Ocular Expression of Type-I Pituitary Adenylate CyclaseActivating Polypeptide (PACAP) Receptors" SEBASTIANO CAVALLARO,~."~ VELIA D'AGATA,~ FILIPPO DRAGO,b SIMONE MUSCO," GIUSEPPE NUCIFORO,' FRANCESCA RICCIARDOLO,' SALVATORE TRAVALI," FRANCA STIVALA," AKIRA ARIMURAf AND PIER LUIGI CANONICOg bInstitute of Pharmacology University of Catania V.le A. Doria 6 Catania 95125, Italy 'Institute of General Pathology University of Catania Via Androne 83 Catania 95125, Italy d
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Catania, Italy 'Department of Pathology S. Bambino Hospital Via Tindaro 2 Cataniu 95100, Italy fU.S.-Japan Biomedical Research Laboratories Tulane University Hebert Center New Orleans, Louisiana 70012 gSchool of Dentistry University of Pavia Pavia, Italy Based on the relative affinities for PACAP and VIP, PACAP binding sites have been divided into at least two types.' Type I PACAP receptors specifically bind to PACAP, whereas type I1 PACAP receptors bind with similar affinity to both PACAP and
'This work was supported by grants from NIH (DK09094; to A.A.), AIRC (F.S.), CNR (S.T.), and a fellowship from AIRC (S.C.). *39-95-313429; fax: 39-95-333219; e-mail:
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FIGURE 1. Immunohistochemical localization of type-I PACAP receptors in rabbit ciliary epithelium. Eyes were fixed in paraformaldehyde and paraffin embedded. Sections were incubated with PACAP receptor antibody and visualized by immunoperoxidase staining. Nuclei are counterstained blue with hematoxylin. [(A) lower and (B) higher magnification.] The PACAP receptor antibody labeled the epithelium covering the ciliary processes, while very little staining was found in the connective tissue.
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VIP. Type I PACAP receptors come from a common gene by an alternative splicing mechanism and diverge from each other due to the presence or absence of either one or two cassettes, designated “hip” and “hop,” inserted in the C-terminal end of the third intracellular 10op.~ Recently, PACAP receptors coupled to adenylate cyclase have been characterized in the anterior uvea, choroid, and retina of different mammalian specie^.^,^ The anterior uvea have mainly type I1 PACAP receptors, while type I PACAP receptors predominate in the choroid and retina. Currently, no information has yet been provided on the anatomical localization of type I PACAP receptors in the eye. To determine the immunohistochemical distribution of type I PACAP receptors in the rabbit eye, we used an antibody raised against a synthetic peptide corresponding to their C-terminal intracellular domain (from Lys4” to Ala435of PACAP-R2).Immunoperoxidase staining of formalin-fixed, paraffin-embedded ocular sections was performed using the streptavidin-biotinylated peroxidase technique. The horseradish peroxidase was visualized using diaminobenzidine tetrahydrochloride (DAB). Sections were counterstained with hematoxylin. PACAP receptor-like immunoreactivity was localized in the retina (ganglionic, inner plexiform, and photoreceptor layer) and ciliary body. Among these tissues, the most staining was found in the epithelium of ciliary processes (FIG.1(A) and (B)). The present results, together with the previous demonstration of PACAP binding sites and PACAP-stimulated adenylate cyclase activity in the retina and ciliary body3suggest that PACAP may have specific functions in these tissues. The demonstration of high levels of PACAP receptor-like immunoreactivity in the epithelium covering the ciliary processes, which is responsible for the formation of aqueous humor, is compatible with the role of PACAP in the secretory process. REFERENCES A. 1992. Trends Endocrinol. Metab. 3: 288-294. 1. ARIMURA, D., C. WAEBER, C. PANTALONI, F. HOLSBOER, J. BOCKAERT, P. H. SEEBURG & 2. SPENGLER, L. JOURNOT. 1993. Nature 365: 170- 175. S. F. E., P. DE NEEF,P. ROBBERECHT & J. CHRISTOPHE. 1994. Exp. Eye Res. 58: 3. NILSSON, 459-461. 4. ONALI,P. & M. C. OLIANAS. 1994. Brain Res. 641: 132-134.
