Division of Gastroenterology, Department of Internal Medicine, University of Arkansas for Medical Sciences and John L. McClellan Memorial Veterans Affairs Hospital, Little Rock, Arkansas 72205-7199
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ABSTRACT |
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Although bile acids damage gastric mucosa, the mechanisms underlying tissue injury induced by these agents are not well understood. To determine whether bile acids alter gastric secretory function, we investigated the actions of sodium cholate, deoxycholate, lithocholate, and their taurine and glycine conjugates on a highly homogeneous population of gastric chief cells. Lithocholyltaurine (LCT), a particularly injurious bile acid, caused a threefold increase in pepsinogen secretion (detectable with 100 nM and maximal with 10 µM LCT). When combined with other secretagogues, increasing concentrations of LCT caused progressive inhibition of carbamylcholine (carbachol)-induced pepsinogen secretion but did not alter CCK- or 8-bromo-cAMP-induced secretion. Taurine and unconjugated lithocholate did not alter basal or carbachol-induced secretion. These observations suggested that LCT is a partial cholinergic agonist. To test this hypothesis, we examined the actions of the cholinergic antagonist atropine on LCT-induced pepsinogen secretion. Atropine (10 µM) abolished carbachol- and LCT-induced pepsinogen secretion. Likewise, carbachol (0.1 mM) and LCT (1 mM) induced an atropine-sensitive, two- to threefold increase in cellular levels of inositol 1,4,5-trisphosphate. We examined the actions of LCT on binding of the cholinergic radioligand [N-methyl-3H]scopolamine ([3H]NMS) to chief cells. Half-maximal inhibition of [3H]NMS binding was observed with ~0.5 mM carbachol and 1 mM LCT. These results indicate that the bile acid LCT is a partial agonist for muscarinic cholinergic receptors on gastric chief cells.
pepsinogen; secretion
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INTRODUCTION |
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BILE ACIDS, which comprise ~85% of biliary solids, play a role in several important physiological processes, including bile formation, the regulation of cholesterol synthesis and elimination, the formation of micelles that promote lipid solubilization and absorption from the gut, and modulation of normal colonic function.1 Anwer et al. (2) reported that in isolated rat hepatocytes bile acids caused a rapid (10-30 s), dose-dependent (0.05-1.0 mM) increase in cytosolic calcium to levels similar to those achieved by other agonists. The rank order for potency of these bile acids was lithocholate = lithocholyltaurine (LCT) > chenodeoxycholate = chenodeoxycholyltaurine = deoxycholate = deoxycholyltaurine (2). Although in this study (2) the actions of bile acids, particularly taurine conjugates, appeared to depend on extracellular calcium, other investigators working in the same area argued, with supporting evidence, that preincubation with EGTA to chelate extracellular calcium also depletes intracellular calcium stores (7-10). They proposed that LCT, LCT sulfate, and lithocholate caused release of calcium from an inositol phosphate-sensitive intracellular store by selective permeabilization of the endoplasmic reticulum. Nevertheless, both groups of investigators hypothesized that the actions of bile acids on cellular calcium in hepatocytes were mediated by specific ionophoretic actions of these hydrophobic agents on cellular membranes, whether it is the plasma membrane or the endoplasmic reticulum (38).
Actions of bile acids on cellular calcium concentration are cell specific. In addition to hepatocytes, similar effects have been reported for cultured kidney cells (LLC-PK1) (18) but not for human platelets or a neuroblastoma cell line (NG108-15), unless cell membranes are first permeabilized with saponin (11). In none of these studies were bile acid-induced increases in calcium related to changes in cell function (e.g., secretion).
Under certain circumstances, bile acids injure gastrointestinal mucosa. Among other clinical manifestations, bile acid-induced gastrointestinal tract injury may result in bile (or alkaline) gastritis and esophagitis (23). Although it has been speculated that unconjugated di- and trihydroxy bile acids cause gastric damage by binding to and crossing cell membranes to enter cells, the intracellular biochemical mechanisms that mediate injury are not completely understood. Furthermore, it is not clear how conjugated, less lipophilic bile acids cause tissue injury.
Many factors, including an increase in the luminal concentration of hydrochloric acid, gastric mucosal ischemia, drugs such as nonsteroidal anti-inflammatory drugs, and infection with Helicobacter pylori, disrupt the mucosal barrier and predispose to ulceration. A common feature of gastric mucosal injury is a drop in intramucosal pH that leads to activation of pepsinogen to pepsin and approaches the pH optimum (1-3) for this acid protease. Several studies indicate that pepsin is a necessary cofactor for acid- and nonsteroidal anti-inflammatory drug-induced gastric mucosal ulceration (see Ref. 30 for review). Increases in chief cell calcium concentration are known to stimulate pepsinogen secretion (6, 9, 22, 24, 33). Because some bile acids cause an increase in cell calcium (see above), we hypothesized that selected bile acids might stimulate secretion of pepsinogen, thereby leading or contributing to mucosal injury.
