Gastrointestinal Diseases Research Unit, Departments of Medicine and Physiology, Queen's University, Kingston, Ontario, Canada K7L 5G2
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ABSTRACT |
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This study examined the neural pathways innervating Brunner's glands using a novel in vitro model of acinar secretion from Brunner's glands in submucosal preparations from the guinea pig duodenum. Neural pathways were activated by focal electrical stimulation and excitatory agonists, and videomicroscopy was used to monitor dilation of acinar lumen. Electrical stimulation of perivascular nerves evoked large dilations that were blocked by TTX (1 µM) or the muscarinic receptor antagonist 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (1 µM). The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (100 µM) had no effect, and the nerve-evoked responses were not inhibited by hexamethonium (200 µM). Dilations were abolished in preparations from chronically vagotomized animals. Activation of submucosal ganglia significantly dilated submucosal arterioles but not Brunner's glands. Effects of electrical stimulation of perivascular and submucosal nerves were not altered by guanethidine. Capsaicin and substance P also dilated arterioles but had no effect on Brunner's glands. Cholinergic (choline acetyltransferase-immunoreactive) nerve fibers were found in Brunner's glands. These findings demonstrate that Brunner's glands are innervated by cholinergic vagal fibers but not by capsaicin-sensitive or intrinsic enteric nerves.
cholinergic innervation; submucosal plexus; vagus; capsaicin-sensitive nerves
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INTRODUCTION |
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THE SECRETION OF MUCIN and bicarbonate from the epithelium and Brunner's glands into the duodenal lumen is thought to play an important role in mucosal defense against gastric acid and proteolytic enzymes (8). The concept that neural reflexes were important in mediating these responses was recognized in the 1940s when it was observed that electrical stimulation of the vagus caused the release of sticky, alkaline fluid into the proximal duodenum of cats in vivo (35) and in the 1960s when the activation of cephalic reflexes by sham feeding caused a similar secretion in dogs (26). It was not clear, however, whether these secretions were derived from the epithelium, Brunner's glands, or both.
Since these early studies (26, 35), considerably more research has focused on duodenal epithelial secretions because, compared with Brunner's glands, the epithelium is much more accessible. Studies that examined regions of the duodenum devoid of Brunner's glands have demonstrated that bicarbonate secretion is regulated by both extrinsic and intrinsic pathways. These include vago-vagal reflexes (2, 7, 15, 16, 23), reflexes within the submucosal plexus of the enteric nervous system (12), C fiber efferent mechanisms (11, 29), and inputs from the sympathetic nervous system (5, 6, 22). The interactions between these neural pathways have also been studied, and the findings suggest that secretomotor reflexes within the submucosal plexus are modified by vagal and sympathetic efferents that provide excitatory and inhibitory inputs, respectively, to submucosal neurons (6,22). These studies demonstrate that neural regulation of epithelial bicarbonate and mucin secretion from intestinal epithelium involves a complex interplay between extrinsic autonomic nerves and intrinsic secretomotor neurons in the submucosal plexus.
There is considerably less known about the neural pathways mediating Brunner's gland secretion. Early studies (35) used the histological identification of mucin glycoproteins to examine the secretory status of the glands after experimentation. It was observed (35) that prolonged stimulation of the vagus leads to depletion of mucin glycoprotein from Brunner's glands in some animals. It was not clear whether secretion was mediated solely by extrinsic neurons or whether neural pathways located within the enteric nervous system were also involved. This lack of understanding persists because it is not possible to separate the neural elements controlling glandular secretion from those of the epithelium in vivo.
We (18) recently established an in vitro model of Brunner's gland secretion and demonstrated that the cholinomimetic carbachol acts directly on acinar cells in guinea pig duodenum to stimulate mucin secretion, suggesting these glands receive cholinergic neural inputs. The current study employs this model and uses focal electrical stimulation and excitatory agonists to selectively activate neural pathways. The aim of the study was to determine whether Brunner's glands receive functional innervation and, if so, whether the neural pathways regulating secretion are mediated by extrinsic and/or enteric nerves.
