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Recent studies indicate that wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase, interferes with bile acid secretion in rat liver; taurocholate induces recruitment of ATP-dependent transporters to the bile canalicular membrane, and PI 3-kinase products are important in intracellular trafficking.We investigated the role of PI 3-kinase in bile acid secretion by studying the in vivo effect of taurocholate, colchicine, and wortmannin on bile acid secretion, kinase activity, and protein levels in canalicular membrane vesicle (CMV) and sinusoidal membrane vesicle (SMV) fractions from rat liver. Treatment of rats or perfusion of isolated liver with taurocholate significantly increased PI 3-kinase activity in both membrane fractions. Taurocholate increased protein content of ATPdependent transporters, which were detected only in CMVs, whereas increased levels of p85 and a cell adhesion molecule, cCAM 105, were observed in both fractions.Colchicine prevented taurocholate-induced changes in all proteins studied, as well as the increase in PI 3-kinase activity in CMVs, but it resulted in further accumulation of PI 3-kinase activity, p85, and cCAM 105 in SMVs. These results indicate that taurocholate-mediated changes involve a microtubular system. Wortmannin blocked taurocholate-induced bile acid secretion. The effect was more profound when wortmannin was administered prior to treatment with taurocholate. When wortmannin was given after taurocholate, the protein levels of each ATP-dependent transporter were maintained in CMVs, whereas the levels of p85 and cCAM decreased in both membrane fractions. Perfusion of liver with wortmannin before taurocholate administration blocked accumulation of all proteins studied in CMVs and SMVs.These results indicate that PI 3-kinase is required for intracellular trafficking of itself, as well as of ATP-dependent canalicular transporters.Bile acids are the predominant organic solutes in bile. Only 5% of the total bile acid pool is produced daily as a result of 7 ␣ hydroxylation of cholesterol in the liver; the remaining 95% of bile acids undergoes enterohepatic circulation and is transferred into the bile from plasma by mechanisms that are only partially understood (1, 2). Three bile acid transporters have been identified in the basolateral plasma membrane of hepatocytes, but none are known to associate with vesicular trafficking of bile acids to the canaliculus (3-5). Although bile acids bind with varied affinity to several hepatocellular cytoplasmic proteins (6), the role of protein binding in intracellular movement is uncertain. Because administration of microtubular inhibitors, such as colchicine, reduces bile acid secretion but not uptake from plasma, a microtubule-based vesicular mechanism of transcellular bile acid transport has been proposed (7,8).In recent years, several ATP-dependent transporters have been functionally identified in CMVs 1 for bile acids, organic cations, phosphatidylcholine, and nonbile acid organic anions (9 -13). Cloning identified the respons...

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Bile acid transport and secretion in hepatocytes require phosphatidylinositol (PI) 3-kinase-dependent recruitment of ATP-dependent transporters to the bile canalicular membrane and are accompanied by increased canalicular PI 3-kinase activity. We report here that the lipid products of PI 3-kinase also regulate ATP-dependent transport of taurocholate and dinitrophenyl-glutathione directly in canalicular membranes. ATP-dependent transport of taurocholate and dinitrophenyl-glutathione in isolated canalicular vesicles from rat liver was reduced 50-70% by PI 3-kinase inhibitors, wortmannin, and LY294002, at concentrations that are specific for Type I PI 3-kinase. Inhibition was reversed by addition of lipid products of PI 3-kinase (PI 3,4-bisphosphate and, to a lesser extent, PI 3-phosphate and PI 3,4,5-trisphosphate) but not by PI 4,5-bisphosphate. A membranepermeant synthetic 10-mer peptide that binds polyphosphoinositides and leads to activation of PI 3-kinase in macrophages doubled PI 3-kinase activity in canalicular membrane vesicles and enhanced taurocholate and dinitrophenylglutathione transport in canalicular membrane vesicles above maximal ATP-dependent transport. The effect of the peptide was blocked by wortmannin and LY294002. PI 3-kinase activity was also necessary for function of the transporters in vivo. ATP-dependent transport of taurocholate and PI 3-kinase activity were reduced in canalicular membrane vesicles isolated from rat liver that had been perfused with taurocholate and wortmannin. PI 3,4-bisphosphate enhanced ATPdependent transport of taurocholate in these vesicles above control levels. Our results indicate that PI 3-kinase lipid products are necessary in vivo and in vitro for maximal ATP-dependent transport of bile acid and nonbile acid organic anions across the canalicular membrane. Our results demonstrate regulation of membrane ATP binding cassette transporters by PI 3-kinase lipid products.The bile canalicular membrane of mammalian hepatocytes contains at least four types of transmembrane proteins that belong to the multidrug resistance or multidrug resistance associated families and that require hydrolysis of ATP for transport of taurocholate [by sister of P-glycoprotein (spgp)], nonbile acid organic anions (by multidrug resistance associated protein 2), organic cations (by multidrug resistance protein 1) and translocation of phosphatidylcholine (PC) from the inner to the outer membrane layer (by multidrug resistance protein 3) (1-5). It recently was demonstrated that taurocholate administration to rats intravenously or by perfusion of isolated rat liver significantly increased biliary secretion and recruitment of each of these transporters from Golgi to the canalicular membrane (6, 7). This process also was associated with an increase in phosphatidylinositol (PI) 3-kinase activity in membrane fractions (7). Administration of wortmannin after perfusion with taurocholate rapidly reduced bile acid secretion Ͻ50% of control values but did not affect taurocholate-induced recruitment...