Modulation of submucosal cholinergic neurons by 5-hydroxytryptamine and neuropeptides WILLIAM M. YAU, JEANNE A. DORSETT, AND MICHAEL L. YOUTHER Department of Physiology, School of Medicine, Southern Illinois University, Carbondale, Illinois 62901-6512

YAU, WILLIAM M., JEANNE A. DORSETT, AND YOUTHER. Modulation of submucosal cholinergic

MICHAEL L. neurons by 5hydroxytryptamine and neuropeptides. Am. J. Physiol. 259 (Gastrointest. Liver Physiol. 22): G1019-G1024, 1990.-Release of [3H]acetylcholine ( [3H]ACh) was examined in a submucous plexus preparation obtained from the guinea pig small intestine in vitro. Constant-current field stimulation evoked ACh output; this output was dependent on the stimulus frequency applied. Maximal release was observed at IO Hz; this release was blocked by tetrodotoxin (1 x 10M6 M) or in Ca”free buffer. Serotonin [&hydroxytryptamine (&HT)] stimulated the release of ACh dose dependently, with an EDs0 of 5 X 10V7 M. Substance P was ineffective, while vasoactive intestinal peptide weakly stimulated ACh secretion. Several neuropeptides were tested on their ability to modulate 5-HT-evoked ACh release. Dynorphin A inhibited 5-HT-stimulated ACh release, while Met-enkephalin was without any effect. Both somatostatin and galanin were effective modulators, with an inhibitory effect in the submicromolar range and an excitatory effect at higher concentrations. The response characteristics of the cholinergic neurons of submucosal plexus differ markedly from those of the myenteric plexus. These distinct features form an important framework for future functional studies on submucous plexus neurons.

striking features of the submucous plexus is that 54% of its neuronal population (8, 9) contains ACh. ACh is an important neurotransmitter to the epithelial cells (4) and also the submucosal arterioles (20). The preponderance of cholinergic neurons in the submucous plexus has raised an important question about how the activity of these neurons are regulated. Relatively little is known, however, of the excitatory or inhibitory modulators of neurotransmitter release from submucous cholinergic neurons. An examination of the secretion of ACh from the submucous plexus would be of considerable significance to our understanding of the regulatory function of the enteric nervous system. The aim of this study was to characterize the release of ACh from a submucous plexus-muscularis mucosa preparation of the guinea pig small intestine in vitro. In an attempt to examine how the peptide-containing neurons are functionally coupled to the cholinergic neurons, the modulatory effects of neuropeptides on ACh release were studied. In view of the excitability that could be elicited by 5-HT on the submucous neurons (13,23), the effect of 5-HT on the release of ACh was also studied.

acetylcholine; rosecretion

MATERIALS

small intestine;

neuroregulators;

neurons;

neu-

that originate from the submucous plexus are known to predominately innervate the mucosa (4, 5, 15, 16). Their axonal projections do not penetrate into the longitudinal or circular smooth muscle. There is ample evidence to indicate that synaptic inputs for the submucous plexus may come not only from neurons within the submucous plexus or the myenteric plexus but also from the extrinsic ganglia and even the mucosa. Because the submucous neurons receive extensive excitatory and inhibitory inputs (12), they are involved in the regulation of a wide range of physiological functions (4) related to those of the epithelium, the musculature, and even the vasculature (20). Numerous submucosal neurons have been identified immunocytochemically to contain substance P, vasoactive intestinal peptide, neuropeptide Y, somatostatin, galanin (5, 15, 17), and calcitonin generelated peptide (6). In addition to an abundance of different neuropeptidecontaining neurons, there are also neurons that contain acetylcholine (ACh), norepinephrine, and serotonin [5hydroxytryptamine (5-HT)] (5, 15). One of the most NEURONS

