Estrogen and the Ca<sup>2+</sup>-mobilizing agonist ATP evoke acute NO synthesis via distinct pathways in an individual human vascular endothelium-derived cell
Bibliographic record
Abstract
In this study, we have systematically evaluated the signaling mechanisms underlying stimulated nitric oxide (NO) synthesis by estrogen (E2) and other vasoactive agents at the level of a single endothelium-derived cell. To do so, we have characterized and contrasted rapid E2-evoked NO synthesis with that of ATP using single-cell microfluorimetry and patch-clamp recordings to monitor stimulated changes in cellular NO synthesis (via 4-amino-5-methylamino-2',7'-difluorofluorescein), Ca2+ transients (via Fluo-3), and membrane hyperpolarization in cultured human EA.hy926 cells. E2-evoked NO synthesis in single cells (EC50 approximately 0.3 nM) was blocked by the E2 receptor antagonist ICI 182,780 and the NO synthase inhibitor N(omega)-nitro-l-arginine methyl ester. Although both E2 and ATP stimulated comparable Ca2+ transients, E2-induced NO synthesis was insensitive to intracellular BAPTA-AM or removal of external Ca2+. In contrast, ATP-evoked NO production was abolished by either one of these treatments. ATP-evoked hyperpolarizations ( approximately 20 mV) and NO production were both inhibited by the respective small-conductance and intermediate-conductance calcium- activated K+ channel blockers apamin and charybdotoxin. E2 minimally affected membrane potential, and stimulated NO synthesis was insensitive to calcium-activated K+ channel blockers. Exposure to either the phosphatidylinositol 3-kinase inhibitor LY-294002 or the MAP kinase inhibitor PD-98059 abolished the NO response to E2, but not that to ATP. Finally, the NO response evoked by a combined stimulus of E2 plus ATP was similar to that of ATP alone. In conclusion, our data directly demonstrate that an individual human EA.hy926 cell contains at least two distinct mechanisms for stimulated NO synthesis that depend on either calcium or protein kinase signaling events.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 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 source (direct Gemma or distilled Codex), 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".