The long-term objective of the present proposal is to more clearly define cellular mechanisms involved in hepatic solute transport and bile formation. Solute transport from blood to bile is an important function of the liver, with accumulation of (otherwise excreted) endogenous and exogenous solutes in blood being a common feature of cholestasis. The major focus of this proposal will be to define the role of phosphoinositide-3-OH kinase (PII3K), protein kinase B (PKB), mitogen activate protein kinase (MAPK) and protein phosphatases (PP2A and PP2B) in hepatic bile acid transport from blood to bile. Sinusoidal Na+/taurocholate (TC) co-transport is mediated via Na/TC co-transport peptide (ntcp), and canalicular ATP-dependent bile acid transport is mediated via a sister P-glycoprotein (spgp). The following hypothesis will be tested: 1) The short term regulation of Na+/TC co-transport by cAMP involves translocation of ntcp to the sinusoidal membrane; the translocation require PI3K/PKB activity, involves vesicular transport and is facilitated by dephosphorylation, a process stimulated by PP2A and PP2B, and inhibited by MAPK, and 2) PI3K and MAPK stimulate canalicular ATP-dependent bile acid transport by translocating spgp to the canalicular membrane. Effects of various activators and inhibitors of kinases and phosphatases on sinusoidal and canalicular bile acid transport will be studied in isolated perfused rat livers, rat hepatocytes and plasma membrane vesicles using established techniques. The effectiveness of various inhibitors and activators will be assessed by determining the activity of PI3K, PKB, MAPK and PPA2/2B. MDCK cells stably transfected with wild- type and mutant ntcp will be used to determine the role of phosphorylation in translocation and transport activity. Transporter translocation will be evaluated by determining transporter mass in plasma membranes isolated from hepatocytes/livers treated with various activators/inhibitors. Suggestive evidence for the involvement of a kinase/phosphatase will be obtained through transport studies in hepatocytes/perfused livers using known inhibitors and activators. Direct modification of the transporter will be assessed by determining transport activity and transporter mass and phosphorylation in membrane vesicles treated with specific kinases/phosphatases. Collectively, proposed studies should provide further insight into cellular mechanisms involved in the regulation of hepatic bile acid transport.
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