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METHODS |
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Guinea pigs (140-225 g) of either sex were obtained from Charles River Laboratories. Experiments were performed according to the guidelines of the Canadian Council of Animal Care. Animals were anesthetized with isoflurane and immediately killed by decapitation. The abdomen was opened, and the entire duodenum was removed from a point ~1 cm distal to the pyloric junction to the ligament of Treitz.
Preparation of Tissue
The proximal duodenum was opened along the mesenteric border and pinned flat in Sylgaard-lined petri dishes with the mucosal surface facing upward. The tissue was covered with Krebs buffer (in mM: 126 NaCl, 2.5 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 5 KCl, 25 NaHCO3, and 11 glucose) at room temperature, equilibrated with 95% O2-5% CO2. Submucosal preparations containing Brunner's glands were dissected as previously described (34) for guinea pig submucosal ileal preparations. Briefly, the mucosa was dissected to expose the submucosa, which was then dissected from the underlying muscle. Preparations (0.5-1 cm2) were maintained under continuous oxygenation at room temperature, and all preparations were used for experimental study within 4 h of dissection.Chronic Subdiaphragmatic Vagotomy
Guinea pigs (200-250 g) were anesthetized, a laparotomy was performed, and the left and right vagal trunks were visualized just beneath the diaphragm. The vagi were completely severed, and animals were allowed to recover for 7 to 10 days so that distal vagal fibers would degenerate.Experimental Protocol
Submucosal preparations were positioned with the mucosal surface upward in small organ baths (1-2 ml) and continuously superfused at 15 ml/min with oxygenated Krebs buffer warmed to 36 ± 0.5°C (Fig. 1). Videomicroscopy was used to monitor changes in the diameter of the acinus lumen to provide a measure of secretion, as previously described (18). This system was originally developed for in vitro monitoring of changes in blood vessel diameter (20). Briefly, the video camera captures the image seen through the microscope for analysis by computer-driven imaging software (Diamtrak, T.O. Neild). The image is converted to gray scale and displayed on a video monitor. Cursors are superimposed on the image and centered across the acinus lumen, which is visualized as parallel dark lines by the software (Fig. 1B, inset) (18). Our previous studies (18) have shown that exogenous application of secretogogues evokes mucin secretion from acinar cells and results in a concentration-dependent dilation of the lumen. Dilation causes the parallel lines to move apart, and the distance between the lines is tracked by the cursors and recorded on a chart recorder. The resolution of this system is ~1 µm, and the sampling rate is 15 Hz. Data were expressed as the percentage of the maximum response to 10 µM carbachol. All drugs were added by superfusion.
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Glass focal electrodes (tip diam, 20-50 µm) were used to
stimulate submucosal ganglia or perivascular nerves (Fig.
1B). Perivascular nerves were stimulated with electrodes
placed on first- or second-order branches of parent arterioles (Fig.
1). Extrinsic nerve fibers enter the submucosa within the perivascular
connective tissue of submucosal arterioles (9). In
preliminary studies, optimum stimulus parameters were studied by
examining frequency (1-20 Hz), pulse duration (1-10 ms), and
train durations up to 30 s. Maximal dilations, which recovered to
baseline (Fig. 2), were obtained with
20-40 V, 10 Hz, 5-ms pulse, and 30-s train duration, and these
parameters were used in all subsequent studies. Our (18)
previous studies demonstrated histologically and by electron microscopy
that the dilations evoked by exogenous application of the cholinergic
agonist carbachol result from mucin secretion from acinar cells into
the acinar lumen.
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Preliminary studies were also conducted to establish the general distribution of Brunner's glands, which consistently dilated in response to perivascular stimulation. Dilations of large numbers of Brunner's glands could be directly observed through the eyepiece of the microscope (×200 magnification), and a calibrated micrometer eyepiece was used to measure distances between the stimulus electrode and Brunner's glands. Dilations were observed in Brunner's glands on both sides of the stimulated arteriole up to 0.3-0.5 cm distally from the point of stimulus (i.e., toward the antimesenteric border). Dilated acini were rarely observed proximal to (i.e., toward the mesenteric border) or adjacent to the stimulating electrode. Consequently, in all subsequent experiments, single acini chosen for monitoring by videomicroscopy were located within 1-1.5 mm distal to the point of stimulation of the perivascular nerves.
