Human Kidney: Endothelin Isoforms Detected by HPLC with Radioimmunoassay and Receptor Subtypes Detected Using Ligands BQ123 and BQ3020

Abstract

Animal kidneys are exquisitely sensitive to the effects of endothelin (ET), but little is known of its binding characteristics, isoform prevalence, or receptor subtype distribution in human kidney. We investigated these parameters using high-performance liquid chromatography, radioimmunoassay (RIA), and the recently synthesized ETA and ETB receptor-selective peptide ligands BQ123 (cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp-]) and BQ3020 (Ala11,15-Ac-ET-1[6-21]). Fresh-frozen normal segments of kidneys excised for carcinoma were solid-phase-extracted using Amprep C2 columns, and parallel eluates were oxidized. All were subjected to RIA for ET and pro-ET-1, in triplicate. ET isoforms were characterized by RP-HPLC and subsequent RIA of eluates. Total amounts of immunoreactive ET were 6.9 +/- 3.8 and 4.5 +/- 1.6 pmol/g wet weight in medulla and cortex, respectively. Reverse-phase high performance liquid chromatography showed peaks of immunoreactivity with retention times identical to synthetic ET-1 and metsulphoxide ET-1. ET-2, ET-3, and pro-ET-1 were not detected. Saturation assays using 0.01-8.0 nM 125I-ET-1 or 125I-BQ3020, gave Kd values (mean +/- SEM) of 0.17 +/- 0.04 and 0.36 +/- 0.06 nM, respectively, with Bmax values of 57.7 +/- 15.4 and 30.0 +/- 5.0 fmol/mg protein, respectively. Hill coefficients were 0.86 +/- 0.03 and 0.77 +/- 0.04, but a two-site fit was not preferred. Receptor autoradiography has detected both subtypes, mainly present in medulla, with ETB predominating.(ABSTRACT TRUNCATED AT 250 WORDS)