Department of Physiology, University of Melbourne, Parkville, Victoria 3010, Australia
Submitted 22 January 2004 ; accepted in final form 8 July 2004
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ABSTRACT |
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enteric nervous system; electrophysiology; smooth muscle; enteric reflex
The guinea pig small intestine has been an excellent model for studying the circuitry of the enteric nervous system. It is in this system that the best data on projection patterns, chemical code, and electrophysiology have been gathered and correlated to characterize specific functional classes of neuron (8) and to reveal the nature of synaptic transmission at specific classes of synapses (7). The circuitry of the ileum, in particular, provides a template on which to design studies investigating the circuitry in the colon.
Neuroanatomical studies (31, 33, 34) have revealed substantial similarities between the circuitry in the ileum and colon, but have also highlighted some significant differences. For example, one class of somatostatin-containing interneurons appears to have descending projections in the ileum but projects orally in colon (29). There is only a single class of ascending interneuron in the ileum, but there are four such classes in the distal colon. Interestingly, recent work in the colon has identified one class of neurons with the morphology of ascending interneurons, but which was also distension-sensitive (4042). Finally, the electrophysiological characteristics of colonic neurons (10, 28, 30, 45) and the whole organ pharmacology (e.g., see Ref. 50) are subtly different from those of the ileum. For example, 5-HT or GABAA receptor antagonists inhibit colonic reflexes (e.g., see Refs. 21 and 25), but have little effect in the ileum (for review, see Ref. 6).
As might be expected, the peristaltic reflex differs between the ileum and the colon. For example, when the muscle is free to contract, a stationary distension of the gut wall causes a descending excitatory reflex in ileum (39) but an initial descending inhibitory reflex in the colon (12, 15, 38). In guinea pig ileum, there is very little cholinergic transmission at any of the functionally identified synapses within the descending inhibitory reflex pathway (4, 24) or in the descending excitatory reflex pathway (35). In rat and guinea pig colon, when nicotinic receptors were blocked at all synapses, ascending and descending reflexes were abolished (19); when each functional class of synapse in the descending inhibitory pathway of rat colon was examined in detail, these were all found to be nicotinic (5). Many other transmitters, such as GABA, somatostatin and vasoactive intestinal peptide, are important and are likely to have a role in the circuitry of the colon (e.g., see Refs. 20 and 25). However, it is our understanding of the fundamentals in this region that is lacking most. For example, the role of nicotinic transmission at identified synapses in the ascending excitatory or descending inhibitory reflexes is still unknown (for review, see Ref. 6), and little is known about the lengths of the functional neural projections underlying these reflexes.
In the present study, we analyzed the nature of transmission at specific classes of synapses within the ascending and descending reflex pathways of the guinea pig distal colon to identify how such synapses may contribute to the overall behavior of this organ. The divided organ bath methodology, which allows functional classes of synapse to be isolated and studied pharmacologically, was identical to that which has already provided detailed information about transmission at analogous classes of synapses in guinea pig ileum (e.g., see Refs. 4, 22, 24, and 49) and in the descending inhibitory pathway of rat distal colon (5), thereby allowing direct comparisons between systems.
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MATERIALS AND METHODS |
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Guinea pigs of either sex (200 to 350 g) were killed by a blow to the head and severing of the carotid arteries, a procedure approved by the University of Melbourne Animal Experimentation Ethics Committee in accordance with the guidelines of the National Health and Medical Research Council of Australia. A segment of colon (510 cm long, taken 510 cm from the rectum) was taken and placed in a physiological saline (composition in mM: 118 NaCl, 4.8 KCl, 1.0 NaH2PO4, 25 NaCO3, 1.2 MgSO4, 11.1 D-glucose, and 2.5 CaCl2) and bubbled with 95% O2-5% CO2 at room temperature. After the lumen was flushed with physiological saline solution, the segment was cut open along the mesenteric border. The full-thickness segment (50 mm length and 15 mm diameter, with myenteric and submucous plexes intact) was opened and pinned flat with the mucosal surface uppermost in a three-chambered organ bath and slowly warmed to 35°C (for schematic see Ref. 4). Each chamber had an
5-ml volume and was separately perfused at 5 ml/min. Drugs or solutions added to one chamber did not affect tissues located in the other chambers. This bath was used for experiments on ascending or descending reflexes; the tissue was simply pinned in the orientation required.
