Reversibility of deoxycholate-induced cellular hypercalcemia in rabbit gastric mucosal cells.

BACKGROUND: Bile acid exposure produces cellular hypercalcemia in gastric and hepatic cells. It is not known, however, whether this event contributes to cell injury or if it results from passive equilibration of calcium ion concentrations across the membranes of irreversibly damaged cells. This study was performed to determine whether the cellular hypercalcemia produced by bile acid exposure in gastric cells is reversible and to determine whether the source of this hypercalcemia is from intracellular stores of calcium, extracellular sources, or both. METHODS: Cytosolic free calcium concentrations ([Ca]i) were measured in rabbit gastric mucosal cells that had been loaded with the intracellular probe FURA-2. Measurements were performed in suspensions of dispersed cells by using standard spectrofluorometry and in primarily cultured cells by using fluorescence videomicroscopy. Measurements were made before and after exposure to 0.2, 0.5, and 1.0 mmol/L deoxycholic acid (DC). These measurements were made in the presence of 1 mmol/L extracellular calcium and in the absence of any extracellular calcium (0.5 mmol/L EGTA). RESULTS: In experiments with dispersed cells and spectrofluorometry, [Ca]i increased from a pretreatment level of 194 +/- 8 nmol/L to 396 +/- 21 nmol/L within 3 minutes of exposure to 0.2 mmol/L DC. When these cells were washed and resuspended in DC-free medium, [Ca]i] decreased to 180 +/- 5 nmol/L. In experiments with cultured cells and fluorescence videomicroscopy, rapid, reversible hypercalcemia was observed after exposure to 0.5 and 1.0 mmol/L DC. Removal of extracellular calcium from the incubating medium reduced both the magnitude and duration of the observed hypercalcemia. CONCLUSIONS: These data show that the cellular hypercalcemia that accompanies DC-induced injury in gastric cells is a reversible event. The initial increase in [Ca]i appears to come from both intracellular and extracellular sources, although sustained hypercalcemia requires a source of extracellular calcium. As a reversible event, cellular hypercalcemia may be an important pathophysiologic feature of bile acid induced injury of the upper gastrointestinal tract.

Effect of growth factors on cell proliferation and epithelialization in human skin.

The failure of chronic wounds to heal remains a major medical problem. Recent studies have suggested an important role for growth factors in promoting wound healing. We investigated the mitogenic effect of basic fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF), comparing their effects with those of media alone (MEM) in a human skin explant model. A stable organ culture system for maintaining the histologic structure of human epidermis for 10 days in vitro was developed. DNA synthesis was measured on Days 1, 3, and 7 of organ culture using [3H]thymidine ([3H]thy) uptake and expressed as cpm/mg dry weight (mean +/- SEM). FGF, IGF-1, and EGF were each capable of stimulating [3H]thy uptake on Day 1 of culture (2372 +/- 335 FGF, 2226 +/- 193 IGF-1, 4037 +/- 679 EGF vs 1108 +/- 70 MEM, P < 0.05). IGF-1 and EGF also stimulated [3H]thy uptake on Days 3 and 7 of culture. The organ culture system was further employed to observe epidermal outgrowth. Longest keratinocyte outgrowth from the explant periphery (simulating epithelial regeneration from the wound edge) was observed on Day 7. EGF resulted in maximum stimulation of epithelial outgrowth (440 +/- 80 microns), followed by FGF (330 +/- 56 microns), IGF-1 (294 +/- 48 microns), and MEM (189 +/- 50 microns). We postulate, therefore, that FGF, IGF-1, and EGF are important mitogens for wound healing and that EGF in particular is capable of stimulating epithelialization.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular hypercalcemia is an early event in deoxycholate injury of rabbit gastric mucosal cells.

Ca2+ entry into the cell may be an early event in the pathophysiology of bile salt-induced gastric mucosal injury. The aim of this study was to characterize the rise in cytosolic free Ca2+ associated with bile salt injury and its association with cell injury and death. Rabbit gastric mucosal cells were preloaded with the Ca2+ indicator fura 2-acetoxymethyl ester (fura 2-AM) for 20 min at 37 degrees C and then exposed to graded concentrations of the bile salt deoxycholate (DC). Cytosolic free Ca2+ concentration ([Ca2+]i) was estimated by spectrofluorometry. The resting [Ca2+]i in gastric cells was 177 +/- 15 nM (n = 6). When cells were subjected to 0.5 mM DC, there was a time-dependent rise in [Ca2+]i. An increase in [Ca2+]i was observed within 2 min, at which time [Ca2+]i rose from 177 +/- 15 to 480 +/- 30 nM. The maximal increase in [Ca2+]i was observed after 20 min of exposure to 0.5 mM DC (639 +/- 49 nM), and [Ca2+]i remained unchanged for at least 2 h. The increase in [Ca2+]i depended on the concentration of DC. The minimum effective dose of DC was 0.2 mM, with which [Ca2+]i was increased by 1.6-fold (from 177 to 285 nM). At 0.5 mM DC also caused a rise in 45Ca2+ influx into the cells and reduced the viability of gastric cells from 96% to 58% at 2 h. The DC-induced rise in cytosolic free Ca2+ depended on the presence of extracellular Ca2+. In the absence of extracellular Ca2+ there was no rise in cytosolic Ca2+ and gastric cells were protected from cell death caused by DC. The DC-induced cell death was reduced from 26% to 10% and from 37% to 16% at 60 and 90 min, respectively, by removal of extracellular Ca2+. The association of DC with gastric cells was not altered by removing extracellular Ca2+. This suggests decreased DC-induced injury in the absence of extracellular Ca2+ is due to the protection from cellular hypercalcemia rather than some other mechanism related to reduced binding and/or association of DC to gastric cells. These experiments show that rising [Ca2+]i appears to be an early pathophysiological event in bile salt-induced cellular injury and that extracellular Ca2+ is critical to produce this effect.

