Endothelin-1-induced proliferation of human endothelial cells depends on activation of K+ channels and Ca2+ influx

Aims: Endothelin-1 (ET-1) promotes endothelial cell growth. Endothelial cell proliferation involves the activation of Ca2+-activated K+ channels. In this study, we investigated whether Ca2+-activated K+ channels with big conductance (BKCa) contribute to endothelial cell proliferation induced by ET-1. Methods: The patch-clamp technique was used to analyse BKCa activity in endothelial cells derived from human umbilical cord veins (HUVEC). Endothelial proliferation was examined using cell counts and measuring [H-3]-thymidine incorporation. Changes of intracellular Ca2+ levels were examined using fura-2 fluorescence imaging. Results: Characteristic BKCa were identified in cultured HUVEC. Continuous perfusion of HUVEC with 10 nmol L-1 ET-1 caused a significant increase of BKCa open-state probability (n=14; P<0.05; cell-attached patches). The ETB-receptor antagonist (BQ-788, 1 mumol L-1) blocked this effect. Stimulation with Et-1 (10 nmol L-1) significantly increased cell growth by 69% (n=12; P<0.05). In contrast, the combination of ET-1 (10 nmol L-1) and the highly specific BKCa blocker iberiotoxin (IBX; 100 nmol L-1) did not cause a significant increase in endothelial cell growth. Ca2+ dependency of ET-1-induced proliferation was tested using the intracellular Ca2+-chelator BAPTA (10 mumol L-1). BAPTA abolished ET-1 induced proliferation (n=12; P<0.01). In addition, ET-1-induced HUVEC growth was significantly reduced, if cells were kept in a Ca2+-reduced solution (0.3 mmol L-1), or by the application of 2 aminoethoxdiphenyl borate (100 mumol L-1) which blocks hyperpolarization-induced Ca2+ entry (n=12; P<0.05). Conclusion: Activation of BKCa by ET-1 requires ETB-receptor activation and induces a capacitative Ca2+ influx which plays an important role in ET-1-mediated endothelial cell proliferation.