RETINAL CGMP METABOLISM IN SITU BY 180 LABELING

总的目标是定义生物化学和分子事件 视网膜光感受器功能的基础光激发和 光适应。这一概念与经典假设形成了鲜明对比，即 环核苷酸只起变构效应(“第二信使”)的作用。 需要改变它们的细胞浓度。相反，水解物 某些种类的磷酸二酯酶对cGMP的作用被视为一种生化 为耗能的蜂窝过程服务的事件。为了支持这一点 假设，已有证据表明cGMP的合成速率， 与cGMP水解紧密相连，在大小上与 光刺激的强度和频率、幅度和极性 光感受器的电反应和视紫红质的量 光致异构化。CGMP的光感受器浓度变化很小 尽管有这些光诱导的cGMP通量的大的漂移。另一方面 手持式连续照明，光线强度自适应 行为导致光感受器cGMP水平显著下降和 抑制早期升高的cGMP代谢率。两国之间的关系 CGMP代谢通量成分对光激发和适应行为的影响 由光感受器的电输出决定，将通过 CGMP代谢动态监测及其反应调控 对不同适应状态下的明暗刺激 化学改变光感受器cGMP的浓度。这项技术 鸟嘌呤核苷酸α-磷酸18-0标记率的测定 由磷酸二酯酶催化的光感受器cGMP的水解所致 将被用来监测代谢成分的行为并识别 参与光反应的酶调节部位。参与其中 将对cGMP的代谢和变构成分进行评估 电定义的从暗适应到光激发，再到 光适应状态和反向转换。人类的生物能量学 CGMP代谢通量将与光感受器高能磷酸有关 利用率、02消耗量和产热量来确定是否 光诱导cGMP通量组分是主要的能量利用过程 在光激发下。光感受器的光激活机制 将在体外和原位检测鸟苷环化酶的活性和 将使用光亲和标记来鉴定光感受器成分 与cGMP特异地相互作用。

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.