CHARACTERIZATION OF VASOACTIVE-INTESTINAL-PEPTIDE RECEPTORS IN RABBITCILIARY PROCESSES

Purpose. To demonstrate a potential role for vasoactive intestinal pep tide (VIP) in the regulation of ciliary process function, VIP receptor s on rabbit ciliary process membranes were identified and characterize d in biochemical and immunochemical studies. Methods. Membranes were i solated from rabbit ciliary processes, and VIP receptors were characte rized by competition binding, affinity cross-linking, and N-glycanase digestion. A site-specific polyclonal antibody directed against the NH 2-terminal end of the deduced sequence of the recently cloned rat VIP receptor was generated and used to identify the VIP receptor by immuno blot analysis. Results. Membranes isolated from rabbit ciliary process es exhibited a high-affinity VIP binding site (K-D approximate to 1 nM ). Secretin and glucagon, which possess considerable primary sequence homolog with VIP, were ineffective in inhibiting I-125-VIP binding to ciliary process membranes. In conjunction with the chemical cross-link ing agent disuccinimidyl suberate, I-125-VIP specifically labeled a 63 -kd protein in membranes from ciliary processes. This apparent size wa s confirmed by immunoblot analysis of ciliary body membranes using a s ite-specific polyclonal antibody that recognizes residues 92 to 104 of the rat VIP receptor. Digestion of the affinity-labeled receptor with N-glycanase generated an N-linked oligosaccharide free core protein o f (-)50 kd. Conclusions. These findings demonstrate the presence of sp ecific VIP receptors in rabbit ciliary processes. The differences in l igand specificity and structure of the ciliary process VIP receptor, c ompared to VIP receptors on peripheral tissues, suggest either a speci fic role(s) for VIP that may be unique to the anterior segment or the existence of VIP receptor isoforms.