Xu K Y, Huso D L, Dawson T M, Bredt D S, Becker L C
Department of Medicine, Division of Cardiology, Johns Hopkins Medical Institutions, Baltimore, MD 21224, USA.
Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):657-62. doi: 10.1073/pnas.96.2.657.
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-nNOS mu 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.
一氧化氮（NO）是一种调节心脏功能和代谢的自由基。我们报告，一种神经元型一氧化氮合酶（NOS）位于心肌肌浆网（SR）膜囊泡上，并且由与SR相关的NOS产生的内源性NO抑制SR对Ca2+的摄取。在存在NOS底物和辅因子的情况下，从分离的兔心肌SR囊泡中观察到L-精氨酸向L-瓜氨酸的钙依赖性生化转化。内源性NO从囊泡中产生，并通过电子顺磁共振自旋捕获测量进行检测。免疫电子显微镜显示，使用抗神经元NOS（nNOS）抗体可标记心肌SR囊泡，但抗内皮NOS（eNOS）或抗诱导型NOS（iNOS）抗体则不能，而骨骼肌SR囊泡没有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。蛋白质印迹法表明，心肌SR NOS比脑NOS大（160 kDa对155 kDa）。在nNOS基因敲除小鼠的心肌SR囊泡中或使用抗nNOSμ抗体未观察到免疫检测信号，提示可能存在一种新的nNOS型同工型。由Ca2+-ATP酶催化的心肌SR囊泡对45Ca的摄取受到心肌SR NOS内源性产生的NO的抑制，而选择性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，一种心肌nNOS同工型位于心肌细胞的SR上，在那里它可能对细胞内Ca2+浓度作出反应并调节心脏中SR Ca2+离子的主动转运。
