N -Monomethyl-£-arginine

A comprehensive, randomized, placebo-controlled trial of infused bolus L-arg and its enantiomer (D-arg) included healthy subjects, non-insulin dependent diabetics, hypertensive subjects, and normotensives with primary hypercholesterolemia [147]. A blood-pressure drop and an acute inhibition of ADP-induced aggregation in platelet-rich plasma were observed in all subjects after L-arg administration (<5 g). Both responses to L-arg infusion closely correlated in magnitude, were weaker in noninsulin dependent diabetics and hypercholesterolemics, and declined with increasing age. Notably, D-arg did not elicit any of the L-arg effects, which were reduced by some 70% when superimposed upon ongoing, nonselective NOS inhibition with infused L-N-monomethyl-arginine (L-NMMA). Since D-arg is not a NOS substrate, and L-NMMA is a substrate-competitive NOS inhibitor, the L-arg effects observed in this study were theorized to reflect a rise in vascular NO production by eNOS. In contrast, the inhibition of platelet aggregation observed in vitro after a 5 min L-arg infusion (160 mg total dose) into healthy subjects and patients with angiographic... 

Toda, N., and Okamura, T. (1990a). Modification of L-N -monomethyl arginine (L-NMMA) of the response to nerve stimulation in isolated dog mesenteric and cerebral arteries. Jpn. J. Pharmacol. 52, 170-173. 

Stefrmovic-Racic M, Meyers K, Meschter C et al. N-monomethyl arginine, an inhibitor of nitric oxide synthase, suppresses the development of adjuvant arthritis in rats. Arthritis Rheum 1994 37 1062-9. 

L-NMMA=N -monomethyl-L-arginine L-NAME=N -monomethyl-L-arginine LTP=long-term potentia-... 

Ramagopal, M. W., and Leighton, H. J. (1989). Effects of N -monomethyl-L-arginine on field stimulation-induced decreases in cytosolic Ca levels and relaxation in the rat anococcygeus muscle. Eur. J. Pharmacol. 174, 297-299. 

Bergmann, L., Kroncke, K.-D., Suschek, C., Kolb, H., and Kolb-Bachofern, V. (1992). Cytotoxic action of lL-1/3 against pancreatic islets is mediated via nitric oxide formation and is inhibited by N -monomethyl-L-arginine. FEES Lett. 299, 103-106. 

L-arginine analogues, such as N -monomethyl-L-arginine (Hibbs et al., 1987b), which acts as an extremely effective and competitive inhibitor of -NO formation. 

C. T Cell Cytokine Production in Presence and Absence of N°-Monomethyl-L-Arginine... 

Granger, D. L., Hibbs, J. B., Jr., and Broadnax, L. M. (1991). Urinary nitrate excretion in relation to murine macrophage activation. Influence of dietary L-arginine and oral N - -monomethyl-L-arginine.], Immunol. 146, 1294-1302. 

Figure 2 Stoichiometry of the enzymatic mechanism of formation of NO, and the structure of a competitive inhibitor, N -monomethyl-L-arginine (NMMA). NO is synthesized by all NOS s by a similar mechanism, involving the NADPH-dependent mixed-function oxidation of a guanidino nitrogen of the amino acid L-arginine (L-arg) to produce L-citrulline (L-cit) and -NO. The nonintegral stoichiometries are explained in the text. NMMA inhibits NOS as a competitive inhibitor...

Calver A, Collier J, Moncada S, Vallance P (1992) Effect of local infusion of N -monomethyl-L-arginine in patients with hypertension. The nitric oxide dilator mechanism appears abnormal J Hypertens 10 1025-1031. 

Heritac ndcaOedro K, LembowiczK, Pytel B (1991) N -monomethyl-L-arginine increases platdet deposition on danuiged endothdium in vivo. A scanning electron microscopy study Thromb Res 64 1-9. 

Billiar, T. R., Curran, R. D., Harbrecht, B. G., Stuehr, D. J., Demetris, A. J., and Simmons, R. L. (1990). Modulation of nitric oxide synthesis in vivo N -Monomethyl-L-arginine inhibits endotoxin-induced nitrite/nitrate biosynthesis while promoting hepatic damage. J. Leukocyte Biol. 48, 568-569. 

Weinberg, J. B., Granger, D. L., Pisetsky, D. S., Seldin, M. F., Misukonis, M. A., Mason, S. N., Pippin, A. M., Ruiz, P., Wood, E. R., and Gilkeson, G. S. (1994). The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease Increased nitric oxide production and nitric oxide synthase expression in MRL-/pr//pr mice, and reduction of spontaneous nephritis and arthritis by orally administered N -monomethyl-L-arginine. J. Exp. Med. 179, 651-660. 

The addition of NBT, which is known to interact with the superoxide anion and is reduced to formazan, led to the concentration-dependent inhibition of NOS activity, with an apparent K of 3-4 p.M (Mittal et al., 1993). The inhibitory effect of NBT was compared with N -monomethyl-L-arginine (NMA), a structural analog of L-arginine. Inclusion of NMA or NBT at 100 fjiM led to the complete inhibition of L-arginine-dependent stimulation of guanylate cyclase activity without any effect on the basal cGMP produc-... 

