The sheep kidney contains a novel unidirectional, high affinity NADP+-dependent 11β-hydroxysteroid dehydrogenase (11β-HSD-3)

Abstract

The 11β-hydroxysteroid dehydrogenase (11β-HSD) enzymes convert corticosterone and cortisol to 11-dehydrocorticosterone and cortisone, and are thought to convey extrinsic specificity to the mineralocorticoid receptor by limiting access of the relatively more abundant glucocorticoids to it. Two different 11β-hydroxysteroid dehydrogenases (11β-HSD) have been described and cloned. The liver-type, NADP +-dependent 11β-HSD-1, has an affinity in the micromolar range and bidirectional activity. The NAD +-dependent 11β-HSD-2 has a higher affinity, in the nanomolar range, and exhibits only oxidase activity. 11β-HSD-2, because of its affinity and co-localization with the mineralocorticoid receptor, is likely to serve as the “gatekeeper” for the mineralocorticoid receptor in the kidney. Although the rat kidney expresses both isoforms, only the high-affinity, NAD +-dependent 11β-HSD-2 has been reported in the sheep kidney. We found both 11β-HSD NAD +- and NADP +-dependent activities in sheep kidney to be present. The NAD +-dependent activity exhibited a Km similar to that reported in the literature, 3.85 ± 1.28 nM for corticosterone and 21.3 ± 5.8 for cortisol, was distributed in approximately equal amounts between microsomes and nuclei, and was unidirectional, converting corticosterone to 11-dehydrocorticosterone. The enzyme exhibited prominent substrate inhibition. The NADP +-dependent activity had a Km for corticosterone of 4 ± 1.3 nM and a Km for cortisol of 35.2 ± 2 nM, 100-fold lower than that described for the 11β-HSD-1 in the liver of sheep and other species, and was more prevalent in the microsomes than the nuclei. This enzyme was not inhibited by its substrate. The NAD +-dependent activity was approximately 3–10 times greater than the NADP +-dependent activity when incubated with 5nM corticosterone substrate, but had similar activity when incubated with 100 nM substrate concentrations. CHOP cells (a modified Chinese hamster ovary cell line) transiently transfected with the sheep 11β-HSD-2 plasmid exhibited a marked preference for NAD + as co-factor. Oxidation of corticosterone by transfected cells in the presence of NADP + was present, but minimal; NADP + did not support the metabolism of cortisol, the primary glucocorticoid of sheep. These data suggest the existence of another NADP +-dependent enzyme, 11β-HSD-3, which, because of its high affinity and unidirectional oxidase activity, may play a physiological role in the modulation of glucocorticoid binding to both the mineralocorticoid and glucocorticoid receptors.