Department of Pharmacology and Toxicology and the Neuroscience
Program, Michigan State University, East Lansing, Michigan
Experiments were designed to test the hypothesis that nicotinic
acetylcholine receptors (nAChRs) are present at sites of
neurotransmission to the guinea pig ileum circular smooth muscle.
Circular smooth muscle preparations, from which the myenteric plexus
had been removed (circular muscle-axon preparation), were used for this purpose. Nicotine and dimethylphenyl piperazinium iodide (10-100 µM)
induced contraction of the circular smooth muscle. Agonist-induced contraction was inhibited by 1 µM scopolamine and abolished in the
combined presence of 1 µM scopolamine and 0.3 µM CP 96,345-01, a
neurokinin-1 receptor antagonist. Contractions induced by
electric field stimulation (30 pulses, 0.5 ms, 70 V, 10 Hz) were
abolished by 0.3 µM tetrodotoxin (TTX); in contrast,
agonist-induced contraction was attenuated but not abolished by 0.3 µM TTX. Mecamylamine (3 or 30 µM), an nAChR antagonist, blocked
agonist-induced contractions. Frequency-response curves for both
"ON" and "OFF" electric field stimulation contractions
were abolished by the combined presence of 1 µM scopolamine and 0.3 µM CP 96,345-01 or by 0.3 µM TTX. At stimulation frequencies
greater than 2 Hz, the ON contraction was increased in the presence of
100 µM nitro-L-arginine. Mecamylamine (3 µM) was used
to block the stimulatory prejunctional nAChRs located near sites of
neurotransmitter release to the circular smooth muscle; however, ON and
OFF contractions were not affected by mecamylamine. Although the
prejunctional nAChRs are not targets for endogenously released
acetylcholine under the conditions tested here, these receptors may be
targets for the development of new prokinetic agents.
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Introduction |
In
the enteric nervous system (ENS), acetylcholine (ACh) acting at
nicotinic ACh receptors (nAChRs) is the principal excitatory neurotransmitter in the myenteric plexus. Intracellular
electrophysiological studies have shown that fast excitatory
postsynaptic potentials recorded from myenteric neurons are at least
partly inhibited by nAChR antagonists (Galligan and Bertrand, 1994
;
LePard and Galligan, 1999). Excitatory and inhibitory intestinal
reflexes caused by distension of the gut wall or mucosal stimulation
are also inhibited by nAChR antagonists (Costa and Furness, 1976; Holzer, 1989; Johnson et al., 1996). In vivo studies have shown that
gastrointestinal motility is also inhibited by nAChR antagonists (Galligan et al., 1986). These observations highlight the central role
of nAChRs in the control of gastrointestinal motor function.
Presynaptic regulation is an important mechanism governing
neurotransmission, causing either inhibition or facilitation of transmitter release. A variety of receptor-mediated presynaptic mechanisms inhibit synaptic transmission in the ENS, including 
2-adrenergic receptors (Wood and Mayer, 1979;
Morita et al., 1982), M2 muscarinic ACh receptors (mAChRs) (Kilbinger
and Wessler, 1980; Morita et al., 1982; North et al., 1985),
5-HT1A receptors (North et al., 1980; Pan and
Galligan, 1994), histaminergic receptors (Tamura et al., 1988), opioid
receptors (Cherubini et al., 1985; Johnson et al., 1988), and
-aminobutyric acid receptors (Cherubini and North, 1984). In
contrast, only presynaptic 5-HT4 receptors have
been shown to facilitate enteric neurotransmitter release (Kilbinger et
al., 1995) and fast synaptic transmission (Pan and Galligan, 1994). In
the central nervous system and autonomic ganglia, however, activation
of presynaptic nAChRs facilitates or induces neurotransmitter release
(Giorguieff-Chesselet et al., 1979; Grady et al., 1992; Sacaan et al.,
1996; Lena and Changeux, 1997). Until recently, nAChRs in the ENS were
only known to participate in fast synaptic transmission.
