Introduction

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The intestinal mucosa has evolved a diverse array of innate and acquired mechanisms to protect the vast surface area it encompasses from infection. In the early stages of infection and tissue injury, the proinflammatory peptide kallidin (lysyl-bradykinin) is produced by the kallikrein-catalyzed cleavage of low molecular weight kininogen (Kaplan et al., 2002). Kallikrein-type proteases have been localized in mast cells and goblet cells along the length of the intestinal tract (Hinterleitner and Powell, 1991). Kallidin and its des-lysyl homolog bradykinin act as agonists at kinin B₁ and B₂ receptors, which are members of the G protein-coupled receptor superfamily (Regoli et al., 2001). The kinin B₂ receptor is constitutively expressed, whereas the B₁ receptor is induced by proinflammatory cytokines released in the course of tissue injury (Couture et al., 2001). These peptides produce pain, vasodilatation, and in the digestive tract, evoke active, transepithelial chloride secretion in the small intestine and colon (Gaginella and Kachur, 1989; Cuthbert and Huxley, 1998) or transepithelial bicarbonate secretion in gallbladder and duodenum (Baird and Margolius, 1989; Chen et al., 1997). The actions of kinins on ion transport are attributed to their combined effects on enteric neurons and non-neuronal cells, including intestinal epithelial cells. Moreover, these peptides can induce the formation of arachidonic acid metabolites that in turn act upon both neurons and enterocytes. For example, kinins activate primary afferent nerves in peripheral tissues, including the small intestine, through direct effects on neurons and the formation of eicosanoids such as prostaglandin E₂ and 12-lipoxygenase metabolites (Maubach and Grundy, 1999; Shin et al., 2002).

Natural and synthetic opioids can alleviate diarrhea and produce constipation, actions that have been attributed in part to their intestinal antipropulsive and antisecretory actions. In most species examined, the latter effect is mediated by inhibitory -opioid receptors expressed in submucosal neuronal circuits that are linked to active anion secretion (DeLuca and Coupar, 1996). Indeed, -opioid receptor immunoreactivity has been colocalized with immunoreactivity to the cholinergic neural marker choline acetyltransferase in submucosal neurons and nerve fibers of the porcine ileum. Subpopulations of these -opioid receptor-positive neurons also express immunoreactivities for the sensory neural markers calcitonin gene-related peptide and vanilloid VR1 receptor (Poonyachoti et al., 2002). In addition, the selective -opioid agonist [D-Pen^2,5]-enkephalin (DPDPE) inhibits active, neurogenic anion secretion mediated by type 2 proteinase-activated receptors, H₁-histamine receptors, and serotonin receptors in muscle-stripped sheets of porcine ileal mucosa (Green et al., 2000; Poonyachoti and Brown, 2001; Green and Brown, 2002). These studies suggest that submucosal -opioid receptors may function to limit neurogenic secretion associated with intestinal inflammation.

In the present investigation, we addressed the hypothesis that kallidin-induced anion secretion, like that evoked by histamine, serotonin, or trypsin, is mediated by opioid-sensitive enteric neural circuits in the porcine ileum. Cannabinoids produce antipropulsive actions in the intestine that are similar to opioids, but their ability to alter intestinal secretion has not been clearly defined (Izzo et al., 2001). Because immunoreactivity for cannabinoid CB₁ receptors has been detected in the porcine submucosal neurons (Kulkarni-Narla and Brown, 2000), it was of additional interest to compare the effects of the cannabinoid agonist HU-210 with those of the selective -opioid agonist DPDPE on kallidin-stimulated neurogenic ion transport in the porcine ileum.

