K. Hillsley: Department of Anatomy and Neurobiology, C-423 Given Building, University of Vermont, Burlington, VT 05405, USA.
Corresponding authors D. Grundy: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK. Email: firstname.lastname@example.org
1This study was performed to elucidate the actions of 5-hydroxytryptamine (5-HT) on mesenteric afferent discharge and to determine the receptor-mechanisms responsible for these effects. The activity of mesenteric afferents innervating the mid-jejunum of urethane-anaesthetized rats was recorded with extracellular microelectrodes. The discharge of single nerves within the whole nerve recording was monitored using waveform discriminator software.
2The intravenous injection of 5-HT produced a complex pattern of afferent activation with two distinct components which could be distinguished both in terms of the response characteristics and the receptors involved. Initially, in 64 % of nerve bundles, there was a brief (2.0 ± 0.1 s) but intense activation of afferent discharge with peak afferent firing increasing with incremental doses of 5-HT. The discharge frequency in seventeen single units from these bundles during the initial response to 10 μg 5-HT was 13.0 ± 1.8 impulses s−1 from a baseline discharge of 1.0 ± 0.1 impulses s−1.
3This initial response was mimicked by the 5-HT3 receptor agonist, 2-methyl-5-HT, whereas 5-methoxytryptamine (5-MEOT, 10–100 μg) had no comparable effect. Similarly, the initial 5-HT response was completely abolished by the 5-HT3 receptor antagonist, granisetron (0.5 mg kg−1).
45-HT also evoked, in approximately 35 % of nerve bundles, a delayed response that single unit analysis showed to be mediated by an entirely different population of afferents from those activated during the initial response. This secondary response to 5-HT was characterized by a more prolonged (> 30 s) but less intense period of afferent activity which was coincident with an increase in intrajejunal pressure, and was mimicked by 5-MEOT (10–100 μg).
5The secondary response to 5-HT and the response to 5-MEOT were significantly attenuated by the 5-HT2A receptor antagonist, ketanserin (0.5 mg kg−1), which had no effect on the initial response.
6The initial response to 5-HT was unaffected by the L-type calcium channel inhibitor nifedipine (1 mg kg−1) or the N-type calcium channel inhibitor ω-conotoxin GVIA (25 μg kg−1). However, the secondary response to 5-HT was significantly reduced after treatment with nifedipine.
7These results demonstrate that 5-HT activates different populations of afferent fibres innervating the rat jejunum. One population of afferents is activated directly via stimulation of 5-HT3 receptors, while another population responds to 5-HT with a time course consistent with secondary activation of mechanosensitive afferents following 5-HT2A-mediated contractile activity.
5-Hydroxytryptamine (5-HT) released from gastrointestinal enterochromaffin cells is implicated in mediating many gastrointestinal reflexes including those controlling peristalsis and ion transport (Bülbring & Lin, 1958; Beubler, Schirgi Degan & Gamse, 1993) and as a peripheral trigger for vomiting (Andrews, Rapeport & Sanger, 1988). With the identification and cloning of multiple 5-HT receptors and the advent of specific 5-HT receptor ligands (see Hoyer et al. 1994), several functional studies have attempted to elucidate the receptors involved in these actions of 5-HT on gastrointestinal nerves, muscle and enterocytes. Such studies suggest that 5-HT1A, 5-HT1P, 5-HT2A, 5-HT3 and 5-HT4 receptors may mediate different actions within the gut. All of these receptors, with the exception of the 5-HT2A receptor, have been found to act on neuronal targets within the enteric nervous system (for review see Galligan, 1995).
With respect to the intrinsic afferent innervation of the small intestine, evidence suggests that the 5-HT1P receptor, which evokes a slow depolarization of myenteric and submucosal AH (after-hyperpolarization) neurons (Takaki, Branchek, Tamir & Gershon, 1985; Freiling, Cooke & Wood, 1991), is located on intrinsic sensory neurons that may be involved in peristalsis and secretion (see Gershon, Kirchgessner & Wade, 1994). Both mucosal deformation by nitrogen puffs and mucosal exposure to cholera toxin have been shown to activate c-fos in submucosal neurons, and these effects were antagonized in the presence of the 5-HT1P receptor antagonist N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (Kirchgessner, Tamir & Gershon, 1992). Recent studies have attempted to characterize electrophysiologically the signal transduction mechanisms that occur in the mucosal processes of these intrinsic sensory neurons (Wang, Fiorica-Howells, Pan, Gershon & Friedman, 1996), and while 5-HT is a potent stimulus to these afferent endings a role for 5-HT in the transduction of luminal signals remains to be elucidated (Bertrand, Kunze, Bornstein & Furness, 1996).