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METHODS

Female guinea pigs (1400 g) were stunned with a blow on the head and bled. The small intestine (jejunum and ileum, but excluding the terminal 10 cm of ileum) was removed and placed in Krebs-bicarbonate buffer on ice. Submucous plexus preparation. A 2cm segment of small intestine was distended over a glass rod with the mucosa facing inward. The longitudinal muscle layer innervated with myenteric plexus was first dissected free and detached from the intestine as described earlier (26, 30). The intestinal segment was then removed from the rod, everted, and cut open on both sides along the mesenteric border. The mesenteric attachments and the blood vessels were trimmed by fine scissors and discarded. The sheet of intestine was laid flat with the mucosa facing upward on a Petri dish under a dissecting microscope. The mucosa layer was gently dissected away from the remaining tissue with a squared-end spatula, leaving a preparation containing the circular muscle layer attached with the submucous plexus and the muscularis mucosa. Releaseof r3H]ACh f rom submucous plexus. The tissue preparation containing the submucous plexus was anchored to platinum-wire electrodes at both ends as pre-

0 1990 the American

Physiological

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viously described for [3H]ACh release studies (30). The tissues were labeled in 2 ml Krebs-bicarbonate buffer containing 5 &i/ml [3H]choline (New England Nuclear, Boston, MA) and 5 PM choline for 30 min at 37°C under continuous oxygenation. After the completion of labeling, there was a 60-min period of washout before the collection of tritium samples was begun. During the collection period, the tissues were perfused with Krebs containing 10 PM hemicholinium-3. Each collected fraction represented the efflux of ACh during a 5-min period. Six 5-min samples were collected as basal control before the tissues were subjected to stimulation by test substances. The exposure time to each test substance was always 5 min. Aliquots of 0.2 ml volume were taken and counted for tritium radioactivity in a liquid scintillation spectrometer (Packard 300) at a counting efficiency of 50%. Electrical field stimulation. The tissue strips were each anchored at both ends to electrodes made of platinum wire. Rectangular pulses of 20 V and 1-ms duration at a frequency varying from 0.1 to 20 Hz were generated to stimulate ACh release by a Grass stimulator (SSS) via a power amplifier (Hewlett-Packard 6826A). In this arrangement, a constant current of 0.3 mA was delivered to each strip of tissue. The duration of the current applied to the tissues was always 5 min. To minimize electrode polarization, reversing pulses of the same current strength but opposite polarity were generated and applied alternately to the tissue preparation. The stimulus was constantly monitored throughout the experiment by the use of an oscilloscope (Tektronix 5103N). Data analysis. Release (peak response) was expressed in moles of [3H]ACh per gram wet weight per minute. Changes in ACh output in response to agonists were also normalized as a percent of [3H]ACh release at basal level. A one-way variance was performed followed by a Scheffe’s post hoc test. The level of statistical significance was established at P < 0.05. Chemicals. All solutions were made up with standard laboratory grade chemicals purchased from Fisher (Pittsburg, PA). Neuropeptides were purchased from either Peninsula (Belmont, CA) or Bachem (Torrance, CA). Tetrodotoxin (TTX), 5-HT, and ethylene glycol-bis(Paminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) were obtained from Sigma (St. Louis, MO). RESULTS

r3H]ACh release from submucous plexus evoked by electrical field stimulation. In the initial phase of the study, ACh release from two different submucosal preparations, one with the mucosa intact and the other with the mucosa detached, was compared. Both preparations were electrically stimulated by rectangular pulses of 0.3 mA (see MATERIALS AND METHODS for detailed stimulus parameters) at the frequency of 10 Hz. There was only a 27.6 t 6.19% (n = 12) increase in ACh output above the basal level from the submucous plexus preparation with the mucosa left intact vs. a 56.5 t 12.1% (n = 12) increase from the second preparation in which the mucosa had been removed. In all subsequent studies, experiments were performed with the submucous preparation without the mucosa.