Submucosal ganglia were stimulated by placing the glass stimulating electrode directly on ganglia adjacent to Brunner's glands. Previous studies (21, 33) have shown that stimulation at 20 Hz for several seconds is sufficient to activate large numbers of submucosal motoneurons and evoke effector responses. In the current study, ganglia were stimulated at 20 Hz with 0.7-ms pulse duration for a 30-s train. In a few experiments, intracellular recordings were made from submucosal neurons using standard 2 M KCl glass electrodes. Synaptic inputs were elicited using bipolar tungsten electrodes (20 Hz, 0.8-ms pulse duration, 400-ms train duration) positioned on adjacent fiber tracts running to the ganglia.
In some experiments, the videomicroscopy system (Diamtrak) was also
employed to monitor dilations of duodenal submucosal arterioles, as
previously described (20, 21, 33). These studies were conducted to demonstrate that chemical and electrical stimuli were
sufficient to activate enteric neurons and capsaicin-sensitive nerves.
Vasodilations were examined by first constricting the vessels to
80-95% of the maximum with PGF2 (400 nM); maximal constriction of these vessels causes complete occlusion of the lumen
(21,33).
Immunohistochemistry
The distribution of a goat polyclonal antiserum directed against the biosynthetic enzyme choline acetyltransferase (ChAT; 1:300, Bio/Can Scientific) was examined in whole mount preparations of guinea pig duodenal submucosa. Tissues were fixed in Zamboni's fixative (2% paraformaldehyde and 15% picric acid in 0.2 M sodium phosphate buffer), and ChAT immunoreactivity was visualized using a donkey anti-goat secondary antibody conjugated to Cy3 (1:500, Bio/Can Scientific). Whole mounts were viewed on an argon laser scanning confocal microscope (Olympus IMT2, Meridian Insight) at an excitation wavelength of 514 nm. Images of Cy3-labeled profiles were obtained using up to 20 serial optical sections (z-sectioning, 4 µm/section) that were captured using MCID-M4 imaging software (Medical Imaging Research) and electronically superimposed. When ChAT antibody was preabsorbed with ChAT (Sigma Chemical), no immunoreactivity was observed.Statistical Analysis
Results are expressed as means ± SE. Data were compared using the two-tailed Student's t-test for paired values or by ANOVA followed by Tukey's post-test where appropriate. P < 0.05 was considered significant.Materials
Carbachol was purchased from Aldrich, and TTX, hexamethonium, guanethidine, capsaicin, PGF2 ![]() |
RESULTS |
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Electrical Stimulation of Perivascular Nerves and Submucosal Ganglia
Perivascular nerves. Stimulation of perivascular nerve fibers elicited large dilations (Fig. 2A; n = 6) that averaged 47 ± 6% of the maximum response to superfusion of 10 µM carbachol. These dilations typically returned to baseline within 30-60 s after the stimulus train. After a 10-min waiting period, reproducible responses could be obtained two to three times with repeated stimulation. After pretreatment of the preparation for 3 min with TTX (1 µM; n = 4), a blocker of sodium-dependent action potentials, electrical stimulation failed to elicit detectable luminal dilations (Fig. 2A). After a 10-min washout of TTX, dilations could be evoked with perivascular nerve stimulation.
Submucosal ganglia. Submucosal neurons were stimulated (20 V, 20 Hz, 0.7-ms pulse duration for 30s) with electrodes placed on ganglia located adjacent to the Brunner's gland selected for videomicroscopy monitoring. Stimulation failed to elicit any dilation of Brunner's gland acini (Fig. 2B) in 70% of the submucosal neurons (5 of 7 neurons) and only very small dilations (<10% of maximum) in the other 30% (2 of 7 neurons). Previous studies (21) in the guinea pig ileum have demonstrated that electrical stimulation of submucosal ganglia activates submucosal vasodilator neurons that innervate submucosal arterioles. In the current study, nerve-evoked dilations of submucosal arterioles in the Brunner's glands preparations in the duodenum (Fig. 1) were examined to directly demonstrate that the stimulus parameters were adequate to activate submucosal motoneurons and evoke an effector response. Stimulation of submucosal ganglia in the duodenum, using the same parameters examined for Brunner's gland secretion (20 V, 20 Hz, 0.7-ms pulse duration for 30 s) evoked large dilations of adjacent submucosal arterioles (Fig. 2B; n = 3).