Rubber balloons (5 mm diameter, embedded in the base of the bath) were used to distend the intestinal wall in the central chamber 15 mm from the recording site, and in the far chamber 30 mm away (e.g., Fig. 1A, left). The volume of the distension stimulus was increased progressively by 0.05 ml until a maximal amplitude junction potential was initiated. The volume used to obtain this initial maximal response was then used for the rest of the stimuli for that tissue; thus all later stimuli were assumed to be maximal or nearly maximal. For the same distension, the amplitude of the junction potential was decreased with an increasing distance of the balloon from the recording site. In some experiments, only the central and recording chambers were used. Intervals of >23 min between distension stimuli were used to avoid rundown of responses (48).
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In some experiments on the descending inhibitory pathway, notably those in which drugs were added to the recording chamber, electrical stimulation of neurons within this chamber was used to provide an extra control. A pair of silver wires (0.5 mm diameter, 2 cm length) was placed in the recording chamber above and below the preparation and either parallel or perpendicular to the circular muscle, 4 mm oral to the recording site so that transmural electric field stimulation (EFS) could be applied to the pathways and directly to motor neurons (e.g., see Ref. 4). Junction potentials evoked in response to EFS could be graded in amplitude according to stimulus intensity. The stimulus intensity of EFS was chosen to evoke junction potentials with amplitudes matching those of physiologically evoked junction potentials in the same preparation.
Electrophysiology
Glass microelectrodes were filled with 2 M KCl (80- to 100-M tip resistance) and mounted on a mechanical micromanipulator (Leitz; Wetzlar, Germany) to record intracellularly from the circular smooth muscle. We found that impaling circular muscle through either the mucosa or the serosa was difficult in tissue from some animals. Thus for some preparations, a patch of mucosa and submucosa at one end was dissected away to allow direct impalement of the circular muscle. Responses recorded under these conditions did not differ from those seen when the circular muscle was impaled from the serosal side through the longitudinal muscle as has been done in previous studies. The potential difference across the cell membrane was recorded onto a personal computer using a computerized polygraph system (Axotape 2.0.2, Axon Instruments, Foster City, CA) and then analyzed with Origin 6.0 (MicroCal, Northampton, MA).
In each experiment, tissue from one animal was used to derive one n value for purposes of statistical comparison. Within each experiment, a set of three control excitatory (EJPs) or inhibitory junction potentials (IJPs) was evoked by distension or EFS (5), and the amplitudes were averaged. Blockers or receptor antagonists were then added to the superfusing solution and allowed to equilibrate for 5 to 10 min in one or more of the chambers. During the next 30 min, a second set of junction potentials was evoked in the presence of this changed solution. Finally, these solutions were washed out for 3060 min, and a third set of junction potentials was evoked.
Statistics
Unless noted otherwise, statistical analyses were performed by using Students paired t-tests to compare junction potential amplitudes (mean of 3 replicates) before and during drug superfusion. ANOVA was used to determine the significance of changes in junction potential amplitudes when more than one drug was applied in sequence. When ANOVA revealed a significant difference, the significance of individual differences was determined with a post hoc Tukey-Kramer multiple comparisons test. Statistical comparisons of relative changes in responses with different stimulation regimens were made by using the Wilcoxon signed-rank test. In all statistical comparisons performed, differences were considered significant if P < 0.05. All data are expressed as means ± SE.
Drugs
The drugs used in the present study were hexamethonium, hyoscine (scopolamine), nicardipine, and pyridoxal-phosphate-6-azophenyl-2'4'-disulphonic acid (PPADS) (all from Sigma, St. Louis, MO). Granisetron and SB-204070 were kindly supplied by SmithKlineBeecham Pharmaceuticals (Middlessex, UK). SR-142801 was kindly supplied by Dr. Emonds-Alt (Sanofi Recherche, Montpellier, France). A low-Ca2+/high-Mg2+ saline was used to block synaptic transmission composed of (in mM) 118 NaCl, 4.8 KCl, 1.0 NaH2PO4, 25 NaCO3, 12 MgSO4, 11.1 D-glucose, and 0.25 CaCl2.