	Materials and Methods

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Drugs and Chemicals. Kallidin, D-arginyl-L-arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-(2,3,7)-octahydro-1H-indole-2-carbonyl-L-arginine (HOE-140), and [des-Arg⁹,Leu⁸]-bradykinin (DALBK) were obtained from Bachem (Torrance, CA). Atropine, bumetanide, carbamylcholine chloride (carbachol), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), histamine, indomethacin, naltrindole, and saxitoxin were obtained from Sigma-Aldrich (St. Louis, MO). DPDPE was purchased from Peninsula Laboratories (Belmont, CA). (6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol (HU-210) was purchased from Tocris Cookson Inc. (Ballwin, MO). HU-210 and indomethacin were solubilized in dimethyl sulfoxide (DMSO); this solvent had no effect on baseline or kallidin-stimulated intestinal ion transport. DPDPE was solubilized in 0.01 M acetic acid with 0.1% bovine serum albumin, aliquoted at stock concentrations of 100 µM, and stored until use at 65°C. All other drugs and reagents were dissolved in distilled water and added to the contraluminal bathing medium unless otherwise noted.

Animals and Tissue Preparation. Intestinal tissues were obtained from Yorkshire pigs (6-10 weeks of age; 10-18 kg b.wt.) of each sex that were not fasted before sacrifice. Animals were sedated with an intramuscular injection of tiletamine hydrochloride-zolazepam (Telazol, 8 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA), in combination with xylazine (8 mg/kg). The animals were subsequently euthanized by barbiturate overdose in accordance with approved University of Minnesota Institutional Animal Care and Use Committee protocols. A midline laparotomy was performed to expose the intestine and a portion of the ileum, identified by its attachment to the ileo-cecal ligament, was removed and placed in an oxygenated organ preservation solution (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 0.5 mM MgCl₂, 25 mM NaHCO₃, 1.0 mM NaH₂PO₄, and 11 mM D-glucose, pH 7.4).

Measurement of Transepithelial Ion Transport. Short-circuit current (I_sc, in microamperes per square centimeter) across each mucosa-submucosal sheet was monitored continuously by an automatic voltage-clamp apparatus (model TR100; JWT Engineering, Overland Park, KS; model EVC-4000, WPI, Sarasota, FL). Experiments were initiated after the basal I_sc had stabilized (approximately 25-35 min). Tissue conductance [G_t, in milliSiemens (mS) per square centimeter] was calculated by Ohm's law from the current change produced by the periodic delivery of a bipolar 5-mV pulse measured throughout each experiment. Data were acquired using a PowerLab data acquisition unit and analyzed with Chart data analysis software (AD Instruments, Grand Junction, CO). Both I_sc and G_t were determined immediately before drug administration and at the peak of drug action. At the end of each experiment, when I_sc had returned to baseline, mucosal I_sc responses to 10 µM carbachol (contraluminal addition) and 10 mM glucose (luminal addition) were measured in each tissue to assess tissue viability.

pH Stat Titration. Tissue sheets were mounted in Ussing chambers under short-circuit conditions and bathed luminally or contraluminally in physiological salt solution and a salt solution with an equimolar substitution of sodium gluconate for sodium bicarbonate and sodium phosphate on the opposite side. The salt solutions were maintained at 39°C and gassed continuously with 5% CO₂ in O₂ or 100% O₂, respectively. In a second set of experiments, tissues bathed with HCO-containing physiological salt solution that was gassed continuously with 5% CO₂ in O₂ and maintained at their individual stable pH baseline (average pH 7.43-7.44) by continuous titration as measured with a PHM-82 pH meter, model TTT-80 titrator, and model ABU-80 autoburette (Radiometer Corp., Copenhagen, Denmark). After the pH baseline had been established, drugs were added to the contraluminal bathing medium and the bathing medium was titrated with 0.005 N HCl for the HCO-substituted salt solution experiments and 0.001 N HCl for the normal physiological salt solution experiments. Changes in luminal or contraluminal pH were monitored and 10 µM carbachol was added to the contraluminal bathing medium at the end of each experiment to determine its action in alkalinizing the bathing medium.

Data Analysis. Data are expressed as mean I_sc or G_t under baseline conditions, or as mean changes in peak I_sc elevations occurring in response to kallidin or other substances. Data from tissues that, at the end of the experimental period, did not respond to carbachol and glucose with elevations in I_sc were omitted. In the case that the tissues serving as controls manifested poor responses to glucose and carbachol, data from all tissues obtained from the donor animal were excluded from further analysis. Statistical analyses of data were performed using the PRISM computer software program (version 3.0; GraphPad Software Inc., San Diego, CA). Comparisons between a control mean and a single treatment mean were made with a two-tailed, paired, or unpaired Student's t test when appropriate. Comparisons of a control mean with multiple treatment means were made by one-way analysis of variance followed by Dunnett's test. In all cases, the limit for statistical significance was set at P < 0.05.

