Attenuation of NMDA receptor activity and neurotoxicity by nitroxyl anion, NO-.

Recent evidence indicates that the NO-related species, nitroxyl anion (NO), is produced in physiological systems by several redox metal-containing proteins, including hemoglobin, nitric oxide synthase (NOS), superoxide dismutase, and S-nitrosothiols (SNOs), which have recently been identified in brain. However, the chemical biology of NO- remains largely unknown. Here, we show that NO- -unlike NO*, but reminiscent of NO+ transfer (or S-nitrosylation)- -reacts mainly with Cys-399 in the NR2A subunit of the N-methyl-D-aspartate (NMDA) receptor to curtail excessive Ca2+ influx and thus provide neuroprotection from excitotoxic insults. This effect of NO- closely resembles that of NOS, which also downregulates NMDA receptor activity under similar conditions in culture.

Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor.

Severely elevated levels of total homocysteine (approximately millimolar) in the blood typify the childhood disease homocystinuria, whereas modest levels (tens of micromolar) are commonly found in adults who are at increased risk for vascular disease and stroke. Activation of the coagulation system and adverse effects of homocysteine on the endothelium and vessel wall are believed to underlie disease pathogenesis. Here we show that homocysteine acts as an agonist at the glutamate binding site of the N-methyl-D-aspartate receptor, but also as a partial antagonist of the glycine coagonist site. With physiological levels of glycine, neurotoxic concentrations of homocysteine are on the order of millimolar. However, under pathological conditions in which glycine levels in the nervous system are elevated, such as stroke and head trauma, homocysteine's neurotoxic (agonist) attributes at 10-100 microM levels outweigh its neuroprotective (antagonist) activity. Under these conditions neuronal damage derives from excessive Ca2+ influx and reactive oxygen generation. Accordingly, homocysteine neurotoxicity through overstimulation of N-methyl-D-aspartate receptors may contribute to the pathogenesis of both homocystinuria and modest hyperhomocysteinemia.

Diethyl dithiocarbamate-induced decomposition of S-nitrosothiols.

Diethyl dithiocarbamate (DDC) has been used extensively as an inhibitor of CuZn superoxide dismutase (SOD) in the study of superoxide and nitric oxide. Addition of DDC to solutions of the endogenous NO adduct S-nitrosoglutathione (GSNO) causes a rapid decrease in GSNO with concomitant formation of nitrite, nitrate, disulfuram, oxidized glutathione, and mixed disulfide. Nitric oxide and superoxide appear to be produced in the process. Product formation is best explained by a radical mechanism in which S-nitrosation of DDC facilitates disulfide formation following homolytic cleavage. S-Nitrosocysteine and S-nitroso-N-acetylpenicillamine are likewise unstable in the presence of DDC. These findings may complicate interpretation of experiments in which DDC is used to alter NO-mediated responses. Some biological actions of DDC may result from SNO elimination rather than SOD inactivation. Moreover, apparent DDC-induced potentiation of superoxide effects may derive from O2- produced during the conversion of SNO to NO.

Nitric oxide in Tanzanian children with malaria: inverse relationship between malaria severity and nitric oxide production/nitric oxide synthase type 2 expression.

Nitric oxide (NO)-related activity has been shown to be protective against Plasmodium falciparum in vitro. It has been hypothesized, however, that excess NO production contributes to the pathogenesis of cerebral malaria. The purpose of this study was to compare markers of NO production [urinary and plasma nitrate + nitrite (NOx)], leukocyte-inducible nitric oxide synthase type 2 (NOS2), and plasma TNF-alpha and IL-10 levels with disease severity in 191 Tanzanian children with and without malaria. Urine NOx excretion and plasma NOx levels (corrected for renal impairment) were inversely related to disease severity, with levels highest in subclinical infection and lowest in fatal cerebral malaria. Results could not be explained by differences in dietary nitrate ingestion among the groups. Plasma levels of IL-10, a cytokine known to suppress NO synthesis, increased with disease severity. Leukocyte NOS2 antigen was detectable in all control children tested and in all those with subclinical infection, but was undetectable in all but one subject with cerebral malaria. This suppression of NO synthesis in cerebral malaria may contribute to pathogenesis. In contrast, high fasting NOx levels and leukocyte NOS2 in healthy controls and asymptomatic infection suggest that increased NO synthesis might protect against clinical disease. NO appears to have a protective rather than pathological role in African children with malaria.

NO+, NO, and NO- donation by S-nitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation.