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Constant-current (0.3 mA) electrical field stimulation at a frequency below 0.1 Hz was found to be ineffective in causing any measurable ACh release. Field-evoked release of ACh at a range from 0.1 to 20 Hz was frequency dependent. Figure 1 shows the [3H]ACh release profile (n = 12) from the collected fractions after the washout period in response to electrical stimulation at 10 Hz. Figure 2 shows an increase in ACh output (peak response above basal level) with increasing stimulus frequency, reaching a maximum at 20 Hz. The ACh output generated at 10 Hz was not significantly different from that at 20 Hz. The actual release of ACh at 10 and 20 Hz stimulation was 4.2 & 0.4 X lo-l3 and 4.6 t 0.5 X lo-l3 mol g-l min-l, respectively. Effect of calcium removal and TTX on field-stimulated ACh output. Release of field-stimulated ACh was subsequently tested in the presence of either calcium-free Krebs-bicarbonate buffer or TTX. No significant increase (7.1 t 5.7%, n = 4) in ACh release from basal level was observed (Fig. 3) when the preparation was stimulated at 10 Hz in a calcium-free Krebs buffer containing 2.4 mM EGTA plus 4.8 mM MgC12. When the tissue was pretreated with TTX (10B6M) for 5 min, there l

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FRACTION FIG. 1. Release of [3H]ACh from submucous neurons by constantcurrent electrical stimulation. Each fraction represents a collection over a 5-min period. Arrow, 5-min period during which tissues were continuously stimulated by reversing rectangular pulses (0.3 mA, 10 Hz, 1 ms duration). Each value represents mean ACh release & SE (n

= 12). 80 K 5 -I ii

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1 STIMULATION

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2. Frequency-dependent release of [“H]ACh by electrical ulation. Values for each stimulation frequency are peak response above basal release & SE (n > 12 animals). FIG.

stimmeans

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the submucous plexus, the modulatory action of Metenkephalin ( [Met’] enkephalin), somatostatin (cyclic), galanin (porcine), and dynorphin (porcine dynorphin A l-17) were examined. The modulatory actions of neuropeptides were tested on ACh efflux selectively evoked by 5-HT, in view of the excitation observed with 5HT on the submucous cholinergic neurons (13, 23). Metenkephalin, when tested in the dose range between 8.7 X 10B8 and 2.2 X 10v5 M, did not significantly alter the basal spontaneous output of ACh. In the same concentration range, Met-enkephalin was also unable to alter the release of ACh induced by 6.2 x 10B6 M 5HT (Table 1). The modulatory action of somatostatin was tested over a dose range between 6.1 x lo-’ and 1.2 x 10B5 M (Fig. 5). Somatostatin exhibited both an inhibition and a stimulation, depending on the concentrations used. There was a significant inhibition of 5-HT-evoked ACh release at 6.1 X low8 M somatostatin, but at a higher concentration (1.2 x 10s5 M) somatostatin caused a significant increase in 5-HT-induced ACh secretion. There was also a 10.9 t 5.1% (n = 8) increase in ACh efflux at this high concentration when somatostatin was given alone, further suggesting that a complex modulatory mechanism may be involved in the action of somatostatin.

AC h release, % increase

SEROTONIN point from

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1. 5-HT-induced ACh release in the presence of Met-enkephalin and galanin

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5-HT (6.2 x + Met-enkephalin + Met-enkephalin + Met-enkephalin + Galanin (5 + Galanin (5 + Galanin (5

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4. Release of [3H]ACh in response represents mean peak response above 17-42 animals.

FIG.

