Bile secretion results from coordinately regulated transport processes located at the sinusoid and bile canalicular domains of hepatocytes. Sodium-dependent taurocholate, and sodium-independent organic anion transporters as well as NaK-ATPase are localized to the sinusoidal domain. The long-range goal of this laboratory has been to identify and characterize the regulation of membrane transporters involved in bile formation and pathogenesis of cholestasis. Studies from our laboratory indicate that disorders of sinusoidal membrane transport processes may be the primary site for the pathogenesis of estrogen-induced cholestasis. The sinusoidal transporters are expressed in sexually dimorphic pattern, and decreased transporter expression by estrogens is mediated by the hypothalamic-pituitary-liveraxis. The first hypothesis we propose to test is: The sexually dimorphic expression of sodium-dependent taurocholate transporter is transcriptionally regulated by DNA elements in the 5'flanking region of the gene. Hormone replacement studies will be conducted in hypophysectomized rats and genetically deficient mice (lit/lit). We will also determine the specific hormone effects using cultured hepatocytes and unique cell lines which express the transporter. The promoter will be cloned and used for transient transfection assays and identification of DNase I sensitive sites, DNA-binding proteins, and the development of transgenic mice.
The second aim i s to examine the cell specific insulin regulation of hepatic NaK-ATPase, which plays a pivotal role in regulation of bile salt transport and bile flow. Preliminary data has shown that diabetes selectively increased NaK-ATPase beta-subunit. We propose to examine the hypothesis that beta-subunit, in part, regulates plasma membrane NaK-ATPase activity, and its expression is transcriptionally regulated by insulin. The effect of insulin deficiency on NaK-ATPase will be examined in diabetic rats, and transcriptional regulation of beta1 subunit by insulin will be determined using transient transfection assays in hepatoma cells. Taken together, these studies will provide an understanding of the hormonal control of the molecular events regulating sinusoidal membrane transporters which are involved in generation of bile flow.
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