Shmuel Batzri

Bile acid accumulation by rabbit esophageal mucosa

Bile acids are one of the noxious components of the gastroduodenal contents which may injure the esophageal mucosa in clinical reflux esophagitis. Animal models of esophagitis have shown that exposure to low luminal bile acid concentrations can cause increased mucosal permeability to a variety of ions and molecules without causing dramatic gross morphologic damage. In order to explore the mechanism by which bile acids alter mucosal permeability, we measured the esophageal mucosal concentration of taurocholic acid and chenodeoxycholic acid after exposure to these bile acids in anesthetized New Zealand white rabbits. We found that bile acids can accumulate in the esophageal mucosa to levels as high as seven times the initial luminal concentration. Thin-layer chromatography showed that this accumulation was not due to bile acid degradation in the mucosa. Since butyric acid also showed some mucosal accumulation, and is a weak acid like taurocholic acid, intracellular ionization may account for some of the accumulation. Mucosal accumulation of these molecules is not a nonspecific phenomenon, since the four-carbon polyol erythritol did not accumulate at all. Bile acid accumulation occurred under the same conditions and in a parallel temporal relationship to the bile-induced permeability changes. It is hypothesized, therefore, that the presence of high concentrations of bile acids in the esophageal mucosa may be pathophysiologically related to the alterations in mucosal permeability which occur after exposure to bile acids.

Bile acid accumulation in gastric mucosal cells

Abstract Bile acids are one of the components of the gastric contents capable of disrupting the mucosal barrier to diffusion. The mechanism by which bile acids can damage the gastric epithelium is not completely understood. Several studies have emphasized mucosal lipid solubilization by bile acids in the pathogenesis of mucosal injury. Bile acid entry into gastric mucosal cells may be a critical and early step in the genesis of mucosal injury, but this possibility has not yet been investigated. The present study was designed to explore the interaction of bile acids with dispersed gastric mucosal cells isolated from the rabbit and guinea pig stomach. Results showed that both glycocholic and deoxycholic acid rapidly associated with the gastric cells and reached a steady state concentration by 30 min. Glycocholic acid accumulated in the cells to a concentration approximately eight times greater than that in the surrounding medium. The amount of bile acid associated with the cells was greater at an acidic than at a neutral pH, and was a function of the concentration of both the cells and the bile acid. The process did not require cellular energy, was nonsaturable, and was not species specific. Experiments with 86Rb, a cytoplasmic marker, revealed that approximately one half of the cellular glycocholic acid was associated with the cytoplasmic compartment and the rest with the membranes. These findings are consistent with a combination of intracellular entrapment of the bile acids due to intracellular ionization and bile acid binding to cellular membrane components being the mechanisms by which bile acids accumulate in cells. Acid-driven bile acid accumulation may explain how relatively low luminal concentrations of bile acid can be damaging to the gastrointestinal mucosa.

Aminopyrine uptake by guinea pig gastric mucosal cells mediation by cyclic AMP and interactions among secretagogues

The role of cyclic nucleotides in regulating acid secretion by dispersed mucosal cells from guinea-pig stomach was examined by measuring first the ability of histamine and carbachol to stimulate [dimethylamine-14C]aminopyrine uptake and cyclic nucleotide metabolism and secondly, the effect of exogenous cyclic nucleotides on basal and stimulated [14C]aminopyrine uptake. The [14C]aminopyrine was found in an acidic, osmotically sensitive compartment, probably associated with the initial steps in acid secretion by these cells. Although histamine increased [14C]aminopyrine uptake and cyclic AMP synthesis as expected, histamine was approx. 10-fold more potent in inducing [14C]aminopyrine uptake. This dissociation of [14C]aminopyrine uptake and cyclic AMP metabolism process was further manifested by the observation that prostaglandin E1 failed to increase [14C]aminopyrine uptake, although it did cause a rise in cellular cyclic AMP. Furthermore, prostaglandin E1 did not alter the [14C]-aminopyrine uptake caused by histamine. Carbachol was found to increase the [14C]aminopyrine uptake and also to potentiate the ability of histamine to increase [14C]aminopyrine uptake. Carbachol, however, affected neither the histamine-induced increase in cyclic AMP nor the binding of [3H]histamine to the cells. Cimetidine, a histamine H2 receptor antagonist, blocked the [14C]aminopyrine uptake induced either by histamine alone or by the potentiating combination of histamine plus carbachol. These results suggest that cyclic AMP is mediating the action of histamine on [14C]aminopyrine uptake but changes in cyclic AMP per se are not necessarily the cause for the potentiated increase in [14C]aminopyrine uptake. Furthermore, the potentiated response observed with histamine plus carbachol on [14C]aminopyrine uptake occurs at a biochemical step distal to and not obviously related to cyclic AMP generation.