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TABLE

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to serotonin basal release

M (5HT). Each t SE obtained

was an insignificant rise (3.7 t 3.7%, n = 8) in the efflux of ACh at 10 Hz stimulation; however, the ACh release in the presence of TTX to stimulation at 20 Hz was significantly elevated (13.1 t 2.6%, n = 8). Effect of 5-HT and KC1 depolarization on ACh secretion. To examine a possible modulation of submucosal cholinergic activity, release of ACh was measured in the presence of 5-HT. An increase in the secretion of ACh was observed in the concentration range from 10B7 to 10B4M 5-HT (Fig. 4). The ACh release to 5-HT stimulation was concentration dependent, with an ED50 of 5 x 10m7M. The stimulation caused by 6.2 X 10s6 M 5-HT was also sensitive to TTX (1 X 10V6 M), suggesting a direct activation of the submucosal cholinergic neurons by 5-HT. There was only a residual increase (15.6 t 3.7%, n = 12) in 5-HT-evoked ACh output in the presence of 1 x 10v6 M TTX. Depolarization by elevated KC1 caused a significant rise in the ACh output. There was a 510 t 44% (n = 8) increase in ACh release above basal level when stimulated by 100 x 10m3M KCl. Modulation of 5-HT-evoked ACh release by neuropeptides. In an attempt to assessthe functional interactions between the peptidergic and cholinergic neurons within

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FIG. 5. Modulation of 5-HT-evoked [“H]ACh release by somatostatin. Each somatostatin value represents mean peak response above basal t SE taken from 8-24 animals. Serotonin concentration equals 6.2 x 10e6 M.

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Galanin was subsequently examined in the concentration range between 5 x 10W8 and 5 x low6 M for its effect on the submucosal neurons. In a manner similar to that observed with somatostatin, the modulatory actions of galanin on 5-HT-induced ACh secretion was inhibitory at 5 X 10B7 M and stimulatory at 5 X 10M6 M galanin (Table 1). The magnitude of both the inhibition and excitation achieved in the dose range applied with either galanin or somatostatin was also the same. Figure 6 shows that when dynorphin (2.3 X 10-l' M to 1.2 x 10D6 M) was examined, there was a persistent significant suppression of ACh release induced by 6.2 x 10m6 M 5HT. Unlike galanin or somatostatin, there was never any excitation of ACh release exhibited by dynorphin. The level of inhibition observed with dynorphin was found not to be significantly different from that attained by somatostatin or galanin. A comparison of the three neuropeptides on their inhibitory effects has revealed a relative order of potency: 1.2 x 10m8 M for dynorphin, 6~ 10m8M for somatostatin, and 5 X 10m7M for galanin. Vasoactive intestinal peptide (VIP) was relatively weak in comparison to 5-HT in its ability to modulate submucosal cholinergic activity. There was only a 5.8 t 1.7% (n = 16) increase above basal release with 6 X lo-’ M VIP. A graded increase in VIP concentration to 6 x 10B8and 6 X 10e7 M only resulted in an increase of 12.8 t 8.4 (n = 24) and 15 t 11.7% (n = 22), respectively. Substance P was also examined for its possible modulatory action on submucosal ACh output. No ACh release was observed with substance P in the nanomolar to submicromolar range. Even when the substance P concentration was elevated to 1.9 x l.0W6M, there was only a 5 Ifi: 1.2% (n = 3) increase in ACh secretion. DISCUSSION

This is a study that examines the regulation of cholinergic activity in the submucous plexus of the guinea pig small intestine. Our findings indicate that the cholinergic

