Small- and intermediate-conductance Ca<sup>2+</sup>-activated K<sup>+</sup>channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.003 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".