Hence, it is clear that 5-HT has the potential to modulate the activity of both intrinsic and extrinsic afferents supplying the gastrointestinal tract and that these actions are mediated by various 5-HT receptors. In the present study, we sought to examine 5-HT sensitivity within mesenteric afferent nerve bundles supplying the rat small intestine. These mesenteric nerves contain both primary extrinsic afferents of vagal and splanchnic origin, and intrinsic intestinofugal afferents projecting from the myenteric plexus (Kuramoto & Furness, 1989). Therefore, by recording at this level the entire afferent traffic supplying a small intestinal segment can be simultaneously examined. In addition, with the aid of new computer software, individual afferents can be distinguished and simultaneously recorded from. Thus the aim of this study was to investigate the mechanisms involved in the 5-HT-mediated activation of mesenteric afferents supplying the rat jejunum and to characterize the receptor subtypes involved.
Experiments were performed on Sheffield strain male Wistar rats (350–400 g) allowed free access to food and water. Animals were anaesthetized with a single intraperitoneal injection of urethane (1.5 g kg−1). After tracheal cannulation, the left external jugular vein was cannulated to enable the administration of drugs and further anaesthetic if required (0.1 ml of 21 % solution). The exposed right carotid artery was cannulated in order to monitor systemic blood pressure. Body temperature was monitored (via a rectal thermometer) and maintained at around 37°C using radiant heat. The animals were killed by an anaesthetic overdose at the end of the experiment.
A mid-line laparotomy was performed and the caecum was removed to create a greater operating field. A 15 cm loop of mid-jejunum was located, typically 30 cm distally from the pylorus, and cannulated with portex tubing through stab incisions in the left side of the abdominal wall. A single cannula was inserted proximally and a double-bored cannula was inserted distally, with one port connected to a pressure transducer (Elcomatic EM760) to allow the intraluminal pressure to be monitored. The muscle and skin comprising the abdominal wall were then sewn to a circular metal ring to create a well, and the abdominal cavity filled with colourless light liquid paraffin pre-warmed to 37°C. The stab incisions in the side of the abdominal wall were sutured to prevent leakage of the paraffin.
Nerve preparation and recording
A single neurovascular bundle was isolated from the surrounding connective tissue and placed upon a black Perspex platform. Under a viewing microscope a mesenteric nerve was exposed by dissection of the overlying fat and surrounding blood vessels. The nerve was then sectioned at the proximal end of the bundle (approximately 10–15 mm from the jejunal wall) and wrapped around one arm of a bipolar platinum electrode, with connective tissue wrapped around the second indifferent electrode. The electrodes were connected to a Neurolog headstage (NL100) and then via a 500 × pre-amplifier, differentially amplified 50 × (NL103) and filtered with a band width of 100–1000 Hz (NL125). The signal was relayed to a spike processor (Digitimer D130) which discriminates action potentials from noise with a variable amplitude and polarity window. The whole nerve signal was displayed on a storage oscilloscope, (Tektronix 5111A) and a permanent record displayed on a chart recorder (TDM PAR 1000) together with systemic blood pressure and intestinal pressure. The whole nerve signal was also subsequently digitized (PCM-2 A/D VCR Adapter, Medical Systems Corp.), and stored on a VHS video recorder for post- experimental analysis.
