Effect of incubation with NADPH generator on progesterone bound to nuclei of the rat uterus.

Publisher Website
Google Scholar
Abstract
The metabolism of progesterone bound to uterine nuclear progesterone receptors was accelerated in vitro by incubation at 25.degree. C for 30 min with NADPH generator. The characteristics of progesterone metabolites and receptors were examined by 3H-R5020 [promegestone] exchange assay. Progesterone bound nuclei were prepared from uteri of estradiol-primed progesterone-treated ovariectomized rats. The specific nuclear R5020 binding sites examined by exchange assay were identified as specific progesterone receptors by the binding specifity of R5020 to progesterone receptors, their high affinity for progesterone, and the increase in the number of specific binding sites in response to a progesterone injection. Major metabolites of progesterone specifically bound to nuclear receptors by incubation with the cofactor were 5.alpha.-dihydroprogesterone (5.alpha.-DHP) and 3.alpha.-hydroxy-5.alpha.-pregnan-20-one. The relative binding affinity of the progesterone receptor for 5.alpha.-DHP at 0.degree. C was 27.5% of progesterone, but 3.alpha.-hydroxy-5.alpha.-pregnan-20-one had no binding affinity for the receptor. Incubation of the nuceli in the presence of NADPH generator decreased the number of R5020 binding sites from 143 fmol to 25 fmol/100 .mu.g DNA, and increased the Kd of the receptor with progesterone from 2.56 nM to 7.78 nM. After gene activation, the specific binding sites of progesterone to nuclear receptors may be denatured in the presence of NADPH, causing both progesterone and receptors to dissociate from the receptors and chromatins, respectively; the dissociated progesterone may be converted to 5.alpha.-reduced pregnanes which have lower affinity for the receptor.