Immunochemical analysis of the sodium channel in rodent and human eye.

The presence of the amiloride-sensitive sodium channel (ASSC) in ocular tissues was studied with the aid of a polyclonal antiserum raised against the 14 amino acid peptide QGLGKGDKREEQGL. This sequence corresponds to the region 44-58 of the alpha subunit of the channel, termed ENaC, cloned from rat colon. The antibody titers, measured by the ELISA technique, rose to 1∶2560 4 weeks after immunization, and this bleed was used in all subsequent experiments. Immunoblotting with the polyclonal anti-alphaENaC serum, revealed a major band of 82-86 kDa in extracts prepared from whole bovine or rat retina; a minor component of 92 kDa in the extract from bovine ciliary body may represent a glycosylated species. Immunohistochemistry, using the alphaENaC-specific antiserum, revealed strong fluorescence in specific areas of the rat and human eye. Pronounced labelling was observed in the epithelial cell layer of the retina, the lens, as well as both the pigmented and the nonpigmented epithelium of the ciliary body and the iris. All of the cell layers (epithelium, endothelium and fibroblasts) in the cornea, the blood vessels in the iris, and iris epithelium, were also strongly immunopositive. The somatic body of the photoreceptor cells (cones and rods) in the inner and outer segments could be traced to forming a synapse in both the internal and external portions of the internal nuclear layer. The bipolar cells and ganglia in the neuronal compartment also exhibited occasional immunofluorescence. The method of fixation and the source of the tissue were important parameters for the immunochemical localization of the ENaC. The resolution was very poor when rat eye was fixed in Bouin's solution but this method was satisfactory for human tissues. For rat eye, optimum resolution was obtained with AMeX fixation. This widespread distribution of the ENaC generally colocalizes with the previously observed immunopositivity for the mineralocorticoid receptor such that steroid hormone-mediated ion regulation would appear to add a new parameter to the functional expression of ocular tissues. Copyright 1999 Academic Press.