Carbon Monoxide Stimulates the Ca ²⁺ –Activated Big Conductance K Channels in Cultured Human Endothelial Cells

Abstract

Abstract-We used the whole-cell patch-clamp technique to study K channels in the human umbilical vein endothelial cells and identified a 201 pS K channel, which was blocked by tetraethylammonium and iberiotoxin but not by TRAM34 and apamin. This suggests that the Ca 2ϩ -activated big-conductance K channel (BK) is expressed in endothelial cells. Application of carbon monoxide (CO) or tricarbonylchloro(glycinato)ruthenium(II), a water soluble CO donor, stimulated the BK channels. Moreover, application of hemin, a substrate of heme oxygenase, mimicked the effect of CO and increased the BK channel activity. The stimulatory effect of hemin was significantly diminished by tin mesoporphyrin, an inhibitor of heme oxygenase. To determine whether the stimulatory effect of CO on the BK channel was mediated by NO and the cGMP-dependent pathway, we examined the effect of CO on BK channels in cells treated with, N G -nitro-L-arginine methyl ester, 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, or KT5823, an inhibitor of protein kinase G. Addition of either diethylamine NONOate or sodium nitroprusside significantly increased BK channel activity. Inhibition of endogenous NO synthesis with N G -nitro-L-arginine methyl ester, blocking soluble guanylate cyclase or protein kinase G, delayed but did not prevent the CO-induced activation of BK channels. Finally, application of an antioxidant agent, ebselen, had no effect on CO-mediated stimulation of BK channels in human umbilical vein endothelial cells. We conclude that BK channels are expressed in human umbilical vein endothelial cells and that they are activated by both CO and NO. CO activates BK channels directly, as well as via a mechanism involving NO or the cGMP-dependent pathway.

Hydrogen peroxide stimulates the Ca2+-activated big-conductance K channels (BK) through cGMP signaling pathway in cultured human endothelial cells

“…1A is an I/V curve confirming the previous finding that a 201 pS BK channel was expressed in the cultured human endothelial cells and the channel was inhibited by 100 nM iberiotoxin [1]. Fig.…”

“…It has been reported that H 2 O 2 increases nitric oxide (NO) production in endothelial cells in a previous study [12,13]. Because NO has been shown to stimulate BK activity in the cultured human endothelial cells in previous study [1], we examined whether the stimulatory effect of H 2 O 2 on BK channels was mediated by a NO-dependent mechanism. Thus, we treated endothelial cells with L-NAME to block the NOS and examined the effect of 100 μM H 2 O 2 on BK channels in the presence of L-NAME.…”

“…Although H 2 O 2 has been suggested to be involved in mediating the effect of acetylcholine on vasoactivity, the mechanism by which H 2 O 2 causes vasodilation is not completely understood. Several studies indicate that the vasodilation induced by H 2 O 2 is the result of stimulating Ca 2+ -activated K channels in endothelial cells [11], Ca 2+ -activated BK channels [1,10,27] and ATP-sensitive K channels in vascular smooth muscle [28].…”

“…Related to this speculation is the report that H 2 O 2 has been shown to stimulate NOS and increase NO production in vessels [12,13]. Since NO is known to stimulate BK channels in endothelial cells and smooth muscle cells [1,23,40], the synergized effect of H 2 O 2 on BK channels in both smooth muscle cells and endothelial cells should lead to vasodilation. We conclude that H 2 O 2 stimulates BK channels in the cultured endothelial cells and that the effect of H 2 O 2 on BK channels is mediated by PKG.…”

“…Ca 2+ -activated K channels including small-conductance, intermediate-conductance and bigconductance have been shown to be expressed in endothelial cells [1][2][3][4][5]. Although Ca 2+ -activated small conductance and intermediate-conductance K channels are mainly responsible for determining the membrane potential under basal conditions [3], BK channels may also be involved in regulating the membrane potential of endothelial cells because BK channels have a high channel conductance.…”