Supraspinal peroxynitrite modulates pain signaling by suppressing the endogenous opioid pathway.

Peroxynitrite (PN, ONOO(-)) is a potent oxidant and nitrating agent that contributes to pain through peripheral and spinal mechanisms, but its supraspinal role is unknown. We present evidence here that PN in the rostral ventromedial medulla (RVM) is essential for descending nociceptive modulation in rats during inflammatory and neuropathic pain through PN-mediated suppression of opioid signaling. Carrageenan-induced thermal hyperalgesia was associated with increased 3-nitrotyrosine (NT), a PN biomarker, in the RVM. Furthermore, intra-RVM microinjections of the PN decomposition catalyst Fe(III)-5,10,15,20-tetrakis(N-methyl-pyridinium-4-yl)porphyrin (FeTMPyP(5+)) dose-dependently reversed this thermal hyperalgesia. These effects of FeTMPyP(5+) were abrogated by intra-RVM naloxone, implicating potential interplay between PN and opioids. In support, we identified NT colocalization with the endogenous opioid enkephalin (ENK) in the RVM during thermal hyperalgesia, suggesting potential in situ interactions. To address the functional significance of such interactions, we exposed methionine-enkephalin (MENK) to PN and identified the major metabolite, 3-nitrotyrosine-methionine-sulfoxide (NSO)-MENK, using liquid chromatography-mass spectrometry. Next, we isolated, purified, and tested NSO-MENK for opioid receptor binding affinity and analgesic effects. Compared to MENK, this NSO-MENK metabolite lacked appreciable binding affinity for δ, µ, and κ opioid receptors. Intrathecal injection of NSO-MENK in rats did not evoke antinociception, suggesting that PN-mediated chemical modifications of ENK suppress opioid signaling. When extended to chronic pain, intra-RVM FeTMPyP(5+) produced naloxone-sensitive reversal of mechanical allodynia in rats following chronic constriction injury of the sciatic nerve. Collectively, our data reveal the central role of PN in RVM descending facilitation during inflammatory and neuropathic pain potentially through anti-opioid activity.

Inhibitory effects of theophylline on the peroxynitrite-augmented release of matrix metalloproteinases by lung fibroblasts.

The anti-inflammatory effects of theophylline have been reported to include inhibition of the release of proinflammatory mediators from macrophages and neutrophils. Overproduction of reactive nitrogen species (RNS) has been reported in the airways of patients with chronic obstructive pulmonary disease (COPD), and this causes tissue inflammation and injury. We investigated whether peroxynitrite stimulated the release of matrix metalloproteinases 2 and 9 (MMP-2 and -9; gelatinases) from human fetal lung fibroblasts (HFL-1 cell line) and whether theophylline inhibited the peroxynitrite-augmented release of MMPs. HFL-1 cells and primary lung fibroblasts were treated with peroxynitrite (an RNS), and gelatinases levels were evaluated by gelatin zymography. The inhibitory effect of theophylline on the peroxynitrite-augmented release of MMP-2 and MMP-9 was also investigated. To explore the cell signaling pathways involved in the peroxynitrite-induced gelatinases release and the inhibitory effect of theophylline, transforming growth factor-ß(1) (TGF-ß(1)), nuclear factor-κB (NF-κB), and histone deacetylase (HDAC) were measured. Peroxynitrite significantly augmented the release of MMP-2 and MMP-9 by fibroblasts (P < 0.01), as well as TGF-ß(1) release (P < 0.01), NF-κB activation (P < 0.01), and HDAC2 inactivation (P < 0.01). An NF-κB inhibitor diminished the RNS-augmented release of MMPs and TGF-ß(1) (P < 0.01), and a neutralizing TGF-ß antibody also diminished MMP release (P < 0.01). Theophylline significantly inhibited the peroxynitrite-augmented release of MMP-2 and MMP-9 in HFL-1 cells and normal adult lung fibroblasts, and it also inhibited the peroxynitrite-mediated HDAC2 inactivation, NF-κB activation, and TGF-ß(1) release in HFL-1 cells (all P < 0.01). These results suggest that peroxynitrite can influence tissue remodeling by promoting gelatinases release, while theophylline suppresses peroxynitrite-induced tissue remodeling via pathways involving NF-κB/TGF-ß(1) and/or HDAC in the HFL-1 cell line.

