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Nitric oxide (NO), a physiologically important activator of soluble guanylyl cyclase (sGC), is synthesized from Larginine and O 2 in a reaction catalyzed by NO synthases (NOS). Previous studies with purified NOS failed to detect formation of free NO, presumably due to a fast inactivation of NO by simultaneously produced superoxide (O 2 . ). To characterize the products involved in NOS-induced sGC activation, we measured the formation of cyclic 3,5-guanosine monophosphate (cGMP) by purified sGC incubated in the absence and presence of GSH (1 mM) with drugs releasing different NO-related species or with purified neuronal NOS. Basal sGC activity was 0.04 ؎ 0.01 and 0.19 ؎ 0.06 mol of cGMP ؋ mg ؊1 ؋ min ؊1 without and with 1 mM GSH, respectively. The NO donor DEA/NO activated sGC in a GSH-independent manner. Peroxynitrite had no effect in the absence of GSH but significantly stimulated the enzyme in the presence of the thiol (3.45 ؎ 0.60 mol of cGMP ؋ mg ؊1 ؋ min The NO/cGMP pathway involving NO-mediated activation of soluble guanylyl cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.; sGC 1 ) is essential to signal transduction in several biological systems (1). In the vasculature, NO/cGMP signaling is important for the regulation of blood pressure and platelet function (2); in the brain, this pathway controls the release of neurotransmitters such as glutamate and acetylcholine (3). Biosynthesis of NO is triggered by autacoids increasing the intracellular concentration of free Ca 2ϩ , resulting in activation of Ca 2ϩ /calmodulin-dependent NOS (EC 1.14.13.39), complex homodimeric enzymes that catalyze the synthesis of NO from the guanidino moiety of the amino acid L-arginine (4 -7). The oxidation of L-arginine is catalyzed by a cytochrome P 450 -type heme iron in the oxygenase domain of NOS with O 2 serving as a cosubstrate. The electrons required for reduction of O 2 are shuttled from the cofactor NADPH to the heme via a flavincontaining cytochrome P 450 reductase that forms the C-terminal half of the NOS protein. This electron transport chain only operates when Ca 2ϩ /calmodulin is bound to the enzyme, which then effects the Ca 2ϩ regulation of endothelial and neuronal NO synthesis.At low concentrations of L-arginine or in its absence, the enzymatic reduction of O 2 uncouples from substrate oxidation and results in the generation of superoxide anions and H 2 O 2 (8 -12). The effective coupling of the reaction requires not only saturation with L-arginine but also the pteridine cofactor H 4 biopterin (13). Since the two subunits of neuronal NOS bind H 4 biopterin in a highly anticooperative manner, the purified enzyme always contains Յ1 molecule of H 4 biopterin/dimer, i.e. it consists of a H 4 biopterin-containing and a H 4 biopterin-free subunit (14). In this state, the enzyme can form L-citrulline and is stimulated about 2-fold upon binding of H 4 biopterin to the low affinity site of the pteridine-free subunit. Together with our recent findings that the two NOS subunits function independently (15), this...

1 The L-arginine derivatives N0-nitro-L-arginine (L-NOARG) and NG-nitro-L-arginine methyl ester (L-NAME) have been widely used to inhibit constitutive NO synthase (NOS) in different biological systems. This work was carried out to investigate whether L-NAME is a direct inhibitor of NOS or requires preceding hydrolytic bioactivation to L-NOARG for inhibition of the enzyme. 2 A bolus of L-NAME and L-NOARG (0.25 Mmol) increased coronary perfusion pressure of rat isolated hearts to the same extent (21 +0.8 mmHg; n = 5), but the effect developed more rapidly following addition of L-NOARG than L-NAME (mean half-time: 0.7 vs. 4.2 min). The time-dependent onset of the inhibitory effect of L-NAME was paralleled by the appearance of L-NOARG in the coronary effluent. 3 Freshly dissolved L-NAME was a 50 fold less potent inhibitor of purified brain NOS (mean IC50 = 70 gM) than L-NOARG (IC50= 1.4 giM), but the apparent inhibitory potency of L-NAME approached that of L-NOARG upon prolonged incubation at neutral or alkaline pH. H.p.l.c. analyses revealed that NOS inhibition by L-NAME closely correlated with hydrolysis of the drug to L-NOARG. 4 Freshly dissolved L-NAME contained 2% of L-NOARG and was hydrolyzed with a half-life of 365 + 11.2 min in buffer (pH 7.4), 207 ± 1.7 min in human plasma, and 29 + 2.2 min in whole blood (n = 3 in each case). When L-NAME was preincubated in plasma or buffer, inhibition of NOS was proportional to formation of L-NOARG, but in blood the inhibition was much less than expected from the rates of L-NAME hydrolysis. This was explained by accumulation of L-NOARG in blood cells. 5 These results suggest that L-NAME represents a prodrug lacking NOS inhibitory activity unless it is hydrolyzed to L-NOARG. Bioactivation of L-NAME proceeds at moderate rates in physiological buffers, but is markedly accelerated in tissues such as blood or vascular endothelium.

Vascular relaxation to GTN (nitroglycerin) and other antianginal nitrovasodilators requires bioactivation of the drugs to NO or a related activator of sGC (soluble guanylate cyclase). Conversion of GTN into 1,2-GDN (1,2-glycerol dinitrate) and nitrite by mitochondrial ALDH2 (aldehyde dehydrogenase 2) may be an essential pathway of GTN bioactivation in blood vessels. In the present study, we characterized the profile of GTN biotransformation by purified human liver ALDH2 and rat liver mitochondria, and we used purified sGC as a sensitive detector of GTN bioactivity to examine whether ALDH2-catalysed nitrite formation is linked to sGC activation. In the presence of mitochondria, GTN activated sGC with an EC50 (half-maximally effective concentration) of 3.77+/-0.83 microM. The selective ALDH2 inhibitor, daidzin (0.1 mM), increased the EC50 of GTN to 7.47+/-0.93 microM. Lack of effect of the mitochondrial poisons, rotenone and myxothiazol, suggested that nitrite reduction by components of the respiratory chain is not essential to sGC activation. However, since co-incubation of sGC with purified ALDH2 led to significant stimulation of cGMP formation by GTN that was completely inhibited by 0.1 mM daidzin and NO scavengers, ALDH2 may convert GTN directly into NO or a related species. Studies with rat aortic rings suggested that ALDH2 contributes to GTN bioactivation and showed that maximal relaxation to GTN occurred at cGMP levels that were only 3.4% of the maximal levels obtained with NO. Comparison of sGC activation in the presence of mitochondria with cGMP accumulation in rat aorta revealed a slightly higher potency of GTN to activate sGC in vitro compared with blood vessels. Our results suggest that ALDH2 catalyses the mitochondrial bioactivation of GTN by the formation of a reactive NO-related intermediate that activates sGC. In addition, the previous conflicting notion of the existence of a high-affinity GTN-metabolizing pathway operating in intact blood vessels but not in tissue homogenates is explained.