A research program is described which will be directed towards the characterization of growth factor-medicated Ca2+ responses in human parenchymal liver cells using liver biopsy material. This study relates to an extension of the parent grant concerning epidermal growth factor (EGF) and hepatocyte growth factor (HGF) actions on the liver. The physician experience of the Hungarian collaborator and his experience with measurement of growth factor effects on Ca2+ homeostasis in single cells will be combined to apply advanced techniques to human liver cells. First, attention will be focussed on establishing a reliable technique for measurement of intracellular Ca2+ in fura-2-loaded single human hepatocytes to characterize """"""""normal"""""""" Ca2+ homeostasis of these cells with an emphasis on the effects of the recently described HGF. The involvement of a receptor tyrosine kinase in HGF-induced Ca2+ mobilization will be evaluated by tyrosine kinase inhibitors. The amount of releasable Ca2+ from non-mitochondrial and mitochondrial pools of human hepatocytes will be assessed by microperfusion of the specific Ca2+-mobilizing agents such as thapsigargin and mitochondrial uncouplers. Furthermore, purified antibodies raised against different components of the signaling pathway (G-proteins, phospholipase C isozymes) will be microinjected into human hepatocytes. This powerful approach has the potential for giving very specific answers to questions related to hormone-induced signal transduction in human liver. Second, growth factor-induced signal transduction and Ca2+ homeostasis will be investigated throughout the pathological evolution of alcoholic liver disease (ALD). We hypothesize that cellular changes in the hepatocytes are accompanied by specific alterations in cellular Ca2+ homeostasis corresponding in severity to the stage of ALD. A higher basal Ca2+ and a reduced cellular responsiveness to the effect of Ca2+ mobilizing hormones and growth factors is expected in relation to a decrease in the capacity of hormone-sensitive pools to sequester Ca2+. Assessment of the size of non-mitochondrial and mitochondrial Ca2+ pools will allow an evaluation of abnormal redistribution in the intracellular Ca2+. The inhibitory properties of transforming growth factor beta (TGF- beta) as well as the effect of protein kinase C-stimulating phorbol esters on HGF-induced Ca2+ response will be also determined. Knowledge of Ca2+ homeostasis and HGF-related early biochemical events of human parenchymal liver cells obtained throughout this project is of direct relevance to the development of new strategies for the prevention, prognostication, and treatment of alcoholic liver disease.
