Isoforms of 11beta-hydroxysteroid dehydrogenase in human granulosa-lutein cells.

To date, two isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD) have been characterized: a low affinity, NADP+-dependent isoform (11betaHSD1) and a high affinity, NAD+-dependent isoform which metabolizes dexamethasone and is inhibited by cortisone (11betaHSD2). Having previously reported a relationship between ovarian 11betaHSD activities and conception in women undergoing in vitro fertilization (IVF-ET), the objective of the present study was to identify which isoforms of 11betaHSD metabolize glucocorticoids in cultures of human granulosa-lutein cells. In both intact cells and cell homogenates, two distinct 11betaHSD activities were identified with differing affinities for cortisol (Km = 490 nM and 2.6 microM). Even at low concentrations, cortisol oxidation was preferentially supported by NADP+ and was independent of NAD+. Although inhibited by the hemisuccinate ester of glycyrrhetinic acid, carbenoxolone, the predominant 11betaHSD activity in intact cells was resistant to end-product inhibition. Intact cells were also able to reduce [3H]cortisone (Km = 190 nM) but did not metabolize [3H]dexamethasone. 11BetaHSD1 mRNA was expressed in 23 of 28 cell cultures whereas 11betaHSD2 mRNA was not expressed in any of the 22 independent cultures studied by reverse transcriptase-polymerase chain reaction (RT-PCR). We conclude that human granulosa-lutein cells express both type 11betaHSD and a novel isoform of this enzyme. While the low affinity 11beta-dehydrogenase and 11-ketosteroid reductase activities exhibit properties consistent with 11betaHSD1, the high affinity 11beta-dehydrogenase differs from 11betaHSD2 in that it is NADP+-dependent, does not metabolize dexamethasone and is resistant to end-product inhibition.

Follicular variation in ovarian 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) activities: evidence for the paracrine inhibition of 11 beta HSD in human granulosa-lutein cells.

We have previously shown that detectable metabolism of cortisol to cortisone by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) in human granulosa-lutein cells, pooled for each patient from all aspirated ovarian follicles, is associated with failure to conceive by in vitro fertilization and embryo transfer. The aims of the present study were to assess: (1) the variation in the 11 beta HSD activities of granulosa-lutein cells obtained from individual follicles in relation to oocyte maturity and (2) whether the 11 beta HSD activity of pooled granulosa-lutein cells reflects the 11 beta HSD activities of the individual follicles for a given patient. 11 beta HSD activities were measured in intact cells in serum-free medium by a radiometric conversion assay (100 nmol/l [3H]cortisol to [3H]cortisone). Follicular 11 beta HSD activities ranged from < 10 (undetectable) to 514 pmol/mg protein per 4 h (n = 105 follicles from 12 patients) and did not correlate with oocyte maturity. In three separate patients, the follicular 11 beta HSD activities ranged from < 10 to 117 pmol/mg protein per 4 h (n = 8 follicles), 19 to 514 pmol/mg per 4 h (n = 9) and 60 to 390 pmol/mg per 4 h (n = 8). The 11 beta HSD activities of the corresponding multi-follicular pools of cells were < 10, < 10 and 44 pmol/mg per 4 h respectively, all of which were significantly lower (P < 0.05) than the arithmetic means for the activities in the individual follicles (52, 132 and 215 pmol/mg per 4 h respectively). Likewise, the 11 beta HSD activities of two independent multi-patient pools of cells were significantly lower than the mean values of the 11 beta HSD activities of the appropriate individual patients. We conclude that ovarian 11 beta HSD activity varies between follicles and that co-culture of granulosa-lutein cells with low enzyme activity can suppress the ovarian 11 beta HSD activity in cells from different follicles (or patients) with high rates of cortisol metabolism. Hence, these data indicate the potential for paracrine inhibition of ovarian 11 beta HSD activity in human granulosa-lutein cells.

Ovarian 11 beta-hydroxysteroid dehydrogenase: potential predictor of conception by in-vitro fertilisation and embryo transfer.

