RETINAL CGMP METABOLISM IN SITU BY 180 LABELING

本研究方案旨在进一步开发和应用一种新的 基于磷酸二酯酶促进的~(18)O-掺入分析技术 从18O-水到核苷酸α-磷酸的动力学监测 完整个体的环GMP、环AMP及相关5‘-核苷酸代谢 孤立的视觉视网膜。总体目标是定义cGMP的角色 新陈代谢在光传导过程中起作用。初步探索 表明cGMP代谢通量的逐步增加，而不是 CGMP稳态水平的变化与强度直接相关 光刺激的影响。拟议的研究旨在获得 有关光与光之间关系的其他信息 刺激和鸟核苷酸代谢，包括a)对应 ~(18)O-掺入鸟嘌呤核苷酸α-磷酸 覆盖视觉敏感度的整个光强度范围 视网膜；b)识别限速步骤 光接收相关的鸟嘌呤核苷酸代谢，设置感官 光感受器的极限；c)~(18)O标记的直接原位检测 光受体间的偶联与cGMP的“开”/“关”调节 磷酸二酯酶；d)隔室离散性的表征 光感受器鸟嘌呤核苷酸和环核苷酸代谢 由~(180)O-掺入动力学数据建模确定。生物学的 光刺激磷酸二酯酶催化降解cGMP的应用 在ROS中也将从这一水解性事件的角度进行研究 这代表了一种从盘膜蛋白中释放钙的方法。 理论上，这是通过一种机制来实现的，该机制涉及到 质子是磷酸二酯酶反应的产物之一， 和/或钙结合蛋白的构象变化。这些研究 将涉及检查光诱导的钙离子与 释放和cGMP水解，以及对质子的需求 利用质子转运体实现cGMP与钙的解偶联 在隔离的ROS中释放。

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.