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It has been demonstrated in experimental cholecystitis in cats produced by lysophosphatidylcholine that the development of inflammation is associated with the exsorption of a large amount of protein into the gallbladder lumen. It was subsequently demonstrated that in feline experimental cholecystitis the protein produced was albumin and that its production was decreased by vesicular transport inhibitors, suggesting an active secretory process. In the present study, the effect of lysophosphatidylcholine on protein production by fresh, isolated human gallbladder mucosal cells was evaluated. Isolated gallbladder mucosal cells were incubated with [14C]leucine for 24 hr in tissue culture medium. The cells readily incorporated the radioactive label into cellular protein, a process inhibited by cycloheximide. Exposure of the cells to lysophosphatidylcholine for 1 hr in buffer solution resulted in loss of intracellular protein into the buffer solution. Exposure of the cells for 1 hr prior to lysophosphatidylcholine administration to vesicular transport inhibitors, colchicine, and cytochalasin B and to 4 degrees C culture conditions failed to alter the lysophosphatidylcholine-produced passage of the 14C label extracellularly. SDS-PAGE evaluation of the protein produced demonstrated that human gallbladder mucosal cells continuously produced a 66-kDa protein that was not increased by increasing concentration of lysophosphatidylcholine and a 14-kDa protein that increased with increasing concentrations of lysophosphatidylcholine. Employing Western blotting with specific antibodies, the 66-kDa protein was demonstrated to not be albumin but a 66-kDa glycoprotein, and the 14-kDa protein was demonstrated to contain phospholipase A2. Human gallbladder mucosal cells produced a protein and glycoprotein in response to lysophosphatidylcholine by a mechanism not related to vesicular transport.

The development of experimental cholecystitis produced by lysophosphatidylcholine is associated with reversal of the normal absorptive characteristics of gallbladder mucosa, resulting in the intraluminal accumulation of water, glycoprotein, and protein. The purpose of the present study was to attempt to ascertain if the protein leaks into the lumen because of the cytolytic properties of lysophosphatidylcholine or if it is due to an active secretory process and to characterize the protein produced. Experiments were performed on anesthetized cats undergoing gallbladder perfusion with and without lysophosphatidylcholine. The amount of protein in the perfusate was measured and albumin clearance from blood to gallbladder lumen was calculated with and without the administration of vesicular transport inhibitors. In separate experiments, control and lysophosphatidylcholine (LPC) produced gallbladder perfusates were collected and the protein subjected to SDS-PAGE to ascertain the nature of the protein secreted. Inhibitors of both microtubular and microfilament activity decreased the protein accumulation and clearance produced by lysophosphatidylcholine. Gallbladder white blood cell accumulation and inflammation as evaluated by beta-glucuronidase and prostaglandin E levels were not significantly altered by cytochalasin or colchicine administration. Lysophosphatidylcholine also produced significant increases in perfusate LDH levels. The protein produced was primarily a 66-kDa protein. Transfer of the protein to a nitrocellulose membrane and immunoblotting with anti-albumin antibody demonstrated that the protein was albumin. The results suggest that during the development of cholecystitis, lysophosphatidylcholine produces albumin accumulation in the gallbladder primarily by inducing an active secretory process resulting in gallbladder distension.

The cellular mechanisms associated with the replicative response of hepatocytes to growth factor simulation is incompletely understood. Murine hepatocyte DNA synthesis is altered by cyclic AMP, suggesting that protein kinase A is involved in the cellular mechanisms associated with liver growth. The purpose of this study was to evaluate the role of protein kinase A in human hepatocyte DNA synthesis. human hepatocytes were isolated and maintained in primary culture on rat tail collagen. DNA synthesis was evaluated by determining [3H] thymidine incorporation. Human hepatocytes between 24 and 96 hr following harvest increased DNA synthesis in response to epidermal growth factor but not in response to glucagon, a stimulant of adenyl cyclase, or dibutyryl cyclic AMP. Mitogen-stimulated DNA synthesis was decreased by dibutyryl cyclic AMP. Cyclic AMP isomers that block or stimulate the effect of cyclic AMP on protein kinase A did not significantly alter resting or mitogens-stimulated human hepatocyte DNA synthesis. The results suggest that increased protein kinase A activity does not produce human hepatocyte replicative DNA synthesis.

