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VASCULAR BIOLOGY

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

Smooth Muscle Research Group, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada

Submitted 25 January 2007 ; accepted in final form 18 April 2007

The contribution of small-conductance (SK_Ca) and intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channels to the generation of nitric oxide (NO) by Ca²⁺-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 Ca²⁺ transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca²⁺, 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 BaCl₂ (50 µmol/l) + ouabain (100 µmol/l), depleting intracellular Ca²⁺ stores by thapsigargin or removing external Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca²⁺ stores is to trigger the opening of both K_Ca channels along with Ca²⁺ 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.

calcium; endothelium; hyperpolarization; small-conductance calcium-activated potassium channel; intermediate-conductance calcium-activated potassium channel channel

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