of presynaptic and postsynaptic beta-

adrenoreceptors in the longitudinal smooth muscle/ myenteric plexus of

dog ileum. Journal of Pharmacology and Experimental Therapeutics,

251, 305–310.

Kuemmerle, J. F, & Makhlouf, G. M. (1992). Characterization of opioid

receptors in intestinal muscle by selective radioglands and receptor

protection. American Journal of Physiology, 263, G269–G276.

Kunze, W. A., Clerc, N., Furness, J. B., & Gola, M. (2000). The soma and

neurites of primary afferent neurons in the guinea-pig intestine respond

differentially to deformation. Journal of Physiology (London), 526,

375–385.

E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158156

Kwan, C. Y., Triggle, C. R., Grover, A. K., Lee, R. M. K. W., & Daniel, E.

E. (1983). An analytical approach to the preparation and characteriza-

tion of subcellular membranes from canine mesenteric arteries. Prepa-

rative Biochemistry, 13, 275–314.

Kwan C. Y. (1992). Signal transduction in smooth muscle as studied by the

subcellular membrane approach. Canadian Journal of Physiology and

Pharmacology, 70, 501–508.

Kwan C. Y. (1999). Membrane abnormalities of vascular smooth muscle of

mesenteric arteries of spontaneous diabetic BB rats. Journal of Smooth

Muscle Research, 35, 77–86.

Laio, A., & Torre, V. (1999). Physical origin of selectivity in ionic channels

of biological membranes. Biophysical Journal, 76, 129–148.

Lee, J. C., Thuneberg, L., Berezin, I., & Huizinga, J. D. (1999). Generation

of slow waves in membrane potential is an intrinsic property of inter-

stitial cells of Cajal. American Journal of Physiology, 277, G409–G423.

Leff, P., Dougall, I. G., Harper, D. H., & Dainty, I. A. (1990a). Errors in

agonist affinity estimation: do they and should they occur in isolated

tissue experiments? Trends in Pharmacological Sciences, 11, 64–67.

Leff, P., Prentice, D. J., Giles, H., Martin, G. R., & Wood, J. (1990b)

Estimation of agonist affinity and efficacy by direct, operational model-

fitting. Journal of Pharmacological Methods, 23, 225–237.

Leff, P., & Giles, H. (1992). Classification of platelet and vascular prosta-

glandin D2 (DP) receptors: estimation of affinities and relative effica-

cies for a series of novel bicyclic ligands [with an appendix on

goodness-of-fit analyses]. British Journal of Pharmacology, 106,

996–1003.

Leung, E., Jacobson, K. A., & Green, R. D. (1991). Apparent heterogeneity

of cardiac A1 adenosine receptors as revealed by radioligand binding

experiments on N-ethylmaleimide-treated membranes. Naunyn Schmie-

debergs Archiv Pharmakologie, 344, 639–644.

Lohn, M., Furstenau, M., Sagach, V., Elger, M., Schulze, W., Luft, F. C.,

Haller, H., & Gollasch, M. (2000). Ignition of calcium sparks in arte-

rial and cardiac muscle through caveolae. Circulation Research, 87,

1034–1039.

Low, A. M., Sormaz, L., Kwan, C. Y., & Daniel, E. E. (1997). Actions of 4-

chloro-3-ethyl phenol on internal Ca2+ stores in vascular smooth

muscle and endothelial cells. British Journal of Pharmacology, 122,

504–510.

Lu, G., Sarr, M. G., & Szurszewski, J. H. (1998). Effect of motilin and

erythromycin on calcium-activated potassium channels in rabbit colonic

myocytes. Gastroenterology, 114, 748–754.

Manaka, H., Manaka, Y., Kostolanska, F., Fox, J. E. T., & Daniel, E. E.

(1989). Release of VIP and substance P from isolated perfused canine

ileum. American Journal of Physiology, 257, G182–G190.

Mao, Y. K., Barnett, W., Coy, D. H., Tougas, G., & Daniel, E. E. (1991).

Distribution of vasoactive intestinal polypeptide (VIP)-binding in circu-

lar muscle and characterization of VIP-binding in canine small intestinal

mucosa. Journal of Pharmacology and Experimental Therapeutics, 258,

986–991.

Mao, Y. K., Tougas, G., Barnett, W., & Daniel, E. E. (1992). VIP receptors

on canine submucosal synaptosomes. Peptides, 14, 1149–1152.

Mao, Y. K., Wang, Y. F., & Daniel, E. E. (1995). Characterization of

cholecystokinin receptors in canine intestinal circular muscle. Peptides,

16, 1025–1029.