Recent immunohistochemical studies of myenteric neurons have suggested
that nAChRs may also be located at presynaptic sites (Kirchgessner and
Liu, 1998; Obaid et al., 1999). Indeed, functional evidence for
presynaptic nAChRs within the ENS has been reported for myenteric
excitatory motoneurons innervating longitudinal smooth muscle
(Galligan, 1999). This recent evidence is based on the persistence of
agonist-induced contraction of the longitudinal smooth muscle layer in
the presence of tetrodotoxin (TTX), an approach similar to that used to
demonstrate presynaptic nAChRs in a variety of other neural
preparations (Marshall et al., 1996; Coggan et al., 1997).
The experiments conducted here were designed to determine whether
nAChRs are also located at sites of neurotransmitter release on
myenteric excitatory motoneurons that innervate the intestinal circular
smooth muscle. Such information may be useful for better understanding
of the control of functional movements of the intestinal tract, such as
peristalsis. Furthermore, the preparation used in this study is useful,
as the axonal projections of the motoneurons innervating the circular
smooth muscle were physically separated from their cell bodies by
removal of the myenteric plexus (Johnson et al., 1988; Brookes and
Costa, 1990). The results indicate that nAChRs are also found on
myenteric excitatory motor nerve fibers innervating the circular smooth
muscle. Pharmacologic stimulation of these receptors results in
contraction of circular smooth muscle.
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Materials and Methods |
Tissue Preparation.
Male albino guinea pigs (350-450 g)
obtained from the Michigan Department of Public Health (Lansing, MI)
were used. The care and use of these animals were approved by the
All-University Committee on Animal Use and Care at Michigan State
University (AUCAUC) (East Lansing, MI). Animals were sacrificed by
exsanguination after general anesthesia induced by inhalation of
halothane (Halocarbon Laboratories, River Edge, NJ). A segment of ileum
was removed and placed in a dissection bath lined with Sylgard 184 elastomer (Dow Corning Corp., Midland, MI) and filled with
preoxygenated (95% O2, 5%
CO2) Krebs' solution of the following
composition: 117 mM NaCl, 1.2 mM
NaH2PO4, 2.5 mM
CaCl2, 4.7 mM KCl, 25 mM
NaHCO3, and 11 mM glucose. The lumen of the bowel
segment was opened along the longitudinal axis and pinned mucosa-side
up, and the bowel contents were rinsed away.
The fine dissection technique has been reported previously (Johnson et
al., 1988) and is briefly described here. The mucosa was removed with
fine tissue forceps. A 1-cm-wide preparation, including at least 75%
of the bowel segment circumference, was cut from the mucosa-free
segment and pinned submucosa-side down. Next, the longitudinal smooth
muscle layer with adherent myenteric ganglia was gently stripped away,
leaving behind the circular smooth muscle and submucosal layers.
Attempts at removing the submucosa resulted in preparations that pulled
apart during isometric contraction (Johnson et al., 1988). Therefore,
the preparation consisted of the circular smooth muscle and submucosal
layers; this is referred to as the circular muscle-axon (CM-axon) preparation.
Each preparation was anchored with 4-0 silk thread to individual
Plexiglas tissue holders and vertically mounted in a 20-ml water-jacketed tissue bath containing Krebs' solution maintained at
37°C and continuously bubbled with a 95% O2,
5% CO2 gas. Preparations were stretched to
maintain a baseline tension of 10 mN. The tissue bath solution was
changed every 15 min for the first 90 to 120 min before
experimentation. For each preparation, the free end was attached via
4-0 silk thread to a force displacement transducer (FT03; Grass
Instruments, Quincy, MA). Isometric tension of the circular smooth
muscle layer was continuously monitored by outputting the preamplified
(7P1F; Grass Instruments) transducer signal to an ink-writing
oscillograph (Grass Instruments). Electrical field stimulation (EFS)
was achieved using platinum foil electrodes mounted within each
Plexiglas tissue holder and connected to a stimulator (S88; Grass
Instruments). Trains of square wave pulses (70-V, 0.5-ms DC pulse
delivered at 10 Hz for 3 s) or responses to 30 µM bethanechol
were used to determine the viability of preparations; in some
experiments, 0.3 µM TTX was added to confirm the neural origin of
contractions induced by EFS. In response to 30 µM bethanechol, a peak
contraction of 10 mN or more was used as a cutoff criterion for use in
the study.