Exogenous glucagon administration is associated with stimulation of hepatic bile flow. The physiologic role that glucagon plays in the control of hepatic bile flow remains indeterminant. The purpose of this study was to evaluate amino acid administration, a stimulus of endogenous glucagon release, on canine hepatic bile flow. The experiments were performed utilizing cholecystectomized dogs with chronic biliary fistulas. The enterohepatic circulation of bile salts was artificially maintained by intravenous bile salt administration. Intravenous L-arginine stimulated endogenous glucagon release and hepatic bile secretion. Intravenous amino acid administration produced significant increases in hepatic bile flow and plasma glucagon and was significantly more potent than intravenous arginine. Intravenous amino acid administration produced small but significant increases in serum insulin but did not significantly change plasma concentrations of cholecystokinin. The results of this study suggest that endogenous glucagon secretion produces a choleresis and supports a role for glucagon in the physiologic control of canine hepatic bile flow.

In a randomized double-blind trial, the effect of ibuprofen on the pain produced by gallbladder disease and on gallbladder mucosa and muscle wall tissue PGE and PGF production was evaluated to determine if the pain of cholecystitis and prostaglandin formation were altered by administration of a prostaglandin synthetase inhibitor. To ascertain potential differences in extracellular and intracellular prostaglandin production rates, gallbladder mucosal cells and muscle tissues were maintained in tissue culture medium and then subsequently homogenized. PGE and PGF concentrations were measured in culture medium and homogenates utilizing radioimmunoassay. Gallbladder mucosa and muscle tissue produced nanogram per milligram protein amounts of PGE and PGF. As the histological estimation of the degree of inflammation increased, so also did the production of PGE. Increased inflammation was associated with unchanged PGF levels, resulting in an increased ratio of PGE/PGF with increasing inflammation. Oral ibuprofen administration was effective in decreasing PGE production by gallbladder mucosa and muscle and eliminating the significant correlation between PGE levels and the histologic degree of inflammation found in the placebo-treated patients. Ibuprofen significantly decreased the pain of cholecystitis when compared to placebo-treated patients. However, there was poor correlation between pain relief and changes in PGE production by gallbladder mucosa and muscle. PGE may play a mediator role in inflammation associated with cholecystitis. Prostaglandin synthetase inhibition decreases the pain associated with cholecystitis; however, the absence of correlation with decreased PGE formation suggests that other prostanoids may play an important role in producing the symptoms of cholecystitis.

This experiment was performed to determine the effect of theophylline on glucagon and secretin stimulated bile flow. We intended to determine the effect of theophylline, a proposed stimulant of canalicular bile flow, on the bile flow response produced by well-recognized stimulants of canalicular (glucagon) and ductular (secretin) bile flow. Dogs with chronic bile fistulas were used. Dose-response curves for glucagon and secretin were produced by administration of a wide range of glucagon and secretin doses. The effect of 20 mg/kg/hr theophylline on the dose-response curves was determined. Theophylline significantly increased the bile flow and bile electrolyte changes produced by glucagon at low doses but not at maximal doses of glucagon for stimulation of bile flow. Theophylline significantly increased bile volume and bile electrolyte changes at all doses of secretin, including maximal doses. These results suggest that theophylline and glucagon, both purported to be stimulants of canalicular bile flow in dogs, utilize the same receptor mechanism to stimulate bile flow. The effects of secretin, a proposed ductular stimulant, are potentiated by theophylline. On a functional basis, it is possible that there are separate canalicular and ductular loci of hormone action in stimulation of bile secretion.

Continuing evaluation of the pathophysiology of gallbladder disease has demonstrated significant relationships between gallbladder mucosal fluid transport, gallbladder inflammation, and prostanoid formation. Inflamed gallbladder mucosa secretes, rather than absorbs, fluid, a process associated with prostaglandin formation. Bradykinin has been previously implicated in the pathogenesis of cholecystitis and, in the intestine, bradykinin stimulates mucosal fluid secretion by a prostaglandin-mediated mechanism. Bradykinin was infused into the gallbladder lumen and administered intraarterially into the hepatic artery of perfused cat gallbladders. Both methods of bradykinin administration reversed the mucosal absorption present during control experiments as measured by concentration changes in a nonabsorbable marker. Perfusate and gallbladder tissue prostaglandin E concentrations were significantly increased by bradykinin when compared to control values. Concentrations of 6-keto PGF1 alpha in perfusate solutions and in gallbladder tissue were significantly increased, suggesting bradykinin increased prostacyclin formation. Bradykinin administration significantly increased inflammation, as evaluated by a histologic scoring system. Indomethacin was administered intravenously along with luminal perfusion of the gallbladder with bradykinin. Indomethacin significantly decreased gallbladder fluid secretion and prostanoid formation, but not histologic inflammation, when compared to values produced by bradykinin alone. An increase in systemic vascular and bile kinin concentrations produces gallbladder mucosal water secretion, a process which may be mediated by prostanoids. Histologic inflammation produced by bradykinin was not prevented by indomethacin.