	Results

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Mediators of Kallidin Action. Baseline I_sc and G_t in isolated sheets of ileal mucosa-submucosa averaged 4 ± 3 µA/cm² and 23 ± 1 mS/cm² (n = 224 tissues from 37 pigs). At a contraluminal concentration of 1 µM, kallidin produced a monophasic rise in I_sc that attained a peak elevation of 80 ± 6 µA/cm² relative to mean baseline values (n = 97 tissues from 37 pigs). The duration of kallidin action on I_sc was 11.2 ± 0.5 min (8 tissues from four pigs). The kallidin-induced increase in I_sc was 70% of that produced by 10 µM carbachol (I_sc produced by carbachol = 114 ± 10 µA/cm², n = 65 tissues from 28 pigs). Tissue conductance nearly doubled to 43 ± 3 mS/cm² when determined at the time of peak change in I_sc produced by kallidin (n = 65 tissues from 28 pigs).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Effects of inhibitors on mucosal Isc responses to kallidin. Tissues were pretreated by contraluminal addition of 0.1 µM saxitoxin or atropine, or 10 µM indomethacin before contraluminal addition of 1 µM kallidin. Control tissues were not treated with inhibitors before kallidin addition. Columns represent the mean ± S.E. of peak change in Isc produced by kallidin measured in 26 tissues from 13 pigs that served as controls, 14 tissues from 7 pigs pretreated with saxitoxin, 9 tissues from 6 pigs pretreated with atropine, and 7 tissues from 6 pigs pretreated with indomethacin. *, P < 0.05 versus control mean, Dunnett's test.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Effect of kinin B1 and B2 receptor blockers on mucosal Isc responses to kallidin. Tissues were pretreated by contraluminal addition of 1 µM DALBK or HOE-140 before contraluminal addition of 1 µM kallidin. Control tissues were not treated with blockers before kallidin addition. Columns represent the mean ± S.E. peak change in Isc produced by kallidin measured in six tissues from three pigs that served as controls, five tissues from three pigs pretreated with DALBK, and six tissues from three pigs pretreated with HOE-140. *, P < 0.05 versus control mean, Dunnett's test.

Ionic Basis for Kallidin-Evoked Short-Circuit Current. Previous reports have indicated that kinins induce anion secretion across the intestinal epithelium (Gaginella and Kachur, 1989; Cuthbert and Huxley, 1998). Therefore, tissues were pretreated with bumetanide, a blocker of the Na⁺/K⁺/Cl cotransporter that represents one Cl entry pathway in intestinal epithelial cells that is of importance in active anion secretion. Bumetanide did not significantly change baseline I_sc or G_t after its contraluminal addition at 10 µM. At this concentration, it did not significantly decrease the peak I_sc elevations occurring in response to kallidin (mean I_sc after kallidin in untreated and bumetanide-pretreated tissues = 107 ± 14 and 97 ± 31 µA/cm², respectively; P > 0.05, Student's t test, n = 26 and 5 tissues from 13 and 3 pigs, respectively).