FIGURE I Effects of superoxide dismutase (SOD), catalase (CAT), heated catalase (CAT-H), nitroblue tetrazolium (NBT), and N"-monomethyl-L-arginine (NMA). Rat brain cytosol was incubated with 100 juM L-arginine and 100 /zM NADPH for 10 min at 37°C. Nitric oxide (NO) synthase activity was determined as an increase in cGMP formation. (Adapted from Mittal, 1993.)... 

Table I presents the results of a study of the effects of endogenous -NO synthesis on catalase levels. Twelve hours after the addition of CME, there is a dramatic (74%) loss in catalase activity, coincident with appreciable NO synthesis. Addition of the NOS inhibitor N -monomethyl-L-arginine (NMMA) prevents -NO formation as well as the loss in catalase activity. These results indicate that increased -NO production may predispose cells and tissues to increased oxidative injury by compromised defense against H2O2.

Hepatocytes were incubated in vitro without (control) or with (CME) a mixture of cytokines (tumor necrosis a, interleukin-16, and interferon-y) plus endotoxin for 12 hr, after which time catalase activity was measured. NMMA, Same experiment as CME except 0.5 mM N°-monomethyl-L-arginine (NMMA) was added at the same time as CME. Detailed methods were as described previously (Kim etal., 1995). NO , Serum nitrite plus nitrite. 

FIGURE I In vivo effects of endotoxin [lipopolysaccharide (LPS) on hepatic microsomal total cytochrome P-450 (Cyt P450) heme and total extractable heme. Rats were injected with LPS (10 mg/kg intraportally) and 12 hr later serum nitrite plus nitrate and microsomal cytochrome P-450 heme and total extractable heme were determined as described previously (Kim etal., 1995). Where indicated, N -monomethyl-L-arginine (NMMA) or aminoguanidine (AG) were injected intraportally beginning 4 hr after LPS injection and continuing at 3-hr intervals. CTRL, Control NOx, nitric oxide. 

Rats were injected with killed C. parimm (intraportally 9 mg/kg). For the in vivo experiments the serum nitrite plus nitrate (NO level was determined 4.5 days after C. parvutn injection, at which time hepatic microsomes were isolated and cytochrome P-450 heme and total heme content were determined as described previously (Kim et al., 1995). For the after culture experiments hepatocytes were isolated, also 4.5 days after C. parvutn, and cultured for 24 hr, without or with 0.5 mM N -monomethyl-L-arginine (NMMA). Medium NO was determined, and microsomal cytochrome P-450 heme and total heme were measured. 

Conditions were similar to those in Table I, except that microsomal total protein, cytochrome P-450 heme, and total heme were measured as described previously (Kim et al., 1995). For the S-nitrosoacetylpenicillamine (SNAP) experiments hepatocytes were incubated with 1 mM SNAP for 9 hr. NMMA, N -monomethyl-L-arginine. 

Hepatocytes were treated as described in Table III. The microsomal fraction was then dialyzed for 6 hr versus buffer without ( - Heme ) or with ( + Heme ) 60 p,M heme and cytochrome P-450 heme was determined (Kim et al., 1995). NMMA, N -monomethyl-L-arginine SNAP, S-nitrosoacetylpenicillamine. 

Bastian, N. R., Xu, S., Shao, X. L., Shelby, J., Granger, D. L., and Hibbs, J. B., Jr. (1994a). N"-monomethyl-L-arginine inhibits nitric oxide production in murine cardiac allografts but does not affect graft rejection. Biochim. Biophys. Acta 1226, 225-231. 

However, NO may also have a role in inhibiting infectious processes. Neutralization of TNF and down-regulation of iNOS promote induction of acute cerebral toxoplasmosis and enhanced pathology in mice chronically infected with Toxoplasma gondii (Gazzinelli et al., 1993). However, N-monomethyl-L-arginine (NMMA) does not affect the antitoxoplasma activity of TNF and IL-6 produced by human microglia in vitro (Chao et al.,... 

Many studies have explored the effects of inhaled NO in humans. A fundamental basis for these studies is the demonstration of an active L-arginine-NO pathway in humans. Kobzik et al. (1993) demonstrated that a variety of cells in the human respiratory system, including endothelial cells, contain NO synthase. Blockade of this pathway by an arginine analog (N°-monomethyl-L-arginine) causes an increased PVR, as demonstrated in healthy adults by Stamler et al. (1994) and in children studied during heart catheterization by Celermajer et al. (1994). Human HPV can be reversed by the addition of NO to inspired gas (Frostell et al 1993). 

BH4 = Tetrahydrobiopterin CAM = Cytotoxic activated macrophage cNOS = Constitutive nitric oxide synthase CPR = Cytochrome P450 reductase EDRF = Endothelial-derived relaxation factor EPR = Electron paramagnetic resonance spectroscopy IL-1 = Interleukin-1 iNOS = Inducible nitric oxide synthase LPS = Lipopo-lysaccharide, or endotoxin NMMA = N -monomethyl-L-arginine NOS = Nitric oxide synthase ROS = Reactive oxygen species SOD = Superoxide dismutase TNF = Tumor necrosis factor. 

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