The multi-unit mesenteric afferent recordings contained action potentials which were of sufficiently different amplitudes and waveforms to be discriminated accurately from each other using computerized waveform analysis. The different spike shapes were not consistently associated with a particular pattern of sensitivity and presumably reflected the position of the fibre within the nerve bundle relative to the recording electrode. This off-line analysis of the afferent activity in the mesenteric nerves was performed using an Elonex PC-425x running Spike 2 software (CED) via a 1401+ plus interface board (CED). The nerve signal was digitally sampled at 23 kHz which was sufficient to allow accurate spike discrimination. Each spike above a given amplitude was used to set up templates for the individual action potentials. Action potential waveforms were automatically averaged, DC offset arising from noise removed, and the resulting spike shapes assigned to different waveform templates. The afferent recording was subsequently analysed such that each action potential was compared with the waveform template and either matched to one or left unclassified. The timing of each template-matched spike was then used to calculate firing frequency and to plot discharge frequency against time. Tolerance was variable but typically the allowed amplitude error was set at between 1 and 2 % and for a spike to be matched at least 85 % of the data points had to fall within the template shape. These relatively rigid parameters were shown empirically to discriminate action potentials accurately but at high firing frequencies a small proportion of individual spikes, typically < 5 %, could be missed because of summation. However, this under-estimate of spike frequency was obviously preferable to less rigorous discrimination configurations in which cross- contamination could occur. The software allowed as many as eight templates to be simultaneously sampled but in our experience the system was most reliable dealing with fewer (typically one to four templates) of the relatively larger amplitude spikes. Once stored on computer, spike discrimination was checked manually by overlaying templates and spikes and compared by eye. Ambiguous spikes could be reassigned to a different template or ignored. The technique was validated as described previously (Richards, Hillsley, Eastwood & Grundy, 1996).
Once the nerve recording had been established an initial test stimulus of 10 μg 5-HT or 10 μg 2-methyl-5-HT (2Me-5-HT) was administered. The intravenous cannula was preloaded with a 50 μg ml−1 solution of either drug, thus avoiding the need to flush in the drug with saline. Experiments were performed on bundles which responded to this initial test. If a nerve bundle failed to respond to 5-HT, that bundle was discarded and a different nerve dissected out for subsequent inspection. Thus, in the majority of experiments there was an initial selection in favour of bundles which contained at least one 5-HT-sensitive afferent fibre. However, since the nerve bundle contained other afferents which were not sensitive to 5-HT it was possible to characterize both 5-HT-sensitive and 5-HT-insensitive populations of mesenteric afferents. No desensitization to successive doses of 5-HT agonists was observed when a minimum interval of 5 min was employed. Five minutes was also allowed following administration of 5-HT antagonists whereas 15 min was allowed following treatment with the calcium channel inhibitors, nifedipine and ω-conotoxin GVIA.
The following drugs were obtained from Sigma (UK): ω-conotoxin GVIA, 5-hydroxytryptamine (5-HT), 5-methoxytryptamine (5-MEOT), ethyl carbamate (urethane), nifedipine and sodium chloride (NaCl). 2-Methyl-5-hydroxytryptamine (2Me-5-HT) and ketanserin tartrate were purchased from RBI (SEMAT, UK). Granisetron (BRL43694a) was kindly provided by SmithKline Beecham. All drugs were dissolved in 0.9 % saline except nifedipine which was dissolved at a concentration of 1 mg ml−1 in 25 % dimethyl sulphoxide (v/v) in 0.9 % saline.
Baseline spike discharge (in impulses s−1) was obtained by averaging thirty consecutive 1 s bins immediately prior to agonist adminstration. Values for discharge frequency following drug administration represent the mean discharge within the time frame of the delineated response boundaries. In the absence of a response the mean discharge was calculated in the immediate period following agonist administration. Data are shown as the mean ±s.e.m. Significance tests were carried out using the appropriate Student's paired or unpaired t test with Bonferroni corrections applied when multiple comparisons were made.
5-HT response characteristics
Approximately 64 % (23/36) of mesenteric nerve bundles contained at least one fibre that responded to systemically administered 5-HT (5–10 μg), while other fibres within the bundle were unresponsive; indeed 36 % of bundles showed no response to 5-HT at all. 5-HT sensitivity is, therefore, not a general property of mesenteric afferents. In those bundles that responded to 5-HT, the response was dominated by a brief but intense burst of action potentials which in eight of twenty-three recordings was followed by a delayed and more prolonged but less intense secondary activation of afferent firing (Fig. 1). Single unit analysis of the 5-HT response clearly indicated that the initial and secondary activation were mediated by different afferent fibres within the mesenteric nerve bundle.
