IN-VITRO EXPRESSION OF ENDOTHELIN-1 (ET-1) AND THE ET(A) AND ET(B) ETRECEPTORS BY THE PROSTATIC EPITHELIUM AND STROMA

RT-PCR analysis of total RNA prepared from the prostate cancer cell li nes DU145 and PC3 and from primary epithelial cells indicated the pres ence of endothelin-1 (ET-1) messenger RNA (mRNA). Neither the LNCaP ce ll line nor primary prostatic stromal cells possess ET-1 mRNA transcri pts. Seventy-two-hour-conditioned media derived from DU145, PC3, and p rimary epithelia contain immunoreactive ET concentrations equivalent t o 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 exhibi ted by LNCaP (0.029+/-0.009 fmol/mL/10(6) cells after 72 h) and stroma l 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 recept or mRNA is almost completely undetectable in the prostate cancer cell lines. Both ET(A) and ET(B) mRNAs are detectable in primary cultures o f prostatic epithelia and stroma. Competitive binding studies demonstr ate a single class of binding site in both primary benign epithelia (d issociation constant =1.85x10(-10) mol/L; maximal binding capacity=2.7 x10(4) binding sites/cell), and stroma (dissociation constant=1.93x10( -10) mol/L; maximal binding capacity=3.7x10(5) binding sites/cell). Us e Of selective ETreceptor antagonists confirmed that the predominant s tromal receptor subtype expressed in vitro is ET(B). This receptor see ms not to be coupled to mitogenic pathways because no growth response to exogenous ET-1 or cooperation between ET-1 and bFGF could be observ ed. 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.