Nicotinic Acetylcholine Receptors at Sites of Neurotransmitter Release to the Guinea Pig Intestinal Circular Muscle 1

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1 /00/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 294, No. 1 Copyright 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 294: , 2000 /2237/ Nicotinic Acetylcholine Receptors at Sites of Neurotransmitter Release to the Guinea Pig Intestinal Circular Muscle 1 DAVID A. SCHNEIDER, MIKE PERRONE, and JAMES J. GALLIGAN Department of Pharmacology and Toxicology and the Neuroscience Program, Michigan State University, East Lansing, Michigan Accepted for publication April 5, 2000 This paper is available online at ABSTRACT 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 ( M) induced contraction of the circular smooth muscle. Agonist-induced contraction was inhibited by 1 M scopolamine and abolished in the combined presence of 1 M scopolamine and 0.3 M 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 M tetrodotoxin (TTX); in contrast, agonist-induced contraction was attenuated but not abolished by 0.3 M 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 Received for publication October 27, This work was supported by Grants 1 F32 DK and NS33289 and by an American Society of Pharmacology and Experimental Therapeutics Summer Institutional Fellowship awarded to the Department of Pharmacology and Toxicology, Michigan State University (to M.P.). TTX. Mecamylamine (3 or 30 M), 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 M scopolamine and 0.3 M CP 96, or by 0.3 M TTX. At stimulation frequencies greater than 2 Hz, the ON contraction was increased in the presence of 100 M nitro-l-arginine. Mecamylamine (3 M) 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. 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-HT 1A 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-HT 4 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 motoneu- ABBREVIATIONS: 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. 363

2 364 Schneider et al. Vol. 294 rons 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. Materials and Methods Tissue Preparation. Male albino guinea pigs ( 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% O 2,5%CO 2 ) Krebs solution of the following composition: 117 mm NaCl, 1.2 mm NaH 2 PO 4, 2.5 mm CaCl 2, 4.7 mm KCl, 25 mm NaHCO 3, 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 waterjacketed tissue bath containing Krebs solution maintained at 37 C and continuously bubbled with a 95% O 2,5%CO 2 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 M bethanechol were used to determine the viability of preparations; in some experiments, 0.3 M TTX was added to confirm the neural origin of contractions induced by EFS. In response to 30 M 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 M) 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 M TTX. Based on preliminary experiments, nachr agonists were tested between 3 and 3000 M, and the order of agonist-concentration application was randomized. At the start of both experiments, all preparations were maximally contracted with 30 M 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 M mecamylamine, 1 M scopolamine, 3 M mecamylamine and 1 M scopolamine, and 100 M nitro-l-arginine (NLA). Response to 30 M 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 M) 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, was a gift from Pfizer (Groton, CT). Results Agonist-Induced Contraction of CM-Axon Preparations. Bethanechol (30 M) induced a 19 2 mn contraction in 16 CM-axon preparations. Nicotine ( mm) caused contractions of the CM-axon preparations; however, contractions induced by 1 and 3 mm nicotine were not blocked by 30 M mecamylamine (Fig. 1, left graph). All contractions caused by DMPP were blocked by mecamylamine (30 M) (Fig. 1, right). Further analysis was restricted to nachr agonist concentrations between 1 and 100 M, a range producing contractions dependent on activation of nachrs. The contractions induced by 100 M nicotine and 100 M DMPP were inhibited, respectively, by and 60 15% in the presence of 1 M scopolamine and were abolished in the combined presence of 1 M scopolamine and 0.3 M CP

