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.
Specific aims of this proposal are to better define cellular mechanisms involved in the endocrine regulation of two hepatic solute transporters; Na+/H+ exchange being integrally involved with the regulation of intracellular pH, and Na+/taurocholate (TC) cotransport being involved in hepatic uptake of bile acids, which play an important role in file formation. The following hypotheses will be tested: 1) hormonal regulation of Na+/H+ exchange may involve kinase-mediated phosphorylation of the transporter, and 2) hormones may stimulate Na+/TC cotransport directly by translocating the transporter from intracellular organelles to the plasma membrane, and/or indirectly by changing membrane potential. Effects of hormones and kinases on Na+/TC cotransport and Na+/H+ will be studied in isolated rat hepatocytes and plasma membrane vesicles using established techniques. Intracellular [Ca++] pH and [Na+] will be quantitated using fluorometric methods. Suggestive evidence for the involvement of kinase will be obtained through transport studies in hepatocytes using known inhibitors and activators of specific kinases. Direct modification of the transporter will be assessed by studying transport in membrane vesicles treated with specific kinases. Transport studies in membrane vesicles prepared from hormone-treated hepatocytes would indicate if hormone action involves stable alteration (phosphorylation/translocation) of transport proteins. Regulation of phosphorylation of the Na+/H+ exchanger or translocation of the Na+/TC cotransporter will be evaluated by protein phosphorylation and immunoblotting studies, respectively. Subcellular fractions (microsomes and golgi complex) isolated from hormone-treated hepatocytes will be immunoblotted to determine the source(s) of the translocated transporter. Whether a hormone action is mediated indirectly via changes in membrane potential will be evaluated by correlating effects of the hormone on Na+/TC cotransport and membrane potential under different experimental conditions. Studies will also be conducted to determine the role of Na+,K+ -ATPase in hormone-induced changes in membrane potential. Collectively, proposed studies should provide further insight into cellular mechanisms involved in the hormonal regulation of hepatic Na+/H+ exchange and Na+/TC cotransport.
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