The broad aim of this project is to understand at the cellular and molecular level the mechanisms by which surface membrane receptors for hormones, neurotransmitters and growth factors modify cellular responses through mobilization of cellular Ca2+. An early event in the action of receptors of this class is the hydrolysis of a membrane phospholipid, phosphatidylinositol 4,5-bisphosphate to yield two putative second messengers, diacylglycerol (DG) and inositol, 1,4,5-trisphosphate ((1,4,5)IP3). DG activates a specific kinase in cells, designated protein kinase C, and (1,4,5)IP3 releases Ca2+ from an intracellular store, presumably a component of the endoplasmic reticulum. Recently, a number of novel inositol polyphosphates have been discovered, including (1,3,4)IP3, (1,3,4,5)IP4 and (cyclic 1:2,4,5)IP3. Each of these has been shown to have biological activity in in vitro assay systems. These studies will attempt to combine in vivo (cells) assays for the amounts and kinetics of these and other important phosphates with real time measurements of cytosolic C- using rationing Ca2+ indicators, such as fura-2, and with in vitro assays of their actions on subcellular Ca2+ metabolism, in order to the relative contributions of these various species to cellular signalling mechanisms. In a related project, the mechanisms by which the phospholipase C-linked receptors are regulated is being investigated, using the parotid substance P receptor as a model. This receptor appears to be regulated by both heterologous and homologous mechanisms, with the latter being the quantitatively more important. The heterologous mechanism involves feed back inhibition at a step subsequent to receptor activation by the protein kinase C. Since Ca2+ is believed to play a central role in mechanisms of chemically-induced cell injury, these studies should provide insights into the mechanisms underlying the pathophysiological consequences of exposure to toxins and other environmental agents.