In the course of investigating the actions of bile acids on dispersed chief cells from guinea pig stomach, it became apparent that at micromolar concentrations LCT, but not other bile acids, is a partial cholinergic agonist, thereby stimulating a modest increase in pepsinogen secretion and inhibiting both the secretory actions of carbamylcholine and the binding of a radiolabeled muscarinic ligand. Further experiments demonstrated that these specific actions were inhibited by the cholinergic antagonist atropine. The data in the present report indicate that in gastric chief cells LCT stimulates pepsinogen secretion by acting as a specific, partial muscarinic agonist.
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MATERIALS AND METHODS |
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Materials. Male Hartley guinea pigs (150-200 g) were obtained from Charles River (Wilmington, MA); HEPES, collagenase (type I), 8-bromo-cAMP, EGTA, bacitracin, and DMSO from Sigma Chemical; BSA (fraction V) from Fisher Biotech; carbamylcholine (carbachol), bile acids, and CCK 26-33 (CCK-8) from Calbiochem; basal medium (Eagle's) amino acids and essential vitamin solution from Cellgro (Herndon, VA); Percoll from Pharmacia Biotechnology; 125I-albumin (1.1 mCi/mg) from ICN; [N-methyl-3H]scopolamine ([3H]NMS, 83 Ci/mmol), 125I-CCK-8 (2,200 Ci/mmol), cAMP antiserum, and 125I-cAMP (2,200 Ci/mmol) from NEN; myo-[2-3H]inositol from Amersham; Soluene-350 from Packard (Meriden, CT); and Ecoscint A from National Diagnostics (Manville, NJ).
Incubation solution. For the preparation of chief cells, the standard incubation solution contained (in mM) 24.5 HEPES (pH 7.4), 98 NaCl, 6 KCl, 2.5 KH2PO4, 1 MgCl2, 11.5 glucose, 5 sodium fumarate, 5 sodium glutamate, 5 sodium pyruvate, 1.5 CaCl2, and 2 glutamine, in addition to 0.1% (wt/vol) albumin, 1% (vol/vol) amino acid mixture, and 1% (vol/vol) essential vitamin mixture. The standard incubation solution was equilibrated with 100% O2, and all incubations were performed with 100% O2 as the gas phase.
Tissue preparation. Dispersed chief cells from guinea pig stomach were prepared by mucosal digestion with collagenase and cell fractionation on a Percoll density gradient as described previously (32) and suspended in standard incubation solution. The composition of the cell suspension was monitored by light microscopy using morphological criteria (32). In this preparation, chief cells constitute >90% of the total cell population and trypan blue exclusion is >95% (32). Cells were allowed to equilibrate in incubation solution at 37°C for at least 10 min before starting an experiment.
Pepsinogen secretion. Peptic activity was determined as described previously (31) using 125I-albumin as substrate. Pepsinogen secretion was expressed as the percentage of total cellular pepsinogen at the start of the incubation that was released into the medium during the incubation. Total pepsinogen content was determined by freezing and thawing an aliquot of cells. The assay was linear over a range that was at least twofold that of the maximal value assayed.
LDH assay. The lactate dehydrogenase (LDH) assay was performed according to the directions provided with the Sigma LDH assay kit. For these experiments, pyruvate was omitted from the incubation solution.
Binding of [3H]NMS. Binding of [3H]NMS to dispersed chief cells was performed as described previously (35) with the following modifications. Dispersed chief cells (107 cells/ml) were incubated for 45 min at 37°C with 0.9 nM [3H]NMS alone or with unlabeled ligands. Nonspecific binding was determined in the presence of 1 µM unlabeled NMS. The reaction was terminated by centrifuging 0.5 ml of cell suspension (10,000 g) for 6 min at room temperature. Supernatant (100 µl) was sampled for determination of free ligand concentration, and the remaining liquid was carefully decanted. The cell pellet was washed, drained, and dissolved in 100 µl Soluene 350. Ecosint A was added, and the radioactivity in the tubes was measured in a liquid scintillation counter (1214 Rackbeta, LKB/Wallac, Gaithersburg, MD).
Binding of 125I-CCK. Binding of 125I-CCK to dispersed chief cells was performed as described previously (34). Briefly, cells (107 cells/ml) were incubated with 30 pM 125I-CCK-8 alone or with LCT for 60 min. Bound radioactivity was measured by layering a sample (200 µl) over 600 µl of standard incubation solution containing 4% (wt/vol) BSA and 0.1% bacitracin and centrifuging for 30 s at 10,000 g. The cell pellet was washed with the same solution and assayed for radioactivity in a gamma counter. Nonsaturable binding was determined using 1 µM unlabeled CCK-8. In all experiments, nonsaturable binding was <10% of total binding.
