The aim of the proposed project is to investigate cellular mechanisms that regulate rod and cone outer segment (ROS and COS) shedding. We will expand upon current findings by the PI, concerning ROS and COS shedding in cultured frog and lizard eyecups. This expansion logically involves studies of cyclic nucleotide metabolism, in which we will utilize the Co-Investigator's experience and developed techniques. We will: I. Investigate the likely means by which strophanthidin activates ROS shedding in Xenopus eyecups; II. Develop an in vitro eyecup preparation suitable for studying COS shedding and begin pharmacological tests with it; III. (a) Following pretreatment of retinae with specific inhibitors of Ca2+-activated thiol proteases, describe ROS and COS cytoskeletal components that are sensitive to these proteases and consequently would not be apparent in conventionally-fixed material; (b) Test if these proteases are required for OS shedding, by seeing if their specific inhibitors block OS shedding; IV. Test for circadian rhythmicity of cAMP metabolism by measuring cAMP levels and the activities of cAMP synthetic and degradative enzymes over a daily cycle. The long-term objectives of this research involve ascertainment of the conditions necessary for ROS and COS shedding to occur, and the chain of events responsible for eliciting this response. OS shedding is critically essential to the viability of the photoreceptors--in RCS rats, an inherited defect in this process causes most of their photoreceptors to die by 2 months after birth. Consequently, elucidation of these basic objectives may provide a necessary foundation for designing treatments for a number of retinal diseases that are characterized by photoreceptor degeneration.
