Nitric oxide synthase in cardiac sarcoplasmic reticulum

NO. is a free radical that modulates heart function and metabolism. We repo rt that a neuronal-type NO synthase (NOS) is located on cardiac sarcoplasmi c reticulum (SR) membrane vesicles and that endogenous NO. produced by SR-a ssociated NOS inhibits SR Ca2+ uptake. Ca2+- dependent biochemical conversi on 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 reso nance spin-trapping measurements. Immunoelectron microscopy demonstrated la beling of cardiac SR vesicles by using anti-neuronal NOS (nNOS), but not an tiendothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies, whereas s keletal muscle SR vesicles had no nNOS immunoreactivity. The nNOS immunorea ctivity also displayed a pattern consistent with SR localization in confoca l micrographs of sections of human myocardium. Western blotting demonstrate d that cardiac SR NOS is larger than brain NOS (160 vs. 155 kDa). No immuno detection was observed in cardiac SR vesicles from nNOS knockout mice or wi th an anti-nNOS mu antibody, suggesting the possibility of a new nNOS-type isoform, Ca-45 uptake by cardiac SR vesicles, catalyzed by Ca2+-ATPase, was inhibited by NO. produced endogenously from cardiac SR NOS, and 7-nitroind azole, a selective nNOS inhibitor, completely prevented this inhibition. Th ese 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.