Mao, Y. K., Wang, Y. F., & Daniel, E. E. (1996a). Characterization of

neurokinin type 1 receptor in canine small intestinal circular muscle.

Peptides, 17, 839–843.

Mao, Y. K., Wang, Y. F., Moogk, C., Fox-Threlkeld, J. E. T., Xiao, Q.,

McDonald, T. J., & Daniel, E. E. (1998). Locations and molecular

forms of PACAP and sites and characteristics of PACAP receptors in

canine ileum. American Journal of Physiology, 274, G217–G225.

Mao, Y. K., Wang, Y. F., Ward, G., Cipris, S., Daniel, E. E., & McDonald,

T. J. (1996b). Peptide YY Receptor in submucosal and myenteric

plexus synaptosomes of canine small intestine. American Journal of

Physiology, 271, G36–G41.

Matlib, M. A., Crankshaw, J., Garfield, R. E., Crankshaw, D. J., Kwan, C. Y.,

Branda, L. A., & Daniel, E. E. (1979). Characterization of membrane

fractions and isolation of purified plasma membranes from rat myome-

trium. Journal of Biological Chemistry, 254, 1834–1840.

Mitsui, M., & Karaki, H. (1990). Dual effects of carbachol on cytosolic

Ca2+ and contraction in intestinal smooth muscle. American Journal of

Physiology, 258, C787–C793.

Miyoshi, H., Boyle, M. B., MacKay, L. B., & Garfield, R. E. (1996).

Voltage-clamp studies of gap junctions between uterine muscle cells

during term and preterm labor. Biophysical Journal, 71, 1324–1334.

Mo, F. M., & Kwan, C. Y. (1998). a-adrenoceptor subtypes of dog saphe-

nous vein: another unusual property. Life Sciences, 62, 1455–1459.

Moore, J. E., & Abercrombie, R. F. (1996). Calcium chelators enhance

45Ca accumulation in permeablized synaptosomes and in microsomes.

American Journal of Physiology, 270, C628–C635.

Munsch, T., & Deitmer, J. W. (1995). Maintenance of fura-2 fluorescence in

glial cells and neurons of the leech central nervous system. Journal of

Neuroscience Methods, 57, 195–204.

Murthy, K. S., Yee, Y. S., Grider, J. R., & Makhlouf, G. M. (2000). Phor-

bol-stimulated Ca(2+) mobilization and contraction in dispersed intes-

tinal smooth muscle cells. Journal of Pharmacology and Experimental

Therapeutics, 294, 991–996.

Nagasaki, M., Kobayashi, T., & Tamaki, H. (1991). Effects of trimebutine

on cytosolic Ca2+ and force transitions in intestinal smooth muscle.

European Journal of Pharmacology, 195, 317–321.

Nickerson, M. (1967). New developments in adrenergic blocking drugs.

Annals of the New York Academy of Sciences, 139, 571–579.

Northway, M. G., & Burks, T. F. (1979). Indirect intestinal stimulatory

effects of heroin: direct action on opiate receptors. European Journal

of Pharmacology, 59, 237–243.

Oh, S. T., Yedidag, E., Conklin, J. L., Martin, M., & Bielefeldt, K. (1997).

Calcium release from intracellular stores and excitation-contraction cou-

pling in intestinal smooth muscle. Journal of Surgical Research, 71,

79–86.

Parekh, A. B., & Penner, R. (1997). Store depletion and calcium influx.

Physiological Reviews, 77, 901–930.

Power, R. F., Bishop, A. E., Wharton, J., Inyama, C. O., Jackson, R. H.,

Bloom, S. R., & Polak, J. M. (1988). Anatomical distribution of vaso-

active intestinal peptide binding sites in peripheral tissues investigated by

in vitro autoradiography. Annals of the New York Academy of Sciences,

527, 314–325.

Prasad, M., Matthews, J. B., He, X. D., & Akbarali, H. I. (1999). Mono-

chloramine directly modulates Ca(2+)-activated K(+) channels in rabbit

colonic muscularis mucosae. Gastroenterology, 117, 906–917.

Quinn, K., & Beech, D. J. (1998). A method for direct patch-clamp record-

ing from smooth muscle cells embedded in functional brain microves-

sels. Pflugers Archives, 435, 564–569.

Rainbow, T. C., Biegon, A., & Berck, D. J. (1984). Quantitative receptor

autoradiography with tritium-labeled ligands: comparison of biochemi-

cal and densitometric measurements. Journal of Neuroscience Methods,

11, 231–241.

