SK channels are involved in the stimulation of intracellular Ca 2+ signals by reactive oxygen species (ROS) in intact endothelium

Abstract

Increased production of ROS such as superoxide anions (O2−) is associated with endothelial dysfunction encountered in cardiovascular pathologies. An elevation of intracellular Ca2+ levels ([Ca2+]i) has been reported in cultured endothelium exposed to high concentration of ROS. However, the impact of O2− on [Ca2+]i dynamics remains to be determined in native endothelium. Therefore, the effects of O2− on Ca2+ signals in the endothelium of cut-open mesenteric arteries from mouse were investigated, using fluo-4 and laser scanning confocal microscopy. Endothelial cells in this preparation exhibit a variety of spontaneous Ca2+ signals (waves and oscillations). Catalase (500 U/ml) had little, if any, effect on basal [Ca2+]i or Ca2+ signals. In contrast, exposure to superoxide dismutase (SOD) (100 U/ml) decreased by 50% the frequency of spontaneous activity. Exposure to O2− caused a rise in global Ca2+ levels, along with a 2- to 3-fold increase in amplitude and frequency of Ca2+ waves, sustained for at least 10 min. The stimulatory effects of O2− on Ca2+ signals did not require extracellular Ca2+, but its maintenance did, suggesting a dependence on the Ca2+ electrochemical gradient. Consistently, blocking SK channels with apamin caused a membrane depolarization, and decreased the long term elevation of Ca2+ signaling by O2−. Therefore, basal production of O2−, but not H2O2, stimulates intracellular Ca2+ signaling in intact endothelium. Furthermore, sustained effects of O2− on [Ca2+]i and its dynamics requires extracellular Ca2+ entry, which is modulated by SK channel-regulation of endothelial membrane potential. Supported by NIH (HL63722 & HL44455) and CIHR.