Biomedical subjects

D G Romero

Publications and source records attributed to D G Romero.

6 recordsLinked to original sources

PubMed2000

Cloning and expression of the bovine 11beta-hydroxysteroid dehydrogenase type-2.

The bovine 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11beta-HSD-2) cDNA was cloned from three overlapping PCR fragments using primers based on the human and ovine 11beta-HSD-2 cDNA sequences. Both cDNA ends were obtained by a modified RACE (Rapid Amplification of cDNA Ends) method. The bovine 11beta-HSD-2 cDNA is 1878 bp long, excluding the poly(A) tail. It consists of a 5'-untranslated region of 133 bp, an open reading frame of 1215 bp and a 3'-untranslated region of 530 bp. Bovine 11beta-HSD-2 cDNA is highly homologous to that of the sheep (92%) and less related to the human (67%), rabbit (65%), rat (52%) and mouse (45%) cDNA. The predicted bovine 11beta-HSD-2 protein contains 404 amino acid residues with a calculated mol wt of 43,985. It is homologous to the sheep (98%) and human (88%) protein, and less related to that of the rabbit (76%), rat (80%) and mouse (77%). The cloned 11beta-HSD-2 cDNA was transfected into CHOP cells and the enzymatic characteristics determined. The enzyme functions primarily as an oxidase, uses NAD(+) and is more active with corticosterone as a substrate than with cortisol or dexamethasone. It is expressed in high concentrations in kidney, adrenal and colon, and in small concentrations in liver, heart and lung. In conclusion, the 11beta-HSD-2 enzyme of cattle is very similar to that of other species in its structure and enzymatic characteristics.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed1998

Mechanisms of action of endothelin-1 in rat adrenal.

Displacement curves of 125I-Endothelim-1 (ET-1) binding to rat adrenal cells with unlabeled ET-1, and the ET-1 receptor-related peptides sarafotoxin and BQ-123, show that rat adrenal cortex possess, as its bovine counterpart, two different receptors to ET-1 named ET-A and ET-B. Binding of ET-1 to its rat adrenal receptors stimulates i) aldosterone production, in vivo and in vitro ii) calcium influx, which is mediated through voltage dependent- and receptor operated- calcium channels, iii) cholesterol uptake, iv) stimulation of Na+/K+-ATPase and iv) diacylglycerol production. While the last effect is mediated through ET-A receptors the others involve binding of ET-1 to ET-B receptors. Finally, ouabain potentiates the ET-1-mediated stimulation of aldosterone production, suggesting that the effect of the peptidic hormone on Na+/K+-ATPase could act as a negative feedback mechanism.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Regulation of the 11 beta-hydroxysteroid dehydrogenase in the rat adrenal. Decrease enzymatic activity induced by ACTH.

Patients with ectopic ACTH syndrome often develop hypertension and hypokalemic alkalosis with an abnormal increase in the ratio of plasma cortisol to cortisone, indicating that 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) activity is inhibited. Inhibition of 11 beta HSD allows access of cortisol or corticosterone to the mineralocorticoid receptor where it act as a mineralocorticoid. Two isozymes, 11 beta HSD-1 and 11 beta HSD-2, have been cloned and characterized. The rat adrenal expresses the mRNAs for 11 beta HSD-2 and, in lesser amounts, 11 beta HSD-1. We investigated the effect of ACTH on the 11 11 beta HSD-2 activity in the rat adrenal. Rat adrenal cells zone fasciculata (ZF) were dispersed and incubated separately with increasing concentrations of ACTH for 90 min, and secretion of corticosterone (B) and 11-dehydrocorticosterone (A) in the media was measured by enzyme-linked immunoabsorbent assays (ELISA). The conversion of [3H]B to [3H]A in the presence of 0.5 mM NAD+ was evaluated in microsomes prepared from dispersed cells preincubated for 30 min with cyanoketone and metyrapone followed by incubation for 30 min with the same inhibitors, with and without 10 nM ACTH. The dispersed cells of the ZF produced significant amounts of A which increased with ACTH. The basal B/A ratio was 0.97 +/- 0.05. ACTH caused a concentration-dependent increase in the ratio of B/A with a maximum ratio of 9.58 +/- 0.20. ACTH also inhibited the conversion of [3H]B to [3H]A in microsomes in which endogenous B production was inhibited by cyanoketone and metyrapone. ACTH did not change the K(m) for B conversion, but the Vmax was reduced significantly (1.73 +/- 0.43 pmol/min. mg protein), indicating that ACTH suppressed the 11 beta HSD-2 in a noncompetitive fashion. Dibutyryl cyclic AMP (dcAMP) also produced a concentration-dependent increase in the B/A ratio, but various concentrations of calcium did not affect the enzyme activity. In summary, adrenal cells treated with ACTH results in a significant increase in the ratio of B/A in the ZF owing a noncompetitive inhibition of the 11 beta HSD-2 via the ACTH receptor.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1997

