Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.     

Influence of bile salts on the endogenous excretion of bile pigments

Effect of the infusion of glycodeoxycholate (GDC), taurocholate (TC) and dehydrocholate (DHC) on bile flow and on bile salt, biliary lipid and bile pigment secretion, has been studied in pentobarbital-anesthetized rabbits. GDC increased bile flow the most, while DHC increased it more than TC. The different choleretic actions of these bile salts cannot be explained by means of variations in their capacity to form micelles. Only GDC and TC were able to stimulate biliary lipid secretion, which suggests that both bile salts increase the formation of mixed micelles. GDC and TC to a lesser extent increased bile pigment excretion, DHC being without effect. These results favour the hypothesis that micellar binding could be an important factor responsible for the effect of bile acids on bile pigment excretion and should not be completely ruled out.     

Effect of metyrapone on bile flow and bile acid excretion in Wistar rats

The influence of metyrapone on bile flow and excretion of mono-(MBA), di-(DBA) and trihydroxy-(TBA)-bile acids was investigated in adult male Wistar rats after single and repeated pretreatment. MBA were not found in the rat bile. Metyrapone administration (200 mg/kg b.w. i.p.) 1 h before onset of a 3-hour bile collection period diminished bile flow and excretion of DBA and TBA. The relation TBA/DBA was changed towards DBA. Similar results were found after repeated administration 12 h after the last metyrapone injection (4 x 50 mg/kg b.w. i.p. per day for 4 consecutive days). But 60 h after the last metyrapone administration bile flow and the excretion of TBA were enhanced and the TBA/DBA ratio was changed towards TBA. The possible influence of metyrapone on bile acid hydroxylation is discussed and compared with metyrapone action on hydroxylation of foreign compounds.     

Comparative aspects of bile pigment formation and excretion.

Breakdown of haem which is of key importance in most organisms, involves oxidative CO-evolving cleavage of the macrocyclic ring with formation of biliverdin-IX. In two major pathways established so far formation of biliverdin-IX alpha is followed by (a) biliary secretion or (b) reduction to bilirubin-IX alpha, formation of more hydrophilic derivatives (usually glycosidic conjugates) and biliary secretion. The scattered comparative information available indicates marked species variation with regard to the methin-bridge carbon atom removed from haem and the metabolic site of cleavage, the nature of bilirubin conjugates and the developmental sequence of maturation of enzyme activities and transport proteins involved in the chain of events leading from breakdown of haem to the excretion of the final end products.     

Mechanisms of bacterial resistance and response to bile

Enteric bacteria are resistant to the bactericidal effects of intestinal bile, but these resistance mechanisms are not completely understood. It is becoming increasingly apparent that enteric bacteria have evolved to utilize bile as a signal for the temporal production of virulence factors and other adaptive mechanisms. A greater understanding of the resistance and response of bacteria to bile may assist the development of novel therapeutic, prevention, and diagnostic strategies to treat enteric and extraintestinal infections.     

The determination and excretion of individual human fecal bile acids

A fluorometric method, using resazurin, for the analysis of individual fecal bile acids separated by thin layer chromatography of crude fecal extracts is described. The method is precise and accurate. The assay was used to investigate the constancy of excretion of individual fecal bile acids in small random stool samples collected over three weeks in six humans. Relatively small but significant variations were found in each case, most were random but one was progressive.     

Disposition and metabolism of 2-fluoro-beta-alanine conjugates of bile acids following secretion into bile

Since 2-fluoro-beta-alanine (FBAL) conjugates of bile acids (BA), the primary biliary metabolites of fluoropyrimidine (FP) drugs, have been suggested to be related to the hepatotoxicity which develops in patients receiving FP chemotherapy by intrahepatic arterial infusion (Proc. Natl. Acad. Sci. USA 84, 5439-5443, 1987), it was important to determine whether they undergo enterohepatic circulation and hence accumulate in the liver and biliary system. In initial studies, sensitivity of FBAL-BA conjugates to hydrolysis by pancreatic enzymes was examined. In subsequent in vivo studies, a model FBAL-BA conjugate, FBAL-chenodeoxycholate (FBAL-CDC), was introduced into the lumen of the small intestine of anesthetized rats with biliary fistulas to quantitate the intestinal absorption, metabolism and tissue distribution of the conjugate. The results indicated that: (1) FBAL-BA conjugates were resistant to hydrolysis by pancreatic enzymes (carboxypeptidase A, carboxypeptidase B and trypsin) and by human pancreatic juice, but were completely hydrolyzed by cholyglycine hydrolase. (2) At least one-half of the administered FBAL-CDC was deconjugated during the process of intestinal absorption, as shown by HPLC analysis of the radioactivity in portal venous blood. (3) Deconjugated FBAL or CDC was reconjugated in liver with other bile acids or amino acids (glycine and taurine), respectively, as shown by radiochromatography of bile. (4) FBAL, formed as a result of hydrolysis of FBAL-CDC, had a wide tissue distribution. In conclusion, FBAL-CDC has a rapid turnover during its enterohepatic circulation due to deconjugation in the intestine and reconjugation in the liver.