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

Abstract

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 (BK(Ca)) contribute to endothelial cell proliferation induced by ET-1.

Methods: The patch-clamp technique was used to analyse BK(Ca) activity in endothelial cells derived from human umbilical cord veins (HUVEC). Endothelial proliferation was examined using cell counts and measuring [3H]-thymidine incorporation. Changes of intracellular Ca2+ levels were examined using fura-2 fluorescence imaging.

Results: Characteristic BK(Ca) were identified in cultured HUVEC. Continuous perfusion of HUVEC with 10 nmol L(-1) ET-1 caused a significant increase of BK(Ca) open-state probability (n = 14; P < 0.05; cell-attached patches). The ET(B)-receptor antagonist (BQ-788, 1 micromol 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 BK(Ca) 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 micromol 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 micromol L(-1)) which blocks hyperpolarization-induced Ca2+ entry (n = 12; P < 0.05).

Conclusion: Activation of BK(Ca) by ET-1 requires ET(B)-receptor activation and induces a capacitative Ca2+ influx which plays an important role in ET-1-mediated endothelial cell proliferation.