Immunochemical Demonstration of the Mineralocorticoid Receptor in Ocular Tissues

Abstract

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.

Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines

“…Figure 1 show that the cytosol from the proerythroblastic TF1 (a) and the HEL (c) cell lines were resolved as a single band of about 102 kDa in Western blots developed with the aid of the polyclonal antibody directed against the entire mineralocorticoid receptor (MCR), but the myeloblastic cell line (U937) did not exhibit this protein at all (b). This is comparable to the molecular weight in a number of other organs that are known to contain the MCR, [22][23][24][25][26] and correlates well with the theoretical molecular mass of 107 kDa calculated from the cloned product.…”

“…The MCR in the HEL cells was readily recognized by an antibody directed against the rat kidney protein purified in the presence of RU 26752 that binds specifically to this receptor. 4,22 The estimated molecular size of about 102 kDa in Western blots is compatible with the MCR in a number of peripheral organs, [22][23][24][25][26] and with the theoretical mass of 107 kDa deduced from the cloned sequence.…”

“…[22][23][24][25][26] The complete elimination of the radiophotochemical labelling obtained with 3 H-Promegestone (R-5020) by radioinert RU 26752 attests to the technical specificity for the MCR [22][23][24][25][26] and furthermore suggests that the hormone binding domain of the MCR in the HEL cells is structurally similar to that in the receptor from other sources. [22][23][24][25][26] The size estimate for the ENaC (a member of the ASSC superfamily) revealed a protein of approximately 82 kDa, as in studies by other investigtaors. 21,32,33 Immunocytochemistry demonstrated the presence of the MCR in the nucleocytoplasmic compartment of the HEL and TF1 cell lines, much as in other target organs.…”

Oxysterol 7α-Hydroxylase (CYP39A1) in the Ciliary Nonpigmented Epithelium of Bovine Eye

“…Immunohistochemical localization of the mineralocorticoid receptor (Mirshahi et al, 1996(Mirshahi et al, , 1997Stokes et al, 2000;Suzuki et al, 2001) and the glucocorticoid receptor (Stokes et al, 2000;Suzuki et al, 2001) was demonstrated in NPE cells. Corticosteroid Fluorescence observation by confocal laser microscopy.…”

“…The NPE cells that face the posterior chamber are involved in the active secretion of aqueous humor (Usukura et al, 1988) and have tight junctions that act as a blood-aqueous barrier (Hirsch et al, 1977;Raviola, 1974Raviola, , 1977. In addition, the NPE cells have various steroid hormone receptors (Mirshahi et al, 1996(Mirshahi et al, , 1997Stokes et al, 2000;Suzuki et al, 2001) and are considered to modulate the aqueous humor in the maintenance of ocular homeostasis.…”