Agonist Concentration-Response Curves.
Noncumulative
concentration-response curves for nicotine and dimethyl-phenyl
piperazinium iodide (DMPP) were constructed in the absence or presence
of an antagonist by randomly assigning one of four daily preparations
to an agonist/antagonist combination. Antagonist was added at least 10 min before the addition of any agonist. In the first set of
experiments, a specific nAChR antagonist (mecamylamine, 30 µM) was
used to test the specificity of agonist-induced contractions. In a
second set of experiments, axonal conduction of action potentials was
blocked with 0.3 µM TTX. Based on preliminary experiments, nAChR
agonists were tested between 3 and 3000 µM, and the order of
agonist-concentration application was randomized. At the start of both
experiments, all preparations were maximally contracted with 30 µM
bethanechol, in the absence or presence of subsequently used antagonists.
Electric-Field Stimulus-Response Curves.
EFS was used to
induce contraction of CM-axon preparations. A frequency-response curve
was constructed for each preparation, using trains of 30 pulses (0.5-ms
pulse duration, 70 V) delivered at frequencies of 0.1, 0.25, 0.5, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, and 3 Hz. From each guinea pig,
four CM-axon preparations were tested, and each was assigned to one of
the following treatment groups: control, 3 µM mecamylamine, 1 µM
scopolamine, 3 µM mecamylamine and 1 µM scopolamine, and 100 µM
nitro-L-arginine (NLA). Response to 30 µM bethanechol and
to supramaximal EFS (30 pulses at 10 Hz) was recorded before the
addition of antagonists. Preparations in which the tone induced by
bethanechol did not achieve at least 10 mN were not used.
Data Handling and Statistical Analysis.
The maximum
contraction force (in millinewtons) was recorded and normalized to that
induced by bethanechol (30 µM) or supramaximal EFS (30 pulses, 0.5 ms, 70 V, at 10 Hz). The maximum concentrations of the nAChR agonists
that induce CM-axon contractions dependent on nAChR activation were
identified by testing the null hypothesis that the least squared mean
contraction was equal to 0. The interaction of frequency and treatment
group for frequency-response data was analyzed by two-factor ANOVA; a
random-effects model was chosen to avoid losing all data from
frequency-response curves that contained a missing data point. Post
hoc, the simple effect of treatment group was examined at each frequency.
Drugs.
Bethanechol, scopolamine, nicotine, DMPP, NLA, and
TTX were purchased from Sigma Chemical Co. (St. Louis, MO).
Mecamylamine was purchased from Research Biochemicals (Natick, MA). CP
96,345-01 was a gift from Pfizer (Groton, CT).
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Results |
Agonist-Induced Contraction of CM-Axon Preparations.
Bethanechol (30 µM) induced a 19 ± 2 mN contraction in 16 CM-axon preparations. Nicotine (0.01-3 mM) caused contractions of the
CM-axon preparations; however, contractions induced by 1 and 3 mM
nicotine were not blocked by 30 µM mecamylamine (Fig.
1, left graph). All contractions caused
by DMPP were blocked by mecamylamine (30 µM) (Fig. 1, right). Further
analysis was restricted to nAChR agonist concentrations between 1 and
100 µM, a range producing contractions dependent on activation of
nAChRs. The contractions induced by 100 µM nicotine and 100 µM DMPP
were inhibited, respectively, by 72 ± 12 and 60 ± 15% in
the presence of 1 µM scopolamine and were abolished in the combined
presence of 1 µM scopolamine and 0.3 µM CP 96,345-01 (Table
1; also Fig.
2 for DMPP). At concentrations of 30 and
100 µM, nicotine and DMPP induced contraction even in the presence of
0.3 µM TTX (Fig. 3). The percentage
inhibition of agonist-induced contractions by mecamylamine,
scopolamine, CP 96,345-01, and TTX is summarized in Table 1.
