Signaling mechanisms that mediate nitric oxide production induced by acetylcholine exposure and withdrawal in cat atrial myocytes
Signaling mechanisms that mediate nitric oxide production induced by acetylcholine exposure and withdrawal in cat atrial myocytes
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DOI：
10.1161/01.res.0000106133.92737.27
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发表时间：
2003-12-12
影响因子：
20.1
通讯作者：
Lipsius, SL
中科院分区：
文献类型：
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作者：
Dedkova, EN;Ji, X;Lipsius, SL
用荧光显微镜和NO敏感指示剂4，5-二氨基荧光素观察了乙酰胆碱（ACh）对猫心房肌细胞内NO（NOi）的影响。场刺激（1 Hz）的细胞或接触静止细胞ACh（1至10 μ mol/L）对NOi没有影响。然而，在现场刺激的细胞，乙酰胆碱暴露增加NOi，乙酰胆碱撤退引起了额外的，显着增加NOi生产。在ACh暴露过程中，加入1 μ mol/L阿托品可增加NOi的产生，与ACh停药相似。预先暴露于1 mmol/L细胞外Ca ~（2+）可降低ACh诱导的NOi升高0.5 mmol/L [Ca ~（2+）]（o）、1 mumol/L维拉帕米、1 mumol/L阿托品、10 mumol/L L-N-5-（1-亚氨基乙基）鸟氨酸、10 μ mol/ L W-7，或在百日咳毒素或10 μ mol/ L LY 294002（抑制磷脂酰肌醇3-激酶）中孵育细胞。在ACh戒断期间切换到0.5 mmol/ L [Ca 2 +]（o）防止了NOi的额外增加。ACh暴露可使蛋白激酶B（Akt）磷酸化（Ser（473））增加，此作用可被LY 294002阻断，而在低[Ca 2 +]（0. 5 mmol/L）时不受影响。共聚焦显微镜显示，乙酰胆碱暴露增加NOi在当地的subsarcolemal网站，和乙酰胆碱撤退另外增加NOi招募额外的subsarcolemal释放网站。用2 mmol/L甲基-β-环糊精阻断Caveolae可抑制乙酰胆碱诱导的NOi产生。我们的结论是，在猫心房肌细胞，乙酰胆碱刺激NOi释放局部肌膜下网站。乙酰胆碱诱导的NOi增加需要毒蕈碱受体介导的G（i）蛋白/磷脂酰肌醇3-激酶/Akt信号和电压激活的Ca 2+内流来刺激钙调蛋白依赖性内皮NO合酶活性。ACh戒断引起的NOi增加是由于ACh抑制后Ca ~（2+）内流恢复所致。乙酰胆碱戒断引起的NO信号刺激胆碱能心房抑制的快速恢复。
Fluorescence microscopy and the NO-sensitive indicator 4,5-diaminofluorescein were used to determine the effects of acetylcholine (ACh) on intracellular NO (NOi) in cat atrial myocytes. Field stimulation ( 1 Hz) of cells or exposure of quiescent cells to ACh (1 to 10 mumol/L) had no effect on NOi. However, in field-stimulated cells, ACh exposure increased NOi, and ACh withdrawal elicited an additional, prominent increase in NOi production. During ACh exposure, addition of 1 mumol/L atropine increased NOi production similar to ACh withdrawal. ACh-induced increases in NOi were reduced by prior exposure to 1 mmol/L extracellular Ca2+ ([Ca2+](o)) and prevented by 0.5 mmol/L [Ca2+](o), 1 mumol/L verapamil, 1 mumol/L atropine, 10 mumol/L L-N-5-(1- iminoethyl) ornithine, 10 mumol/ L W-7, or incubating cells in pertussis toxin or 10 mumol/ L LY294002 (inhibits phosphatidylinositol 3-kinase). Switching to 0.5 mmol/ L [Ca2+](o) during ACh withdrawal prevented the additional increase in NOi. ACh exposure increased phosphorylation (Ser(473)) of protein kinase B (Akt), and this effect was blocked by LY294002 and unaffected in low (0.5 mmol/L) [Ca2+](o). Confocal microscopy revealed that ACh exposure increased NOi at local subsarcolemmal sites, and ACh withdrawal additionally increased NOi by recruiting additional subsarcolemmal release sites. Disruption of caveolae by 2 mmol/L methyl-beta-cyclodextrin abolished ACh-induced NOi production. We conclude that in cat atrial myocytes, ACh stimulates NOi release from local subsarcolemmal sites. ACh-induced increases in NOi requires both muscarinic receptor-mediated G(i) protein/phosphatidylinositol 3-kinase/Akt signaling and voltage-activated Ca2+ influx for stimulation of calmodulin-dependent endothelial NO synthase activity. Increases in NOi elicited by ACh withdrawal result from the recovery of Ca2+ influx after ACh inhibition. NO signaling elicited by ACh withdrawal stimulates rapid recovery from cholinergic atrial inhibition.