Cellular cAMP. Chief cell cAMP was determined by RIA using the procedure described previously (36). The concentration of chief cells in the incubate was adjusted to maintain cAMP on the linear portion of the standard curve. Results for cyclic nucleotide content were expressed as picomoles of cAMP per 106 cells.
IP3. Chief cell inositol 1,4,5-trisphosphate (IP3) was determined by the method of Hu and El-Fakahany (15), as modified by Labarca et al. (18). Briefly, chief cells (107 cells/ml) were incubated with myo-[2-3H]inositol for 1 h at 37°C, washed, and resuspended in incubation solution containing 10 mM LiCl. Cells were then incubated with secretagogues for 45 min at 37°C. The IP3 fraction was separated by ion-exchange chromatography (AG2 X8 resin), and dpm in this fraction were counted by liquid scintillation.
Bile acids. With the exception of lithocholic acid conjugates, bile acids were dissolved in water. LCT and lithocholylglycine stock solutions (100 mM) were prepared in a 60% DMSO-water solution. The highest final concentration of DMSO in any experiment was 0.6%. Stock solutions were stored at room temperature.
Statistical analysis. Significance between two means was determined by Student's unpaired t-test. Differences among several means were determined by ANOVA, followed by Dunnett's test. Values of P < 0.05 were considered significant.
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RESULTS |
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To determine whether bile acids altered pepsinogen secretion from dispersed chief cells, we incubated cells with primary and secondary bile acids and their glycine and taurine conjugates (Table 1). Because of difficulties with solubility, lithocholylglycine and LCT stock solutions (100 mM) were prepared in 60% DMSO-water. Whereas LCT was dissolved, the lithocholylglycine stock was in suspension. The greatest final concentration of DMSO in any assay mixture was 0.6%. We determined that this concentration of DMSO did not alter the characteristics of the pepsin assay, pepsinogen secretion, radioligand binding, or cellular cAMP (data not shown). Furthermore, preliminary experiments determined that none of the bile acids examined altered the characteristics of the 125I-albumin assay for peptic activity (data not shown).
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As shown in Table 1, LCT caused a significant, approximately threefold increase in pepsinogen secretion from dispersed chief cells. None of the other bile acids tested increased pepsinogen secretion. Of interest, but not examined further in the present studies, was the observation that unconjugated and taurine-conjugated deoxycholic acid inhibited basal pepsinogen secretion (Table 1).
We examined the dose-response curve for LCT on pepsinogen secretion for concentrations from 10 nM to 1 mM. As shown in Fig. 1A, stimulation of secretion was detectable with 100 nM LCT and maximal with 10 µM LCT. Maximal pepsinogen secretion with LCT was ~30% of that observed with a maximal concentration of the cholinergic agonist carbachol (Fig. 1A).
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To investigate further the actions of LCT on pepsinogen secretion, we examined its interaction with other secretagogues and observed that LCT decreased carbachol-induced secretion. From this observation, we hypothesized that LCT was a partial cholinergic agonist. To test this hypothesis, we examined the effects of increasing concentrations of LCT on a fixed concentration of carbachol (Fig. 1A) and the noncholinergic agonist CCK (Fig. 1B). As shown in Fig. 1A increasing concentrations of LCT progressively inhibited the increase in pepsinogen secretion caused by 100 µM carbachol. Pepsinogen secretion observed with 1 mM LCT plus 100 µM carbachol was the same as that observed with LCT alone (Fig. 1A). Moreover, we observed near superimposition of the curves when we compared the results of adding increasing concentrations of LCT with a fixed concentration of carbachol with a hypothetical curve calculated assuming interaction of LCT and carbachol with the same receptors (Fig. 1A) (5).
As shown in Fig. 1B, increasing concentrations of LCT did not alter pepsinogen secretion caused by 1 nM CCK-8. To examine further the specificity of effects of LCT on carbachol-induced pepsinogen secretion, we determined the effects of taurine and cholyltaurine on secretion. Increasing concentrations of cholyltaurine and taurine did not alter basal or carbachol-induced pepsinogen secretion (Fig. 1, C and D).
To confirm that LCT is a cholinergic agonist, we examined the effect of adding atropine, a cholinergic antagonist. Whereas atropine (10 µM) alone did not alter basal pepsinogen secretion, the cholinergic antagonist inhibited carbachol- (not shown) and LCT-induced secretion (Fig. 2). These results support the hypothesis that LCT-induced pepsinogen secretion is mediated by a cholinergic mechanism.