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Fig. 1.
Nicotine- and DMPP-induced contractions of the
CM-axon preparation are mediated by nAChRs. The concentration-response
curve induced by nicotine appeared to be biphasic ( ,
n = 4), but only the first phase of the curve
(1-100 µM) was blocked by 30 µM mecamylamine ( ). The
concentration-response curve induced by DMPP appeared to be monophasic
( , n = 4), and all contractions induced by DMPP,
except at 1 mM, were abolished in the presence of 30 µM mecamylamine
( ). (Data represent the mean ± S.E. For experiments conducted
in the presence of 30 µM mecamylamine, * identifies agonist
concentrations resulting in a least-square mean contraction greater
than 0. The vertical axis represents contraction relative to that
induced by 30 µM bethanechol.)
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Fig. 2.
Contractions of the CM-axon preparation induced by
DMPP are mediated by mAChRs and NK-1 receptors. A, contraction induced
by 100 µM DMPP (left) was reduced in the presence of 1 µM
scopolamine (center) and abolished in the combined presence of 1 µM
scopolamine and 0.3 µM CP 96,345-01 (right). B, contraction induced
by 100 µM DMPP was stable (time control) over the same time period as
in A ( , addition of 100 µM DMPP).
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Fig. 3.
Contractions of the CM-axon preparation induced by
nicotine and DMPP were reduced, but not abolished, by TTX. Traces of
agonist-induced contraction are shown in A and B. , addition of
agonist at 10, 30, or 100 µM nicotine (A) or DMPP (B) in the absence
(control) or presence of 0.3 µM TTX. C, summary of the experiments
conducted in the absence (solid symbols) or presence of 0.3 µM TTX
(open symbols). Each concentration-response curve represents the
mean ± S.E. contraction of four CM-axon preparations relative to
maximum contraction induced by 30 µM bethanechol. (* identifies
agonist concentrations at which the contraction induced in the presence
of TTX is significantly less than the control contraction.)
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Frequency-Response Curves.
The amplitude of baseline
contractions induced by 30 µM bethanechol or supramaximal EFS (30 pulses, 0.5 ms, 70 V, at 10 Hz) was not different between control and
treatment groups, and the resting tension did not change over time or
after the addition of antagonist. In the control group
(n = 8), the maximum contraction induced by 30 µM
bethanechol was 38 ± 5 mN and that induced by supramaximal EFS
was 32 ± 8 mN; in the presence of 0.3 µM TTX, the contraction
induced by bethanechol was unchanged, but the contraction induced by
EFS was reversibly abolished. In the following experiments, 3 µM
mecamylamine was used to provide maximum inhibition of nAChRs [see
Table 1 and Galligan and Bertrand (1994)] while avoiding the potential
for nonspecific antagonism by higher concentrations of mecamylamine
(Martin et al., 1989). Contraction induced by 30 µM bethanechol was
unaffected by 3 µM mecamylamine (18 ± 7 versus 20 ± 7 mN;
P > .05, n = 6).
EFS caused "ON" and "OFF" contractions (Fig.
4). ON contractions were observed after
each pulse but were variable in amplitude and intermittent during each
train of 30 pulses. ON contractions fuse and sum with increasing
stimulus frequency, but the peak ON contraction decreased and was
typically absent at frequencies greater than 2.0 Hz (Figs. 4 and
5). At frequencies greater than 1.25 Hz,
ON contractions in the presence of 100 µM NLA were greater than those
of controls (Fig. 5). The peak amplitude of ON contractions in the
presence of 1 µM scopolamine was half that of controls, but this
difference was not significant because of the inherent variation in
this response to EFS. ON contractions, in the absence or presence of 1 µM scopolamine, were unaffected by 3 µM mecamylamine (Fig. 5). At
stimulus frequencies greater than 2 Hz, ON contractions were
occasionally replaced by a small tonic relaxation that was blocked by
100 µM NLA (n = 6). In a separate experiment, ON
contractions induced at 0.25 Hz were reduced by 25 ± 26% in the
presence of 1 µM scopolamine and abolished in the combined presence
of 1 µM scopolamine and 0.3 µM CP 96,345-01 (n = 4).
