Ziolo M T, Katoh H, Bers D M
Department of Physiology, Loyola University Medical Center, Maywood, IL 60153, USA.
Circulation. 2001 Dec 11;104(24):2961-6. doi: 10.1161/hc4901.100379.
beta-adrenergic hyporesponsiveness in many cardiomyopathies is linked to expression of inducible nitric oxide synthase (iNOS) and increased production of NO. The purpose of this study was to examine whether iNOS expression alters the function of the sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR) during beta-adrenergic stimulation.
Expression of iNOS was induced by lipopolysaccharide (LPS) injection (10 mg/kg) 6 hours before rat myocyte isolation. Confocal microscopy (fluo-3) was used to measure Ca(2+) spark frequency (CaSpF, reflecting resting RyR openings) and Ca(2+) transients. CaSpF was greatly increased by the adenylate cyclase activator forskolin (100 nmol/L) in normal myocytes (iNOS not expressed), but this effect was suppressed (by 77%) in LPS myocytes (iNOS expressed). When NO production by iNOS was inhibited by aminoguanidine (1 mmol/L), there was a further increase in the forskolin-induced CaSpF in LPS myocytes (to levels similar to the forskolin-stimulated CaSpF in normal myocytes). This effect was also seen in myocytes isolated from a failing human heart. There was no effect of aminoguanidine on forskolin-stimulated CaSpF in normal myocytes. ODQ (10 micromol/L), an inhibitor of NO stimulation of guanylate cyclase, did not restore the forskolin-induced rise in CaSpF in LPS myocytes. Aminoguanidine also increased twitch Ca(2+) transient amplitude in LPS myocytes after forskolin application (independent of changes in SR Ca(2+) load).
iNOS/NO depresses beta-adrenergic-stimulated RyR function through a cGMP-independent pathway (eg, NO- and/or peroxynitrite-dependent redox modification). This mechanism limits beta-adrenergic responsiveness and may be an important signaling pathway in cardiomyopathies, including human heart failure.
许多心肌病中的β-肾上腺素能反应低下与诱导型一氧化氮合酶（iNOS）的表达及一氧化氮（NO）生成增加有关。本研究的目的是检测在β-肾上腺素能刺激过程中，iNOS表达是否会改变肌浆网（SR）钙释放通道（兰尼碱受体，RyR）的功能。
在大鼠心肌细胞分离前6小时，通过注射脂多糖（LPS，10mg/kg）诱导iNOS表达。利用共聚焦显微镜（fluo-3）测量钙火花频率（CaSpF，反映静息状态下RyR开放情况）和钙瞬变。在正常心肌细胞（未表达iNOS）中，腺苷酸环化酶激活剂福斯高林（100nmol/L）可使CaSpF显著增加，但在LPS处理的心肌细胞（表达iNOS）中，这种效应受到抑制（降低77%）。当用氨基胍（1mmol/L）抑制iNOS产生NO时，LPS处理的心肌细胞中福斯高林诱导的CaSpF进一步增加（达到与正常心肌细胞中福斯高林刺激的CaSpF相似的水平）。从衰竭的人类心脏分离的心肌细胞中也观察到了这种效应。氨基胍对正常心肌细胞中福斯高林刺激的CaSpF没有影响。ODQ（10μmol/L），一种NO刺激鸟苷酸环化酶的抑制剂，不能恢复LPS处理的心肌细胞中福斯高林诱导的CaSpF升高。在应用福斯高林后，氨基胍还增加了LPS处理的心肌细胞中的收缩期钙瞬变幅度（与SR钙负荷变化无关）。
iNOS/NO通过一条不依赖环鸟苷酸的途径（如依赖NO和/或过氧亚硝酸盐的氧化还原修饰）抑制β-肾上腺素能刺激的RyR功能。这一机制限制了β-肾上腺素能反应性，可能是包括人类心力衰竭在内的心肌病中的一条重要信号通路。