ON

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neurons from the submucous plexus can be characterized by their responses to 1) electrical field stimulation and 2) modulation by neuropeptides. These two key features are distinctly dissimilar from the cholinergic neurons of the myenteric plexus (27). Table 2 gives a comparison of the different response characteristics for the cholinergic neurons of these two neuronal plexi. Although the cholinergic neurons of the submucous plexus are activated by field stimulation at a high frequency range (lo-20 Hz), their myenteric counterparts are known to be more responsive at a relatively lower frequency range (0.1-l Hz). In addition, there are numerous distinguishable features in their responses to neuropeptide modulation. One of the major differences lies in their ability to respond specifically to substance P and Met-enkephalin. There is no evidence that the submucous cholinergic neurons examined in our experiments are regulated by substance P or Met-enkephalin. Despite the presence of substance P binding sites in the submucous plexus neurons (3), there is no evidence for a mediation of substance P action by way of the cholinergic neurons. Met-enkephalin is found to be ineffective in altering the basal or 5HT-evoked release of ACh. Immunohistochemical evidence suggests an absence of Met-enkephalin in submucous neurons (14), whereas 15-24% of myenteric nerve cells contain Met-enkephalin (10, 21). There are, however, Met-enkephalin-containing varicosities in submucous ganglia, but presumably they are derived from nerve cells outside of submucous plexus. Although Metenkephalin has been demonstrated to hyperpolarize certain submucous neurons (18, 24), it is difficult to correlate these electrophysiological observations with our release data. In a separate study, the inhibitory synaptic potentials still persisted in some submucous neurons even in the presence of naloxone (2), suggesting that Met-enkephalin was not responsible for such an action. In view of the complexity of neuronal types within the submucous plexus, it is possible that Met-enkephalin might hyperpolarize certain neurons other than those containing ACh. There is already some evidence to suggest that inhibitory synaptic inputs are received by VIPcontaining neurons (1). The identity of those submucous 2. Response characteristics of cholinergic neurons in submucous and myenteric plexus of guinea pig small intestine

TABLE

Submucous Plexus

Electrical [“HJACh

field stimulation output (10-l”

10 Hz mol.

4.2k0.4

Myenteric Plexus

0.1 Hz 7.3kO.4

g -l. mindl)

Neuropeptide modulation Substance P VIP Somatostatin Galanin Met-enkephalin Dynorphin A CONTROL

2.3E-10 1.2E -9 1.2E-8 DYNORPHIN CONCENTRATION,

FIG. 6. Modulation of &MT-evoked [3H]ACh A. Each dynorphin value represents mean peak release t SE taken from 119-23 animals. Serotonin 6.2 x 1O-6 M.

l.2E-7

1.2E-6 M

release by dynorphin response above basal concentration equals

NR + ++NR +

+++ +++ * ---------

Data for myenteric plexus cholinergic neurons are obtained from previous works in the literature (see Ref. 27 for pertinent references). NR, no response observed; + and - indicate relative degree of excitatory or inhibitory modulation of cholinergic activity based on an arbitrary scale of 1 to 3. * Indicates only a generalized action; may not be active in some conditions.

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5HT

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nerve cells that are susceptible to hyperpolarization by Met-enkephalin is an important question that has yet to be answered. The ability for the submucous cholinergic neurons to respond to dynorphin but not Met-enkephalin suggests a high degree of opioid receptor specificity. Dynorphincontaining nerve cells are present in the submucous plexus (525). An inhibition of ACh release by dynorphin is also consistent with the reported action of opioid in the intestinal mucosa (11). In addition to dynorphin, two other neuropeptides have been identified in our studies that either inhibit or stimulate cholinergic activity. In the myenteric plexus, both somatostatin (29) and galanin (28) have been recognized for their ability to depress ACh release. However, in the submucous plexus, these two neuropeptides retain their inhibitory action only at submicromolar concentrations. When tested at higher concentrations, they behaved as stimulators, causing the release of ACh to rise above control levels. The exact mechanism for the modulatory actions of these two neuropeptides is unclear. The hyperpolarization of somatostatin appears to be mediated by an increase in inwardly rectifying potassium conductance (19). The mechanism by which an inhibitory neuropeptide can trigger an increase in ACh secretion is still not understood. One possibility is that the increase in ACh output could be ascribed to a selective suppression by high concentration of somatostatin or galanin of a tonically active inhibitory input to the cholinergic neurons. 5HT appeared to be the most potent of all the modulators examined. Because there are no histochemically demonstrable 5-HT-containing nerve cells within the submucous plexus, the nerve projections presumably originate from the myenteric plexus (7). Earlier electrophysiological studies have demonstrated an excitation by 5-HT of every submucous neuron examined (13). Release data from our present study provide the direct evidence for its stimulation of submucous cholinergic neurons. This further supports the current view of a cholinergitally mediated action of 5-HT in submucous plexus (4, 11,13,23). It remains to be further investigated, however, as to the precise mechanism through which the 5-HT receptors on the cholinergic neurons are activated. In contrast to 5-HT, VIP has a relatively weak stimulation on the release of ACh. Given that close to 50% of submucous neurons contain VIP, our results seem to imply that the cholinergic population receives only minor inputs from neurons secreting VIP. This is consistent with the view that submucous VIP-containing neurons, which may receive synaptic inputs from other neurons, are likely to serve as an important effector neurons to the mucosa (1, 4, 15). About 50% of neurons in submucous plexus contain ACh, but they are quite heterogeneous. Substance P is known to be colocalized in a subpopulation of submucous cholinergic neurons (5, 8, 9). One subset of cholinergic neurons presumably contains a mixture of transmitters consisting of neuropeptide Y, somatostatin, and the COOH-terminal octapeptide of cholecystokinin (CCK8). The remaining group contains exclusively ACh. These cholinergic subsets are also likely to receive different