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Fig. 4.
Two types of contraction of the CM-axon preparation
were observed in response to EFS. ON contractions occurred after
individual pulses but were variable in amplitude and intermittent in
appearance (best seen at 0.1 Hz). ON contractions fuse and sum, but the
peak amplitude declined to 0 at higher stimulus frequencies (see 3.0 Hz, for example). A single OFF contraction was observed at each
stimulus frequency greater than 0.5 Hz. Horizontal bars indicate the
period of EFS (30 pulses, 0.5 ms, 70 V) at the stimulus frequencies
indicated; chart speeds between 10 and 50 mm/min were used.
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Fig. 5.
Frequency-response curve for ON contractions of the
CM-axon preparation in response to EFS. The mean ± S.E. ON
contractions induced by EFS ( = 6-8 control preparations) are
repeated in each graph for comparison with contractions in the presence
of antagonist (open symbols). A, at stimulus frequencies greater than
1.25 Hz, ON contractions were greater in the presence of 100 µM NLA
( = 5 preparations). B, ON contractions in the presence of 3 µM
mecamylamine ( = 6-10 preparations) were not different from
control. C, at frequencies less than 2.25 Hz, however, ON contractions
in the presence of 1 µM scopolamine ( = 6-9 preparations) were
approximately half that of control contractions, but these differences
were not significant. ON contractions in the combined presence of 3 µM mecamylamine and 1 µM scopolamine ( = 6-9 preparations) also
were not different from control, nor were they different from
contractions in the presence of 1 µM scopolamine (). (* indicates a frequency at which the contraction in the presence of an
antagonist is different from control.)
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A large OFF contraction was first observed at the end of a 0.75-Hz
train (Figs. 4 and 6) and increased in
amplitude with increases in stimulus frequency. OFF contractions were
unaffected by 100 µM NLA or 3 µM mecamylamine but were reduced in
the presence of 1 µM scopolamine (Fig. 6). In the presence of
scopolamine, 3 µM mecamylamine had no additional effect (Fig. 6). In
a separate experiment, the OFF contraction induced at 3 Hz was reduced
by 75 ± 5% by 1 µM scopolamine and abolished in the combined
presence of 1 µM scopolamine and 0.3 µM CP 96,345-01 (n = 4).
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Fig. 6.
Frequency-response curve for OFF contractions
of the CM-axon preparation in response to EFS. The mean ± S.E.
for OFF contractions induced by EFS ( = 6-8 control preparations)
is repeated in each graph for comparison with contractions in the
presence of antagonist (open symbols). A, OFF contractions in the
presence of 100 µM NLA ( = 5 preparations) and (B) in the presence
of 3 µM mecamylamine ( = 6-10 preparations) were not different
from control. C, at frequencies greater than 1.0 Hz, OFF contractions
in the presence of 1 µM scopolamine ( = 6-9 preparations) and in
the combined presence of 3 µM mecamylamine and 1 µM scopolamine
( = 6-9 preparations) were approximately half that of control
contractions. OFF contractions in the combined presence of 3 µM
mecamylamine and 1 µM scopolamine were not different from
contractions in the presence of 1 µM scopolamine (). (* indicates a frequency at which the contraction in the presence of an
antagonist is different from control.)
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Discussion |
The purpose of this investigation was to test the hypothesis that
nAChRs are present at sites of neurotransmitter release along myenteric
excitatory motor axons innervating circular smooth muscle. We found
that nicotine and DMPP (up to 100 µM) induced circular smooth muscle
contraction via a nAChR-dependent mechanism in preparations devoid of
myenteric ganglia. The nAChR-mediated contraction involved activation
of muscarinic and neurokinin-1 (NK-1) receptors, consistent with the
neuromuscular physiology of this smooth muscle layer (Brookes et al.,
1991). Furthermore, activation of voltage-gated sodium channels in the
axons contribute to, but were not required for, nAChR agonist-induced contraction.