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We used several approaches to exclude the possibility that LCT-induced pepsinogen secretion was caused by chief cell injury. Release of the cytosolic enzyme LDH has been used previously to examine the integrity of the chief cell plasma membrane (28, 32). As demonstrated in Table 2, none of the agents used in the present study altered the release of LDH from chief cells. Whereas Triton X-100, used as a positive control, released almost 80% of cellular LDH, LDH release with the other agents tested was not significantly different from release with no agents added. Likewise, when we examined trypan blue exclusion, another measure of cell membrane integrity, incubation with 1 mM LCT for up to 30 min did not alter cell staining compared with control cells [89.8 ± 0.1 and 90.0 ± 0.2% of LCT-treated and control cells, respectively, excluded the dye (means ± SE of 4 experiments)].
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A functional approach to determining chief cell integrity after exposure to LCT involved incubating cells with the combination of LCT (1 mM) and carbachol (0.1 mM) for 30 min, measuring pepsinogen secretion, washing the cells three times with fresh incubation solution, and performing a second 30-min incubation, with and without 0.1 mM carbachol. Whereas the addition of LCT in the first incubation reduced carbachol-induced secretion from 9.7 ± 1.3 to 2.5 ± 0.5% (basal secretion 2.4 ± 0.5%, means ± SE of 4 separate experiments); in the second incubation there was no difference in the response to 0.1 mM carbachol between washed cells that had been incubated in the absence or presence of LCT in the first incubation (7.8 ± 1.1 vs. 7.3 ± 0.4%, respectively). These data indicate that the inhibitory effects of LCT on carbachol-induced secretion are reversible and that chief cells retain their responsiveness after a 30-min incubation with LCT, washing, and a second 30-min incubation with another secretagogue. The latter observation provides further evidence that LCT treatment does not damage chief cells.
To examine directly the potential interaction of LCT with chief cell cholinergic receptors, we compared the effects of LCT, CT, taurine, and carbachol on [3H]NMS binding. Binding of [3H]NMS to chief cell muscarinic receptors is time and temperature dependent, reversible, saturable, and specific (35). Nonspecific binding was determined in the presence of 1 µM unlabeled NMS. A cholinergic agonist and antagonist, carbachol and atropine, respectively, caused a dose-dependent inhibition of binding of radiolabeled NMS (Fig. 3). Like carbachol, LCT inhibited binding of [3H]NMS in a dose-dependent manner (Fig. 3). The highest concentration of LCT used (1 mM) inhibited specific binding of [3H]NMS by 48%. Cholyltaurine, taurine, and sulfated LCT (3-Sul-LCT), an LCT metabolite, did not alter the binding of [3H]NMS (Fig. 3). Moreover, LCT did not alter binding of another radioligand, 125I-CCK-8, to dispersed chief cells. In two separate experiments, specific binding of 125I-CCK-8 in the presence of 1 µM to 1 mM LCT was the same as that observed with no additions (not shown). These observations indicate the specificity of the interaction of LCT with chief cell cholinergic receptors.
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As reported previously, muscarinic cholinergic receptor agonists are more potent at stimulating pepsinogen secretion than at inhibiting binding of radiolabeled cholinergic ligands to dispersed chief cells (35). Comparison of the carbachol and LCT dose-response curves for stimulation of pepsinogen secretion and inhibition of [3H]NMS binding indicates that the concentrations of these agonists that inhibit binding are ~200-fold greater than those that stimulate secretion (Fig. 4). This discrepancy has been attributed to the presence of spare cholingergic receptors on dispersed chief cells (35). This discrepancy does not result from the different chief cell concentrations used in the secretion (1.5 × 105 chief cells/ml, Fig. 1) and binding (107 chief cells/ml, Fig. 3) experiments. When we examined the effect of increasing concentrations of LCT on the carbachol dose-response curve for pepsinogen secretion using 107 chief cells/ml, the pattern of inhibition was the same as that observed with the lower concentration of chief cells (data not shown).
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Previous analysis of [3H]NMS binding to dispersed chief cells indicated the presence of high- and low-affinity binding sites, with secretion being stimulated by binding of cholinergic agonists to the high-affinity sites (35). In the present studies, solubility constraints prevented us from examining LCT concentrations greater than 1 mM, a value that results in ~50% inhibition of [3H]NMS binding (Figs. 3 and 4). Hence, we cannot analyze quantitatively the interaction of LCT with chief cell cholinergic receptor sites. Nevertheless, similar configurations of the dose-response curves for carbachol- and LCT-induced stimulation of pepsinogen secretion and inhibition of [3H]NMS binding support the hypothesis that LCT interacts with chief cell muscarinic receptors.