View larger version (7K): [in this window] [in a new window]   Fig. 3.   Dependence of neurogenic and prostanoid components of kallidin-induced Isc elevations on extracellular anions. Responses to 1 µM kallidin (contraluminal addition) were measured in mucosal sheets bathed (top) in Cl-deficient media or (bottom) in HCO-deficient media and either untreated (control) or pretreated with contraluminally administered saxitoxin (STX, 0.1 µM) or indomethacin (INDO, 10 µM). Columns represent the mean ± S.E. peak change in Isc produced by kallidin under chloride-free conditions in 22 tissues from 16 pigs that served as controls, 14 tissues from 6 pigs pretreated with saxitoxin, and 14 tissues from 7 pigs pretreated with indomethacin. Under bicarbonate-free conditions, the peak change in Isc produced by kallidin was measured in 16 tissues from 9 pigs that served as controls, 4 tissues from 4 pigs pretreated with saxitoxin, and 4 tissues from 3 pigs pretreated with indomethacin. *, P < 0.05 versus control mean, Dunnett's test.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Luminal alkalinization produced by kallidin in comparison with that produced by with histamine. The chart records shown are representative tracings of luminal buret pH-stat experiments from tissues treated contraluminally with either 1 µM kallidin (top) or 2 µM histamine (bottom). The kallidin tracing is representative of eight tissues from seven pigs. The histamine tracing is representative of one tissue from each of two pigs; in these two tissues, histamine increased Isc by 52 and 111 µA/cm2. The inset histogram under the kallidin chart record depicts the mean nanomoles ± S.E. of hydrochloric acid delivered into the luminal or contraluminal bathing medium that were necessary to clamp the pH to 7.43 or 7.44, respectively, in tissues treated with kallidin integrated over the period encompassing the peak Isc response to kallidin. Luminal versus contraluminal means determined in experiments using eight tissues from seven pigs and six tissues from six pigs, respectively; *, P < 0.05, Student's unpaired t test.

Effects of Opioid and Cannabinoid Receptor Agonists on Kallidin-Stimulated Ion Transport. The -opioid agonist DPDPE (0.1 µM, contraluminal addition) did not produce significant changes in baseline I_sc or G_t. However, it decreased by 53% the peak I_sc elevation produced by kallidin. Tissues pretreated with both saxitoxin and DPDPE seemed to display an additional decrease in I_sc responses to kallidin. The selective -opioid antagonist naltrindole prevented the inhibitory action of DPDPE (Fig. 5).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Inhibitory actions of the -opioid agonist DPDPE and the nonselective cannabinoid receptor agonist HU-210 on kallidin-induced Isc elevations in porcine ileal mucosa. Responses to kallidin, added to the contraluminal bathing medium at a concentration of 1 µM, were measured in mucosal sheets untreated (control) or pretreated with 0.1 µM DPDPE (contraluminal addition). In some cases, naltrindole (naltrindole/DPDPE) or saxitoxin (saxitoxin/DPDPE) was present in the contraluminal medium at a concentration of 1 or 0.1 µM, respectively, before DPDPE addition. Some tissues were pretreated contraluminally with 1 µM HU-210 before kallidin administration. Control tissues were not treated with drugs before kallidin addition. Columns represent the mean ± S.E. peak change in Isc produced by kallidin measured in 58 tissues from 25 pigs that served as controls, 19 tissues from 9 pigs pretreated with DPDPE, 7 tissues from 3 pigs pretreated with naltrindole and DPDPE, 7 tissues from 3 pigs pretreated with saxitoxin and DPDPE, and 15 tissues from 6 pigs pretreated with HU-210. *, P < 0.05 versus control condition, Dunnett's test.

	Discussion

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Consistent with the results of previous investigations on the intestinal secretory actions of kinins (Gaginella and Kachur, 1989), kallidin transiently increased I_sc in mucosa-submucosa sheets from porcine ileum after its contraluminal administration. This effect was blunted by saxitoxin and indomethacin, providing evidence that it is mediated in part by enteric neurons and cyclooxygenase metabolites, respectively. The enteric neural circuit(s) mediating kallidin-induced secretion does not seem to contain muscarinic cholinergic receptors because of the insensitivity of kallidin action to atropine. This result is in contrast to that obtained by Diener et al. (1988), who reported that atropine decreased significantly mucosal I_sc responses to bradykinin in the rat distal colon. However, our previous studies in porcine ileal mucosa that examined the secretory effects of other inflammatory mediators, including serotonin (Green and Brown, 2002), proteinases (Green et al., 2000), and histamine (Poonyachoti and Brown, 2001), showed a similar pattern of atropine resistance. These results in combination suggest that proinflammatory mediators stimulate active anion secretion in the porcine intestine through a neural mechanism that does not involve cholinergic secretomotor neurons. Kinin B₂ receptors seem to mediate the effect of kallidin on transepithelial ion transport, because kallidin-induced elevations in I_sc were sensitive to the selective B₂ receptor blocker HOE-140, but not to the kinin B₁ antagonist DALBK. This result again is consistent with studies in other intestinal segments and in other mammalian species as well as in intestinal tissues from B₂ receptor knockout mice, which demonstrate that kinin B₂ receptors mediate the secretory effects of kinins (Gaginella and Kachur, 1989; Cuthbert and Huxley, 1998). The present results show that the porcine ileal mucosa seems to be generally similar to analogous intestinal preparations from other species with respect to the kinin receptor type and the neural and eicosanoid influences contributing to kallidin activity in the intestinal mucosa.