NAChRs are located on somatodendritic regions of myenteric neurons
(Kirchgessner and Liu, 1998; Obaid et al., 1999) and participate in
fast synaptic transmission (Galligan and Bertrand, 1994; LePard and
Galligan, 1999). Some of these myenteric neurons are motoneurons that
project to the smooth muscle layers (Brookes et al., 1991, 1992).
However, if nAChRs are only somatodendritic, then isolation of the
motor axon and its varicosities from myenteric ganglia should abolish
nAChR agonist-induced contraction. In a recent study (Galligan, 1999),
axons projecting to the longitudinal smooth muscle were chemically
isolated from myenteric ganglia by use of the voltage-gated sodium
channel blocker TTX. In that study, persistence of nAChR
agonist-induced contraction of the longitudinal smooth muscle in the
presence of TTX was consistent with the hypothesis that nAChRs are
localized to the axons and/or terminals of excitatory motoneurons. In
this study, the use of CM-axon preparations provided a similar
conceptual yet alternative approach to the question of nAChR
localization on myenteric motoneurons. In CM-axon preparations, motor
axons projecting to the circular smooth muscle are physically separated
from myenteric ganglia (Johnson et al., 1988). Because nicotine and
DMPP induce contraction of the circular smooth muscle layer in a manner
codependent on the activation of mAChRs and NK-1 receptors, it is
logical to conclude that the location of nAChRs on myenteric
motoneurons includes axonal or terminal sites of neurotransmitter
release to circular smooth muscle. Alternative sites of acetylcholine
and tachykinin release are unlikely, as choline acetyltransferase-,
Substance P-, and nAChR-specific staining within these preparations is,
to date, reported only for neural structures (Brookes et al., 1991,
1992; Kirchgessner and Liu, 1998).
Furthermore, in this study, addition of nicotine or DMPP (up to 100 µM) resulted in a contraction mediated by nAChRs that was reduced,
but not abolished, in the presence of TTX; TTX, however, did abolish
EFS contractions. Therefore, two processes mediate neurotransmitter
release at these sites: one dependent on, and the other independent of,
the generation of action potentials. It is proposed that activation of
nAChRs on motoneuron varicosities causes sufficient local
depolarization to open voltage-gated sodium channels. Action potentials
propagating along the axons enhance, but are not essential for,
agonist-induced neurotransmitter release (i.e., TTX-sensitive
contraction; see Fig. 7). This conclusion is similar to the mechanism of nAChR agonist-induced release of neurotransmitter from some studies using synaptosome preparations of
the central nervous system (Marshall et al., 1996) and for nAChR-induced release of norepinephrine from sympathetic neurons (Kristufek et al., 1999).
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Fig. 7.
Representation of the mechanism for nAChR-mediated
contraction of CM-axon preparations. Agonist-induced contraction of the
underlying circular smooth muscle is mediated by nAChRs (shaded ion
channel) located near myenteric excitatory motoneuron transmitter
release sites. Ca2+ and Na+ pass through nAChRs
and cause local depolarization and transmitter release. Additional
Ca2+ enters via voltage-gated Ca2+ channels
(unshaded ion channel), most likely activated by an action potential
triggered by the nAChR-induced depolarization. This is the classic
mechanism of action-potential-induced neurotransmitter release, but in
this case, it is locally activated via nAChRs, and it is not essential
for agonist-induced contraction. Both mechanisms may operate within
individual release sites (as represented by the dotted oval varicosity)
and/or may involve action-potential-dependent facilitation brought
about by enhanced release from adjacent varicosities (as represented by
two solid oval varicosities).