The biological effects of S-nitrosothiols have been attributed to homolytic cleavage of the S-N bond with release of nitric oxide (NO.). Rates of NO. release from several S-nitrosothiols were determined by monitoring the oxidation of oxymyoglobin to metmyoglobin at pH 7.4; half-lives for oxymyoglobin oxidation ranged from seconds to hours. Transnitrosation reactions between S-nitrosothiols and thiol-containing amino acids, peptides, and proteins, which indicate the ability of nitrosothiols to act as nitrosyl (NO+) donors, occurred more rapidly than spontaneous NO. release. Decomposition of S-nitrosodithiols were examined as models for the reaction of nitrogen oxides with vicinal thiols on proteins. Rapid disulfide formation was accompanied by formation of hydroxylamine and nitrous oxide, indicative of nitroxyl (NO-) release. Taken together, these model studies demonstrate the ability of S-nitrosothiols to act as NO+, NO., and NO- donors under physiological conditions. Transnitrosation and acceleration of disulfide formation suggest mechanisms of regulation of protein function through the intermediacy of nitrosothiols, and support the notion that biological activities of S-nitrosothiols may be associated with heterolytic as well as homolytic mechanisms of decomposition.

Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways.

Recent discoveries suggesting essential bioactivities of nitric oxide (NO.) in the lung are difficult to reconcile with the established pulmonary cytotoxicity of this common air pollutant. These conflicting observations suggest that metabolic intermediaries may exist in the lung to modulate the bioactivity and toxicity of NO.. We report that S-nitrosothiols (RS-NO), predominantly the adduct with glutathione, are present at nano- to micromolar concentrations in the airways of normal subjects and that their levels vary in different human pathophysiologic states. These endogenous RS-NO are long-lived, potent relaxants of human airways under physiological O2 concentrations. Moreover, RS-NO form in high concentrations upon administration of NO. gas. Nitrite (10-20 microM) is found in airway lining fluid in concentrations linearly proportional to leukocyte counts, suggestive of local NO. metabolism. NO. itself was not detected either free in solution or in complexes with transition metals. These observations may provide insight into the means by which NO. is packaged in biological systems to preserve its bioactivity and limit its potential O2-dependent toxicity and suggest an important role for NO. in regulation of airway luminal homeostasis.

Binding of carbon dioxide to phosphoenolpyruvate carboxykinase deduced from carbon kinetic isotope effects.

Phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] from Chloris gayana Kunth has been purified by a combination of ammonium sulfate fractionation, ion exchange, gel filtration, and affinity chromatography on agarose-hexane-ATP. In the direction of OAA formation, the specific activity of the enzyme was 33 mumol/(min.mg of protein). The carbon isotope effect on carboxylation was measured by successive analysis of remaining CO2 over the course of the reaction. At 22 mM PEP and 1.3 mM MgADP, pH 7.5, the isotope effect is 1.024 +/- 0.001. When the concentration of PEP was reduced to 1 mM, the isotope effect rose to 1.034 +/- 0.004; when the concentration of MgADP was reduced to 60 microM, the value rose to 1.040 +/- 0.006. The variation of the carbon isotope effect on carboxylation with both substrate concentrations indicates that the enzyme operates by a random kinetic mechanism. This in turn requires that the enzyme have a binding site for substrate CO2; this is one of the first enzymes for which such a site has been demonstrated.

Pyridoxal 5'-phosphate levels in children with sickle cell disease.

Pyridoxal 5'-phosphate (PLP), the major coenzyme form of vitamin B6, is known to have antisickling properties in vitro. Recently, low plasma PLP levels were reported in a group of adults with sickle cell anemia. We measured the plasma PLP levels in a group of 55 asymptomatic nontransfused children with sickle cell diseases (SCD) to determine the prevalence of low plasma PLP levels in this population. Comparative studies were made with the measurement of PLP in three other groups serving as controls: Group A (black children, n = 36); Group B (white children, n = 37); and Group C (black adults, n = 13). PLP was measured directly in plasma by a radioenzymatic technique. The results of these comparisons showed that there was no statistically significant difference in plasma PLP of black children with SCD (10.7 +/- 10.0 ng/ml) as compared with black control children (group A, 9.0 +/- 12.3 ng/ml). The low plasma levels PLP in these two groups were significantly lower than that of the plasma PLP of white control children (group B, 15.85 +/- 15.92 ng/ml). This data suggest that a high prevalence of low PLP levels exists in black children seen at Grady Memorial Hospital, both with and without SCD.