IDENTIFICATION OF ENDOTHELIN RECEPTOR SUBTYPES IN RAT-KIDNEY CORTEX USING SUBTYPE-SELECTIVE LIGANDS

I-125-Endothelin (ET)-1 and I-125-ET-3 displayed specific, saturable, and high affinity binding to membranes prepared from rat kidney cortex. Saturation binding experiments using I-125-ET-1 and I-125-ET-3 revealed that I-125-ET-3 binding sites were 40-50% less abundant than I-125-ET-1 binding sites. The dissociation constants (K(d)) and maximum binding (B(max)) for I-125-ET-1 and I-125-ET-3 with these membranes were 218 +/- 23 pM and 275 +/- 20 fmol/mg of protein and 207 +/- 19 pM and 113 +/- 17 fmol/mg of protein, respectively. In the presence of 10 nM sarafotoxin 6c, a selective agonist for ET(B) receptors, I-125-ET-1 binding was decreased by 45-50% and I-125-ET-3 binding was totally abolished, suggesting that approximately 40-50% of kidney cortex ET receptors are of the ET(B) subtype and that I-125-ET-1 binds to both ET(A) and ET(B) receptors with the same high affinity, whereas I-125-ET-3 binds to only ET(B) receptors with high affinity. In addition, in the presence of BQ123 [cyclo(D-Trp,D-Asp,L-Pro,D-Val,L-Leu)], a selective antagonist for ET(A) receptors, I-125-ET-1 binding was decreased by 50%, whereas I-125-ET-3 binding was unaffected. Our results strongly suggest that rat kidney cortex contains ET(A) and ET(B) receptors in a 50:50 ratio and that sarafotoxin 6c and BQ123 are valuable tools in identifying the subtypes of ET receptors in various tissues.