In vitro expression of endothelin-1 (ET-1) and the ET(A) and ET(B) ET receptors by the prostatic epithelium and stroma

RT-PCR analysis of total RNA prepared from the prostate cancer cell lines DU145 and PC3 and from primary epithelial cells indicated the presence of endothelin-1 (ET-1) messenger RNA (mRNA). Neither the LNCaP cell line nor primary prostatic stromal cells possess ET-1 mRNA transcripts. Seventy-two-hour-conditioned media derived from DU145, PC3, and primary epithelia contain immunoreactive ET concentrations equivalent to 0.814+/-0.048, 0.330+/-0.050, and 0.856+/-0.055 fmol/ml/10(6) cells after 72 h, respectively. Basal immunoreactive ET secretion was exhibited by LNCaP (0.029+/-0.009 fmol/mL/10(6) cells after 72 h) and stromal cells (0.067+/-0.007 fmol/mL/10(6) cells after 72 h). Examination of ET(A) and ET(B) gene expression by RT-PCR demonstrates that ET receptor mRNA is almost completely undetectable in the prostate cancer cell lines. Both ET(A) and ET(B) mRNAs are detectable in primary cultures of prostatic epithelia and stroma. Competitive binding studies demonstrate a single class of binding site in both primary benign epithelia (dissociation constant =1.85x10(-10) mol/L; maximal binding capacity=2.7x10(4) binding sites/cell), and stroma (dissociation constant=1.93x10(-10) mol/L; maximal binding capacity=3.7x10(5) binding sites/cell). Use Of selective ETreceptor antagonists confirmed that the predominant stromal receptor subtype expressed in vitro is ET(B). This receptor seems not to be coupled to mitogenic pathways because no growth response to exogenous ET-1 or cooperation between ET-1 and bFGF could be observed. Similarly, no effect of ET-1 or the ET-converting enzyme inhibitor, phosphoramidon, on benign epithelial cells could be observed over a 4-day period.