A variety of peptides which have been localized previously to nerve and gut tissues have also been found in the retina, primarily amacrine cells. The retina is uniquely well suited for study of these neuropeptides because its anatomy, physiology and pharmacology have been characterized extensively and it can be activated by the same light stimuli in vitro that it responds to in vivo. By further examination retinal peptides, specifically cholecystokinin (CCK) a model carboxyl-terminally amidated brain-gut peptide, this proposal aims 1) to shed light on the neurotransmitter function of peptides, 2) to determine the functional significance of retinal amacrine cells, and 3) advance current knowledge regarding the biochemistry and molecular biology of neuropeptides. CCK, in its various molecular forms as well as in its various stages of post-translational processing, will be purified from frog brain and retinal tissues by the use of affinity chromatography with region specific antisera and high pressure liquid chromatography (HPLC). A recombinant clone encoding CCK will be isolated from a cDNA library constructed with frog retinal mRNA. The CCK cDNA will be selected by hybridization to an icosanucleotide specific for an amino acid sequence contained within the octapeptide of CCK as follows: 1Asp-2Tyr-3Met-4Gly-5Trp-GMet-7Asp-8Phe-NH2 3 prime CT-AG AT-AG TAC CCN ACC TAC CT 5 prime The CCK cDNA will be utilized to isolate CCK gene from a cosmid genomic library. Biosynthesis of CCK in cultured from retinas will be examined by pulse-labelling experiments with S35-Met and processing of the post-translational products of CCK synthesis will be followed by chasing the label into intermediates with non-radioactive Met. Cell free synthesis of CCK will be examined in a wheat germ system using mRNA extracted from frog brain and retina. The enzyme responsible for activation of CCK by carboxyl-terminal amidation, the final step in processing, will be characterized with synthetic substrates using HPLC and radioimmunoassays with region specific antisera. Regulation of CCK release from cultured retinas will be examined using light exposure, electrolyte alterations, and putative retinal transmitters as stimuli. Factors influencing cellular CCK synthesis will be studied by examining light-induced alterations of CCK-specific mRNA content, CCK biosynthetic rates, and rates of CCK post-translational processing in cultured retinas.
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