RETINAL CGMP METABOLISM IN SITU BY 180 LABELING

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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.