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There are two possible mechanisms by which activation of nAChRs might
induce TTX-resistant contraction. First, depolarization induced by
nAChRs might be sufficient to activate voltage-gated calcium channels
(Soliakov and Wonnacott, 1996; Tredway et al., 1999). However,
presynaptic nAChR activation in sympathetic ganglia results in
TTX-resistant neurotransmitter release that is not affected by blockade
of voltage-gated calcium channels (Kristufek et al., 1999). Because
nAChRs are ligand-gated ion channels that can pass significant amounts
of calcium ions (Vernino et al., 1992), it is possible that calcium
entering activated nAChRs might also induce neurotransmitter release
(Giorguieff-Chesselet et al., 1979; White, 1982; Sacaan et al., 1996;
Lena and Changeux, 1997). Therefore, in the CM-axon preparation,
activation of nAChRs localized to motoneuron varicosities could induce
neurotransmitter release directly by calcium entering through nAChRs
(TTX-resistant) and indirectly through voltage-gated calcium channels
brought to threshold by propagated action potentials (Fig. 7).
The presence of nAChRs at sites of neuromuscular transmission involving
acetylcholine release implies a possible autoregulatory function. For
example, acetylcholine is a mediator of slow excitatory synaptic
transmission via postsynaptic M1 receptors. But released acetylcholine
also activates presynaptic M2 receptors to inhibit further
acetylcholine release, thus forming a negative feedback mechanism
(North et al., 1985). Because nAChRs cause depolarization, we
anticipated that nAChRs might function as a positive feedback mechanism
when located at sites of acetylcholine release. We tested this
hypothesis by using nerve-mediated contractions induced by EFS in
CM-axon preparations in the absence and presence of the nAChR
antagonist mecamylamine. Use of the CM-axon preparation simplified data
interpretation because these preparations are without the influence of
myenteric synapses also activated by EFS.
Two types of contraction were noted in CM-axon preparations in response
to EFS: ON and OFF contractions. At stimulus frequencies greater than
1.25 Hz, the peak amplitude of the ON contractions was attenuated by
nitric oxide as the amplitude of the contraction was increased in the
presence of NLA. Although the mechanisms by which these two types of
contraction were induced by EFS were not further explored, both
contractions were abolished by TTX and were mediated by mAChRs and NK-1
receptors. But the peak amplitude of the contractions induced by EFS
was not affected by mecamylamine. This may imply that under the
experimental conditions used here, the concentration of acetylcholine
at the neuroeffector junction, which is sufficient to cause contraction
via postjunctional mAChRs, is not sufficient to activate prejunctional
nAChRs. It is possible that acetylcholinesterase activity and distance
of prejunctional nAChRs from release sites may limit the concentration
of endogenous acetylcholine reaching the prejunctional nAChRs. The TTX
sensitivity of a variety of responses induced by nAChR agonists has
resulted in mixed conclusions about the existence of presynaptic nAChRs in the ENS. Acetylcholine release induced by nAChR agonists, for example, is abolished in the presence of TTX (Töröcsik et
al., 1991; Gordon et al., 1992). However, other nAChR-induced responses are clearly TTX-resistant (Romano, 1981; Briggs and Cooper, 1982; White, 1982; Gordon et al., 1992; Borjesson et al., 1997).
In summary, our results demonstrate that nAChRs are present on
myenteric excitatory motor nerve fibers near sites of neurotransmitter release to the circular smooth muscle in the guinea pig ileum. Activation of these receptors causes circular smooth muscle contraction by inducing the release of ACh and tachykinin peptides. Our findings corroborate a recent report of nAChRs located at sites of neuromuscular transmission to longitudinal smooth muscle (Galligan, 1999) and indicate that excitatory motoneurons, irrespective of projection, might
express nAChRs near sites of neurotransmitter release. As with other
studies of the peripheral nervous system, the physiological role of
these prejunctional receptors is not yet known, but they may provide
additional targets for drugs used to stimulate intestinal motility.
ENS, enteric nervous system;
ACh, acetylcholine;
nAChR, nicotinic ACh receptors;
CM-axon, circular
muscle-axon;
TTX, tetrodotoxin;
DMPP, dimethyl-phenyl piperazinium
iodide;
NK-1, neurokinin-1;
mAChR, muscarinic ACh receptor;
EFS, electric field stimulation;
NLA, nitro-L-arginine.
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Abstract
Introduction