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RESULTS |
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Effect of Synaptic Blockade in the Central Chamber on Ascending and Descending Reflexes
In the ascending pathway, EJPs evoked by distension from the central chamber were greatly reduced by blockade of synaptic transmission with a low-Ca2+/high-Mg2+ solution in the central chamber (control, 12.9 ± 1.2 mV; high Mg2+, 0.9 ± 0.3; P < 0.05; one-way ANOVA; n = 5; Fig. 1B); EJPs evoked by distension in the far chamber were also greatly reduced (control, 10.3 ± 1.6 mV; high Mg2+, 0.7 ± 0.5, P < 0.05; one-way ANOVA; n = 6). Thus no distension-sensitive neurons or ascending interneurons project orally >15 mm (the width of the chamber). This implies that drugs applied to the central chamber will act primarily at synapses between ascending interneurons in pathways excited from the far chamber.
In contrast, in the descending pathway, IJPs evoked by distension from either chamber were only reduced by approximately half during blockade of synaptic transmission in the central chamber [central control: 17.3 ± 1.0 mV; high Mg2+: 10.3 ± 1.2 (59% of control); Far control: 7.1 ± 0.6 mV; high Mg2+: 3.0 ± 0.5 (43% of control); P < 0.05; one-way ANOVA; n = 6; Fig. 1C]. IJPs evoked by distension in the far chamber were significantly more depressed than IJPs evoked by central chamber distension (P < 0.01. Wilcoxon signed-rank test). When transmission was blocked in both the far and central chambers, IJPs evoked by distension in the far chamber were not significantly more reduced than when transmission was only blocked in the central chamber. These data suggest that some distension-sensitive neurons project from the far chamber to the recording chamber, but descending interneurons do not. Distension-sensitive neurons and interneurons both project from the central to the recording chamber. Note, elements of the circuitry, which can now be excluded, have been omitted from subsequent figures.
Transmission in the Descending Inhibitory Pathway
Pharmacology of transmission in the central chamber. Transmission within the central chamber was assessed. The nicotinic receptor antagonist hexamethonium (200 µM) when added to the central chamber significantly reduced the peak amplitude of IJPs evoked by central chamber distension from 11.3 ± 0.2 to 5.3 ± 0.3 mV (47%, P < 0.01, ANOVA; n = 6). Further application of a low-Ca2+/high-Mg2+ solution in the same chamber significantly enhanced the peak amplitude of IJPs evoked by distension from 5.3 ± 0.3 to 6.3 ± 0.3 mV (119%, P < 0.01, ANOVA; n = 6, Fig. 2A). This enhancement was similar to that seen in rat distal colon (see DISCUSSION and Ref. 5).
We tested whether ATP also plays a role in transmission at these synapses. The nonselective P2 receptor antagonist PPADS (30 µM) in the central chamber consistently caused a small reduction in IJPs evoked by distension in this chamber [control, 13.6 ± 2.1 mV; PPADS, 12.1 ± 2.3 mV (89% of control); paired t-test, P < 0.05; n = 8, Fig. 2B].
Serotonin plays an important role in the initiation of peristalsis in the guinea pig colon, and it has been reported that both 5-HT3 and 5-HT4 receptors are required (see Ref. 21). We tested a combination of the selective 5-HT3 receptor antagonist granisetron and the selective 5-HT4 receptor antagonist SB-204070. This combination, in the central chamber, significantly reduced IJPs evoked by distension in this chamber [control, 19.4 ± 2.4 mV; combination, 14.4 ± 2.8 mV (74% of control); Fig. 2C]. Similarly, when granisetron or SB-204070 were tested alone, they significantly reduced the size of the IJPs [control, 19.3 ± 1.9 mV; granisetron, 16.8 ± 1.6 mV (87% of control); control, 19.2 ± 2.7 mV; SB-204070, 16.6 ± 2.2 mV (86% of control)]. The combination of granisetron and SB-204070 was not significantly more effective than either drug alone (P > 0.05, one-way ANOVA).
