Endothelin generating pathway through endothelin1-31 in human cultured bronchial smooth muscle cells

1 The effects of endothelin (ET)-1(1-31) and ET-2(1-31), human chymase products of the corresponding big ETs, on the intracellular free Ca2+ concentration ([Ca2+](i)) and [I-125]-ET-1 binding were investigated using human cultured bronchial smooth muscle cells (BSMC). 2 ET-1(1-31) (10(-8) M - 3 X 10(-7) M) and ET-2(1-31) (3 X 10(-8) M - 3 X 10(-6) M) caused an increase in [Ca2+](i) in a concentration-dependent manner. Big ET-1 (3 X 10(-8) M - 10(-6) M) also caused this increase, but not big ET-2 at concentrations up to 10(-6) M. The [Ca2+](i) increase induced by ET-1 was inhibited by both BQ123, an ETA-receptor antagonist, and BQ788, an ETB-receptor antagonist, whereas that induced by ET-3 was inhibited by BQ788 but not by BQ123. 3 Increases in [Ca2+](i) caused by ET-1(1-31), big ET-1 and ET-2(1-31) were completely inhibited by 10(-4) M phosphoramidon, a dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor, and 10(-5) M thiorphan, a NEP inhibitor. 4 Scatchard plot analyses of the saturation curves of [I-125]-ET-1 and [I-125]-ET-3 showed that both ETA- and ETB- receptors at the ratio of 4 : 1 were expressed on BSMC. ET-1(1-31), big ET-1 and ET-2(1-31) inhibited [I-125]-ET-1 binding in a concentration-dependent manner, and these effects were attenuated by treatment with thiorphan. On the other hand, big ET-2 slightly inhibited the binding at a high concentration and this was not affected by thiorphan. 5 These results suggest that ET-1(1-31), big ET-1 and ET-2(1-31) cause an increase in [Ca2+](i) by being converted into the corresponding ET-1 and ET-2 by NEP, but this did not occur with big ET-2 in human BSMC. ET-2(1-31) produced by human chymase from big ET-2 might be important for the generation of ET-2 in human bronchial tissue.