Although LCT has previously been shown to increase intracellular calcium in a number of cell types (2, 7, 11, 20), the actions of this agent on other signal transduction pathways have not been determined. Because the adenylyl cyclase system mediates pepsinogen secretion by agents such as vasoactive intestinal peptide and secretin (36), we examined the actions of bile acids, including LCT, on chief cell cAMP. None of the bile acids tested (all 1 mM), including LCT, altered chief cell cAMP levels (Table 3). In contrast, like carbachol, LCT stimulated an increase in cellular levels of IP3. As shown in Fig. 5, concentrations of carbachol (0.1 mM) and LCT (1 mM) that were maximal for pepsinogen secretion, caused an approximately two- to threefold increase in cellular levels of IP3. Carbachol- and LCT-induced increases in cellular IP3 were inhibited by 10 µM atropine (Fig. 5). These data support the hypothesis that, like carbachol, LCT interacts with chief cell muscarinic cholinergic receptors that trigger the same signal transduction cascade involving activation of phospholipase C, production of IP3, release of calcium from intracellular stores (6, 8-11), and, ultimately, pepsinogen secretion (29).
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DISCUSSION |
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Cholinergic stimulation of cells in the stomach and intestines has been studied for many years. Acetylcholine induces pepsinogen secretion from gastric chief cells (35), acidification of parietal cell canaliculi, and fluid and electrolyte secretion from the small intestine and colon (3, 4, 6, 20, 37). In isolated gastric, small intestinal, and colonic epithelial cells, muscarinic receptors that regulate secretion and ion transport (measured by changes in short-circuit current and potential difference) have been characterized by radioligand binding studies (4, 35, 37). It has been considered that acetylcholine, released from neurons in the central and enteric nervous systems, is the sole physiological ligand for these receptors.
In the course of investigating the actions of bile acids on dispersed chief cells from guinea pig stomach, it became apparent that LCT interacts at micromolar concentrations with cholinergic receptors, thereby stimulating a dose-dependent, threefold increase in pepsinogen secretion. Nonetheless, before accepting the concept that LCT interacts with gastric or intestinal cholinergic receptors in vivo, several issues must be addressed. These include 1) the solubility of LCT and the concentrations used in the present studies, 2) potential cell damage caused by this highly lipophilic bile acid, and, 3) the specificity of these apparently cholinergically mediated effects.
Although solvents such as DMSO or methanol are required to achieve solubilization of high concentrations of LCT (100 mM) at pH 7.4, micromolar concentrations remain in solution in an aqueous environment. Regarding a potential role for peptic activity in the mediation of bile acid-induced gastric mucosal injury, taurine conjugates are soluble at very acidic pH values. Hence, at low pH, damage may be mediated largely by uptake of bile acids by the gastric mucosa, with local stimulation of pepsinogen secretion and activation of pepsin, whereas at higher pH, dissolution of mucosal membrane lipids may be a more important factor.
The concentrations that define the LCT dose-response curve for pepsinogen secretion are within the range found under physiological conditions. In various healthy animals and humans, approximate concentrations for total bile acids are 2-45 mM in bile, 10 mM (7-8 mM in micellar form) in jejunum, 1 mM in the cecum, 3-200 µM in portal vein, and 20 µM in the systemic circulation (the lower values for these ranges represent fasting values) (1, 17, 19, 25, 26). Estimating that lithocholic acid conjugates comprise 5% of total biliary bile acids, their concentration in normal bile would be 0.1-2.5 mM. In cholestatic states, bile acid concentrations are lower in bile, the intestines, and portal circulation, and higher in the systemic circulation. With respect to potential cholinergic actions of bile acids, bradycardia, reversible with atropine, has been observed in advanced cholestasis (13). Hence, some of the systemic toxicity associated with elevated serum bile acid concentrations in cholestasis may be mediated by cholinergic mechanisms.
Regarding potential cell damage by LCT, at concentrations that stimulate pepsinogen secretion from chief cells (1-100 µM), this bile acid does not alter LDH release or trypan blue exclusion. Moreover, the inhibitory actions of LCT on carbachol-induced pepsinogen secretion are reversible after washing the cells in fresh incubation solution. These results indicate that LCT-induced pepsinogen secretion, or inhibition of the actions of carbachol, is unlikely to be explained by altered cell membrane permeability, cell lysis, or cytotoxicity.
Evidence for the specificity of LCT interaction with cholinergic receptors includes the following observations: 1) LCT-induced pepsinogen secretion is inhibited by atropine, 2) increasing concentrations of LCT cause a specific, progressive reduction in carbachol-induced pepsinogen secretion, 3) LCT competes with radiolabeled NMS, a muscarinic ligand, but not radiolabeled CCK, for receptors on chief cells, and 4) like carbachol, LCT increases cellular levels of IP3 and this increase is inhibited by atropine, indicating that the same muscarinic receptor-coupled signal transduction pathway is activated (29). None of these actions was observed with the other bile acids tested. The observation that sulfated LCT does not interact with cholinergic receptors (Fig. 3) suggests that this may be one mechanism whereby sulfation reduces the toxicity of LCT.