Direct action of somatostatin on dispersed mucosal cells from guinea-pig stomach.

In dispersed mucosal cells from guinea-pig stomach, somatostatin inhibited in a noncompetitive fashion (Ki, 2 x 10(-8) M) the increase in cellular cyclic AMP caused by histamine but not by prostaglandin E1 or phosphodiesterase inhibitors. Somatostatin also inhibited the increase in [14C]aminopyrine uptake caused by low concentrations of histamine probably by interfering with the synthesis of cellular cyclic AMP.

Comparison of cimetidine with new H2-antagonists in rabbit and guniea pig gastric cells

We compared the effect of three relatively new H2-antagonists (compounds L-643,411, BL-6341A and SK&F 93479) to cimetidine in two preparations of mucosal cells isolated from rabbit and guinea pig stomachs. The indices for the histamine-stimulated acid secretory response were the changes in [14C]aminopyrine uptake in the rabbit and in cellular cyclic AMP, in the guinea pig. Both functions were mediated by the histamine H2-receptors and hence, can be used to examine antagonist-receptor interaction in vitro. In both rabbit and guinea pig, the new antagonists were highly potent competitive inhibitors of histamine on the H2-receptor, 30- to 200-fold more potent than cimetidine. The Ki values for cimetidine (500-800 nM) and L-643,411 (6-12 nM) were the same in the two animal species, but those for SK&F 93479 and BL-6431A were significantly lower in rabbit than in guinea pig cells. In inhibiting the changes in [14C]aminopyrine uptake in rabbit cells the Ki values for SK&F 93479 and BL-6341A were 2.4-3.5 nM whereas on cyclic AMP in guinea pig cells they were 10-fold higher (25-30 nM). These differences may reflect the structural requirements of the H2-receptors in that in rabbit these antagonists possess higher affinity for the H2-receptors than in guinea pig, or alternatively, uptake or metabolism of histamine by rabbit gastric cells may be responsible for these differences. Furthermore, these preparations appeared to be satisfactory for in vitro assay of gastric acid secretion to test for competitiveness of new H2-receptor antagonists.

Secretion of intrinsic factor from dispersed mucosal cells isolated from guinea pig and rabbit stomach

In dispersed mucosal cells prepared from rabbit and guinea pig stomach, the secretion of intrinsic factor was constant (0.3-0.4%/min) for at least 30 min incubation at 37 degrees C. Histamine or isobutyl methylxanthine increased cyclic AMP and intrinsic factor secretion in both cells preparations. Isobutyl methylxanthine potentiated and cimetidine competitively inhibited (ki = 5.10-7 M) both effects of histamine. Dibutyryl cyclic AMP (1.0 mM), also caused a 3-fold increase in intrinsic factor secretion. These results suggest that in rabbit and guinea pig histamine interacts with H2-receptors to increase cyclic AMP which mediates the rise in the rate of intrinsic factor secretion.

A new in vivo method for repeatedly studying gastric acid secretion and other secretory parameters in awake guinea pig