The initial response to 5-HT
The initial response to 5-HT was fully characterized in seventeen single units. The latency following injection into the jugular vein was 2.9 ± 0.2 s (range, 2.5–4.7 s) which was coincident with the drug reaching the intestinal loop as seen when dye was added to the solution of 5-HT and observed in the mesenteric vasculature. Single units identified from within the whole nerve discharge by waveform discrimination had a low level of spontaneous discharge of 1.0 ± 0.1 impulses s−1 which was increased by >1200 % after 10 μg 5-HT, with a mean response discharge of 13.0 ± 1.8 impulses s−1 (n= 17). The afferent firing returned rapidly to baseline and the overall duration of the response was just 2.0 ± 0.1 s (Fig. 1). It was evident on the arterial and intestinal pressure recordings that 5-HT also produced cardiovascular effects and stimulated intestinal motility. The cardiovascular effects consisted of the well-documented triphasic effect of bradycardia, followed by transient hypertension and prolonged hypotension. These events were used to gauge the efficacy of the various 5-HT agonists and antagonists employed in the present study.
5-HT3 receptor ligands
The response to the 5-HT3 receptor agonist, 2Me-5-HT was essentially the same as the initial response to 5-HT. When tested in the same preparation, 5-HT and 2Me-5-HT always produced responses which, dose for dose, were of comparable magnitude and duration. The response to 2Me-5-HT was dose dependent over the range (2.5–10 μg) tested (Fig. 2), although maximal responses were not obtained due to the cardiovascular consequences of injecting higher doses of 5-HT3 receptor agonists.
Granisetron, the 5-HT3 receptor antagonist, at a dose of 0.5 mg kg−1 completely blocked the von Bezold-Jarisch component of the cardiovascular response to 5-HT and had a marked effect on mesenteric afferent discharge. Spontaneous whole nerve discharge was significantly reduced by granisetron (P < 0.05, n= 9) as shown in Fig. 3. This attenuation arose out of a preferential effect on the spontaneous firing of 5-HT-sensitive afferents, while afferents which were not responsive to 5-HT showed no significant decrease in their spontaneous firing (control, 1.8 ± 0.4 impulses s−1; granisetron, 1.7 ± 0.4 impulses s−1; n= 12). The initial response of mesenteric afferents to 5-HT was completely abolished by granisetron in all experiments (see Fig. 3), as was the response to 2Me-5-HT. Thus, the 5-HT3 receptor subtype is an obligatory mediator of this response to 5-HT. However, the possibility that other 5-HT receptors could modulate either the 5-HT response or the spontaneous afferent discharge was assessed using other 5-HT agonists and antagonists.
Other 5-HT receptor ligands
At a dose of 0.5 mg kg−1, ketanserin abolished the hypertensive response to 5-HT, but had no significant effect on spontaneous whole nerve discharge (control, 13.2 ± 2.5 impulses s−1; ketanserin, 14.0 ± 3.3 impulses s−1; n= 9). The peak discharge in response to 5-HT was similarly unaffected (Fig. 4), indicating that the 5-HT2A receptor played no role in the initial effect of 5-HT on mesenteric afferents.
The effect of 5-MEOT was examined in six experiments. This ligand is effective at all 5-HT receptors with the exception of 5-HT3 receptors, but has the greatest affinity for 5-HT4 receptors. At a dose of 10 μg 5-MEOT had no significant effect on the whole nerve discharge in the six nerve bundles when measured over the 30 s period following administration. However, in seventeen of nineteen nerve bundles sensitive to 5-HT, there was a pronounced response to 100 μg 5-MEOT (Fig. 5). However, the profile of this response to 5-MEOT was markedly different from the initial response to 5-HT and from single unit analysis it was evident that individual afferent fibres that were stimulated by 5-MEOT were not stimulated by 2Me-5-HT and vice versa. Thus, the discharge of nine 2Me-5-HT-sensitive afferents was unaffected by 100 μg 5-MEOT (control, 0.5 ± 0.1 impulses s−1; 5-MEOT, 0.6 ± 0.1 impulses s−1).