It is of interest to compare the observations in the present report using dispersed chief cells with studies investigating the actions of bile acids on other cell types. The actions of bile acids have been examined previously on cultured murine (PT-18) and freshly isolated rat peritoneal mast cells. Mast cells are widely distributed in the stomach and intestines where they come in contact with bile acids. Whereas, in these cells, lipophilic bile acids (chenodeoxycholate and deoxycholate, and their glycine and taurine conjugates) caused dose-dependent (0.3-10 mM) histamine release, less lipophilic or hydrophilic bile acids (cholate, ursodeoxycholate, and ursocholate) caused little or no histamine release (27). Lithocholate and its conjugates were not tested in this study. Because the concentrations of bile acids that caused histamine release from mast cells were similar to those that caused LDH release, Quist et al. (27) concluded that membrane solubilization was responsible for the observed effects. Dharmsathaphorn et al. (12) investigated the effects of bile acids on chloride secretion from cultured colonic epithelial cells (T84). They found that application of deoxycholyltaurine, but not cholyltaurine, to the basolateral surface reversibly increased chloride secretion, paracellular permeability, and cellular calcium concentration in a dose-dependent (0.2-1 mM) manner.
Hence, studies using mast and intestinal cells indicate that bile acids may alter the function of cells that they are likely to contact during normal physiology. Effective concentrations of bile acids were 10- to 100-fold higher in these studies (12,27) than in ours, and the actions of LCT were not investigated. For intestinal cells, it appears likely that changes in cellular calcium mediate the alterations observed in short-circuit current and chloride secretion. In vivo, interruption of enterohepatic circulation of bile acids causes increased secretion of fluid and electrolytes in the colon, resulting in diarrhea. It is certainly conceivable that some, if not many, of these effects result from the interaction of LCT with muscarinic cholinergic receptors.
In gastric chief cells, experimental evidence indicates that the actions of secretagogues are mediated by one of two major signaling pathways. Cholinergic agonists, such as CCK and gastrin, interact with receptors that are linked to activation of phospholipase C and a subsequent rise in cellular calcium. Secretin and vasoactive intestinal peptide interact with receptors that are linked to activation of adenylyl cyclase and a subsequent rise in cAMP (29). Evidence from the present study and others indicates that, similar to other cholinergic agonists, the actions of LCT are mediated by activation of phospholipase C and changes in cell calcium. This includes the observations that LCT increases cell calcium concentration in isolated hepatocytes (2, 7, 11), LCT increases chief cell IP3 (Fig. 5), and (as for carbachol) the actions of the bile acid on pepsinogen secretion are independent of changes in cellular cAMP (Table 3).
Muscarinic cholinergic receptors can be characterized pharmacologically by their interaction with a variety of agonists and antagonists. Molecular cloning studies have revealed the existence of five muscarinic receptor genes, designated m1-5, based on their order of cloning. Studies with chief cells from guinea pig and rabbit, using pharmacological characterization and Western blotting, respectively, indicate that these cells express muscarinic (subtype m3) cholinergic receptors (16). Muscarinic subtype m3 receptors are common in the gastrointestinal tract. Hence, it is likely that LCT binds to chief cell muscarinic (subtype m3) cholinergic receptors that have been characterized previously (35) and were determined to be the only muscarinic receptor subtype expressed in these cells (16).
To our knowledge no endogenous mammalian cholinergic agonist, other than acetylcholine, has been identified. We believe that this apparent paucity of endogenous cholinergic agonists increases the likelihood that interaction of LCT with cholinergic receptors on gastric chief cells is a significant finding. Moreover, this observation raises the intriguing possibility that in humans some physiological and pathophysiological effects of bile acids are caused by cholinergic agonist properties of LCT.
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ACKNOWLEDGEMENTS |
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We acknowledge the technical assistance of Lenna Craft.
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FOOTNOTES |
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1 Although the term bile acid refers to the protonated form and bile salt refers to the ionized form of these molecules, in this report, as is common in the literature, these terms will be used interchangeably, and bile acid nomenclature will conform to recommendations by Hofmann et al. (14).
Address for reprint requests: J.-P. Raufman, Div. of Gastroenterology, Univ. of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 567, Little Rock, AR 72205-7199.
Received 25 November 1997; accepted in final form 17 February 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alpini, G.,
R. Lenzi,
L. Sarkozi,
and
N. Tavaloni.
Biliary physiology in rats with bile ductular hyperplasia.
J. Clin. Invest.
81:
569-578,
1988[Medline].
2.
Anwer, M. S.,
L. R. Engelking,
K. Nolan,
D. Sullivan,
P. Zimniak,
and
R. Lester.
Hepatotoxic bile acids increase cytosolic Ca2+ activity of isolated rat hepatocytes.
Hepatology
8:
887-891,
1988[Medline].
3.
Browning, J. G.,
J. Hardcastle,
P. T. Hardcastle,
and
J. S. Redfern.
Localization of the effect of acetylcholine in regulating intestinal ion transport.