Inhibition of Sympathetic Neurotransmitter Release
Perivascular nerve stimulation activates sympathetic nerve fibers and has been shown to release neurotransmitters in the submucosal plexus causing pre- and postsynaptic inhibition (28). The possibility that these actions might have masked excitatory actions in other nerves was studied by comparing control responses with those obtained with guanethidine (10 µM) in the bath. Guanethidine blocks neurotransmitter release from sympathetic nerve terminals (28). Control responses elicited by electrical stimulation of perivascular nerves were not significantly different from those obtained with guanethidine (10 µM) superfused for 3 min into the bath (Fig. 3; n = 8). Similarly, no difference was found with 30 µM guanethidine (control, 63 ± 10.5% vs. guanethidine, 59 ± 7.9%; n = 3). The finding that stimulation of submucosal ganglia did not elicit significant dilations (see Submucosal ganglia) was also not altered when guanethidine (10 µM) was in the bath (n = 8). However, guanethidine (10 µM) completely blocked inhibitory postsynaptic potentials (mean amplitude, 17 ± 4 mV) recorded intracellularly in submucosal neurons (n = 3). These synaptic potentials were elicited by electrical fiber tract stimulation (20 Hz, 0.8-ms pulse duration, 400-ms train). In the same preparations in which the effects of submucosal ganglia stimulation on Brunner's gland dilation were assessed, typical large dilations (Fig. 3; n = 5) were evoked when the electrode was repositioned on the perivascular nerves.
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Effects of Cholinergic Agonists and Antagonists
Pretreatment of preparations with the nicotinic receptor antagonist hexamethonium (200 µM; n = 7) for 3 min had no effect on dilations of glandular acini elicited by stimulation of perivascular nerves, whereas dilations were almost completely blocked by the muscarinic receptor antagonist 4-DAMP (1 µM; n = 7) (Fig. 4A). The activation of submucosal neurons by superfusion of the nicotinic receptor agonist DMPP (100 µM; n = 4) failed to stimulate Brunner's gland acinar dilation. The effects of DMPP on dilation of submucosal arterioles were examined to establish that DMPP was an adequate stimulus to activate submucosal motoneurons and elicit an effector response. Superfusion of DMPP (100 µM; n = 4) evoked large dilations of submucosal arterioles (Fig. 4B). These responses were blocked by TTX (1 µM; n = 3), demonstrating that the site of action of DMPP was nicotinic receptors on submucosal vasodilator neurons (21, 32, 33).
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Effects of Subdiaphragmatic Vagotomy
The possibility that acinar dilation elicited by stimulation of perivascular nerve fibers was mediated by vagal fibers was assessed by studying the effects of subdiaphragmatic vagotomy. In vagotomized animals, stimulus-evoked dilations were almost completely abolished (95%) (Fig. 5; n = 9) compared with controls (n = 5).
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Effects of Capsaicin and Substance P
Nerve terminals of extrinsic sensory afferent neurons can release peptides and play a local effector role in the intestine (13). A potential role for this sensory efferent process in the regulation of Brunner's gland secretion was examined by studying the actions of capsaicin, a selective neurochemical probe, and the putative neurotransmitter substance P (13, 30). Our previous studies (31), in the guinea pig ileum, have shown that capsaicin (2 µM) selectively activates capsaicin-sensitive nerves with ensuing release of neurotransmitter. In the current study, superfusion of capsaicin (2 µM; n = 5) or substance P (30 or 100 nM; n = 4 for each concentration) had no effect on the luminal diameter of Brunner's gland acini (Fig. 6A). In the same preparations, maximal dilations were recorded in response to 10 µM carbachol. The effects of capsaicin on dilation of duodenal submucosal arterioles in the Brunner's gland preparation were examined to establish that capsaicin is a sufficient stimulus to activate extrinsic sensory nerves and evoke an effector response. Both capsaicin (600 nM; n = 3) and substance P (30 nM; n = 3) evoked large arteriolar dilations, similar to those described in the ileum (30, 31).
