MECHANISM OF PHORBOL ESTER-INDUCED LIPID METABOLISM

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The proposed research involves defining proximal intracellular events involved in the action of tumor-promoting phorbol esters. Specifically, the biochemical mechanisms of arachidonic acid release and phosphatidylcholine metabolism will be investigated using GH3 rat pituitary cells. Although it has been suggested that stimulation of a phospholipase A2 and phospholipase C, respectively are causative in these events, conclusive evidence is lacking. These events are important because accumulated evidence from many cell types suggests 1) that arachidonic acid and/or metabolites may mediate tumor promotion and 2) phosphatidylcholine metabolism appears specifically related to phorbol ester action. GH3 cells provide an excellent model for these studies because 1) they are a homogeneous cell population permitting unambiguous assignment of changes to a specific cell type and 2) extensive research into protein kinase C, the putative phorbol ester receptor has been performed in these cells. Suggestions as to the enzymic reactions involved from intact cell studies will be confirmed in subcellular fractions. Whether protein kinase C or another cellular acceptor for phorbol esters mediates these events will be studied directly in crude and purified membranes. The effects of diacylglycerols will be compared to phorbol esters on arachidonic acid release and phosphatidylcholine metabolism, cell proliferation, and protein phosphorylation. A potential role for arachidonic acid as an intracellular mediator of phorbol ester and/or diacylglycerol action will be assessed. These studies should further our understanding of the biochemical mechanisms involved in phorbol ester-induced arachidonic acid release and phosphatidylcholine metabolism. Furthermore, and importantly, these studies into proximal intracellular events in phorbol ester action may help elucidate processes involved in the development of some neoplasias.