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 calcium. Recent findings indicate that 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 messenger molecules, diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DG is believed to activate a specific kinase in cells (protein kinase C) and IP3 is believed to act by releasing stored calcium. IP3 is subsequently metabolized by two distinct pathways, one involving dephosphorylation, and the other phosphorylation and dephosphorylation; the result is a complex mixture of different inositol phosphates which might have significant biological actions. The formation and metabolism of these compounds is being investigated in intact and broken cell systems to determine the quantities formed of each these substances, and the kinetics of their formation and degradation. Some of the studies involve the use of tissues from euthanized laboratory rats, while others use immortal cell lines (rat pancreatoma). These techniques involve the use of tritium- labeled precursors, and separation of compounds by a combination of anion exchange chromatography and HPLC. Cellular calcium is being investigated in the same model cell systems with fluorescent indicators to determine correlations between calcium and the various inositol phosphates. These studies should provide insights into mechanisms of action of calcium mobilizing agents, and may indicate vulnerable sites of interaction with environmental toxins such as heavy metals.