Bile acid accumulation in gastric mucosal cells.

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.

Inhibition of gastric secretion in guinea pig by relatively low dose ionizing radiation.

We evaluated the effect of a single dose of ionizing radiation on gastric secretion in awake guinea pigs equipped with a permanent gastric cannula. Changes in gastric secretion were measured using a dye dilution technique. Infusion of histamine increased acid and fluid output and there was a positive correlation (r = 0.93) between the two. Total body irradiation with 400 cGy, like cimetidine, suppressed acid and fluid secretion under basal conditions and during histamine stimulation by 50-90%. Recovery from the radiation damage was only partial after one week. Irradiation inhibited the rise in gastric juice volume during histamine stimulation and also reduced the normal gain in body weight of the guinea pig. These results demonstrate that ionizing radiations have an immediate and long lasting effects on the gastric mucosal function of the guinea pig.

Inhibition of acid secretion in guinea pigs by tricyclic antidepressants: comparison with ranitidine and omeprazole.

The antisecretory properties of imipramine on gastric secretion in guinea pig in comparison with other antisecretory agents was determined. In awake guinea pigs s.c. infusion of histamine (30 micrograms/kg/hr) increased acid and fluid secretion by 3- to 4-fold. When acid output peaked, a bolus administration of the tricyclic anti-depressant imipramine inhibited acid and fluid secretion. Imipramine and other agents, such as ranitidine and omeprazole, inhibited gastric secretion in a dose-dependent fashion. The most potent was the H2-antagonist ranitidine (IC50, 0.2-0.3 mumol/kg), followed by the gastric H-K-adenosine triphosphatase inhibitor, omeprazole (IC50, 0.5-0.6 mumol/kg). Imipramine (IC50 1-2 mumol/kg) was the least potent of the inhibitors. Both ranitidine and omeprazole could abolish acid secretion, but maximal inhibition with imipramine was 60% of initial. Promethazine (25 mumol/kg), an H1 antagonist, and atropine (12 mumol/kg), a muscarinic antagonist, inhibited gastric secretion by 40 to 50%. Imipramine and atropine also inhibited basal acid secretion. In dispersed gastric cells comparison between imipramine and omeprazole showed that imipramine was about 5-fold more potent than omeprazole in blocking histamine or dibutyryl cyclic AMP stimulation of aminopyrine accumulation. Imipramine probably acts as a protonophore by increasing the rate of proton-gradient dissipation rather than by interfering with the hydrogen-pump system because, in gastric membranes, imipramine was 20-fold less potent than omeprazole in inhibiting the gastric H-K-adenosine triphosphatase activity. These results suggest that imipramine administered s.c. in guinea pigs is a potent antisecretory drug. Its action may be due to a combination of anticholinergic and antihistamine H2 activities.

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.

Lipid solubilization during bile salt-induced esophageal mucosal barrier disruption in the rabbit.

Bile salts in the gastric contents are one of the harmful agents that may contribute to the mucosal injury found in clinical reflux esophagitis. To clarify the mechanism by which bile salts can injure the esophageal mucosa, we studied the effects of exposure to the bile salts taurocholate and deoxycholate in an in vivo perfused rabbit model of esophagitis. Specifically we examined the roles of accumulation of bile salt by the mucosa and solubilization of mucosal lipid into the lumen during bile salt-induced mucosal injury. Results showed a consistent association between bile salt accumulation by the esophageal mucosa and bile salt-induced disruption of the physiologic barrier to diffusion. Under certain conditions, bile salts caused barrier disruption with no release of lipid from the mucosa. Whenever exposure to bile salt did cause release of mucosal lipid into the lumen, a significant amount of morphologic injury was also observed by light microscopy. The findings suggest that the presence of bile salt in the mucosa is an important aspect of the mechanism of bile salt-induced barrier disruption. Solubilization of mucosal lipids into the lumen occurs when bile salts cause an injury that is severe enough to result in light microscopic evidence of morphologic damage.