J. Physiol. (Lond.)
281:
15-27,
1978[Medline].
4.
Carey, H. V.,
X.-Y. Tien,
L. J. Wallace,
and
H. J. Cooke.
Muscarinic receptor subtypes mediating the mucosal response to neural stimulation of guinea pig ileum.
Am. J. Physiol.
253 (Gastrointest. Liver Physiol. 16):
G323-G329,
1987
5.
Cherner, J. A.,
V. E. Sutliff,
D. M. Grybowski,
R. T. Jensen,
and
J. D. Gardner.
Functionally distinct receptors for cholecystokinin and gastrin on dispersed chief cells from guinea pig stomach.
Am. J. Physiol.
254 (Gastrointest. Liver Physiol. 17):
G151-G155,
1988
6.
Chew, C. S.,
and
M. R. Brown.
Release of intracellular Ca2+ and elevation of inositol trisphosphate by secretagogues in parietal and chief cells isolated from rabbit gastric antrum.
Biochim. Biophys. Acta
888:
116-125,
1986[Medline].
7.
Combettes, L.,
B. Berthon,
and
M. Claret.
Selective permeabilization of the endoplasmic reticulum by monohydroxylated bile acids in liver.
Hepatology
9:
663-664,
1989[Medline].
8.
Combettes, L.,
M. Dumont,
B. Berthon,
S. Erlinger,
and
M. Claret.
Release of calcium from the endoplasmic reticulum by bile acids in rat liver cells.
J. Biol. Chem.
263:
2299-2303,
1988
9.
Combettes, L.,
M. Dumont,
B. Berthon,
S. Erlinger,
and
M. Claret.
Effect of the bile acid taurolithocholate on cell calcium in saponin-treated rat hepatocytes.
FEBS Lett.
227:
161-166,
1988[Medline].
10.
Combettes, L.,
M. Dumont,
E. Doucet,
S. Erlinger,
and
M. Claret.
Characteristics of bile acid-mediated Ca2+ release from permeabilized liver cells and liver microsomes.
J. Biol. Chem.
264:
157-167,
1989
11.
Coquil, J.-F.,
B. Berthon,
N. Chomiki,
L. Combettes,
P. Jourdon,
C. Schteingart,
S. Erlinger,
and
M. Claret.
Effects of taurolithocholate, a Ca2+-mobilizing agent, on cell Ca2+ in rat hepatocytes, human platelets and neuroblastoma NG108-15 cell line.
Biochem. J.
273:
153-160,
1991[Medline].
12.
Dharmsathaphorn, K.,
P. A. Huott,
P. Vongkovit,
G. Beuerlein,
S. J. Pandol,
and
H. V. Ammon.
Cl secretion induced by bile salts.
J. Clin. Invest.
84:
945-953,
1989[Medline].
13.
Harvey, S. C.
Gastric antacids and digestants.
In: The Pharmacological Basis of Therapeutics, edited by L. S. Goodman,
and A. Gilman. New York: Macmillan, 1975, p. 960-975.
14.
Hofmann, A. F.,
J. Sjsvall,
G. Kurz,
A. Radominska,
C. D. Schteingart,
G. S. Tint,
Z. R. Vlahcevic,
and
K. D. R. Setchell.
A proposed nomenclature for bile acids.
J. Lipid Res.
33:
599-604,
1992[Abstract].
15.
Hu, J.,
and
E. E. El-Fakahany.
Selectivity of McN-A-343 in stimulating phosphoinositide hydrolysis mediated by M1 muscarinic receptors.
Mol. Pharmacol.
38:
895-903,
1990[Abstract].
16.
Kajimura, M.,
M. A. Reuben,
and
G. Sachs.
The muscarinic receptor gene expressed in rabbit parietal cells is the m3 subtype.
Gastroenterology
103:
870-875,
1992[Medline].
17.
Klos, C.,
G. Paumgartner,
and
J. Reichen.
Cation-anion gap and choleretic properties of rat bile.
Am. J. Physiol.
236 (Endocrinol. Metab. Gastrointest. Physiol. 5):
E434-E440,
1979[Medline].
18.
Labarca, R.,
A. Janowsky,
and
S. Paul.
Neurotransmitter-stimulated inositol phosphate accumulation in hippocampal slices.
Methods Enzymol.
141:
192-201,
1987[Medline].
19.
McJunkin, B.,
H. Fromm,
R. P. Sarva,
and
P. Amin.
Factors in the mechanism of diarrhea in bile acid malabsorption: fecal pH-a key determinant.
Gastroenterology
80:
1454-1464,
1981[Medline].
20.
Mekhjian, H. S.,
S. F. Phillips,
and
A. F. Hofmann.
Colonic secretion of water and electrolytes induced by bile acids: perfusion studies in man.
J. Clin. Invest.
50:
1569-1577,
1971[Medline].
21.