Rainbow, T. C., Biegon, A., & Bleisch, W. V. (1982a). Minireview: quanti-

tative autoradiography of neurochemicals. Life Sciences, 30, 1769–1774.

Rainbow, T. C., Bleisch, W. V., Biegon, A., & McEwen, B. S. (1982b).

Quantitative densitometry of neurotransmitter receptors. Journal of

Neuroscience Methods, 5, 127–138.

Robinson, T. L., Sircar, K., Hewlett, B. R., Chorneyko, K., Riddell, R. H.,

& Huizinga, J. D. (2000). Gastrointestinal stromal tumors may originate

from a subset of CD34-positive interstitial cells of Cajal. American

Journal of Physiology, 156, 1157–1163.

Rodbard, D., Lutz, R. A., Cruciani, R. A., Guardabasso, V., Pesce, G. O.,

& Munson, P. J. (1986). Computer analysis of radioligand data: ad-

vantages, problems, and pitfalls. NIDA Research Monographs, 70,

209–222.

Romero, F., Silva, B. A., Nouailhetas, V. L., & Aboulafia, J. (1998). Acti-

vation of Ca(2+)-activated K+ (maxi-K+) channel by angiotensin II in

myocytes of the guinea pig ileum. American Journal of Physiology,

274, C983–C991.

Rumessen, J. J., Mikkelsen, H. B., & Thuneberg, L. (1992). Ultrastructure

E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158 157

of interstitial cells of Cajal associated with deep muscular plexus of

human small intestine. Gastroenterology, 102, 56–68.

Rumessen, J. J., Thuneberg, L., & Mikkelsen, H. B. (1982). Plexus mus-

cularis profundus and associated interstitial cells. II. Ultrastructural

studies of mouse small intestine. Anatomical Record, 203, 129–146.

Sakai, Y. & Kwan, C. Y. (1993). Calcium regulation and contractile dys-

function of smooth muscle. Biological Signals, 2, 305–312.

Salapatek, A. M. F., Wang, Y. F., Mao, Y. K., Mori, M., & Daniel, E. E.

(1998). Myogenic NOS in canine lower esophageal sphincter: enzyme

activation, substrate recycling and product actions. American Journal of

Physiology, 274, C1145–C1157.

Sanders, K. M. (1996). A case for interstitial cells of Cajal as pacemakers

and mediators of neurotransmission in the gastrointestinal tract. Gastro-

enterology, 111, 492–515.

Sato, K., Ozaki, H., & Karaki, H. (1988). Changes in cytosolic calcium

level in vascular smooth muscle strip measured simultaneously with

contraction using fluorescent calcium indicator fura-2. Journal of Phar-

macology and Experimental Therapeutics, 246, 294–300.

Scaramellini, C., & Leff, P. (1998). A three-state receptor model: predic-

tions of multiple agonist pharmacology for the same receptor type.

Annals of the New York Academy of Sciences, 861, 97–103.

Schild, H. O. (1969). Drug receptors and drug classification. Triangle, 9,

132–137.

Schild, H. O. (1975). An ambiguity in receptor theory. British Journal of

Pharmacology, 53, 311.

Schild, H. O. (1997). pA, a new scale for the measurement of drug antag-

onism [originally published in 1947]. British Journal of Pharmacology,

120 (4 Suppl.), 29–46 discussion 27–28.

Shuttleworth, C. W., & Sanders, K. M. (1996). Involvement of nitric oxide

in neuromuscular transmission in canine proximal colon. Proceedings

of the Society for Experimental Biology and Medicine, 211, 16–23.

Smith, T. K., Bornstein, J. C., & Furness, J. B. (1991). Interactions between

reflexes evoked by distension and mucosal stimulation: electrophysio-

logical studies of guinea-pig ileum. Journal of the Autonomic Nervous

System, 34, 69–75.

Steffan, R., & Heinemann, S. H. (1997). Error estimates for results of

nonstationary noise analysis derived with linear least squares methods.

Journal of Neuroscience Methods, 78, 51–63.

Stevens, R. J., Publicover, N. G., & Smith, T. K. (2000). Propagation and

neural regulation of calcium waves in longitudinal and circular muscle

layers of guinea pig small intestine. Gastroenterology, 118, 892–904.

Stewart, J. J., & Burks, T. F. (1977). Actions of cholecystokinin octapeptide

on smooth muscle of isolated dog intestine. American Journal of

Physiology, 232, E306–E310.

Stewart, J. J., & Burks, T. F. (1980). Actions of pentagastrin on smooth

muscle of isolated dog intestine. American Journal of Physiology, 239,

G295–G299.

