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.
Sheng
Jian-Zhong
JZ
Department of Pharmacology and Therapeutics, University of Calgary, Alberta, Canada T2N 4N1.
Braun
Andrew P
AP
D000255:Adenosine Triphosphate / Q000378:metabolism; D001030:Apamin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D003592:Cytophotometry; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006632:Histamine / Q000378:metabolism; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000819:agonists / Q000378:metabolism*; D008564:Membrane Potentials; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000819:agonists / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014664:Vasodilation / Q000187:drug effects; D014665:Vasodilator Agents / Q000378:metabolism* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D026902:Potassium Channel Blockers; D011720:Pyrazoles; D051640:Small-Conductance Calcium-Activated Potassium Channels; C411671:TRAM 34; D014665:Vasodilator Agents; D001030:Apamin; D009569:Nitric Oxide; D019284:Thapsigargin; D006632:Histamine; D000255:Adenosine Triphosphate; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D000252:Calcium-Transporting ATPases; D002118:Calcium