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ChAT Immunoreactivity in Brunner's Glands
Previous studies (10) have demonstrated dense projections of ChAT-immunoreactive and cholinesterase-positive nerve fibers within the guinea pig duodenal submucosal plexus. In the present study, confocal microscopy was used to examine the distribution of ChAT-immunoreactive nerves in Brunner's glands. Numerous ChAT-immunoreactive fibers were evident within the periglandular connective tissue of Brunner's glands and between individual acini (Fig. 7; n = 3 animals). Immunoreactivity was also localized within cell bodies and processes of a subpopulation of submucosal neurons and on nerve fibers within the perivascular connective tissue of submucosal arterioles (Fig. 7), as previously described (10).
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DISCUSSION |
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This study employed a novel in vitro model of Brunner's gland secretion that enabled intrinsic and extrinsic neural pathways to be selectively activated and their role in the regulation of glandular secretion to be examined. We (18) have previously shown that cholinomimetics are potent activators of Brunner's gland secretion in this model. These agents acted directly on the acinar cell to stimulate concentration-dependent exocytosis of mucin-containing secretory granules, suggesting these glands may receive cholinergic innervation. In the current study, ChAT-immunoreactive nerve fibers were found in close association with Brunner's glands, and studies of perivascular electrical stimulation provided direct evidence that Brunner's glands receive functional cholinergic innervation.
Several lines of evidence demonstrated that electrically evoked dilations were mediated by cholinergic neural mechanisms directly activating acinar cells. The neural origin of the responses was confirmed by demonstrating that TTX, the selective blocker of sodium-dependent action potentials, completely abolished all responses. Dilations were also blocked by the muscarinic receptor antagonist 4-DAMP. Our (18) previous study showed that carbachol-evoked dilations were also blocked by 4-DAMP but not by TTX, demonstrating that cholinergic responses could be mediated by muscarinic receptors on the acinar cell. Taken together, these data suggest that electrically evoked dilations observed in the current study are mediated by the release of ACh from nerve terminals innervating Brunner's glands with the ensuing activation of muscarinic receptors on the acinar cell. Further studies are required to determine if this secretion results from the acute release of preformed granules or whether there is continuous secretion in the presence of ongoing stimulation.
Two cholinergic pathways, involving extrinsic autonomic nerves and/or intrinsic enteric nerves, were likely candidates for the cholinergic-mediated dilations observed in this study. Vagal efferents have been implicated in previous in vivo studies that have shown vagal stimulation causes depletion of Brunner's gland mucin (35), although it was unclear whether this action resulted from direct innervation of the glands or was mediated through activation of submucosal secretomotor neurons. A role for submucosal secretomotor neurons has also been suggested (10), because submucosal ganglia are closely associated with Brunner's glands (Fig. 1), there are dense projections of cholinergic nerves around the glands (Fig. 7), and submucosal secretomotor neurons stimulate epithelial mucin and electrolyte secretion (3,19). In the current study, we provided direct evidence that the cholinergic perivascular fibers were vagal in origin, because these responses were abolished in animals that had been surgically vagotomized (see Fig. 5). The possibility that these vagally evoked responses were mediated through the activation of submucosal secretomotor neurons or, alternatively, that there was a separate secretomotor pathway mediated by submucosal secretomotor neurons, was examined by selective activation of these neurons both electrically and with exogenous application of the nicotinic receptor agonist DMPP. Neither of these stimuli had a significant effect on Brunner's gland secretion. In a small number of preparations, very small dilations (<10% maximum) were elicited by electrical stimulation. Given the lack of effect of DMPP and the small size of the dilations, these responses most likely represent activation of vagal fibers passing "en passe" or in close proximity to the ganglia. However, both DMPP stimulation and electrical stimulation appear to be adequate to activate submucosal motoneurons (see Figs. 2 and 4), because we were able to demonstrate in the same preparation that they evoke large dilations of submucosal arterioles. In addition, the vagally evoked Brunner's gland dilations were also hexamethonium insensitive, providing further evidence that submucosal neurons did not participate in these actions. These data suggest that vagal fibers directly innervate duodenal Brunner's glands and that submucosal secretomotor neurons do not play a significant role in the innervation of these glands.
