This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

bound pterin to the tetrahydro form (BH4). Cit, citrulline; cyt c, cytochrome c.

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, l-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

A soluble enzyme obtained from rat forebrain catalyzes the NADPH-dependent formation of nitric oxide (NO) and citrulline from L-arginine. The NO formed stimulates the soluble guanylate cyclase and this stimulation is abolished by low concentrations of hemoglobin. The synthesis of NO and citrulline is dependent on the presence of physiological concentrations of free Ca2+ and is inhibited by NG-monomethyl-L-arginine, but not by its enantiomer NGmonomethylDarginin e or by Lcanavanine. L-Homoarginine, L-argyl-L-apartate, or L-arginine methyl ester can replace L-ginine as substrates for the enzyme. These results indicate that NO is formed from Larginine in the brain through an enzymic reaction similar to that in vascular endothelial cells, neutrophils, and macrophages, adding support to our hypothesis that the formation of NO from L-arginine is a widespread transduction mechanism for the stimulation of the soluble guanylate cyclase. Activated macrophages also synthesize NOj and NO3 from the terminal guanido nitrogen atom(s) of L-arginine (5). This reaction, which occurs via the formation of NO (6), is involved in the cytotoxic activities of these cells (7). We have demonstrated recently that rat peritoneal neutrophils form NO from L-arginine (8).The endothelial cell and macrophage enzyme that forms NO from L-arginine is soluble, is NADPH-dependent, forms citrulline as a coproduct, and is inhibited by NI-monomethyl-L-arginine refs. 6 and 9). Furthermore, in both cells the enzyme requires a divalent cation, which in the case of the macrophage has been suggested to be Mg2+ (6).Some years ago, L-arginine was identified as an endogenous activator of the soluble guanylate cyclase in brain tissue (10). Since this activation resembled that of the nitrovasodilators (11) and NO is known to stimulate soluble guanylate cyclase in the brain (12), we have investigated the existence in the central nervous system of an enzymic system capable of converting L-arginine into NO and citrulline.While this work was in progress Garthwaite et al. (13) Preparation of Crude Synaptosomal Cytosol. Male rats (200-300 g, four for each preparation) were killed by cervical dislocation and the forebrains were rapidly removed and cooled in ice-cold washing buffer (0.32 M sucrose/10 mM Hepes/0.1 mM EDTA, pH 7.4); subsequent procedures were carried out at 0-40C. The tissue was placed in fresh washing buffer, finely minced, washed once with 50 ml of washing buffer and twice with homogenization buffer (0.32 M sucrose/10 mM Hepes, 1 mM DL-dithiothreitol, pH 7.4) to remove contaminating erythrocytes, and homogenized with 20 strokes of a Dounce homogenizer. The homogenate was diluted to 50 ml with homogenization buffer and centrifuged (1400 x g, 10 min); the supernatant obtained was centrifuged (18,000 x g, 10 min) to obtain a crude synaptosomal pellet. After aspiration of the supernatant, 8 ml of 1 mM DLdithiothreitol (dissolved in distilled water) was added to the pellet to cause hypotonic swelling of the synaptosomes, which were then lysed by ho...

N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was a slow, tight binding inhibitor of human inducible nitricoxide synthase (iNOS). The slow onset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s ؊1 and a binding constant of 2.0 M. Inhibition was dependent on the cofactor NADPH. LArginine was a competitive inhibitor of 1400W binding with a K s value of 3.0 M. Inhibited enzyme did not recover activity after 2 h. Thus, 1400W was either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS with a K d value 7 nM. In contrast, inhibition of human neuronal NOS and endothelial NOS (eNOS) was relatively weaker, rapidly reversible, and competitive with L-arginine, with K i values of 2 M and 50 M, respectively. Thus, 1400W was at least 5000-fold selective for iNOS versus eNOS. This selectivity was similar to that observed in rat aortic rings, in which 1400W was greater than 1000-fold more potent against rat iNOS than eNOS. Finally, 1400W was greater than 50-fold more potent against iNOS than eNOS in a rat model of endotoxin-induced vascular injury. Thus, the potency and selectivity of 1400W inhibition of iNOS both in vitro and in vivo were far greater than of any previously described iNOS inhibitor.

We have ex ed the effects of pregnancy and sex hormones on calciumdependent and m-independent nitric oxide synthas (NOSs) in the guinea pig. Pregnancy (near term) caused a >4-fold iase in the activity of cacium-dependent NOS in the uterine artery and at least a doubling in the heart, kidney, skeletal muscle, esophagus, and cerebeflum. The increase in NOS activity in the cerebellum during pregnanc was nhibited by the estn-receptor antagonittam len. Treatment with a ol (but not p rone) also i ed calcium-dependent NOS activity in the tissues examined from both females and males. Testosterone a calcium-dependent NOS only in the cerebeflum. No sign nt change in calcium-independent NOS acv was observed either during pregency or after the admiration f any sex hormone. Both pregan and estradol treatment increased the amount of mRNAs for NOS Isogymes eNOS and nNOS in skeletal muscle, 8 that the ireases in NOS activity result from enzyme induction. Thus both eNOS and nNOS are subject to rution by esten, an action that could explain some of the changes that occur during pegn and some gender differences in physiology and pa polg.Nitric oxide (NO) synthases (NOSs) constitute a family of isozymes that catalyze the oxidation of L-arginine to NO and citrulline. First identified in the vascular endothelium (1, 2), NO synthesis has subsequently been shown to play important roles in the regulation of vascular and gastrointestinal tone, in cell-mediated cytotoxicity against bacteria and tumors, and in a variety of central and peripheral nervous system activities (for review, see ref.3). NOSs can be divided into two functional classes based on their sensitivity to calcium (3). The cytokine-or bacterial product-inducible isoenzyme iNOS binds calmodulin tightly at resting intracellular calcium concentrations. The constitutive forms, isozymes eNOS (originally described in endothelial cells) and nNOS (originally described in neuronal tissue), bind calmodulin in a reversible and calcium-dependent fashion. The mechanisms by which their synthesis is controlled are unknown.The cDNA species encoding the rat, mouse, and human nNOS, the human and bovine eNOS, and iNOS from several species and cell types have been cloned and sequenced (4-17). ref. 28). This protection is lost with the onset of menopause or after surgical castration. This observation could be linked to reports that NO might slow the development ofatherosclerosis by inhibiting the proliferation of smooth muscle cells while stimulating endothelial cell proliferation (29,30).We therefore decided to investigate the hypothesis that sex hormones could regulate constitutive NOSs. We tested this in female and male guinea pigs by determining the effect of pregnancy on NOS activity and by measuring both NOS activity and NOS mRNA before and after sex hormone therapy. MATERIALS AND METHODSHartley guinea pigs of similar chronological age were obtained from a commercial breeder (Charles River Breeding Laboratories): males and nonpregnant females (600-700 g) and pregnant females of >5...

The distribution of Ca2'-dependent and &"-independent nitric oxide synthase (NOS) was studied in rabbits and in conteol and endotoxin-treated rats and guinea-pigs. There was a widespread localization of NOS which differed for the two forms of the enzyme and which showed marked differences between species. Endotoxin induced the activity of the Ca2'-independent NOS in many tissues and also inceeased the activity of Ca2'-dependent NOS in the rat ileum and caecum. These results demonstrate the differential distribution of NOSs in control and endotoxin-treated animals and emphasize the widespread biological role of nitric oxide (NO).

Clustering based on clinicophysiologic parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.