EXTRANUCLEAR LOCALIZATION OF ENDOGENOUS 11-BETA-HYDROXYSTEROID DEHYDROGENASE-2 IN ALDOSTERONE TARGET-CELLS

Type 2 11 beta-hydroxysteroid dehydrogenase (11 beta HSD2) plays a key role in conferring aldosterone selectivity on the mineralocorticoid r eceptor (MR) by inactivating intracellular glucocorticoids before they can occupy the MR. 11 beta HSD2 is a microsomal enzyme expressed in a ldosterone target cells, although its subcellular distribution is stil l unclear. The goal of this study was to determine the subcellular loc alization of the endogenous 11 beta HSD2 in renal aldosterone target c ells. We generated an antibody against rabbit 11 beta HSD2 and used it in combination with a nuclear marker and confocal laser scanning micr oscopy. On Western blots the antibody recognized a single band of appr oximately 41 kDa in the renal cortical collecting duct, outer medullar y collecting duct, submandibular gland and adrenal cortex, whereas the colon, liver, renal medulla, and heart were negative. Immunohistochem istry showed specific reaction in the known aldosterone target cells o f the kidney (connecting tubule, cortical collecting duct, and outer m edullary collecting duct) with no signals over glomeruli, proximal nep hron segments, and blood vessels. Staining for 11 beta HSD2 was very w eak in rabbit colon, and no immunoreactivity could be detected in the heart and brain. Confocal microscopy of kidney sections costained with the 11 beta HSD2 antibody and the nuclear marker propidium iodide dem onstrated that 11 beta HSD2 is in the cytoplasmic compartment with no evidence for nuclear localization. Subcellular localization of 11 beta HSD2 to a cytoplasmic compartment seems ideal for fulfilling its biol ogical function, i.e. the efficient inactivation of intracellular gluc ocorticoids before they occupy MRs, which are predominantly cytoplasmi c in the absence of hormone.