Gene expression, localization, and characterization of endothelin A and B receptors in the human adrenal cortex.
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 125I 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 (Bmax) 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, ET-3, S6C, and BQ-123 for each receptor subtype resulted to be within the range reported for human tissue for the ETA and ETB receptors. In the APA tissue the Bmax tended to be lower (1.33 and 0.8 pmol/mg protein, for the ETA and ETB, respectively) but the Kd were similar. Autoradiographic studies confirmed 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 ETA and ETB are actively transcribed in the human adrenal cortex. Furthermore, both receptor subtypes are translated into proteins in ZG and APA cells.
Rossi
G
G
Department of Clinical Medicine, University of Padova, Italy.
Albertin
G
G
Belloni
A
A
Zanin
L
L
Biasolo
M A
MA
Prayer-Galetti
T
T
Bader
M
M
Nussdorfer
G G
GG
Palù
G
G
Pessina
A C
AC
D000302:Adrenal Cortex / Q000378:metabolism*; D001345:Autoradiography; D001483:Base Sequence; D001667:Binding, Competitive; D017931:DNA Primers; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism; D016232:Endothelins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D016232:Endothelins; D007457:Iodine Radioisotopes; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin