Formation of bile is a major function of the liver. Bile secretion results from coordinately regulated transport processes for bile salts and electrolytes. These transports are for the most part asymmetrically distributed at opposite poles of the liver plasma membrane. Many of the transporters, such as Na+-dependent taurocholate carrier, NaK-ATPase, Na/H exchanger and Na-HCO3 Cotransporter have been identified on the sinusoidal membrane surface. It is the long range goal of this laboratory to identify and characterize the regulation of membrane transport processes involved in bile formation and cholestasis. We propose to examine the following hypotheses: 1) the sinusoidal domain is the primary site for regulation of bile formation and alterations in intrahepatic cholestasis, and 2) hepatic NaK-ATPase activity is regulated by both genomic and nongenomic mechanisms. In animal models of cholestasis we will isolate liver plasma membrane subfractions and determine taurocholate transport in vesicles from the sinusoidal and bile canalicular domain. NaK-ATPase activity, Na/H exchange, and Na-HCO3 co-transport will be measured in sinusoidal vesicles. Hepatocytes will be isolated and in addition to transport of bile acids and electrolytes, we will determine intracellular pH and potential difference. Because the NaK-pump is the key regulator for many of the secondary active transporters involved in vectorial translocation of bile salts and electrolytes, we propose to examine its genomic and nongenomic (membrane lipid fluidity) regulation. Hepatic NaK-ATPase is uniquely located at the apical as well as the sinusoidal surface. With the use of biochemical, pharmacological and genetic techniques, we will determine differences between the pumps located at each domain. Transcriptional and post-transcriptional events in the control of hepatic NaK-ATPase will be determined by measurement of mRNA levels and the turnover and immunoquantitated of NaK-ATPase isoforms for alpha and beta subunits. Nongenomic control of NaK-ATPase activity will be examined following 2/3 hepatectomy and regeneration. This model shows selectively increased canalicular fluidity and NaK-ATPase activity. NaK-ATPase will be immunoquantitated, while fluidity and lipid composition measurements will be determined in plasma membrane subfractions. Finally, agents (phenobarbital, T 3 and glucocorticoids) known to increase NaK-ATPase activity and bile flow will be similarly analyzed. Taken together, these studies will provide an understanding of the molecular and cellular steps involved in regulation of bile flow and the primary sites of alteration in bile secretory failure.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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General Medicine A Subcommittee 2 (GMA)
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University of Colorado Denver
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Qadri, Ishtiaq; Hu, Ling-Jia; Iwahashi, Mieko et al. (2009) Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2). Toxicol Appl Pharmacol 234:281-92
Qadri, Ishtiaq; Iwahashi, Mieko; Kullak-Ublick, Gerd A et al. (2006) Hepatocyte nuclear factor (HNF) 1 and HNF4 mediate hepatic multidrug resistance protein 2 up-regulation during hepatitis C virus gene expression. Mol Pharmacol 70:627-36
Simon, Francis R; Iwahashi, Mieko; Hu, Ling-Jia et al. (2006) Hormonal regulation of hepatic multidrug resistance-associated protein 2 (Abcc2) primarily involves the pattern of growth hormone secretion. Am J Physiol Gastrointest Liver Physiol 290:G595-608
Qadri, Ishtiaq; Iwahashi, Mieko; Capasso, Juan M et al. (2004) Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1. Biochem J 378:919-28
Simon, Francis R; Fortune, John; Iwahashi, Mieko et al. (2004) Multihormonal regulation of hepatic sinusoidal Ntcp gene expression. Am J Physiol Gastrointest Liver Physiol 287:G782-94
Reichard, John F; Doorn, Jonathan A; Simon, Franz et al. (2003) Characterization of multidrug resistance-associated protein 2 in the hepatocellular disposition of 4-hydroxynonenal. Arch Biochem Biophys 411:243-50
Qadri, Ishtiaq; Iwahashi, Mieko; Simon, Francis (2002) Hepatitis C virus NS5A protein binds TBP and p53, inhibiting their DNA binding and p53 interactions with TBP and ERCC3. Biochim Biophys Acta 1592:193-204
Simon, Francis R; Fortune, John; Iwahashi, Mieko et al. (2002) Sexual dimorphic expression of ADH in rat liver: importance of the hypothalamic-pituitary-liver axis. Am J Physiol Gastrointest Liver Physiol 283:G646-55
Fiedler, J; Simon, F R; Iwahashi, M et al. (2001) Effect of peroxisome proliferator-activated receptor alpha activation on leukotriene B4 metabolism in isolated rat hepatocytes. J Pharmacol Exp Ther 299:691-7
Shiao, T; Iwahashi, M; Fortune, J et al. (2000) Structural and functional characterization of liver cell-specific activity of the human sodium/taurocholate cotransporter. Genomics 69:203-13

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