Nitric oxide synthase in cardiac sarcoplasmic reticulum

Abstract

NO ⅐ 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 ⅐ produced by SR-associated NOS inhibits SR Ca 2؉ uptake. Ca 2؉ -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 ⅐ 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 orwith an anti-nNOS antibody, suggesting the possibility of a new nNOS-type isoform. 45 Ca uptake by cardiac SR vesicles, catalyzed by Ca 2؉ -ATPase, was inhibited by NO ⅐ 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 Ca 2؉ concentration and modulate SR Ca 2؉ ion active transport in the heart. NO synthase (NOS) isoforms [neuronal NOS (nNOS), inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS )], have been identified in cardiac and skeletal muscle (1-7). NO ⅐ , the product of NOS, has emerged as an important physiological regulator of calcium homeostasis (8) and myocardial contractile function (5) through interactions with both L-type calcium channels in cardiac myocytes (8) and the ryanodine receptor (RyR) (9). The sarcoplasmic reticulum (SR) in cardiomyocytes sequesters and releases graded levels of calcium ions near cytosolic contractile proteins to regulate contraction and relaxation of the heart. Although eNOS has been recently reported to copurify with the cardiac muscle RyR Ca 2ϩ release channel (10), little is known about intracellular NOS sources or NO ⅐ targets in cardiomyocytes and their roles in cardiac function. Here we report that cardiac muscle SR clearly expresses endogenous NOS activity and that endogenous NO ⅐ can modify SR calcium uptake by inhibiting Ca 2ϩ -ATPase. Furthermore, our data suggest that the predominant isoform present on the SR of cardiomyocytes is an nNOS isoform.

Roles of CHOP/GADD153 in endoplasmic reticulum stress

“…NO was reported to activate ryanodine receptors or to inhibit SERCA activity by protein nitration. [82][83][84] Overexpression of calreticulin, which is a major Ca 2 þ -binding protein in the ER, prevents b-cells from NO-mediated apoptosis. 56 It should be noted that the expression of SERCA2b and calreticulin is induced by ER stress, which means that increased ER Ca 2 þ stores are likely to be required to adapt ER stress conditions.…”

A novel method of measuring nitric-oxide-dependent fluorescence using 4,5-diaminofluorescein (DAF-2) in the isolated Langendorff-perfused rabbit heart

“…It is likely that the basal fluorescence signal could originate from a number of sources since NO synthases can be found in endothelial cells (Miethke et al, 2003), neurones and nerve fibres of cardiac ganglia (Sosunov et al, 1996;Sosunov et al, 1995) and subcellularly located within cardiac myocytes e.g. sarcoplasmic reticulum and mitochondria (Xu et , 1999;Kanai et al, 2001). We have performed preliminary experiments applying this novel NO measurement technique to the innervated isolated heart preparation which we developed (Ng et al, 2001).…”

“…Apart from the well-established role of NO in vascular tone (Furchgott & Zawadzki, 1980), NO has been implicated to play a physiological role in heart rate (Herring et al, 2002) and contractile regulation (Cotton et al, 2001) as well having a pathological role in myocardial disease (Massion et al, 2003) and a protective role against ischaemia-reperfusion injury (Jones & Bolli, 2006). NO is synthesised from L-arginine by three distinct isoforms of nitric oxide synthase (NOS): neuronal (nNOS) or NOS 1, inducible (iNOS) or NOS 2 and endothelial (eNOS) or NOS 3, that can be found throughout the heart (Faber-Zuschratter & Wolf, 1994;Hecker et al, 1994;Rothe et al, 1998;Xu et al, 1999). NO may therefore have several distinct roles in cardiac muscle that is highly dependent on cellular localisation.…”

Nitric oxide and hypertension: not just an endothelium derived relaxing factor!

“…This may be due to pre-synaptic inhibition of noradrenaline release from cardiac sympathetic nerves. 71,72 The recent description of nNOS in the cardiac sarcoplasmic reticulum 73 may mean that this isoform also contributes to a post-synaptic inhibition of adrenergic signal transduction possibly by modulating intracellular calcium currents. 74 -76 Not all data on the cardiac effects of NO have been consistent.…”