Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells.

The contribution of small-conductance (SK(Ca)) and intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channels to the generation of nitric oxide (NO) by Ca(2+)-mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining single-cell microfluorimetry with perforated patch-clamp recordings to monitor agonist-evoked NO synthesis, cytosolic Ca(2+) transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca(2+), as judged by 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) and fluo-3 fluorescence, respectively, that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium (10 mmol/l) or BaCl(2) (50 micromol/l) + ouabain (100 micromol/l), depleting intracellular Ca(2+) stores by thapsigargin or removing external Ca(2+) inhibited NO production, as did exposure to high (80 mmol/l) external KCl. Importantly, apamin and charybdotoxin (ChTx)/ triarylmethane (TRAM)-34, selective blockers SK(Ca) and IK(Ca) channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca(2+) transients. Apamin and TRAM-34 also inhibited an agonist-induced outwardly rectifying current characteristic of SK(Ca) and IK(Ca) channels. Under voltage-clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SK(Ca) and IK(Ca) channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca(2+) responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca(2+) stores is to trigger the opening of both K(Ca) channels along with Ca(2+) entry channels at the plasma membrane. Finally, the observed inhibition of stimulated NO synthesis by apamin and ChTx/TRAM-34 demonstrates that SK(Ca) and IK(Ca) channels are essential for NO-mediated vasorelaxation.