NO⋅ is a free radical that modulates heart function and metabolism. We report that a neuronal-type NO synthase (NOS) is located on cardiac sarcoplasmic reticulum (SR) membrane vesicles and that endogenous NO⋅ produced by SR-associated NOS inhibits SR Ca2+ uptake. Ca2+-dependent biochemical conversion of l-arginine to l-citrulline was observed from isolated rabbit cardiac SR vesicles in the presence of NOS substrates and cofactors. Endogenous NO⋅ was generated from the vesicles and detected by electron paramagnetic resonance spin-trapping measurements. Immunoelectron microscopy demonstrated labeling of cardiac SR vesicles by using anti-neuronal NOS (nNOS), but not anti-endothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies, whereas skeletal muscle SR vesicles had no nNOS immunoreactivity. The nNOS immunoreactivity also displayed a pattern consistent with SR localization in confocal micrographs of sections of human myocardium. Western blotting demonstrated that cardiac SR NOS is larger than brain NOS (160 vs. 155 kDa). No immunodetection was observed in cardiac SR vesicles from nNOS knockout mice or with an anti-nNOSμ antibody, suggesting the possibility of a new nNOS-type isoform. 45Ca uptake by cardiac SR vesicles, catalyzed by Ca2+-ATPase, was inhibited by NO⋅ produced endogenously from cardiac SR NOS, and 7-nitroindazole, a selective nNOS inhibitor, completely prevented this inhibition. These results suggest that a cardiac muscle nNOS isoform is located on SR of cardiac myocytes, where it may respond to intracellular Ca2+ concentration and modulate SR Ca2+ ion active transport in the heart.
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机译:NO ⋅ sup>是调节心脏功能和新陈代谢的自由基。我们报告说,神经元型NO合酶(NOS)位于心脏肌质网(SR)膜囊泡上,并且由SR相关NOS产生的内源性NO ⋅ sup>抑制SR Ca 2+ < / sup>吸收。在存在NOS底物和辅因子的条件下,从离体兔心脏SR囊泡中观察到了Ca 2+依赖性的L-精氨酸向L-瓜氨酸的生化转化。从囊泡中产生内源性NO ⋅ sup>,并通过电子顺磁共振自旋俘获测量进行检测。免疫电子显微镜显示使用抗神经元NOS(nNOS)而非抗内皮NOS(eNOS)或抗诱导性NOS(iNOS)抗体标记心脏SR囊泡,而骨骼肌SR囊泡则没有nNOS免疫反应性。在人心肌切片的共聚焦显微照片中,nNOS免疫反应性也显示出与SR定位一致的模式。 Western印迹表明,心脏SR NOS大于大脑NOS(160 vs. 155 kDa)。在来自nNOS基因敲除小鼠或抗nNOSμ抗体的心脏SR囊泡中未观察到免疫检测,提示可能存在新的nNOS型同种型。 Ca 2 + sup> -ATPase催化心脏SR囊泡摄取 45 sup> Ca受到心脏SR NOS内源性产生的NO ⋅ sup>的抑制,并且选择性nNOS抑制剂7-硝基吲唑完全阻止了这种抑制作用。这些结果表明,心肌nNOS同工型位于心肌细胞的SR上,可能对细胞内Ca 2 + sup>的浓度作出反应并调节SR Ca 2 + sup>的离子活性转运。在心里。
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