When distension from the far chamber was investigated, the addition of hexamethonium (200 µM) to the central chamber significantly reduced the peak amplitude of IJPs from 8.6 ± 0.5 to 5.4 ± 0.8 mV (63% of control, P < 0.05, one-way ANOVA; n = 6). Further addition of a low-Ca2+/high-Mg2+ solution to the same chamber did not significantly alter the peak amplitude of IJPs (from 5.4 ± 0.8 to 5.5 ± 0.7 mV, P > 0.05, one-way ANOVA; n = 6, Fig. 3A).
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Pharmacology of transmission in the recording chamber. Hexamethonium (200 µM) in the recording chamber significantly reduced the amplitude of IJPs evoked by distension in the central chamber from 10.4 ± 0.1 to 4.8 ± 0.4 mV (46% of control; P < 0.05, n = 6) and caused a small reduction in IJPs evoked by EFS in the recording chamber from 10.5 ± 0.1 to 9.4 ± 0.4 mV (90% of control; P < 0.05, n = 6). Hexamethonium (200 µM) was significantly more effective on reflex-evoked IJPs than on electrically evoked IJPs (P < 0.05, paired Wilcoxon signed-rank test, Fig. 4A).
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In a separate series of experiments, PPADS (30 µM) in the recording chamber reduced IJPs evoked by distension in the central chamber from 21.4 ± 1.3 to 18.4 ± 1.2 mV (86% of control, P < 0.05; n = 6, Fig. 4C). PPADS did not change IJPs evoked by EFS in the recording chamber (control, 17.7 ± 1.0 mV; PPADS, 18.3 ± 1.2 mV) or by distension in the far chamber (control, 12.3 ± 1.3 mV; PPADS, 12.7 ± 1.9 mV).
Granisetron (1 µM) added to the recording chamber caused a significant increase in the IJPs evoked by EFS in the recording chamber (control, 17.1 ± 1.5 mV; granisetron, 23.6 ± 1.9 mV; 138% of control; P < 0.05, Fig. 4D). Granisetron increased the amplitude of IJPs evoked by distension in the central chamber from 18.4 ± 2.0 to 20.8 ± 2.6 mV (113% of control) and caused an increase in IJPs evoked by distension in the far chamber (control, 14.0 ± 2.1 mV; granisetron, 15.5 ± 2.4 mV; 111% of control). These unexpected enhancements were not significant when assessed with a two-tailed t-test (P = 0.08, 0.37, respectively).
Transmission in the Ascending Excitatory Pathway
Pharmacology of transmission in the central chamber. Transmission within the central chamber was assessed. Hexamethonium (200 µM) in this chamber reduced EJPs evoked by distension in the central chamber from 16.2 ± 0.8 to 12.5 ± 1.1 mV (77% of control, P < 0.05; n = 6; Fig. 5A). The nonselective muscarinic receptor antagonist hyoscine (1 µM) did not reduce the EJPs nor did it potentiate the effect of hexamethonium when used in combination [control, 15.7 ± 0.9 mV; hyoscine, 14.5 ± 1.1 mV (93% of control); control, 15.7 ± 1.1 mV; hyoscine + hexamethonium, 11.8 ± 1.6 mV (75% of control), P > 0.05, one-way ANOVA; n = 6]. Similarly, the neurokinin 3 (NK3) receptor antagonist, SR-142801 (1 µM) in combination with hexamethonium did not cause any greater change in the EJP amplitude than hexamethonium alone [control, 16.4 ± 1.4 mV; SR-142801 + hexamethonium, 13.1 ± 1.6 mV (80% of control); P > 0.05, one-way ANOVA; n = 6].
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Transmission through the central chamber was assessed when EJPs were evoked by distension in the far chamber These EJPs were abolished by hexamethonium (200 µM) in the central chamber (control, 12.1 ± 1.4; hexamethonium, 0 ± 0; P < 0.05; n = 5, Fig. 6A).
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DISCUSSION |
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Projections of distension-sensitive neurons.
Synaptic blockade in the central chamber greatly reduced ascending excitatory reflexes evoked in this chamber as it does in the ileum (24), demonstrating that, if there are ascending distension-sensitive neurons in the distal colon, they project for less than the width of the chamber (15 mm). In contrast, descending inhibitory reflexes were only reduced by half, even when synaptic transmission was blocked in both the far and central chambers, suggesting that some distension-sensitive neurons have long axons that project anally for 30 mm. Identical methods have revealed long anal projections of distension-sensitive neurons in the guinea pig ileum (4, 22, 24) and in rat colon (5), although in the ileum, these projections appeared to be substantially shorter (
15 mm). In the ileum < 10% of the descending inhibition evoked by stimulation in the far chamber persists after synaptic blockade in the central chamber (e.g., 22), whereas in the colon, we have found the resistant response is approximately five times as large.
