**2. Canonical function of NO**

NO, initially known as the endothelium-derived relaxing factor (EDRF), is a gas molecule and free radical with an unpaired electron which has been shown to be involved in an ever-growing list of biological processes. NO generated in the tissue binds to major physiological target, haem moiety of GC, activating the cGMP cascade. The GC family is composed of two members including membrane-bound GC and soluble GC (sGC). Membrane-bound GC is a receptor responsive to atrial natriuretic peptide (ANP), and sGC acts as the NO sensor. NO exerts its biological

effects by activating sGC to increase the cGMP level and vascular effects known to be mediated by cGMP such as vasodilation, inhibition of platelet aggregation, and inflammatory reaction. Cyclic GMP modulates a number of signaling processes downstream of NO. The NO-cGMP cascade can be regulated by pharmacological modulation of protein kinases, phosphodiesterases (PDE), and ion channels to alter vascular tones as well as endothelial and vascular smooth muscle cell growth. Pharmacological alteration of the NO level has been a major strategy to develop therapeutic agents for cardiovascular diseases.

Deguchi and his colleagues found that GC activity in the supernatant of neuroblastoma and brain preparations were activated by L-arginine which has been identified as an endogenous activator of sGC [3]. Hibbs et al. noted the latter that the cytotoxic properties of macrophages in co-cultures with tumor cells could be enhanced with L-arginine but suppressed by N-N-methyl-arginine (LNMA), an inhibitor of nitric oxide synthase (NOS) [4]. This cytotoxicity action was accompanied by accumulation of nitrite in the conditioned medium. These important studies provide the insight to identify a pathway of L-arginine metabolism that could produce NO and nitrite.

NOS is a group of isozymes which convert L-arginine to L-hydroxyarginine and subsequently to NO and L-citrulline through cofactors including reduced nicotinamide-adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and tetrahydrobiopterin (BH4) [5]. The first NOS isoform to be identified was the neuronal NOS (nNOS or named as NOS-1) [6]. This was followed shortly thereafter by inducible NOS (iNOS), also known as type II NOS (NOS-2) [7], and then by endothelial NOS (eNOS or named as NOS-3) [8]. NOS can also be inactivated by asymmetric dimethyl arginine (ADMA), an endogenous and competitive inhibitor of NOS [9–11].
