CLONING AND EXPRESSION OF AN ENDOTHELIN RECEPTOR SUBTYPE-B FROM HUMANPROSTATE THAT MEDIATES CONTRACTION

Recent evidence suggests a role for endothelin (ET) in contraction of human prostate [J. Urol. 149:495-499 (1993)]. Although both ET(A) and ET(B) receptors have been shown to mediate contraction of smooth muscl e, the molecular identity of the contractile ET(B) receptor is controv ersial. The aim of this study was to examine the receptor subtype that mediates ET-induced contraction in prostate from patients with benign prostatic hyperplasia. Saturation binding with I-125-ET-1 and I-125-E T-3 in prostate stromal cells (PSC) indicated the presence of receptor s with subnanomolar affinity for these radioligands, with equivalent r eceptor densities. Inhibition of specific I-125-ET-1 or I-125-ET-3 bin ding in PSC revealed a rank order of potency of ET-1 = ET-3 = sarafoto xin S6c much greater than BQ-123. These data are consistent with a pre dominance of ET(B) receptors in PSC. The functional effects of ET stim ulation of PSC were examined in a collagen gel contraction assay. ET-1 and ET-3 caused contraction of underlying collagen gel matrices with EC(50) values of 0.4 +/- 0.04 and 0.7 +/- 0.2 nM, respectively. To det ermine the molecular nature of the contractile ET(B) receptor in PSC, reverse transcription-polymerase chain reactions were conducted with o ligonucleotide primers to the 5' and 3' ends of the coding sequence of the full length human ET(B) receptor. DNA sequence analysis of the 1. 3-kilobase DNA product showed 99% homology to other human ET(B) recept or cDNAs. The encoded protein has a deduced amino acid sequence identi cal to that of other human ET(B) receptors, with the exception of two conservative substitutions. Expression of the PSC ET(B) cDNA in COS-7 cells resulted in a binding profile similar to that observed in parent cells. Polymerase chain reaction analysis revealed the presence of pr epro-ET-1 mRNA in PSC. Collectively, these data indicate that PSC from patients with benign prostatic hyperplasia express ET(B) receptors th at mediate ET-induced contraction.