It has been known since 1983 that the hyrdolysis of a plasma member phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), yields two intracellular second messengers, inositol 1,4,5- trisphosphate (IP3) and 1,2-diacylglycerol (DG). 1,4,5-IP3 releases calcium from intracellular stores and 1,2,-DG activates protein kinase C in a variety of cells, including photoreceptors. We have shown that light accelerates the rate of PIP2 hydrolysis in frog outer segments (ROS). These findings were confirmed in retinas from other animals, both vertebrate and invertebrate. Electrophysiological studies, primarily in invertebrates, have provided evidence that 1,4,5-IP3 may play a role in excitation and/or adaptation of these retinas. This proposal contains two specific aims. The first is to study the metabolism of phosphoinositides and their products in the retina. Specifically, we will: determine the mechanisms of activation of phospholipase C following photolysis of rhodopsin, isolate and characterize phospholipase C(s) from ROS, characterize the synthetic and degradative enzymes for the phosphoinositides in ROS, describe the metabolism of the water soluble inositol phosphates, search for a phosphoinositide-specific exchange protein in photoreceptor cells, and isolate and characterize protein kinase C(s) and its (their) phosphorylated products in ROS. The second objective is to continue our long-term studies on overall lipid metabolism in the retina, of which the phosphoinositide problem is a small but significant part. We propose to describe the biosynthesis and turnover of individual molecular species of the phospholipids of ROS membranes by following the fate of injected 2-3H-glycerol and 2-3H-acetate. To accomplish these goals, we will take advantage of technological advances in recent years to fractionate ROS phospholipids into individual molecular species by high performance liquid chromatography (HPLC). HPLC will also be used for separation and quantification of water soluble products of PIP2 hydrolysis for the purification of protein kinase C and phospholipase C in ROS. Gas-liquid chromatography, high voltage electrophoresis, and thin layer chromatography will also be used extensively in these studies. These results will provide information on the role of phosphoinositides in the vertebrate retina. Hopefully, by the end of the grant period, we will have a greater insight into the interrelationships between cyclic nucleotide and phosphoinositide metabolism in photoreceptors. The net result of these studies will be a better understanding of the physiological significance of the rapid biochemical changes that follow photolysis of rhodopsin.
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