Hydrogen Peroxide Stimulates the Ca^2+-activated Big-Conductance K Channels (BK) Through cGMP Signaling Pathway in Cultured Human Endothelial Cells

We used the whole cell patch-clamp technique to examine the effect of hydrogen peroxide (H(2)O(2)) on the Ca2(+)-activated BK channels in human endothelial cells. We confirmed the previous finding that a 200 pS BK channel activity was detected when the cell membrane potential was clamped at 50 mV. Application of H(2)O(2) or adding glucose oxidase (GO) stimulated BK channels. The stimulatory effect of H(2)O(2) and GO was absent in cells treated with ebselen, a scavenger of reactive oxygen species (ROS). To determine whether the stimulatory effect of H(2)O(2) and GO on BK channels is the result of increasing NO production in the endothelial cells, we examined the effect of H(2)O(2) and GO on BK channels in the presence of 0.1 mM L-NAME which inhibits NO synthase (NOS). Inhibition of NOS completely abolished the stimulatory effect of H(2)O(2) on BK channels. In contrast, treatment of endothelial cells with D-NAME did not block the effect of H(2)O(2) on BK channels. Moreover, inhibiting soluble guanylate cyclase (sGC) with ODQ mimicked the effect of L-NAME and abolished the effect of H(2)O(2). Addition of 8-bromo-cGMP stimulated BK channels and further application of H(2)O(2) did not increase BK channel activity in the presence of cGMP analog. The notion that the effect of H(2)O(2) on BK channels was the result of stimulating NO-cGMP pathway is further indicated by the observation that inhibition of PKG with KT5823 also abolished the stimulatory effect of H(2)O(2) on BK channels. We conclude that H(2)O(2) stimulates the Ca2(+) BK channels through NO/sGC/cGMP pathway in cultured human endothelial cells.