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.

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.

Tricyclic antidepressants and acid secretory response of rabbit gastric cells.

Tricyclic antidepressants have been tested for their effect on the H2-receptor-mediated changes in cAMP, O2 consumption, and acid secretion in mucosal cells isolated from rabbit stomach. Amitriptyline (AMT) inhibited the action of histamine on both cAMP generation and O2 consumption in an apparent competitive fashion without altering these parameters in unstimulated cells. The onset of this inhibition was rapid and the time at which the increases in these functions had reached steady state was not changed. The Schild regression line for AMT was close to unity, and its Ki values for cAMP production or O2 consumption were 0.75 microM AMT. Imipramine and nortriptyline also caused a rightward shift in the dose-response curve of histamine-induced cAMP generation. The inhibitory action of AMT was specific to histamine in that AMT neither altered the effect of isobutylmethylxanthine on cAMP and respiration nor inhibited the increase in respiration caused by carbachol. However, at relatively high concentrations (250-500 microM), AMT inhibited the increase in cAMP caused by 5 and 100 microM prostaglandin E1 and inhibited the increase in respiration caused by both dibutyryl cAMP (DBcAMP) and by the combination of DBcAMP plus carbachol. Antidepressant drugs were also very potent inhibitors of acid formation as measured by [14C]aminopyrine accumulation (Ki, 1 microM). They inhibited acid formation in control cells, in cells stimulated by histamine, carbachol, and DBcAMP, and also the potentiated response to carbachol plus histamine or DBcAMP. The onset of this inhibition was also rapid, and adding 5 microM AMT to the cell suspension either together with histamine or after stimulation by histamine caused a rapid decline in aminopyrine accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Bile acid efflux precedes mucosal barrier disruption in the rabbit esophagus.

In a rabbit model of esophagitis, bile acids are one of the agents capable of disrupting the normal esophageal mucosal barrier to diffusion. They are of particular interest because they may play a central role in causing reflux esophagitis, a major clinical problem. Bile acid efflux from the lumen of the esophagus occurs when bile acids disrupt the normal esophageal barrier. Whether this entry of bile acids into the mucosa is a critical step in the pathophysiology of bile acid injury to the mucosa or a result of the injury that is occurring by some other means is unknown. To differentiate these two possibilities, the temporal relation between bile acid efflux from the esophageal lumen and disruption of the mucosal barrier was studied with an in vivo perfused model of esophagitis in anesthetized New Zealand White rabbits. Solutions containing taurocholic acid or chenodeoxycholic acid at pH 2 or 7 were perfused for 4 h while bile acid, hydrogen ion, glucose, and erythritol flux rates were measured. In each case in which the bile acid caused barrier disruption, the highest rate of bile acid efflux occurred during the 1st h of perfusion. In contrast, the increase in flux rates of hydrogen ion, glucose, and erythritol, indicators of mucosal barrier disruption, were the highest during the 2nd or 3rd h of exposure. These data suggest that entry of bile acids into the esophageal mucosa is a cause rather than an effect of the bile-induced increase in mucosal permeability.

Action of tricyclic antidepressants on cyclic AMP in mucosal cells from guinea pig stomach.

In dispersed mucosal cells from guinea pig stomach, tricyclic antidepressants (amitriptyline and imipramine) inhibited the increases in cyclic AMP caused by histamine or dimaprit. The inhibition by low concentrations of amitriptyline was competitive, with a pA2 value of 5.8 +/- 0.15 (Ki, 1.5 +/- 0.8 microM). At high concentrations, amitriptyline reduced the maximal response to histamine as well as inhibited the increase in cyclic AMP caused by theophylline and prostaglandin E1, and also reduced the basal respiratory rate of gastric cells. This inhibition was non-competitive (IC50, 100-250 microM). These results suggest that at low concentrations tricyclic antidepressants interact with the H2-receptors on gastric cells in a fashion similar to their interaction with the brain histamine H2-receptors.

Comparison of cimetidine with new H2-antagonists in rabbit and guinea 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.

Direct action of D-lysergic acid diethylamide on dispersed mucosal cells from guinea pig stomach.

In dispersed mucosal cells from guinea pig stomach, D-lysergic acid diethylamide (LSD) was a partial agonist with respect to histamine. LSD, like histamine, inhibited [3H]histamine binding and increased both cellular cyclic AMP and [14C]aminopyrine uptake by interacting with histamine H2-receptors on parietal cells. These processes were blocked by both histamine H1- and H2-antagonists and, thus, provide evidence that histamine H2-receptors on guinea pig parietal cells resemble those in brain tissue in that they interact with LSD as well as with both classes of histamine antagonists.