Metalloprotein-dependent decomposition of S-nitrosothiols: studies on the stabilization and measurement of S-nitrosothiols in tissues.

The stabilization of S-nitrosothiols is critical for the development of assays to measure their concentration in tissues. Low-molecular-weight S-nitrosothiols are unstable in tissue homogenates, even in the presence of thiol blockers or metal-ion chelators. The aim of this study was to try and stabilize low-molecular-weight S-nitrosothiols in tissue and gain insight into the mechanisms leading to their decomposition. Rat tissues (liver, kidney, heart, and brain) were perfused and homogenized in the presence of a thiol-blocking agent (N-ethylmaleimide) and a metal-ion chelator (DTPA). Incubation of liver homogenate with low-molecular-weight S-nitrosothiols (L-CysNO, D-CysNO, and GSNO) resulted in their rapid decomposition in a temperature-dependent manner as measured by chemiluminescence. The decomposition of L-CysNO requires a cytoplasmic factor, with activity greatest in liver > kidney > heart > brain > plasma, and is inhibitable by enzymatic proteolysis or heating to 80 degrees C, suggesting that a protein catalyzes the decomposition of S-nitrosothiols. The ability of liver homogenate to catalyze the decomposition of L-CysNO is up-regulated during endotoxemia and is dependent on oxygen, with the major product being nitrate. Multiple agents were tested for their ability to block the decomposition of L-CysNO without success, with the exception of potassium ferricyanide, which completely blocked CysNO decomposition in liver homogenates. This suggests that a ferrous protein (or group of ferrous proteins) may be involved. We also show that homogenization of tissues in ferricyanide-containing buffers in the presence of N-ethylmaleimide and DTPA can stabilize both low- and high-molecular-weight S-nitrosothiols in tissues before the measurement of their concentration.

Heart rate dynamics in iNOS knockout mice.

Nitric oxide has both an inhibitory and excitatory role in the regulation of pre-ganglionic sympathetic neurons, involving the iNOS and nNOS systems respectively. The aim of the present study was to examine cardiovascular autonomic activity in iNOS knockout mice using spectral analysis of heart rate variability (HRV), and to determine the role of iNOS in altered HRV in endotoxaemia. Electrocardiograms were recorded in anaesthetised mice, and the R-R intervals digitized for spectral analysis of HRV and cardiac rhythm regularity using sample entropy analysis. The basal heart rate was higher in iNOS knockout mice compared with controls (465+/-8 vs 415+/-13 beat/min P<0.05), with a significant increase in the low frequency power of HRV spectra in iNOS knockout mice compared with controls (49.4+/-4.3 vs 33.8+/-5.6 normalized units, P<0.05), consistent with increased cardiac sympathetic activity. Endotoxaemia is known to decrease HRV, but the role of iNOS is unknown. LPS (20 mg/kg i.p) increased basal heart rate in both wild type and iNOS knockout mice, but caused a depression of HRV and sample entropy in both groups. Studies in isolated beating atria showed that the changes of HRV under basal or post-LPS conditions disappeared in vitro, suggesting that the autonomic system is responsible for altered HRV. We conclude that disruption of iNOS gene leads to an increase in the low frequency power of HRV consistent with increased cardiac sympathetic activity. These data also demonstrate that LPS-induced decrease of HRV is independent of iNOS.

Nitration of endogenous para-hydroxyphenylacetic acid and the metabolism of nitrotyrosine.

Reactive nitrogen species, such as peroxynitrite, can nitrate tyrosine in proteins to form nitrotyrosine. Nitrotyrosine is metabolized to 3-nitro-4-hydroxyphenylacetic acid (NHPA), which is excreted in the urine. This has led to the notion that measurement of urinary NHPA may provide a time-integrated index of nitrotyrosine formation in vivo. However, it is not known whether NHPA is derived exclusively from metabolism of nitrotyrosine, or whether it can be formed by nitration of circulating para -hydroxyphenylacetic acid (PHPA), a metabolite of tyrosine. In the present study, we have developed a gas chromatography MS assay for NHPA and PHPA to determine whether or not NHPA can be formed directly by nitration of PHPA. Following the injection of nitrotyrosine, 0.5+/-0.16% of injected dose was recovered unchanged as nitrotyrosine, and 4.3+/-0.2% as NHPA in the urine. To determine whether or not NHPA could be formed by the nitration of PHPA, deuterium-labelled PHPA ([(2)H(6)]PHPA) was injected, and the formation of deuterated NHPA ([(2)H(5)]NHPA) was measured. Of the infused [(2)H(6)]PHPA, 78+/-2% was recovered in the urine unchanged, and approx. 0.23% was recovered as [(2)H(5)]NHPA. Since the plasma concentration of PHPA is markedly higher than free nitrotyrosine (approx. 400-fold), the nitration of high-circulating endogenous PHPA to form NHPA becomes very significant and accounts for the majority of NHPA excreted in urine. This is the first study to demonstrate that NHPA can be formed by nitration of PHPA in vivo, and that this is the major route for its formation.

