RETINAL CGMP METABOLISM IN SITU BY 180 LABELING

总的目标是定义生物化学和分子事件， 光激发的视网膜感光器功能的基础， 光适应 这一概念与传统的假设相反， 环核苷酸仅作为变构效应物（“第二信使”） 需要改变它们的细胞浓度。 相反，水解 cGMP的某些种类的磷酸二酯酶被视为一种生化 一个需要能量的细胞过程。 为支持这一 假设，已经获得的证据表明，cGMP合成的速率， 与cGMP水解紧密耦合，在幅度上与 光刺激的强度和频率、振幅和极性 光感受器的电反应和视紫红质的量 光异构化的 cGMP的光感受器浓度变化最小， 尽管cGMP通量存在这些大的光诱导偏移。 另 手，光强度的连续照明引发自适应 行为导致光感受器cGMP水平显著降低， 抑制早期升高的cGMP代谢率。 的关系 cGMP代谢通量分量对光激发和适应行为影响 由光感受器电输出确定的光信号将在原位进行研究， 监测cGMP代谢的动力学及其响应调节 在不同的适应状态下， 光感受器cGMP的化学改变浓度。 技术 测量鸟嘌呤核苷酸α-磷酰基的18-0标记率 由磷酸二酯酶催化的光感受器cGMP水解产生 将用于监测代谢成分的行为， 调节光反应的酶位点。 参与 cGMP代谢和变构成分的评估将在 从暗适应到光激发， 光适应状态和反向转换。 的生物能量学 cGMP代谢通量与光感受器高能磷酸盐有关 利用率、O2消耗量和产热量，以确定 光诱导cGMP通量组分是主要的能量利用过程 在光激发中。 感光细胞的光激活机制 将在体外和原位检测鸟苷酸环化酶活性， 光亲和标记将用于识别感光器组件 与cGMP特异性相互作用。

The overall goal is to define the biochemical and molecular events that underlie retinal photoreceptor functions of photoexcitation and photoadaptation. The concept contrasts with the classical hypothesis that cyclic nucleotides act solely as allosteric effectors ("second messengers") requiring changes in their cellular concentration. Instead, the hydrolysis of cGMP by certain species of phosphodiesterase is viewed as a biochemical event subserving an energy-requiring cellular process. In support of this hypothesis, evidence has been obtained that the rates of cGMP synthesis, tightly coupled to cGMP hydrolysis, correspond in magnitude with the intensity and frequency of photic stimulation, the amplitude and polarity of the photoreceptor electrical response, and the amount of rhodopsin photoisomerized. Photoreceptor concentrations of cGMP change minimally in spite of these large, light-induced excursions in cGMP flux. On the other hand, continuous illumination at light intensities elicting adaptive behavior result in marked decreases in photoreceptor cGMP levels and suppression of earlier elevated cGMP metabolic rates. The relationship of the cGMP metabolic flux component to photoexcitation and adaptive behavior determined by photoreceptor electrical output will be studied in situ by monitoring the dynamics of cGMP metabolism and its regulation in response to light and dark stimuli under different states of adaptation and chemically altered concentrations of photoreceptor cGMP. The technology of measuring the rate of 18-0 labeling of guanine nucleotide Alpha-phosphoryls resulting from phosphodiesterase-catalyzed hydrolysis of photoreceptor cGMP will be used to monitor behavior of the metabolic component and identify enzymic sites of regulation mediating the photoresponse. The involvement of the cGMP metabolic and allosteric components will be assessed in electrically defined transitions from dark-adapted, to photoexcited, to light-adapted states and in the reverse transitions. The bioenergetics of cGMP metabolic flux will be related to photoreceptor high energy phosphate utilization rates, 02 consumption, and heat production to establish whether the light-induced cGMP flux component is the major energy-utilizing process in photoexcitation. The mechanism of light activation of photoreceptor guanylate cyclase activity will be examined in vitro and in situ and photoaffinity labeling will be used to identify photoreceptor components that interact specifically with cGMP.