A new model for measuring gastric secretory parameters in awake guinea pigs is described. A chronic cannula was surgically implanted in the stomach of each guinea pig. The rates of gastric secretion and changes in intragastric volume were measured using a dye dilution technique. In contrast to previous techniques in small laboratory animals, there was no collection of gastric juice via drainage, no oral intubation for aspiration was involved, no special or sophisticated equipment was used, no anesthesia was employed, and there was no stress associated with acute surgery. This method offers a valuable advantage by combining the chronic gastric cannula with a dye dilution technique in that the same animal can be used several times and finally, several gastric secretory parameters can be measured simultaneously. The animals were used from 3 weeks to 10 months after surgery and as many as 15 studies were performed on the same guinea pig. Samples were collected at 10-min intervals and analyzed for acid and dye concentration from which the onset and kinetics of gastric secretion were followed. Basal gastric secretion (11.8 +/- 1.6 mueq/kg/min; all mean +/- 1 SEM) was increased within 20 min after subcutaneous infusion of histamine (30 micrograms/kg/hr) and peaked by 40-60 min at a mean acid output rate of 41 +/- 3 mueq/kg/min. Histamine also increased the intragastric volume from 6.3 to 13.4 ml as it increased fluid output from 1.6 +/- 0.1 ml/10 min to 3.4 +/- 0.2 ml/10 min. The increase in acid output caused by histamine was inhibited by the H2-antagonists cimetidine (3 mumole/kg) and ranitidine at 0.5 mumole/kg. Omeprazole (1.2 mumole/kg), an H-K-ATPase inhibitor, almost abolished acid output under both basal and histamine-stimulated conditions. Thus, the present method is simple and suitable to study the physiology and pharmacology of gastric secretion in the guinea pig with a particular emphasis on the action of histamine. Furthermore, because of the species involved, there is also a significant economical advantage and the guinea pig can also be used as a potential model for studying experimental ulcer.

Is [3H]-tiotidine a specific ligand for the H2-receptor?

The H2-antagonist tiotidine inhibited the H2-receptor-mediated, histamine-induced increase in cyclic AMP in dispersed mucosal cells from guinea pig stomach (Ki, 4 X 10(-8) M). The radiolabeled [3H]-tiotidine bound specifically and reversibly to the same cells with a half-maximal binding occurring at 5 X 10(-7) M tiotidine. The dissociation of bound [3H]-tiotidine from gastric cells and the Scatchard analysis of the binding binding data suggest the existence of additional binding sites for tiotidine. Eight other antagonists which inhibited the H2-receptor-mediated increase in cyclic AMP also inhibited [3H]-tiotidine binding. However, the potencies of these agents for binding did not agree with their effects on cyclic AMP. The selective H2-agonists impromidine and dimaprit which increased cyclic AMP caused only partial inhibition of [3H]-tiotidine binding. These results demonstrate that [3H]-tiotidine has limited binding to the H2-receptors and as such [3H]-tiotidine is not a suitable ligand for labelling the H2-receptor on gastric mucosal cells.

Identification of [3H] histamine binding sites on gastric mucosal cells unrelated to histamine H2-receptors

Binding of 3H-histamine to guinea pig gastric mucosal cells was inhibited by various imidazole and indole derivatives as well as by histamine and its H2-receptor agonists and antagonists. In contrast to the H2-agonists or H2-antagonists, these derivatives (e.g. imidazole, 4-hydroxymethyl imidazole, tryptamine, serotonin), neither increased cellular cyclic AMP nor altered the increase in cyclic AMP caused by histamine. The imidazole derivatives were more potent in inhibiting [3H]histamine binding than the corresponding indole derivatives. These results confirm our earlier observations suggesting that gastric cells posses a class of binding sites for 3H-histamine that is not linked to adenylate cyclase and is unrelated to the histamine H2-receptor.

Distortion of H2-Antagonist Equilibrium Constants by Uptake in Rabbit Gastric Mucosal Cells

In rabbit gastric cells the new H2-antagonists, BL-6341A, SK&F 93479 and L-643,441 were highly potent inhibitors of the H2-receptor mediated action of histamine as monitored by 14C-aminopyrine uptake and cyclic AMP formation. BL-6341A and SK&F 93479 acted as competitive antagonists to histamine and dimaprit but they were less potent against dimaprit (Ki, 8.9 nM) than against histamine (Ki, 3.5-4.4 nM). Furthermore, the Schild slope for L-643,441 against histamine was significantly higher than unity (1.69-1.79), which is inconsistent with competitive antagonism, whereas against dimaprit it was close to unity. In contrast to these antagonists, cimetidine was an equally potent competitive antagonist of both histamine and dimaprit. 3H-histamine was taken up by gastric cells as evidenced by the loss of 60-70% of the cell-associated radioactivity upon hypotonic lysis. These results suggest that uptake and possibly metabolism of histamine by rabbit gastric cells is partially responsible for the distortion of the estimated equilibrium constants for these H2-antagonists.