Nitration of cardiac proteins is associated with abnormal cardiac chronotropic responses in rats with biliary cirrhosis.

Acceleration of the heart rate in response to catecholamines is impaired in cirrhosis. In this study, we tested the hypothesis that increased formation of reactive nitrogen species in biliary cirrhosis causes nitration of cardiac proteins and leads to impaired chronotropic function. Bile duct-ligated (rats with cirrhosis) or sham-operated rats were injected daily with either saline, N(G)-L-nitro-arginine methyl ester (L-NAME), or N-acetylcysteine for 7 days from week 3 to week 4 after surgery. Cardiac chronotropic responsiveness to beta-adrenergic stimulation was assessed in vitro using spontaneous beating isolated atria. Nitration of cardiac proteins was measured by mass spectrometry and located by immunogold electron microscopy. Marked impairment of chronotropic responses of isolated atria to isoproterenol was seen in rats with cirrhosis, which normalized after the administration of N-acetylcysteine or L-NAME. The levels of protein-bound nitrotyrosine in atrial tissue increased from 16 +/- 1 to 23 +/- 3 pg/microg tyrosine in rats with cirrhosis, and decreased to 15 +/- 1 and 17 +/- 1 pg/microg after treatment with L-NAME and N-acetylcysteine, respectively (P < .05). Immunogold electron microscopy demonstrated increased nitration of mitochondrial proteins in the atria of rats with cirrhosis. The plasma nitrite/nitrate levels were elevated in rats with biliary cirrhosis, and decreased after administration of L-NAME but were unchanged by N-acetylcysteine. In conclusion, abnormal cardiac chronotropic function in cirrhosis is associated with increased nitration of cardiac proteins. Two independent treatments (N-acetylcysteine and L-NAME) that decrease nitration of cardiac proteins led to normalization of cardiac responses. Nitration of critical proteins in cardiac tissue may lead to abnormal cardiac function.

Oxidation of nitric oxide by oxomanganese-salen complexes: a new mechanism for cellular protection by superoxide dismutase/catalase mimetics.

Manganese-salen complexes (Mn-Salen), including EUK-8 [manganese N,N'-bis(salicylidene)ethylenediamine chloride] and EUK-134 [manganese 3-methoxy N,N'-bis(salicylidene)ethylenediamine chloride], have been reported to possess combined superoxide dismutase (SOD) and catalase mimetic functions. Because of this SOD/catalase mimicry, EUK-8 and EUK-134 have been investigated as possible therapeutic agents in neurological disorders resulting from oxidative stress, including Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis. These actions have been explained by the ability of the Mn-Salen to remove deleterious superoxide (O(2)(-)) and H(2)O(2). However, in addition to oxidative stress, cells in models for neurodegenerative diseases may also be subjected to damage from reactive nitrogen oxides (nitrosative stress), resulting from elevated levels of NO and sister compounds, including peroxynitrite (ONOO(-)). We have been examining the interaction of EUK-8 and EUK-134 with NO and ONOO(-). We find that in the presence of a per-species (H(2)O(2), ONOO(-), peracetate and persulphate), the Mn-Salen complexes are oxidized to the corresponding oxo-species (oxoMn-Salen). OxoMn-Salens are potent oxidants, and we demonstrate that they can rapidly oxidize NO to NO(2) and also oxidize nitrite (NO(2)(-) to nitrate (NO(2)(-)). Thus these Mn-Salens have the potential to ameliorate cellular damage caused by both oxidative and nitrosative stresses, by the catalytic breakdown of O(2)(-), H(2)O(2), ONOO(-) and NO to benign species: O(2), H(2)O, NO(2)(-) and NO(3)(-).

Novel role for low molecular weight plasma thiols in nitric oxide-mediated control of platelet function.

Nitric oxide (NO) is a powerful antiplatelet agent, but its notoriously short biological half-life limits its potential to prevent the activation of circulating platelets. Here we used diethylamine diazeniumdiolate (DEA/NO) as an NO generator to determine whether the antiplatelet effects of NO are prolonged by the formation of a durable, plasma-borne S-nitrosothiol reservoir. Preincubation of both platelet rich plasma (PRP) and washed platelets (WP) with DEA/NO (2 microm) for 1 min inhibited collagen-induced platelet aggregation by 82 +/- 5 and 91 +/- 2%, respectively. After 30 min preincubation with DEA/NO, NO was no longer detectable in either preparation, but aggregation remained markedly inhibited (72 +/- 7%) in PRP. In contrast, the inhibitory effect in WP was almost completely lost at this time (5 +/- 3%) but was partially restored (39 +/- 10%) in WP containing human serum albumin (1%) and fully restored by co-incubation with albumin and the low molecular weight (LMW) thiols, glutathione, (5 microm), cysteinyl-glycine (10 microm), or cysteine (10 microm). This NO-mediated effect was not seen with LMW thiols in the absence of albumin and was associated with S-nitrosothiol formation. Our results demonstrate that LMW thiols play an important role in both the formation and activation of an S-nitrosoalbumin reservoir that significantly prolongs the duration of action of NO.