GENE-EXPRESSION, LOCALIZATION, AND CHARACTERIZATION OF ENDOTHELIN-A AND ENDOTHELIN-B RECEPTORS IN THE HUMAN ADRENAL-CORTEX

Compelling evidence indicates that the endothelium-derived potent vaso constrictor endothelin-1 (ET-1) stimulates aldosterone secretion by in teracting with specific receptors. Although two different ET-1 recepto rs have been identified and cloned, the receptor subtype involved in m ediating aldosterone secretion is still unknown. Accordingly, we wishe d 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 specif ic primers for ET-1 and the ET(A) and ET(B) receptors genes and develo ped a reverse transcription polymerase chain reaction (RT-PCR) with ch emiluminescent quantitation of the cDNA. In addition, we carried out I -125 ET-1 displacement studies with cold ET-1, ET-3 and the specific E T(A) and ET(B) ligands BQ123 and sarafotoxin 6C. Localization of each receptor subtype was also investigated by autoradiography. Binding exp eriments were first individually analyzed by Scatchard and Hofstee plo t and then co-analyzed by the nonlinear iterative curve fitting progra m Ligand. Histologically normal adrenal cortex tissue, obtained from k idney cancer patients (n = 7), and an aldosterone-producing adenoma (A PA), which is histogenetically derived from the zona glomerulosa (ZG) cells, were studied, Results showed that the ET-1, ET(A) and ET(B) mRN A can be detected by RT-PCR in all adrenal cortices as well as in the APA. The best fitting of the I-125 ET-1 displacement binding data was consistently provided by a two-site model both in the normal adrenal c ortex (F = 22.1, P < 0.0001) and in the APA (F = 18.4, P < 0.0001), In the former the density (B-max) of the ET(A) and ET(B) subtype was 2.6 +/-0.5 pmol/mg protein (m+/-SEM) and 1.19+/-0.6, respectively. The dis sociation constant (K-d) of ET-1, ET-3, S6C, and BQ-123 for each recep tor subtype resulted to be within the range reported for human tissue for the ET(A) and ET(B) receptors. In the APA tissue the B-max tended to be lower (1.33 and 0.8 pmol/mg protein, for the ET(A) and ET(B), re spectively) but the K-d were similar. Autoradiographic studies confirm ed the presence of both receptor subtypes on the ZG as well as on APA cells. Thus, the genes of ET-1 and both its receptor subtypes ET(A) an d ET(B) are actively transcribed in the human adrenal cortex. Furtherm ore, both receptor subtypes are translated into proteins in ZG and APA cells.