Change in the cytosolic free Ca2+ concentration functions as an impOrtant intracellular signaling mechanism whereby hormones and growth factors regulate many different cellular processes such as muscle contraction, secretion, metabolism, cell growth and differentiation. A central question is how these diverse processes, often occurring within the same cell, are regulated by a common intracellular signaling mechanism, namely Ca2+. Studies on single cells have revealed that the Ca2+ signal is a highly complex and variable signal consisting of both temporal and spatial information. It is likely that the generation of a hormone-specific Ca2+ signal results from the differential activation of an intricate network of positive and negative controls by the various receptor systems. The objective of this research project is to elucidate this network of control mechanisms that coordinate the Ca2+ signal into hormone- specific responses. Ca2+ signaling will be studied in its physiological relevant environment of the intact cell by utilizing fluorometric monitoring of Ca2+ in combination with microinjection, flash photolysis and patch clamp techniques. Hepatocytes will be utilized as a model system for a non-excitable cell. Specifically, the aims of this study are to characterize Ins 1,4,5-P3-mediated Ca2+ entry, to determine the function of protein kinase A- and C-mediated phosphorylation of the Ins I,4,5-P3 receptor, and to determine the importance of Ins 1,4,5- P3-insensitive Ca2+ stores, particularly as to their relation to Ca2+- induced Ca2+ release and Ca2+ oscillations. Preliminary data are presented that demonstrate a direct activation of Ca2+ entry by Ins 1,4,5-P3 and that the Ins 1,4,5-P3 receptor is phosphorylated and regulated in vivo by hormones that activate protein kinase A. In addition, preliminary """"""""knockout"""""""" studies using antisense DNA indicate that expression of the hepatic Ins 1,4,5-P3 receptor can be suppressed, providing a unique model system to study the mechanisms coordinating hormone-mediated Ca2+ signaling. The information gained will directly aid our understanding of how Ca2+ mobilizing hormones elicit their specific responses, a fundamental stimulus-response mechanism common to almost every type of cell.
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