Sympathetic nerve fibers also enter the submucosa via the perivascular connective tissue where they project to multiple effector systems, including enteric neurons and the submucosal arterioles (9). There is evidence, at least in some species, that they may also project to Brunner's glands (27) and that activation of the splanchnic nerves can inhibit Brunner's gland secretion (24). In the current study, activation of perivascular sympathetic nerves could potentially inhibit mucin secretion by release of inhibitory neurotransmitter onto enteric secretomotor neurons (28) and/or pre- or postsynaptic inhibition at the acinar cell, potentially masking simultaneous activation of additional secretomotor pathways. However, we found no evidence for such actions, because the responses to perivascular stimulation and submucosal ganglia stimulation were not altered when guanethidine, which blocks release of sympathetic neurotransmitter(s) (4), was added to the bath.
Several noncholinergic neural pathways have also been proposed to activate Brunner's glands. Previous studies (14), including our (18) own, have shown that exogenous application of vasoactive intestinal polypeptide (VIP) stimulates mucin secretion from Brunner's glands in this model and in the rat and that VIP-immunoreactive fibers form a dense network around the acini of Brunner's glands in the rat. VIP immunoreactivity has also been found in vagal nerve fibers (17) and in enteric secretomotor neurons (10), implying both of these neural pathways could be a source of this putative neurotransmitter. Despite these findings, we found no evidence for a significant noncholinergic component to the electrically evoked Brunner's gland dilations observed in this study. Another noncholinergic effector system that has been suggested to innervate Brunner's glands was capsaicin-sensitive sensory nerves. These nerves release neuropeptides, such as substance P and calcitonin gene-related peptide (30), from nerve terminals within the intestine, which evokes multiple effector responses (13), including duodenal bicarbonate secretion (29) and mucosal blood flow (1). Capsaicin is a selective neurochemical probe for activation of these nerves but failed to stimulate Brunner's gland secretion in our study. Similarly, the putative neurotransmitter substance P had no effect. Both capsaicin and substance P appear to be adequate stimuli to evoke effector responses in this model, because we were able to demonstrate that they can dilate submucosal arterioles in the same preparation. (see Fig. 6). Consequently, despite the potential role for several noncholinergic neural pathways to modulate Brunner's gland secretion, there is little evidence that they play a direct role in regulating secretion in this model.
In summary, this study provides direct evidence that Brunner's glands in the guinea pig duodenum are functionally innervated by cholinergic nerves but not by noncholinergic and capsaicin-sensitive nerves. This cholinergic neural pathway is mediated by vagal fibers within the duodenum that directly innervate the Brunner's gland. Unlike other secretory glands (25), in which vagal fibers activate intrinsic secretomotor neurons in periglandular ganglia, secretomotor neurons in periglandular duodenal submucosal ganglia played no role in stimulating adjacent Brunner's glands.
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ACKNOWLEDGEMENTS |
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This study was supported by a grant from the Medical Research Council of Canada (MRC) (S. Vanner) and the MRC and Pharmaceutical Manufacturer's Association of Canada Health Program Fellowship Award with the Canadian Association of Gastroenterology (CAG)/Astra (B. A. Moore). David Kim was supported by a summer studentship from the CAG.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Vanner, 166 Brock St., Hotel Dieu Hospital, Kingston, Ontario, Canada K6L 5G2 (E-mail: vanners{at}hdh.kari.net).
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.
Received 9 November 1999; accepted in final form 25 April 2000.
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