Generation of polyclonal antibodies against the mineralocorticoid receptor and analysis of mineralocortin in rat myocardium by immunophotochemistry.

ABSTRACT

Fawn, Burgundy rabbits were immunized with the mineralocorticoid receptor (MCR) purified biochemically from rat kidney by a simple, two step procedure. High anti-MCR titers were observed in radioimmunoassays just 3 weeks after the initial injection and increased further with time. Western blot analysis revealed a single band of 94-98 kDa in renal and cardiac cytosol from the rat, like the antigen prepared biochemically. The two atria from beef heart exhibited far greater MCR-positivity compared to the two ventricles, suggesting physiological relevance. The receptor was also photolabelled for the first time with promegestone in this very 94-98 kDa region which could be displaced by the antagonist RU 26752 specific to MCR. The immune IgG precipitated 3H-aldosterone or 3H-RU 26752-MCR complexes from rat heart, and displaced the MCR-antagonist complex to high molecular weight regions during gel permeation chromatography on Sephacryl columns. Immunofluorescent labelling showed that MCR was widely distributed in the cytoplasm in rat myocardium with limited staining in what appeared to be the nuclear compartment. These open up the possibility of large scale purification of the endogenous mineralocorticoid binding protein, mineralocortin, for detailed physicochemical characterization. The technique of photoaffinity labelling presented here should also help delineate the nature of the steroid binding domain in the MCR.