Compelling evidence indicates that the endothelium-derived potent vasoconstrictor endothelin-1 (ET-1) stimulates aldosterone secretion by interacting with specific receptors. Although two different ET-1 receptors have been identified and cloned, the receptor subtype involved in mediating aldosterone secretion is still unknown. Accordingly, we wished to investigate whether the genes of ET-1 and of its receptors A and B are expressed in the normal human adrenal cortex. We designed specific primers for ET-1 and the ETA and ETB receptors genes and developed a reverse transcription polymerase chain reaction (RT-PCR) with chemiluminescent quantitation of the cDNA. In addition, we carried out 'I ET-1 displacement studies with cold ET-1, ET-3 and the specific ETA and ETB ligands BQ123 and sarafotoxin 6C. Localization of each receptor subtype was also investigated by autoradiography. Binding experiments were first individually analyzed by Scatchard and Hofstee plot and then coanalyzed by the nonlinear iterative curve fitting program Ligand. Histologically normal adrenal cortex tissue, obtained from kidney cancer patients (n = 7), and an aldosterone-producing adenoma (APA), which is histogenetically derived from the zona glomerulosa (ZG) cells, were studied. Results showed that the ET-1, ETA and ETB mRNA can be detected by RT-PCR in all adrenal cortices as well as in the APA. The best fitting of the 125I ET-1 displacement binding data was consistently provided by a two-site model both in the normal adrenal cortex (F = 22.1, P < 0.0001) and in the APA (F = 18.4, P < 0.0001). In the former the density (B..n) of the ETA and ETB subtype was 2.6±0.5 pmol/mg protein (m±SEM) and 1.19±0.6, respectively. The dissociation constant (Kd) of ET-1, S6C,
We investigated the gene expression and localization of endothelin-1 (ET-1) receptor subtypes ET(A) and ET(B) in the rat adrenal cortex as well as their involvement in the corticosteroid secretagogue effect of ET-1 in vitro. Reverse transcription-polymerase chain reaction with primers specific for ET(A) and ET(B) cDNAs demonstrated the expression of both receptor genes in homogenates of adrenocortical tissue. However, in isolated zona glomerulosa and zona fasciculata cells, only ET(B) mRNA was detected. Autoradiographic examination of the selective displacement of 125I-ET-1 binding by BQ-123 and BQ-788 (specific ligands for ET(A) and ET(B), respectively) indicated that zona glomerulosa possesses both ET(A) and ET(B), whereas zona fasciculata is exclusively provided with ET(B). ET-1 enhanced in a concentration-dependent manner aldosterone and corticosterone secretions of dispersed zona glomerulosa and zona fasciculata cells, respectively. The ET(B) antagonist BQ-788 markedly reduced the secretory response of zona glomerulosa cells and completely suppressed that of zona fasciculata cells, whereas the ET(A) antagonist BQ-123 was ineffective. These findings indicate that in the rat, the adrenocortical secretagogue action of ET-1 is mediated by the ET(B) receptor subtype and that the ET(A) receptor is not directly involved in such an effect.
Collectively, these findings are consistent with the contention that endothelial damage occurs in most patients with atherosclerosis and/or hypertension and that ET-1 is synthesized in VSMCs of these patients.
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