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synaptic inputs from a wide variety of intramural or extrinsic neurons. Whether neurons in such a mixed population are all capable of responding to the same extent to different modulators is a crucial question that remains to be answered. There are distinct submucous neurons that do not receive any synaptic inputs (22); however, the identity of these neurons remains unclear. Furthermore, the tissue preparations used in our study might affect the overall picture of ACh release because of 1) the inclusion of myenteric terminal varicosities on the circular muscle layer and 2) the absence of the mucosa and the cholinergic projections into the epithelial cells. Therefore, based on the experimental data presented here, it is not possible to discern the exact subset of cholinergic neurons or varicosities from which the released ACh might have originated. In conclusion, striking response characteristics have been obtained from submucous cholinergic neurons that differ significantly from those of the myenteric plexus. Besides 5-HT there are only a few neuropeptides that can weakly modulate the secretion of ACh. Our data seem to suggest that in the submucous plexus, most of the peptide-containing neurons mediate their actions independently of the cholinergic neurons. These observations may provide an important framework for functional studies of submucous plexus in the future. This work was supported by National Institute Digestive and Kidney Diseases Grant DK-26860. Address reprint requests to W. M. Yau. Received

6 March

1990; accepted

in final

form

13 July

of Diabetes

and

1990

REFERENCES 1. BORNSTEIN, J. C., M. COSTA, AND J. B. FURNESS. Synaptic inputs to immunohistochemically identified neurones in the submucous plexus of the guinea-pig small intestine. J. Physiol. Lond. 381: 46% 482, 1986. 2. BORNSTEIN, J. C., M. COSTA, AND J. B. FURNESS. Intrinsic and extrinsic inhibitory synaptic inputs to submucous neurones of the guinea-pig small intestine. J. Physiol. Lond. 398: 371-390, 1988. 3. BURCHER, E., AND J. C. BORNSTEIN. Localization of substance P binding sites in submucous plexus of guinea pig ileum, using wholemount autoradiography. Synapse 2: 232-239, 1988. 4. COOKE, H. J. Neurobiology of the intestinal mucosa. Gastroenterology 90: 1057-1081, 1986. 5. COSTA, M., AND J. B. FURNESS. Structure and neurochemical organization of the enteric nervous system. In: Handbook of Physiology. The Gastrointestinal System. Neural and Endocrine Biology. Bethesda, MD: Am. Physiol. Sot., 1989, sect. 6, vol. II, chapt. 5, p. 97-109. 6. FEH~R, E., G. BURNSTOCK, I. M. VARNDELL, AND J. M. POLAK. Calcitonin gene-related peptide-immunoreactive nerve fibers in the small intestine of the guinea pig: electron-microscopic immunocytochemistry. CeZZ Tissue Res. 245: 353-358, 1986. 7. FURNESS, J. B., AND M. COSTA. Neurons with 5-hydroxytryptamine-like immunoreactivity in the enteric nervous system. Their projections in the guinea pig small intestine. Neuroscience 7: 341349, 1982. 8. FURNESS, J. B., M. COSTA, AND F. ECKENSTEIN. Neurons localized with antibodies against choline acetyltransferase in the enteric nervous system. Neurosci. Lett. 40: 105-110, 1983. 9. FURNESS, J. B., M. COSTA, AND J. R. KEAST. Choline acetyltransferase and peptide immunoreactivity of submucous neurons in the small intestine of the guinea pig. CeZl Tissue Res. 237: 329-336, 1984. 10. FURNESS, J. B., M. COSTA, AND R. J. MILLER. Distribution and projections of nerves with enkephalin-like immunoreactivity in the guinea pig small intestine. Neuroscience 8: 653-664, 1983.

