Connexin distribution in the rabbit and rat ciliary body. A case for heterotypic epithelial gap junctions.

PURPOSE To evaluate the distribution of different alpha- and beta-type connexins (Cx) present in the dual layered ciliary body epithelia (CBE) of both rabbit and rat. METHODS Immunocytochemical detection of Cx26, Cx32, Cx43, and Cx50 was performed on frozen sections of rabbit and rat ciliary body using indirect immunofluorescent methods. The identity of the antigens recognized by the monoclonal primary antibodies was further confirmed by Western immunoblots. Double labeling experiments based on either conventional or confocal microscopy were carried out to establish the exact spatial relationship between different connexins. RESULTS Connexin 50 was found only in the nonpigmented epithelium (NPE) at apical and basolateral membranes, whereas Cx43 was observed exclusively and at a very high concentration in the pigmented epithelium (PE), primarily in the apical cell membrane, with minimal extension to the proximal lateral zone. The correct antigenicity of the antibodies was confirmed by Western blots of rabbit ciliary body membranes. In rabbit, the Cx26 antibody detected an antigen that was abundant in the NPE and was weakly expressed in the PE. In rat, however, the Cx26 staining was confined to capillary wall endothelia. Western blots of ciliary body and liver membranes and liver immunohistology indicated that the Cx26 antibody used does not recognize rabbit Cx26. Cx32 did not yield any substantial epithelial labeling in either species. CONCLUSIONS The distribution of Cx50 around the entire NPE cell perimeter suggests its involvement in NPE-NPE cell homotypic gap junctions. The concentration of Cx43 and Cx50 at the apical membranes of the PE and NPE cells, respectively, and their complete absence from the opposite cell suggest that these connexins may participate in the formation of heterotypic gap junctions, either with each other or with other yet unidentified connexins.