THE SHEEP KIDNEY CONTAINS A NOVEL UNIDIRECTIONAL, HIGH-AFFINITY NADP-DEPENDENT 11-BETA-HYDROXYSTEROID DEHYDROGENASE (11-BETA-HSD-3)()

The 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) enzymes convert corticosterone and cortisol to 11- dehydrocorticosterone and cortison e, and are thought to convey extrinsic specificity to the mineralocort icoid receptor by limiting access of the relatively more abundant gluc ocorticoids to it. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described and cloned. The liver-type, NADP(+) -dependent 11 beta-HSD-1, has an affinity in the micromolar range and bidirectional activity. The NAD(+)-dependent 11 beta-HSD-2 has a highe r affinity, in the nanomolar range, and exhibits only oxidase activity . 11 beta-HSD-2, because of its affinity and co-localization with the mineralocorticoid receptor; is likely to serve as the ''gatekeeper'' f or the mineralocorticoid receptor in the kidney. Although the rat kidn ey expresses both isoforms, only the high-affinity, NAD(+)-dependent 1 1 beta-HSD-2 has been reported in the sheep kidney. We found both 11 b eta-HSD NAD(+)- and NADP(+)-dependent activities in sheep kidney to be present. The NAD(+) -dependent activity exhibited a Km similar to tha t reported in the literature, 3.85 +/- 1.28 nM for corticosterone and 21.3 +/- 5.8 for cortisol, was distributed in approximately equal amou nts between microsomes and nuclei, and was unidirectional, converting corticosterone to 11-dehydrocorticosterone. The enzyme exhibited promi nent 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 11 beta-HSD-1 in the live r 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,vas approximately 3-10 times greater than th e NADP(+)-dependent activity when incubated with 5nM corticosterone su bstrate, but had similar activity when incubated with 100 nM substrate concentrations. CHOP cells (a modified Chinese hamster. ovary cell li ne) transiently transfected with the sheep 11 beta-HSD-2 plasmid exhib ited a marked preference for NAD(+) as co-factor. Oxidation of cortico sterone by transfected cells in the presence of NADP(+) was present, b ut minimal, NADP(+) did not support the metabolism of cortisol, the pr imary glucocorticoid of sheep. These data suggest the existence of ano ther NADP(+)-dependent enzyme 11 beta-HSD-3, which, because of its its high affinity and unidirectional oxidase activity may play a physiolo gical role in the modulation of glucocorticoid binding to both the min eralocorticoid and glucocorticoid receptors. (C) 1997 by Elsevier Scie nce Inc.