Projections of interneurons. The ascending interneurons were found to have an effective projection length of no greater than the width of the central chamber (i.e., 15 mm) within which synaptic transmission had been blocked. This is similar to what is seen in the ileum (8) but is surprising in the colon, because some ascending interneurons have projections that appear considerably longer in lesion studies (34).
Blockade of synaptic transmission in the central chamber also indicated that descending interneurons play as important a role in the inhibitory reflex pathway in the colon as they do in the ileum. However, the reduction in IJP amplitude was no different when synaptic transmission was blocked simultaneously in the far and central chambers. This indicates that the effective projection length of the descending interneurons is similar to that of ascending interneurons, 15 mm or less. In contrast, tracing studies of the descending interneurons in the ileum have found the different classes or descending interneuron have projections of
120 mm (8, 32).
Nature of the distension-sensitive neurons. There is still some discussion over the identity of the sensory elements in the intestine. The multipolar AH/Dogiel type II neurons are clearly sensitive to some stimuli (e.g., maintained stretch, certain chemicals), but this does not preclude other enteric neuronal types or extrinsic sensory nerves (e.g., vagal) from participating in reflexes evoked by these or other stimuli (e.g., for review, see Refs. 7 and 14). Indeed, in the rat colon, the extrinsic sensory nerves appear to be solely responsible for reflexes evoked by distension (17), whereas Spencer et al. (40, 41, 43) have recently shown that a subset of neurons with the morphology of ascending interneurons in the myenteric plexus of the guinea pig colon respond directly to maintained increases in length.
In the present study, the identity of the distension-sensitive neurons is unclear. The functional projections were asymmetrical, with long descending projections but no, or very short, ascending projections. Similar projections have been found for distension-sensitive Dogiel type II neurons (9) in the guinea pig ileum. Furthermore, the projections of extrinsic nerves would be predicted to be symmetrical, with equal lengths in the oral and anal direction, which suggests that the asymmetrical sensory pathways seen here are of intrinsic origin (i.e., an enteric neuron). That the long pathways were only descending tends to rule out a simple participation of any distension-sensitive ascending interneurons (40). In guinea pig ileum, the firing of AH/Dogiel type II neurons during maintained stretch is blocked by nicardipine (27), but firing in response to rapid mechanical distortion of their terminal is not (26). In contrast, the firing of some neurons with the morphology of ascending interneurons in the distal colon during maintained distension is not blocked by nicardipine or a 0.25 mM Ca2+/12 mM Mg2+ solution (41). Finally, studies of the projections of AH/Dogiel type II neurons in distal colon (e.g., see Refs. 30 and 44) reported no long descending projections; however, our data indicate these projections are easy to overlook and are indeed found in the distal colon (J. C. Bornstein, unpublished observations).
Nicotinic Transmission plays a Major Role in Distension-Evoked Ascending and Descending Reflexes
Transmission from sensory neurons.
Our data indicate that there are no long ascending sensory neurons. Thus, in the ascending pathway, transmission in the stimulus chamber should be primarily from sensory neurons to ascending interneurons. When the nicotinic receptors were blocked in this chamber, the EJPs were reduced by 30%. Thus some transmission was via nicotinic receptors, but the majority was through nonnicotinic receptors. In the guinea pig ileum, nonnicotinic transmission from sensory neurons to ascending interneurons is blocked by a combination of muscarinic and NK3 receptor antagonists (22, 24, 47). However, the nonnicotinic component of transmission in the distal colon is resistant to both of these receptor antagonists. It appears that transmission from sensory neurons is mixed, with ACh and another unidentified transmitter each playing a role. Alternatively, transmission from sensory neurons may be exclusively nonnicotinic, with the apparent nicotinic component being due to ascending interneurons with synapses in the stimulus chamber. Because transmission between ascending interneurons is unambiguously nicotinic, blockade of such synapses would appear as partial blockade of the total response. In either case, it is clear that transmission from the sensory neurons in the ascending pathway is largely via nonnicotinic receptors.