3 2000 nachrs on Circular Muscle Excitatory Motoneuron Axons 365 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 (f, n 4), but only the first phase of the curve (1 100 M) was blocked by 30 M mecamylamine ( ). The concentration-response curve induced by DMPP appeared to be monophasic (F, n 4), and all contractions induced by DMPP, except at 1 mm, were abolished in the presence of 30 M mecamylamine (E). (Data represent the mean S.E. For experiments conducted in the presence of 30 M 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 M bethanechol.) Fig. 2. Contractions of the CM-axon preparation induced by DMPP are mediated by machrs and NK-1 receptors. A, contraction induced by 100 M DMPP (left) was reduced in the presence of 1 M scopolamine (center) and abolished in the combined presence of 1 M scopolamine and 0.3 M CP 96, (right). B, contraction induced by 100 M DMPP was stable (time control) over the same time period as in A (Œ, addition of 100 M DMPP). 96, (Table 1; also Fig. 2 for DMPP). At concentrations of 30 and 100 M, nicotine and DMPP induced contraction even in the presence of 0.3 M TTX (Fig. 3). The percentage inhibition of agonist-induced contractions by mecamylamine, scopolamine, CP 96,345-01, and TTX is summarized in Table 1. Frequency-Response Curves. The amplitude of baseline contractions induced by 30 M 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 M bethanechol was 38 5mN and that induced by supramaximal EFS was 32 8 mn; in the presence of 0.3 M TTX, the contraction induced by bethanechol was unchanged, but the contraction induced by EFS was reversibly abolished. In the following experiments, 3 M 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 M bethanechol was unaffected by 3 M 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 M NLA were greater than those of controls (Fig. 5). The peak amplitude of ON contractions in the presence of 1 M 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 M scopolamine, were unaffected by 3 M 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 M NLA (n 6). In a separate experiment, ON contractions induced at 0.25 Hz were reduced by 25 26% in the presence of 1 M scopolamine and abolished in the combined presence of 1 M scopolamine and 0.3 M CP 96, (n 4). 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 M NLA or 3 M mecamylamine but were reduced in the presence of 1 M scopolamine (Fig. 6). In the presence of scopolamine, 3 M 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 M scopolamine and abolished in the combined presence of 1 M scopolamine and 0.3 M CP 96, (n 4). 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 M) 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 myen-

4 366 Schneider et al. Vol. 294 TABLE 1 Percentage inhibition of nachr agonist-induced CM-axon contraction (mean S.E.) Nicotine DMPP 30 M 100 M 30 M 100 M 3 M Mecamylamine 97 2 N.D N.D. (n 6) 30 M Mecamylamine (n 4) 1 M Scopolamine N.D N.D (n 4) 1 M Scopolamine and N.D N.D M CP 96; (n 4) 0.3 M TTX (n 6) N.D., not done. 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. Œ, addition of agonist at 10, 30, or 100 M nicotine (A) or DMPP (B) in the absence (control) or presence of 0.3 M TTX. C, summary of the experiments conducted in the absence (solid symbols) or presence of 0.3 M 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 M bethanechol. ( identifies agonist concentrations at which the contraction induced in the presence of TTX is significantly less than the control contraction.) 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. teric ganglia by use of the voltage-gated sodium channel blocker TTX. In that study, persistence of nachr agonistinduced 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 M) 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). There are two possible mechanisms by which activation of nachrs might induce TTX-resistant contraction. First, de-

5 2000 nachrs on Circular Muscle Excitatory Motoneuron Axons 367 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 (f 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 M NLA ( 5 preparations). B, ON contractions in the presence of 3 M 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 M 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 M mecamylamine and 1 M scopolamine ( 6 9 preparations) also were not different from control, nor were they different from contractions in the presence of 1 M scopolamine ( ). ( indicates a frequency at which the contraction in the presence of an antagonist is different from control.) 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 (F 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 M NLA (E 5 preparations) and (B) in the presence of 3 M mecamylamine (E 6 10 preparations) were not different from control. C, at frequencies greater than 1.0 Hz, OFF contractions in the presence of 1 M scopolamine (E 6 9 preparations) and in the combined presence of 3 M mecamylamine and 1 M scopolamine ( 6 9 preparations) were approximately half that of control contractions. OFF contractions in the combined presence of 3 M mecamylamine and 1 M scopolamine were not different from contractions in the presence of 1 M scopolamine (E). ( indicates a frequency at which the contraction in the presence of an antagonist is different from control.) polarization 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 TTXresistant 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

6 368 Schneider et al. Vol. 294 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. Ca 2 and Na pass through nachrs and cause local depolarization and transmitter release. Additional Ca 2 enters via voltage-gated Ca 2 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). 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. 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