The kallidin-induced elevation in I_sc across the porcine ileal epithelium bathed in media containing Cl and HCO ions was not sensitive to the loop diuretic bumetanide, which blocks Cl loading in enterocytes through the Na⁺/K⁺/Cl cotransporter. However, the related loop diuretics furosemide and piretanide have been shown to decrease the actions of kallidin in the mouse and rat intestine (Cuthbert and Margolius, 1982; Cuthbert, 2001). Sensitivity of mucosal I_sc responses to loop diuretics has been taken as presumptive, albeit indirect evidence that active transport of the chloride anion is responsible for the transient I_sc elevations induced by kallidin (Cuthbert and Huxley, 1998). Anion substitution experiments were undertaken to examine in further detail the contributions of the major extracellular anions Cl and HCO to the kallidin-evoked change in I_sc. Kallidin action was reduced by similar magnitudes in tissues bathed in media deficient in either Cl or HCO ions, indicating that it is dependent on both extracellular anions. From the results of these experiments, we propose that kallidin stimulates bumetanide-insensitive, anion-dependent secretion in porcine ileum.

Saxitoxin decreased I_sc responses to kallidin in tissues bathed in Cl-deficient media, but had no significant effect on responses to kallidin in tissues bathed in HCO-free media. Serotonin-evoked changes in I_sc in porcine ileal mucosa sheets bathed in anion-deficient media exhibit a similar pattern of saxitoxin sensitivity (Green and Brown, 2002). The results suggest that neurogenic ion transport induced by kallidin is dependent on extracellular HCO ions. Indomethacin markedly attenuated mucosal I_sc responses to kallidin in tissues bathed in media deficient in either Cl or HCO ions, and we hypothesize that both the neurogenic and non-neurogenic components of kallidin action are dependent on the formation of cyclooxygenase metabolites.

Because the neurogenic actions of kallidin seemed to involve electrogenic HCO secretion, we examined the effects of DIDS at a relatively high luminal concentration on mucosal responses to the peptide. DIDS has been shown to inhibit epithelial HCO transport mediated by both Na⁺-coupled HCO transporters and Cl/HCO exchangers (Boron, 2001). Because DIDS did not significantly alter mucosal I_sc responses to kallidin, it is possible that neither mode of HCO transport contributes to the effects of the peptide. Alternatively, DIDS-resistant HCO transport mechanisms have been reported to exist (Tsuruoka and Schwartz, 1999; Luo et al., 2001). To further clarify a role for HCO secretion in the action of kallidin, the ability of this peptide to alkalinize the luminal medium bathing mucosal sheets was examined through pH-stat titration experiments. Tissues bathed either luminally or contraluminally in HCO-free media and gassed with 100% O₂ sustained a baseline rate of alkalinization into the opposite medium, but kallidin did not evoke an increase in alkalinization rate. On the other hand, kallidin produced a rapid increase in luminal alkalinization and a smaller increase in contraluminal alkalinization in tissues bathed on both sides with HCO-containing media and gassed with carbogen. On the basis of these data, we speculate that kallidin stimulates a transcellular anion transport pathway across the ileal epithelium that delivers HCO ions into the luminal bathing medium in excess of the basal paracellular HCO flux. Carbachol but not histamine evoked luminal alkalinization as well. We have previously reported that carbachol increases luminal alkalinization in porcine ileum (Chandan et al., 1991). Kinins have been found to evoke active HCO secretion in a few other gastrointestinal preparations in vitro. For example, luminal addition of bradykinin to the guinea pig gallbladder mucosa produced increases in I_sc that were abolished in HCO-deficient media (Baird and Margolius, 1989). Moreover, bradykinin increases luminal alkalinization in rat duodenum through an action on kinin B₂ receptors (Chen et al., 1997). The present results suggest that kallidin stimulates HCO-dependent, electrogenic ion transport in the porcine ileum as well.

