Estrogen and the Ca2+-mobilizing agonist ATP evoke acute NO synthesis via distinct pathways in an individual human vascular endothelium-derived cell.
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.
Sheng
Jian-Zhong
JZ
Smooth Muscle Research Group, Libin Cardiovascular Institute and Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
Arshad
Furqan
F
Braun
Janice E
JE
Braun
Andrew P
AP
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001030:Apamin / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D018999:Charybdotoxin / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D003592:Cytophotometry; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D000077267:Fulvestrant; D006801:Humans; D008564:Membrane Potentials; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D018408:Patch-Clamp Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013997:Time Factors; D015854:Up-Regulation
D000255:Adenosine Triphosphate; D001030:Apamin; D018999:Charybdotoxin; D002614:Chelating Agents; D002867:Chromones; D004533:Egtazic Acid; D004791:Enzyme Inhibitors; D004958:Estradiol; D004965:Estrogen Antagonists; D005419:Flavonoids; D000077267:Fulvestrant; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines; D019331:NG-Nitroarginine Methyl Ester; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D026902:Potassium Channel Blockers; D024681:Potassium Channels, Calcium-Activated; D047428:Protein Kinase Inhibitors; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one