The Role of Phosphoinositide 3-Kinase in Taurocholate-induced Trafficking of ATP-dependent Canalicular Transporters in Rat Liver

Abstract

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...

Shared apical sorting of anion exchanger isoforms AE2a, AE2b1, and AE2b2 in primary hepatocytes

“…4). This effect of colchicine on AE2 isoforms resembles what has been reported for transcytotic canalicular proteins such as the cell adhesion molecule cCAM105 [31].…”

Familial intrahepatic cholestasis 1: Studies of localization and function

“…1B, green fluorescence). Because Mdr gene products are restricted to the canalicular plasma membrane domain of hepatocytes, 21,23,24,25 we conclude that Fic1 is also localized in the apical domain of the hepatocyte membrane. By immunoblotting (Fig.…”

Tauroursodeoxycholic acid inserts the bile salt export pump into canalicular membranes of cholestatic rat liver

“…The exact molecular mechanism by which TUD-CA stimulates exocytotic fusion of vesicles at the apical membrane 7,25 and apical carrier insertion 7,12,13 remains elusive. Various signaling molecules including isoforms of PKC, 7,9,13 cytosolic-free calcium (Ca 2 þ ) i , 9,25 mitogen-activated protein (MAP) kinases p38 MAPK6,12,13 and p44/42 MAPK , 11,14 src kinase 6 as well as PI3K 8,[26][27][28] have been shown to be involved in bile acid-dependent hepatobiliary exocytosis and carrier insertion/retrieval in apical membranes under various experimental conditions. 29 In the normal rat liver, TUDCA may trigger insertion of Bsep into canalicular membranes by a dual integrin-dependent signaling pathway which includes activation of p44/42 MAPK on one hand and activation of p38 MAPK on the other hand 6,[11][12][13] Preliminary evidence suggests that this pathway may be less relevant for the anticholestatic effect of TUDCA in bile acid-induced cholestasis 14,15 In TLCA-induced cholestasis, the anticholestatic effect of TUDCA appears to be mediated in part by PKC-dependent mechanisms although the exact isoform involved has not yet been determined.…”