Secondary 5-HT response characteristics
An additional component to the response to 5-HT was observed in 35 % (8/23) of 5-HT-sensitive mesenteric nerve bundles. This secondary 5-HT response occurred following a longer latency period and was characterized by a more prolonged but less intense period of afferent activity (see Figs 1 and 6). After a latency of 5–7 s, there was a gradual increase in afferent activity until a peak was reached within 10–20 s, with nerve discharge returning to control levels usually within 1 min. The onset of the secondary 5-HT response appeared to closely follow jejunal motor events triggered by 5-HT, typically a contraction which raised intraluminal pressure by approximately 1 cmH2O. Single unit analysis revealed that the initial 5-HT response and the secondary response to 5-HT were mediated by entirely separate subpopulations of afferents as shown in Fig. 6.
A secondary response was never observed following administration of 2Me-5-HT. However, the response elicited following administration of 100 μg 5-MEOT as described above was similar to the secondary response to 5-HT, and was also associated with a pronounced motor response (see Fig. 5). The latency and duration of the two nerve responses were similar, although the magnitude of the 5-MEOT response was often greater than that produced during the secondary response to 5-HT. This is probably due to the discrepancy between the two doses administered, and accounts for the differences observed between the incidence of the 5-MEOT response (17/19) and the secondary 5-HT response (8/23).
The overall duration of the response to 5-HT was significantly reduced by 0.5 mg kg−1 ketanserin (P < 0.05, n= 9; see Fig. 4), which resulted from a preferential effect on the secondary response. Ketanserin also significantly reduced the response to 100 μg 5-MEOT by 46 % (P < 0.01, n= 9; see Fig. 7). Thus 5-HT2A receptors play an important role in mediating both the secondary response to 5-HT and the 5-MEOT response. In contrast, granisetron (0.5 mg kg−1) had no significant effect on either the 5-MEOT response or the secondary 5-HT response (see Fig. 3), for which the secondary increase in whole mesenteric afferent firing was 11.7 ± 1.5 impulses s−1 under control conditions and 11.9 ± 1.9 impulses s−1 after granisetron (n= 9).
Response to 5-HT after nifedipine and ω-conotoxin GVIA
The effects of nifedipine (1 mg kg−1, i.v.) or ω-conotoxin GVIA (ω-CTX, 25 μg kg−1, i.v.) on the profile of the response to 5-HT (10 μg, i.v.) were examined in thirteen additional nerve bundles, all of which produced a biphasic increase in afferent discharge in response to the amine (Fig. 8A). In these experiments the lumen was distended with saline to a pressure of approximately 5 cmH2O in order to monitor changes in muscle tone after treatment with the calcium (Ca2+) channel inhibitors and 5-HT. This resulted in a significantly greater baseline afferent discharge than in previous experiments and presumably reflects the activation of mechanosensitive afferents within the mesenteric nerve bundles. Treatment with either nifedipine or ω-CTX induced a significant decrease in blood pressure from 134 ± 7 mmHg to 77 ± 3 mmHg (P < 0.001, n= 5) and from 135 ± 5 mmHg to 68 ± 4 mmHg (P < 0.0001, n= 8), respectively, although only ω-CTX induced a significant change in heart rate (from 403 ± 18 to 336 ± 13 beats min−1, P < 0.0001). Nifedipine had no effect on the peak discharge following administration of 5-HT but significantly reduced the secondary increase in firing (Fig. 8B). The intestinal motor response to 5-HT was similarly reduced by nifedipine (pre-nifedipine, 8.3 ± 1.4 cmH2O; post-nifedipine, 2.6 ± 0.6 cmH2O; P < 0.05, n= 5).
