The Na+/K+ ATPase is a ubiquitous cell surface protein which is present in all eukaryotic cells. In the liver, the Na+/K+ ATPase is a plasma membrane protein which creates the electrochemical sodium-gradient required to drive sodium-dependent bile salt transport, as well as other sodium-dependent transport processes. However, little is known about the molecular biology and tissue-specific transcriptional gene regulation of the hepatocyte Na+/K+ ATPase; and the mechanisms responsible for the inhibitory effects of interleukin-6 on Na+/K+ ATPase activity have been virtually unexplored. The objective of this study are to determine the mechanisms by which the cytokine Il-6 inhibits hepatocyte Na+/K+ ATPase activity, to identify and clone the beta-subunit isoform of the hepatic Na/K+ ATPase, and to characterize the transcriptional gene regulation of this critical plasma membrane transport protein. The initial studies will examine the effects of interleukin-6 on hepatocyte Na/K+ ATPase gene transcription and protein expression using cell cultures and an in vivo model. Fluorescence polarization will also be utilized to determine if the IL-6 induced inhibition of hepatocyte Na+/K+ ATPase activity is caused by alterations of membrane fluidity. Subsequently, the hepatocyte beta-subunit isoform of the Na+/K+ ATPase will be identified and cloned, and an isoform-specific polyclonal antibody will be developed. Finally, hepatocyte Na+/K+ ATPase transcriptional gene regulation by cytokines, hormones and bile salts, and transcriptional regulation during hepatic regeneration, will be characterized using gel-retardation assays and transient transfection assays. Collectively, this project will enhance our understanding of the molecular biology, transcriptional gene regulation and protein expression of the hepatocyte Na+/K+ ATPase. These studies can help delineate the pathophysiological mechanisms responsible for the inhibition of bile salt excretion which often occurs in severe inflammatory states such as sepsis and hepatitis. In addition, as the Na+/KI+ ATPase is a critical and ubiquitous cell surface enzyme, these studies may have relevance not only to sodium-dependent transport processes in the liver, but potentially to cellular transport in the intestine, kidney and other organ systems.
