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The capacity of endothelial cells from pulmonary arteries and veins to convert dehydroisoandrosterone (3 beta-hydroxy-5-androsten-17-one) and androstenedione to potent, biologically active steroids was investigated. The metabolites of [3H]dehydroisoandrosterone produced in pulmonary artery endothelial cells were androstenedione and 5-androstene-3 beta, 17 beta-diol. The metabolites isolated from incubation of pulmonary arterial cells with [3H]androstenedione were testosterone, 5 alpha-androstane-3,17-dione, 5 alpha-dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), isoandrosterone (3 beta-hydroxy-5 alpha-androstan-17-one), and androsterone. The products of [3H]androstenedione metabolism in human pulmonary venous cells were the same as those formed in arterial cells, and in addition, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol were formed. The rates of metabolite formation from [3H]androstenedione in pulmonary arterial and venous endothelial cells were linear with incubation time up to 3 h. These findings suggest that the pulmonary endothelium is an important site for the metabolism of dehydroisoandrosterone and androstenedione in the human lung. Endothelial cells produce the same metabolites as human lung tissue, with the exception of hydroxylated steroids.

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The metabolism of tritium-labeled estrone and estradiol-17 beta in slices of lung tissue obtained from an adult human was studied; estrone was identified as the only metabolite of estradiol-17 beta and estradiol-17 beta as the exclusive product of estrone metabolism. Product formation remained linear as a function of time of incubation up to 3 h and of wet lung tissue mass up to 300 mg/ml. At equimolar substrate concentrations, the rates of estrone formation were at least 2-fold greater than those of estradiol-17 beta. The apparent KM of 17 beta-hydroxysteroid oxidoreductase for estrone was 11 microM and that for estradiol-17 beta was 10 microM. These results are suggestive that the human lung enzyme binds estrone and estradiol-17 beta with similar affinities; however, the oxidative pathway is favored as indicated by the greater Vmax attained in the formation of estrone. It is possible that, in vivo, the human lung constitutes a site for estradiol-17 beta inactivation to estrone as well as a site for the conversion of estrone to estradiol-17 beta. This last process may become particularly important in instances in which the ovaries have ceased to function and secrete estradiol-17 beta, e.g. the postmenopausal women.

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The activation of placental AC by either Mg2+ or Mn2+, in the presence and absence of NaF, followed sigmoidal saturation kinetics. Mn2+ enhanced maximally the NaF-stimulated Mg2+-dependent AC activity. The apparent Km of Mg2+-dependent AC for ATP was 0.4 mM, with and without NaF addition. GTP and GMP-P(NH)P stimulated the Mg2+-dependent AC in a dose-dependent manner with half-maximal stimulation taking place at concentrations of approximately 2 microM. In the presence of GMP-P(NH)P (10 microM) the kinetics of the AC dependence on Mg2+ ion concentration changed from sigmoidal to hyperbolic. Most of the AC activity (greater than 83 per cent) was associated with the particulate fractions of placental homogenate. For better reproducibility, the AC assay was performed using sonicated particulate fraction preparations; sonication did not alter the response of AC to stimuli to a variety of agents used in these experiments; freezing and thawing, however, obliterated the stimulation by beta-adrenergic agonists. Placental AC activity was inhibited by p-hydroxymercuriphenyl sodium sulphonate in a dose-dependent fashion, and the inhibition was reversed by dithiothreitol. Mg2+-dependent AC was inhibited by 0.5 mM phenylhydrazine (95 per cent). Mg2+-dependent AC activity was responsive to stimulation by epinephrine, without and with GTP addition, with half-maximal stimulation taking place at a concentration of 2 microM. The stimulatory effect of epinephrine was blocked by propranolol in a dose-dependent manner but was not blocked by phentolamine. Oestrone, oestradiol-17 beta, 2-hydroxyoestreone, 2-hydroxyoestradiol-17 beta, dehydroepiandrosterone sulphate, and progesterone, as well as oxytocin, did not alter either the basal or GMP-P(NH)P-stimulated Mg2+-dependent AC activities. Preincubation of 20 000 g particulate fraction with either NaF or GMP-P(NH)P, followed by washing, resulted in preparations that remained stimulated without the requirement of any further additions.

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An optimal assay system for the measurement of GC activity in subcellular fractions of human placenta homogenates was developed. MN2+ was more effective than Mg2+ as a cofactor and, in addition, it markedly potentiated the Mg2+-dependent GC activity. The apparent optimal temperature for GC activity was 45 degrees C and the pH optimum was in the range of 7.5 to 8.3. The apparent Km of Mn2+-dependent GC for GTP was 0.1 and that of MG2+-dependent GC for GTP was 0.4 mm. More than 95 per cent of GC activity was associated with the 100 000 X g supernatant fraction. ATP at a concentration of 0.5 mm inhibited GC activity by 50 per cent. The Mn2+-dependent GC activity was inhibited by p-hydroxymercuriphenyl sulphonate at concentrations between 10 and 50 micrometers; this inhibition was reversed, in part, by dithiothreitol at concentrations of 150 to 500 micrometers to approximately one-fifth the activity of control values. In addition, we found that dithiothreitol was an inhibitor of GC activity. Studies on the responsiveness of placental GC to nucleophilic compounds indicated that phenylhydrazine was the most potent stimulus, while hydroxylamine, hydrazine, sodium azide, sodium nitrite and sodium nitroprusside were less potent. Placental GC was unaffected by alpha- and beta-adrenergic agonists or by cholinergic agonist, as well as by steroids produced by the placenta.

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