In the descending inhibitory pathway, hexamethonium in the central chamber depressed reflexes evoked in this chamber to the same extent as synaptic blockade, indicating that the residual response in hexamethonium was due to anally projecting axons of distension-sensitive neurons. Thus nicotinic transmission accounts for all transmission from sensory neurons to descending interneurons in the guinea pig distal colon. A similar situation exists in the rat distal colon (5). However, only a minor role for nicotinic or muscarinic receptors has been identified in the guinea pig ileum with a large component of transmission unaccounted for (22).
Although transmission from the sensory neurons to descending interneurons appeared to be purely nicotinic, there was a small effect of blocking purinergic or serotonergic receptors. Both effects were small, which suggests these transmitters do not have an important physiological role between sensory neurons and interneurons. Serotonin may act between the enterochromaffin cells in the mucosal epithelium and the sensory nerve terminals that are nearby (3, 36, 37), as may be purines, because mucosally released ATP excites sensory nerve terminals and so, may contribute to reflexes (1, 2, 11).
Transmission between interneurons. In the ascending pathway, hexamethonium abolished transmission through the central chamber indicating that the distension-evoked reflex is exclusively carried by nicotinic transmission. In the descending inhibitory pathway, hexamethonium significantly depressed transmission through the central chamber, an effect identical to that of blocking synaptic transmission in this chamber. Thus transmission between descending interneurons may be entirely via nicotinic receptors, with the hexamethonium-resistant response being due to the long descending sensory neurons. Together, these data indicate that all transmission between the ascending and descending interneurons is via nicotinic receptors in the guinea pig distal colon. The rat colon also shows purely nicotinic transmission between descending interneurons (5). In guinea pig ileum, transmission between the ascending interneurons is purely nicotinic (24), but there is a large nonnicotinic component between descending interneurons (2224) that may be purinergic (46). In the present study, we also found a small purinergic component to transmission between descending interneurons, but this was not additive with the nicotinic component. Thus any functional purinergic synapses may be in series with the nicotinic synapses in the descending inhibitory pathway of the colon.
Transmission to motor neurons. In the ascending pathways, the excitatory motor neurons in the recording chamber receive input only from ascending interneurons. Input was predominantly via ACh acting at nicotinic receptors, with a residual nonnicotinic component that was not due to tachykinins acting at NK3 receptors. In contrast, input to excitatory motor neurons in guinea pig ileum is partially nicotinic and partially due to tachykinins acting at NK3 receptors (22, 24).
In the descending inhibitory pathway, nicotinic transmission accounts for half of the input to inhibitory motor neurons. When synaptic transmission was blocked in the central chamber and distension was applied in this chamber so that activity entering the recording chamber was only via long descending sensory neurons, nicotinic transmission still accounted for half the input to the motor neurons. This suggests that input to inhibitory motor neurons from both long descending sensory neurons and descending interneurons is approximately half nicotinic and half via an unknown transmitter. In rat colon, input to inhibitory motor neurons from either descending pathway is purely nicotinic (5), but in guinea pig ileum, input is almost completely nonnicotinic (22), with input from descending inter-neurons being wholly purinergic and that from sensory neurons via an unidentified transmitter (4).
PPADS in the recording chamber caused a small reduction of IJPs evoked by central distension but did not reduce IJPs evoked by far chamber distension. Although the reduction to central distension was statistically significant, taken together, these data suggest that, if P2 receptors play a role in transmission to inhibitory motor neurons in guinea pig distal colon, it is a small one.
The nonnicotinic input to the inhibitory motor neurons was not due to 5-HT acting at 5-HT3 receptors. Blockade of 5-HT3 receptors in the recording chamber increased the amplitude of electrically evoked IJPs, and there was a trend for reflex-evoked IJPs to also be enhanced. Granisetron may block a concurrent descending excitatory reflex that interferes with the IJPs; 5-HT3 receptors are important in the descending excitatory pathway of the guinea pig ileum (35).
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of X.-C. Bian: Dept. of Pharmacology and Toxicology, Michigan State Univ., East Lansing, MI 48823.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* X.-C. Bian and L. F. Heffer contributed equally to this work.
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REFERENCES |
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