Heme availability affects corticosterone and aldosterone biosynthesis in rat adrenal.

In this paper, we studied the effect of heme availability on corticosterone and aldosterone synthesis in rat adrenal. We found that hemin stimulated corticosterone and aldosterone production in adrenal homogenates in a dose-dependent fashion. Hemin administration to rats also provoked an increase in both corticosterone and aldosterone content in adrenal. 3,5-Diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), an inhibitor of liver ferrochelatase activity, was able to inhibit this enzyme in rat adrenal. This resulted in an impairment of heme concentration and consequently adrenal ALA-synthase and porphyrin content were increased. Thus, it was proven that DDC inhibits heme biosynthesis in adrenal as it does in liver. In vivo experiments with rats showed that DDC was able to partially blocked ACTH-mediated corticosterone and aldosterone production while hemin administration was able to partially restore it. These data indicate that heme availability affects steroid biosynthesis in rat adrenal.

5-Aminolevulinate Synthetase↗

PubMed1996

Endothelin-1-induced incorporation of cholesterol into rat adrenals.

The effect of endothelin-1 (ET-1) on cholesterol uptake by adrenal cortex was evaluated through several experimental approaches: infusion of ET-1 followed by measurement of endogenous cholesterol in excised adrenals; infusion of ET-1 followed by tritiated cholesterol incorporation into adrenal quarters in vitro; coinfusion of ET-1 with tritiated cholesterol-enriched serum and determination of adrenal-associated radioactivity; and tritiated cholesterol incorporation in incubations of adrenal cells. In all cases ET-1 increased cholesterol uptake. Subcellular fractionation showed an ET-1-mediated augmentation in mitochondrial fraction. This increase was mediated by the subpopulation B of adrenal receptors for ET-1. In addition, ET-1 also increased cytochrome P450-SCC (side-chain cleavage) activity.

Adrenal Glands↗

PubMed1996

Effects of staurosporine on ACTH-mediated stimulation of aldosterone production.

Incubation of rat adrenal glomerulosa cells with low concentrations (up to 50 nM) of the protein kinase (PKC) inhibitor staurosporine (ST) inhibited aldosterone (ALDO) and cyclic AMP (cAMP) production stimulated by adrenocorticotropic hormone (ACTH) and cholera toxin. Only higher concentrations (1.6 microM) of staurosporine inhibited dibutyryl-cAMP- and forskolin-induced stimulation of aldosterone production. cAMP levels were increased only with low concentrations of the PKC inhibitor. This latter increase was avoided by treatment with a maximal concentration of isobutylmethylxanthine (MIX). Our results suggest that: (1) second messengers other than cAMP are involved in ACTH action; (2) staurosporine inhibits different kinases involved in ACTH action in a dose-dependent manner; (3) the protein kinase inhibited by high concentrations of staurosporine appears to be the cAMP-dependent kinase, PKA; and (4) the protein kinase inhibited by low concentrations of staurosporine remains to be identified. This latter species is suggested as being involved in mediating ACTH-induced activation of Gs.

Adrenocorticotropic Hormone↗