ω-CTX also had no effect on either the mean peak afferent discharge elicited by administration of 5-HT (pre-ω-CTX, 104 ± 14 impulses s−1; post-ω-CTX, 96 ± 13 impulses s−1) or the secondary increase in firing (pre-ω-CTX, 50 ± 7 impulses s−1; post-ω-CTX, 48 ± 8 impulses s−1). Treatment with ω-CTX did, however, reduce the peak 5-HT-evoked pressure increase to a comparable extent to that seen with nifedipine (pre-ω-CTX, 8.7 ± 1.3 cmH2O; post-ω-CTX, 2.7 ± 0.3 cmH2O; P < 0.05, n= 8), suggesting that the motor response to 5-HT involved both direct and indirect components. The dissociation of the motor and secondary afferent response to 5-HT after ω-CTX but not nifedipine may indicate that reflex mechanisms may also serve to modulate afferent sensitivity. In this respect nifedipine reduced both the basal afferent discharge (pre-nifedipine, 73 ± 12 impulses s−1; post-nifedipine, 58 ± 7 impulses s−1; P < 0.05, n= 5) and intrajejunal pressure (pre-nifedipine, 5.3 ± 0.6 cmH2O; post-nifedipine, 2.9 ± 0.2 cmH2O; P < 0.05, n= 5) whereas ω-CTX caused a marked increase in baseline afferent activity (pre-ω-CTX, 45 ± 7 impulses s−1; post-ω-CTX, 67 ± 8 impulses s−1; P < 0.001, n= 8) with no associated change in intrajejunal pressure (pre-ω-CTX, 6.4 ± 0.7 cmH2O; post-ω-CTX, 5.9 ± 0.6 cmH2O). No significant effects on baseline parameters or 5-HT responses were produced by the vehicle for either inhibitor (data not shown).
The results from this study indicate that 5-HT has two distinct effects on mesenteric afferents that can be separated by their pharmacological profiles. Here these two responses are termed the initial and the secondary response to 5-HT. The initial response was mediated entirely by 5-HT3 receptors; the response was mimicked by the 5-HT3 receptor agonist 2Me-5-HT and the response to 5-HT was abolished by granisetron. The presence of 5-HT3 receptors on afferent fibres supplying the gastrointestinal tract and the short latency of the activation suggest that this action of 5-HT may be a direct one on the terminations of afferents within the gut wall. This postulate is further supported by the experiments conducted with the Ca2+ channel inhibitors nifedipine and ω-CTX, since neither agent modified the magnitude of the initial response to 5-HT.
This sensitivity of intestinal afferents to 5-HT raises the intriguing possibility that 5-HT released from enterochromaffin cells is involved in afferent signal transduction. Endogenous 5-HT has been suggested to activate 5-HT1P receptors located on the nerve terminals of submucosal neurons within the lamina propria (Gershon et al. 1994). Stimulation of these nerve endings by 5-HT is suggested to underlie afferent signal transduction for luminal signals including mechanical stimulation and chemosensitivity to cholera toxin. Both stimuli cause c-fos activation in submucosal neurons, which are considered to be intrinsic sensory neurons, and subsequent stimulation of second order neurons in the myenteric plexus (Kirchgessner et al. 1992). The present observation on the sensitivity of mesenteric afferents suggests that 5-HT sensitivity may be shared by both intrinsic and extrinsic afferents but that these different afferent populations may be discriminated on the basis of their sensitivity to 5-HT ligands, with 5-HT1P receptors present on intrinsic afferents and 5-HT3 receptors on extrinsic ones. Since the 5-HT1P receptor is coupled to adenylate cyclase activity while the 5-HT3 receptor is a ligand-gated ion channel (see Hoyer et al. 1994), it appears that intrinsic and extrinsic afferents possess different signal transduction pathways.
2Me-5-HT has been suggested to be an agonist for the 5-HT1P receptor in submucosal neurons (Surprenant & Crist, 1988). However, in the present study, the action of 2Me-5-HT appeared to be specific for the 5-HT3 receptor since it was abolished by prior application of granisetron.
5-HT4 receptors have been demonstrated on vagal afferents at the level of the thoracic nerve trunk and the nodose ganglion (Rhodes et al. 1992; Peters et al. 1992). However, the absence of a response to the 5-HT4 agonist 5-MEOT in the present study would suggest either that 5-HT4 receptors are present on vagal afferents not destined for the small intestine or are present but not functionally linked for impulse generation in this model.
