Immunochemical demonstration of the mineralocorticoid receptor in ocular tissues

We studied the presence of the mineralocorticoid receptor (MCR) in the eye with the aid of a number of immunochemical techniques. Immunoblotting with a polyclonal antibody, directed against the rat renal MCR, revealed a single band of about 102 kD in extracts prepared from whole bovine or rat retina similar to that observed in cytosol from the kidney and myocardium from these species. Isolated cells of the bovine retinal pigment epithelium (RPE) similarly exhibited a 98- to 102-kD band in Western blots developed with the aid of anti-MCR antiserum. The 98- to 102-kD band was also obtained following autoradiography of RPE cytosol irradiated in the presence of 3H-R 5020. This fluorographic pattern was abolished when RU 26752, an antagonist specific to the MCR, was allowed to compete with radiolabelled promegestone. The MCR-3H-RU 26752 complex in RPE cytosol underwent heat activation, as judged by binding to DNA cellusose, and could also be precipitated by anti-MCR IgG. In primary cultures, the proliferation of the RPE cells was inhibited by the two MCR-specific antagonists RU 26752 and ZK 91587. The loss of the MCR-specific immunofluorescence in RPE cells after only 3 passages in culture was associated with refractoriness to the inhibitory effect of both of these spironolactones. Immunohistochemistry, using MCR-specific antiserum, revealed strong fluorescence in specific areas of the rat eye. In the retina, immunopositivity was observed in Müller cells, external and internal limiting membranes, the vitreous base lining and in the pigment epithelium. Epithelial cells of the ciliary body, iris and cornea also exhibited strong MCR-specific immunofluorescence. Thus, both the epithelial and the nonepithelial compartments of the ocular tissues form interesting new targets to delineate the mechanism of action of mineralotropic hormones.