Thomas, M. V. (1982). Techniques in calcium research. London:

Academic Press.

Thuneberg, L. (1999). One hundred years of interstitial cells of Cajal.

Microscopy Research and Techniques, 47, 223–238.

Toma,H., Nakamura, K., Kuraoka, A., Tanaka,M.,&Kawabuchi,M. (1999).

Three-dimensional structures of c-Kit-positive cellular networks in the

guinea pig small intestine and colon.Cell TissueResearch,295, 425–436.

Tsien, R. Y. (1989). Fluorescent indicators of ion concentrations. Methods

in Cell Biology, 30, 127–156.

Tsumura, T., Hazama, A., Miyoshi, T., Ueda, S., & Okada, Y. (1998).

Activation of cAMP-dependent C1- currents in guinea-pig paneth cells

without relevant evidence for CFTR expression. Journal of Physiology

(London), 512, 765–777.

Van der Graaf, P. H., & Danhof, M. (1997). On the reliability of affinity

and efficacy estimates obtained by direct operational model fitting of

agonist concentration-effect curves following irreversible receptor in-

activation. Journal of Pharmacological and Toxicological Methods, 38,

81–85.

Van der Graaf, P. H., & Stam,W. B. (1999). Analysis of receptor inactivation

experiments with the operational model of agonism yields correlated

estimates of agonist affinity and efficacy. Journal of Pharmacological

and Toxicological Methods, 41, 117–125.

Vergara, P., Woskowska, Z., Cipris, S., Fox-Threlkeld, J. E. T., & Daniel, E.

E. (1995). Somatostatin excites canine ileum ex vivo: role for nitric

oxide? American Journal of Physiology, 269, G12–G21.

Vergara, P., Woskowska, Z., Cipris, S., Fox-Threlkeld, J. E. T., & Daniel,

E. E. (1996). Mechanisms of action of cholecystokinin in the canine

gastrointestinal tract: role of vasoactive intestinal peptide and nitric

oxide. Journal of Pharmacology and Experimental Therapeutics,

279, 306–316.

Wang, X. Y., Sanders, K. M., & Ward, S. M. (1999). Intimate relationship

between interstitial cells of cajal and enteric nerves in the guinea-pig

small intestine. Cell Tissue Research, 295, 247–256.

Waterfield, A. A., & Kosterlitz, H. W. (1975). Stereospecific increase by

narcotic antagonists of evoked acetylcholine output in guinea-pig ileum.

Life Sciences, 16, 1787–1792.

Wood, J. D. (1989). The handbook of physiology: the gastrointestinal sys-

tem I. Boca Raton, FL: CRC Press, Inc.

Yokoyama, S., & North, R. A. (1983). Electrical activity of longitudinal

and circular muscle during peristalsis. American Journal of Physiology,

244, G83–G88.

Young, W. S., & Kuhar, M. J. (1979). A new method for receptor

autoradiography: [3H]opioid receptors in rat brain. Brain Research,

179, 255–270.

Yuan, S. Y., Bornstein, J. C., & Furness, J. B. (1994). Investigation of the

role of 5–HT3 and 5–HT4 receptors in ascending and descending

reflexes to the circular muscle of guinea-pig small intestine. British

Journal of Pharmacology, 112, 1095–1100.

Zhang, Z. D., Kwan, C. Y., & Daniel, E. E. (1993a). Identification of Ca2+

release channels in smooth muscle and isolated membranes. Biological

Signals, 2, 284–292.

Zhang, Z. D., Kwan, C. Y., & Daniel, E. E. (1993b). Characterization of

[3H] ryanodine binding sites in smooth muscle of dog mesentery

artery. Biochemical Biophysical Research Communications, 194,

1242–1247.

ZhuGe, R., Tuft, R. A., Fogarty, K. E., Bellve, K., Fay, F. S., & Walsh,

J. V. Jr. (1999). The influence of sarcoplasmic reticulum Ca2 + con-

centration on Ca2+ sparks and spontaneous transient outward currents

in single smooth muscle cells. Journal of General Physiology, 113,

215–228.

ZhuGe, R., Fogarty, K. E., Tuft, R. A., Lifshitz, L. M., Sayar, K., & Walsh,

J. V. Jr. (2000). Dynamics of signaling between Ca(2+) sparks and

Ca(2+)- activated K(+) channels studied with a novel image-based

method for direct intracellular measurement of ryanodine receptor

Ca(2+) current. Journal of General Physiology, 116, 845–864.

E.E. Daniel et al. / Journal of Pharmacological and Toxicological Methods 45 (2001) 141–158158