Enteric -like opioid receptors seem to modulate electrogenic ion transport evoked by transmural stimulation of submucosal neurons in the porcine ileum (Poonyachoti et al., 2001). Moreover, I_sc elevations produced by either trypsin, histamine, serotonin, or an immediate hypersensitivity reaction to a food allergen in the porcine ileal mucosa are attenuated by the selective -opioid agonist DPDPE (Green et al., 2000; Poonyachoti and Brown, 2001; Green and Brown, 2002). We tested the hypothesis that -opioid receptors are expressed in a common enteric neuronal circuit that mediates secretory responses to inflammatory mediators, including kallidin. In support of this hypothesis, DPDPE markedly attenuated mucosal I_sc responses to kallidin, and its effects were prevented by the selective -opioid antagonist naltrindole. When tissues were pretreated with both saxitoxin and DPDPE, there seemed to be a small additional decrease in kallidin action. This may be due to a minor action of kallidin on additional enteric neural pathways that do not express -opioid receptors. In previous studies, the combination of the neurotoxin and DPDPE was without any additional effect on mucosal I_sc responses to histamine or serotonin (Poonyachoti and Brown, 2001; Green and Brown, 2002). Based on this result and our previous data, we postulate that the indomethacin-sensitive portion of HCO-dependent, neurogenic ion transport stimulated by kallidin is mediated by an enteric neural circuit that contains inhibitory -opioid receptors. The potent cannabinoid agonist HU-210, like DPDPE, decreased mucosal I_sc responses to kallidin. Unlike DPDPE however, HU-210 does not alter I_sc elevations in sheets of porcine ileal mucosa that are evoked by serotonin or transmural electrical stimulation (Green and Brown, 2002; S. Poonyachoti, H. Albasan, and D.R. Brown, unpublished data). Thus, cannabinoids may act on a more circumscribed enteric neural pathway that contains cannabinoid CB₁ and kinin B₂ receptors, and possibly -opioid receptors as well. Indeed, some myenteric neurons from porcine ileum maintained in primary culture manifest immunoreactivity for both -opioid and cannabinoid CB₁ receptors (Kulkarni-Narla and Brown, 2001). Cannabinoid receptor agonists have been found previously to decrease mucosal ion transport in rat intestine evoked by electrical stimulation in vitro (Tyler et al., 2000) and intestinal fluid accumulation in intact rats (Izzo et al., 1999). A role for cannabinoids and cannabinoid receptors in the antisecretory actions of HU-210 must await confirmation with future studies using cannabinoid antagonists and additional selective agonists.

Proinflammatory kinins seem to stimulate neurogenic HCO secretion in porcine ileum by activating enteric neural circuits expressing inhibitory opioid and possibly cannabinoid receptors. The active secretion of HCO ions in the duodenum and colon is an important contributor to luminal pH in these intestinal segments (Seidler et al., 2000), and HCO transport induced by kallidin or other inflammatory mediators may have a similar role in the ileum. Bicarbonate transport is also linked to absorption of short-chain fatty acids, which seem to alleviate intestinal inflammation; chronic ileitis is associated with a reduction in short-chain fatty acid/HCO exchange (Manokas et al., 2000). Bicarbonate concentrations in the intestinal lumen may influence cytoplasmic pH regulation in phagocytic cells extruded from the epithelium and thus indirectly modulate their defensive functions (Grinstein et al., 1991). Luminal HCO ions also stimulate the expression of bacterial gene products mediating attachment and effacement of enterohemorrhagic Escherichia coli on epithelial cells (Kenny et al., 1997; Abe et al., 2002). By acting through the enteric nervous system to alter bicarbonate secretion induced by kallidin and other inflammatory mediators, opioids and possibly cannabinoids could alter interactions between the intestinal mucosa and enteropathogenic microorganisms.