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11. GAGINELLA, T. S., T. J. RIMELE, AND M. WIETECHA. Studies on rat intestinal epithelial cell receptors for serotonin and opiates. J. Physiol. Land. 335: 101-111, 1983. 12. HIRST, G. D. S., AND H. C. MCKIRDY. Synaptic potentials recorded from neurones of the submucous plexus of guinea pig. J. Physiol. Lond. 249: 369-3851975. 13. HIRST, D. G. S., AND E. M. SILINSKY. Some effects of 5-hydroxytryptamine, dopamine and noradrenaline on neurones in the submucous plexus of guinea-pig small intestine. J. Physiol. Lond. 251: 817-832,1975. 14. KEAST, J. R., J. B. FURNESS, AND M. COSTA. Origins of peptide and norepinephrine nerves in the mucosa of the guinea pig small intestine. Gastroenterology 86: 637-644, 1984. 15. LUNDGREN, O., J. SVANVIK, AND L. JIVEGARD. Enteric nervous system. I. Physiology and pathophysiology of the intestinal tract. Dig. Dis. Sci. 34: 264-283, 1989. 16. MAKHLOUF, G. Enteric neuropeptides: role in neuromuscular activity of the gut. TIPS 6: 214-218, 1985. 17. MELANDER, T., T. HOKFELT, A. ROKAEUS, J. FAHRENKRUG, K. TATEMOTO, AND V. MUTT. Distribution of galanin-like immunoreactivity in the gastrointestinal tract of several mammalian species. CeZZ Tissue Res. 239: 253-270, 1985. 18. MIHARA, S., AND R. A. NORTH. Opioids increase potassium conductance in guinea-pig submucous neurones by activating b-receptors. Br. J. PharmacoZ. 88: 315-322, 1986. 19. MIHARA, S., R. A. NORTH, AND A. SURPRENANT. Somatostatin increases an inwardly rectifying potassium conductance in guineapig submucous plexus neurones. J. Physiol. Lond. 390: 335-355, 1987. 20. NEILD, T. O., K.-Z. SHEN, AND A. SURPRENANT. Vasodilatation of arterioles by acetylcholine released from single neurones in the guinea-pig submucosal plexus. J. Physiol. Lond. 420: 247-265,199O. 21. SCHULTZBERG, M., T. HOKFELT, G. NILSSON, L. TERENIUS, J. F. REHFELD, M. BROWN, R. ELDE, M. GOLDSTEIN, AND S. SAID. Distribution of peptideand catecholamine-containing neurons in the gastrointestinal tract of rat and guinea pig: immunohistochem-

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SUBMUCOUS

PLEXUS

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Modulation of submucosal cholinergic neurons by 5-hydroxytryptamine and neuropeptides.

Release of [3H]acetylcholine ([3H]ACh) was examined in a submucous plexus preparation obtained from the guinea pig small intestine in vitro. Constant-...
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