This research proposal is aimed at further developing and applying a new analytical technique based on phosphodiesterase-promoted 18O-incorporation from 18O-water into nucleotide Alpha-phosphoryls to monitor the dynamics of cyclic GMP, cyclic AMP and related 5'-nucleotide metabolism in the intact, isolated visual retina. The overall goal is to define the role that cGMP metabolism plays in the light transduction process. Initial explorations indicate that graded increases in the metabolic flux of cGMP rather than changes in cGMP steady-state levels are directly related to the intensity of light stimulation. The proposed studies are intended to obtain additional information regarding the relationship between photic stimulation and guanine nucleotide metabolism including a) correspondence of 18O-incorporation into guanine nucleotide Alpha-phosphoryls over the entire range of light intensities covering the sensitivity of the visual retina; b) identification of the rate-limiting step in photoreception-related guanine nucleotide metabolism that sets the sensory limit of the photoreceptors; c) direct assessment in situ by 18O-labeling of the coupling between light receptors and """"""""on""""""""/""""""""off"""""""" regulation of cGMP phosphodiesterase; d) characterization of the compartmental discreteness of photoreceptor guanine nucleotide and cyclic nucleotide metabolism as determined by modeling of 18O-incorporation kinetic data. The biological utility of light-stimulated phosphodiesterase-catalyzed hydrolysis of cGMP in ROS will also be examined from the standpoint of this hydrolytic event representing a means for releasing calcium from disc membrane proteins. This is theorized to occur by a mechanism involving the generation of protons, which is one of the products of the phosphodiesterase reaction, and/or a conformational change in calcium binding proteins. These studies will involve examining the correspondence between light-induced calcium release and cGMP hydrolysis, as well as the requirement for protons by the use of proton translocators to uncouple the hydrolysis of cGMP from calcium release in isolated ROS.
