Dynamic regulation of metabolism and respiration by endogenously produced nitric oxide protects against oxidative stress.

One of the many biological functions of nitric oxide is the ability to protect cells from oxidative stress. To investigate the potential contribution of low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H(2)O(2), spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H(2)O(2) (50-150 microM). Inhibition of nitric oxide synthesis abolished the protective effect against H(2)O(2) exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria as ECV304+eNOS cells with diminished mitochondria respiration (rho(-)) are injured to the same extent as nontransfected ECV304 cells and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H(2)O(2)-induced death. Nitric oxide also found to have a profound effect in cell metabolism, because ECV304+eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H(2)O(2) exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.

Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation.

An elevation in inorganic phosphate (P(i)) concentration activates epiphyseal chondrocyte apoptosis. To determine the mechanism of apoptosis, tibial chondrocytes were treated with P(i), and nitrate/nitrite (NO/NO) levels were determined. P(i) induced a threefold increase in the NO/NO concentration; inhibitors of nitric oxide (NO) synthase activity and P(i) transport significantly reduced NO/NO levels and prevented cell death. Furthermore, a dose-dependent increase in cell death was observed after exposure of chondrocytes to S-nitrosoglutathione. P(i) increased caspase 3 activity 2.7-fold. Both caspase 1 and caspase 3 inhibitors protected chondrocytes from P(i)-induced apoptosis. P(i) caused a significant decrease in the mitochondrial membrane potential, while NO synthase inhibitors maintained mitochondrial function. While P(i) caused thiol depletion, inhibition of P(i) uptake or NO generation served to maintain glutathione levels. The results suggest that NO serves to mediate key metabolic events linked to P(i)-dependent chondrocyte apoptosis.

Plasma proteins modified by tyrosine nitration in acute respiratory distress syndrome.

The present study identifies proteins modified by nitration in the plasma of patients with ongoing acute respiratory distress syndrome (ARDS). The proteins modified by nitration in ARDS were revealed by microsequencing and specific antibody detection to be ceruloplasmin, transferrin, alpha(1)-protease inhibitor, alpha(1)-antichymotrypsin, and beta-chain fibrinogen. Exposure to nitrating agents did not deter the chymotrypsin-inhibiting activity of alpha(1)-antichymotrypsin. However, the ferroxidase activity of ceruloplasmin and the elastase-inhibiting activity of alpha(1)-protease inhibitor were reduced to 50.3 +/- 1.6 and 60.3 +/- 5.3% of control after exposure to the nitrating agent. In contrast, the rate of interaction of fibrinogen with thrombin was increased to 193.4 +/- 8.5% of the control value after exposure of fibrinogen to nitration. Ferroxidase activity of ceruloplasmin and elastase-inhibiting activity of the alpha(1)-protease inhibitor in the ARDS patients were significantly reduced (by 81 and 44%, respectively), whereas alpha(1)-antichymotrypsin activity was not significantly altered. Posttranslational modifications of plasma proteins mediated by nitrating agents may offer a biochemical explanation for the reported diminished ferroxidase activity, elevated levels of elastase, and fibrin deposits detected in patients with ongoing ARDS.

Detection of peroxynitrite in biological fluids.

Peroxynitrite (ONOO(-)) is both an oxidant and a nitrating agent (1-3). However, unlike other strong oxidants, peroxynitrite reacts selectively with biological targets. This selectivity is derived in part from the different second-order rate constants (vary from 10(3)-10(6) M (-1) s(-1)) by which ONOO(-) reacts with biological targets. Competing for peroxynitrite-mediated oxidation of biological targets are two pathways. One is the protonation to form peroxynitrous acid and the second is the reaction with CO(2). Peroxynitrous acid is also an oxidant but it readily isomerizes to nitrate. The reaction with CO(2) results in the formation of the ONO(O)CO(2) (-) adduct that is a more potent nitrating agent but a weaker oxidant than peroxynitrite. Peroxynitrite can diffuse through biological membranes before it encounters and reacts with biological targets. This observation implies that diffusion can effectively compete with the isomerization to nitrate or the reaction with CO(2).

Inhibition of bunyaviruses, phleboviruses, and hantaviruses by human MxA protein.

Viruses of the Bunyaviridae family cause a variety of diseases ranging from uncomplicated fever to potentially lethal encephalitis and hemorrhagic fever. Little is known about the factors determining pathogenicity in the vertebrate host. Interferons have been reported to be inhibitory, but their mode of action against members of the Bunyaviridae has not yet been elucidated. The interferon-induced MxA protein encoded on human chromosome 21 is a large GTPase with antiviral activity against distinct negative-strand RNA viruses, notably influenza viruses. Here we show that MxA inhibits representative members of the Bunyaviridae family by interacting with an early step of virus replication. When constitutively expressed in stably transfected Vero cells, MxA prevented the accumulation of viral transcripts and proteins of Hantaan virus (genus Hantavirus). Other members of the family such as La Crosse virus (genus Bunyavirus) and Rift Valley fever virus and sandfly fever virus (both genus Phlebovirus) were likewise inhibited, and virus titers were reduced up to 10(4)-fold. Our data indicate that humans have evolved a mechanism of controlling these viruses irrespective of differences in viral coding strategies.