Montrose, M. H.,
R. Lester,
P. Zimniak,
M. S. Anwer,
and
H. Murer.
Bile acids increase cellular free calcium in cultured kidney cells (LLC-PK1).
Pflügers Arch.
412:
164-171,
1988[Medline].
22.
Muallem, S.,
C. J. Fimmel,
S. J. Pandol,
and
G. Sachs.
Regulation of free cytosolic Ca2+ in the peptic and parietal cells of the rabbit gastric gland.
J. Biol. Chem.
261:
2660-2667,
1986
23.
Nath, B. J.,
and
A. L. Warshaw.
Alkaline reflux gastritis and esophagitis.
Annu. Rev. Med.
35:
383-396,
1984[Medline].
24.
Okayama, N.,
M. Itoh,
T. Joh,
T. Miyamoto,
T. Takeuchi,
T. Suzuki,
A. Moriyama,
and
T. Kato.
Mediation of pepsinogen secretion from guinea pig chief cells by Ca2+/calmodulin-dependent protein kinase II.
Biochim. Biophys. Acta
1268:
123-129,
1995[Medline].
25.
Poley, J. R.,
and
A. F. Hofmann.
Role of fat maldigestion in pathogenesis of steatorrhea in ileal resection. Fat digestion after two sequential test meals with and without cholestyramine.
Gastroenterology
71:
38-44,
1976[Medline].
26.
Pries, J. M.,
C. A. Sherman,
G. C. Williams,
and
R. F. Hanson.
Hepatic extraction of bile salts in conscious dog.
Am. J. Physiol.
236 (Endocrinol. Metab. Gastrointest. Physiol. 5):
E191-E197,
1979[Medline].
27.
Quist, R. G.,
H.-T. Ton-Nu,
J. Lillienau,
A. F. Hofmann,
and
K. E. Barrett.
Activation of mast cells by bile acids.
Gastroenterology
101:
446-456,
1991[Medline].
28.
Raffaniello, R. D.,
and
J.-P. Raufman.
Pepsinogen secretion from streptolysin O-permeabilized chief cells from guinea pig stomach.
Am. J. Physiol.
263 (Gastrointest. Liver Physiol. 26):
G452-G459,
1992
29.
Raufman, J.-P.
Gastric chief cells: receptors and signal-transduction mechanisms.
Gastroenterology
102:
699-710,
1992[Medline].
30.
Raufman, J.-P.
Peptic activity and gastroduodenal mucosal damage.
Yale J. Biol. Med.
69:
85-90,
1996[Medline].
31.
Raufman, J.-P.,
S. Berger,
L. Cosowsky,
and
E. Straus.
Increases in cellular calcium concentration stimulate pepsinogen secretion from dispersed chief cells.
Biochem. Biophys. Res. Commun.
137:
281-285,
1986[Medline].
32.
Raufman, J.-P.,
V. E. Sutliff,
D. K. Kasbekar,
R. T. Jensen,
and
J. D. Gardner.
Pepsinogen secretion from dispersed chief cells from guinea pig stomach.
Am. J. Physiol.
247 (Gastrointest. Liver Physiol. 10):
G95-G104,
1984
33.
Sakamoto, C.,
T. Matozaki,
M. Nagao,
H. Nishizaki,
and
S. Baba.
Regulation of free cytosolic Ca2+ in the isolated guinea pig gastric chief cells.
Biochem. Biophys. Res. Commun.
142:
865-871,
1987[Medline].
34.
Sutliff, V. E.,
J. A. Cherner,
R. T. Jensen,
and
J. D. Gardner.
Binding of 125I-CCK-8 and 125I-gastrin-I to dispersed chief cells from guinea pig stomach.
Biochim. Biophys. Acta
1052:
9-16,
1990[Medline].
35.
Sutliff, V. E.,
S. Rattan,
J. D. Gardner,
and
R. T. Jensen.
Characterization of cholinergic receptors mediating pepsinogen secretion from chief cells.
Am. J. Physiol.
257 (Gastrointest. Liver Physiol. 20):
G226-G234,
1989
36.
Sutliff, V. E.,
J.-P. Raufman,
R. T. Jensen,
and
J. D. Gardner.
Actions of VIP and secretin on dispersed chief cells from guinea pig stomach.
Am. J. Physiol.
251 (Gastrointest. Liver Physiol. 14):
G96-G102,
1986[Medline].
37.
Zimmerman, T. W.,
and
H. J. Binder.
Muscarinic receptors on rat isolated colonic epithelial cells.
Gastroenterology
83:
1244-1251,
1982[Medline].
38.
Zimniak, P.,
J. M. Little,
A. Radominska,
D. G. Oelberg,
M. S. Anwer,
and
R. Lester.
Taurine-conjugated bile acids act as Ca2+ ionophores.
Biochemistry
30:
8598-8604,
1991[Medline].