Cortisol is converted to the inactive glucocorticoid, cortisone, in several tissues by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). We have recently measured 11 beta HSD activity in cultured human granulosa-lutein cells recovered from patients undergoing in-vitro fertilisation and embryo transfer (IVF-ET). We now report an association between the outcome of IVF-ET and 11 beta HSD activity in these cells. Of the 64 patients studied, 32 had detectable 11 beta HSD activity and none became pregnant; whereas 76% of the remaining "11 beta HSD-negative" patients achieved pregnancies. Hence 11 beta HSD activity may predict the outcome of IVF-ET.

WEB 2086 inhibits neutrophil dependent increases in coronary resistance in blood perfused rabbit heart.

OBJECTIVE: The aim was to investigate the mechanism of the pressor action of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine in the blood perfused Langendorff preparation of the isolated rabbit heart; and in particular to establish whether the response was dependent on the presence of neutrophils and whether the release of platelet activating factor contributed to the pressor effect. METHODS: An isolated rabbit heart was perfused with blood from an anesthetised support rabbit. Formyl-methionyl-leucyl-phenylalanine was injected intra-arterially proximal to the isolated heart and measures of cardiac performance recorded. In some experiments the support animal was depleted of neutrophils by pretreatment with mechlorethamine while in others the specific platelet activating factor receptor antagonist WEB 2086 was given to the support animal before formyl-methionyl-leucyl-phenylalanine. Differential blood cell counts were determined throughout the course of each experiment. Each heart was examined histologically after the experiment. RESULTS: Formyl-methionyl-leucyl-phenylalanine caused a significant rise in perfusion pressure which was virtually abolished by leucocyte depletion of the support animal. The response could also be reduced by about 80% with intravenous WEB 2086. Histological examination of the perfused hearts showed that the number of accumulated neutrophils was very variable and not correlated with the rise in perfusion pressure. There was no significant difference between control hearts and those receiving WEB 2086. CONCLUSIONS: The results confirm previous reports that the response to formyl-methionyl-leucyl-phenylalanine is neutrophil dependent and show that this model of a blood perfused heart can be used successfully to examine the response to a leucocyte dependent stimulus. The results also suggest that the response to formyl-methionyl-leucyl-phenylalanine may not only be due to physical obstruction of the coronary circulation or "neutrophil plugging", but may also be due to the release of platelet activating factor.

Platelet-activating factor (Paf) antagonist, WEB 2086, protects against Paf-induced hypotension in Macaca fascicularis.

1. The actions of intravenously administered platelet-activating factor (Paf) (0.1-3.33 nmol kg-1) and the effect of a recently described Paf antagonist, WEB 2086, were investigated in the anaesthetized open-chest monkey, Macaca fascicularis. 2. Paf dose-dependently reduced blood pressure, left ventricular pressure (LVP) and its first differential LV dP/dt. 3. Mean pulmonary artery pressure, recorded in three animals, was essentially unchanged by any dose of Paf. 4. WEB 2086 (0.22 mumol kg-1, i.v.) attenuated the Paf-induced changes in BP, LVP and LV dP/dt. The dose-response curve for fall in BP was shifted to the right by one order of magnitude. 5. Histamine-induced cardiovascular changes (systemic hypotension and tachycardia) were not affected by prior administration of WEB 2086. 6. WEB 2086 should be of value in assessing the role of Paf in pathophysiological conditions.

Endothelin-1 is a potent constrictor in conductive and resistive coronary arteries.

The action of endothelin-1 (ET-1) in canine and porcine coronary artery ring preparations and perfused canine and porcine myocardium was examined in vitro to determine the site of action of ET-1 in the coronary vasculature. ET-1 had a vasoconstrictor effect that was more potent in smaller diameter ring preparations. The EC50s for both canine and porcine ring preparations decreased with a decrease in vessel diameter and the EC50 of ET-1 in the perfused myocardium was three to four times lower than in the smallest ring preparation. The results suggest that ET-1 may cause cardiac ischemia by constriction of resistive vessels rather than by epicardial coronary artery spasm.