Thus the agonist profile indicates that the initial 5-HT response of mesenteric afferents is solely due to activation of 5-HT3 receptors. However, although the nerves mediating the 5-HT response were clearly stimulated following activation of the 5-HT3 receptors, after granisetron was administered and the 5-HT response was subsequently blocked, the spontaneous activity of these nerves still persisted. Granisetron administration significantly reduced the on-going discharge of 5-HT-sensitive afferents, indicating that in the experimental preparation there was release of endogenous 5-HT which influenced the on-going activity in these afferents. However, spontaneous activity was not completely inhibited by granisetron, suggesting that these remaining afferents are activated by other endogenous stimuli by a pathway not involving the 5-HT3 receptor. Indeed, there may also be endogenous mechanisms which reduce the sensitivity of mesenteric afferents since treatment with ω-CTX resulted in an elevation in baseline afferent discharge. Presumably, on-going activity in enteric reflex circuits or enteroendocrine cells gives rise to mediators which decrease baseline afferent discharge. Whether this activation or inhibition reflects the normal sensitivity of these intestinal afferents or arises as a consequence of the experimental conditions cannot be determined although there were no overt signs of inflammation in these studies. This is particularly relevant to studies in the rat since in this species the mucosal mast cell is a rich source of 5-HT (Enerback & Wingren, 1980).
The secondary response to 5-HT occurred following a longer latency, usually after the initial response had subsided, and was characterized by a prolonged burst of firing that was coincident with an increase in intestinal pressure. Other groups have demonstrated intestinal afferent sensitivity to 5-HT which follows a similar delayed time course (Lew & Longhurst, 1986; Akoev, Filippova & Sherman, 1996) but in the present study we have been able to demonstrate using waveform discrimination of single afferent units that this response to 5-HT is mediated by a different population of afferents from those showing the initial 5-HT response. The pharmacological profile of the secondary response was also different. Administration of 2Me-5-HT up to a dose of 10 μg never elicited a secondary response or an increase in intestinal pressure in these animals, indicating that 5-HT3 receptors are not involved in this secondary excitation. Granisetron similarly had no effect on the secondary response to 5-HT. However, 5-MEOT, especially at high doses, mimicked the secondary increase in afferent firing and the increase in intestinal pressure. Moreover, it seems unlikely, due to the high affinity of 5-MEOT for 5-HT4 receptors, that at this high dose the secondary effect is mediated by activation of 5-HT4 receptors on the nerve terminals. Furthermore, the 5-HT2A receptor antagonist ketanserin significantly reduced both the secondary response to 5-HT and 5-MEOT together with the augmented intestinal contractile activity which generally accompanies the secondary afferent discharge. It is probable, therefore, that the secondary response arises in mechanosensitive afferents as a consequence of the contractile activity evoked by 5-HT and 5-MEOT, a conclusion consistent with the post-junctional location of 5-HT2A receptors on intestinal smooth muscle cells. The observation that the secondary afferent response to 5-HT was markedly attenuated by nifedipine would support this view. Nifedipine, which inhibits L-type Ca2+ channels, caused a reduction in intraluminal pressure and greatly attenuated the contractile response to 5-HT. In association with these events there was a corresponding decrease in baseline afferent discharge and more than 50 % reduction in the secondary afferent response to 5-HT.
The contractile response to 5-HT was also attenuated by the N-type Ca2+ channel inhibitor ω-CTX, which would indicate that nerve-mediated mechanisms contribute to the 5-HT-induced contraction as described in a number of species including the rat (Gershon et al. 1994). Paradoxically, there was no corresponding decrease in the secondary afferent response after ω-CTX. This might argue against the secondary afferent response being linked to a mechanical event as the data with nifedipine clearly show. However, ω-CTX, unlike nifedipine, caused a marked increase in baseline afferent discharge unaccompanied by any change in intraluminal pressure. If reflex mechanisms are capable of modulating mesenteric afferent sensitivity, as these data would suggest, then it is likely that such mechanisms may cause a shift in the relationship between contraction (and the resultant increase in intraluminal pressure) and the activation of mechanosensitive afferents.
In summary, 5-HT stimulates two different populations of mesenteric afferents. The initial effect of 5-HT is characterized by the transient stimulation of 5-HT3 receptors and this is proposed to be a direct action on afferent nerve terminals. The secondary response to 5-HT, however, is a prolonged action mediated predominantly by 5-HT2A receptors. Since this secondary response is attenuated by nifedipine it is probably due to the indirect activation of mechanosensitive afferents following augmented contractile activity.
K. H. was funded by a studentship from the SERC. A. J. K. is a GlaxoWellcome Research Fellow.