Melatonin Attenuates Peroxynitrite-Induced Meiosis Dysfunction in Porcine Oocytes.

A high level of reactive oxygen species (ROS) is widely considered one of the major causes of oocyte quality decline. Peroxynitrite is known as a powerful oxidant, which could induce multiple physical diseases. Recently, emerging pieces of evidences indicate that melatonin effectively promotes the development of oocytes, although the specific work mechanism remains to be further clarified. In this study, it was shown that peroxynitrite increased the level of ROS in porcine oocytes, which induced the apoptosis of oocytes, thereby leading to the obstruction of spindle assembly, depolymerization of actin, and decrease of polar body expulsion. These negative effects contributed to the failure of meiosis and ultimately blocked the maturation of porcine oocytes. As expected, it was found that melatonin effectively removed the accumulated ROS in oocytes, preventing oocytes from peroxynitrite-induced oocyte maturation failure, which might provide a novel approach to improve female livestock reproduction and cure female infertility in clinical practice.

Melatonin reduces hyperalgesia associated with inflammation.

The perception of pain is altered by inflammatory processes. Anti-inflammatory drugs block this by raising the pain threshold and by reducing the inflammatory process. Melatonin is claimed to have anti-inflammatory activity in animal models of acute and chronic inflammation. However, little is known whether melatonin can reverse the hyperalgesia that is secondary to the inflammation. This study assessed the effect of melatonin on in a well-established model of hyperalgesia associated with inflammation in rats. Peroxynitrite, as generated by the interaction between superoxide anion radical exogenously supplied (O(2)(˙-) ) and endogenous nitric oxide (NO), led to the development of hyperalgesia. This subplantar injection of O(2)(˙-) into the right hindpaw evoked potent thermal hyperalgesia measured by changes in withdrawal latency. Melatonin (25-100 mg/kg, given ip 30 min prior to O(2)(˙-) ) dose dependently attenuated the hyperalgesic responses to O(2)(˙-) . Moreover, melatonin (100 mg/kg) significantly improved tissue damage and inflammation, blocked protein nitration affecting cyclooxygenase-2 and inducible nitric oxide synthase expression in paw tissue. To investigate the antinociceptive activity of melatonin and characterize the underlying mechanisms involved in this action, mitogen-activated protein kinase and NF-κB pathways were explored. Moreover, antihyperalgesic effect of melatonin derived partly from the inhibition of superoxide-driven PARP activation. These results suggest that melatonin has ameliorative potential in attenuating the hyperalgesia associated with inflammation.

Peroxynitrite-induced protein nitration is responsible for renal mitochondrial damage in diabetic rat.

Oxidative stress, especially mediated by peroxynitrite (ONOO-), plays a key role in diabetes. Mitochondria, as the generating source of ONOO-, may also be the major damaging target of ONOO-. Whether ONOO--induced protein nitration is responsible for renal mitochondrial damage in diabetes is not fully known. This study was aimed to clarify the relationship between nitration of entire mitochondrial proteins induced by ONOO- and the renal mitochondrial damage in diabetes. Sprague-Dawley male rats were injected ip with streptozotocin to induce diabetes. After 10 weeks, inducible nitric oxide synthase (iNOS) mRNA expression and protein content in renal cortex were detected. Distribution of nitrotyrosine (NT), a specific marker of ONOO-, in renal cortex and NT content in mitochondrial proteins were detected. The ultrastructure of glomerulus was observed. Aminoguanidine was used as a selective inhibitor of iNOS to reduce the derivation of ONOO-. In diabetic rat, increasing levels of iNOS mRNA and protein content, and NT content were observed, in accord with the pathological alterations of glomerulus. In aminoguanidine group, these alterations were attenuated significantly. In conclusion, ONOO- could induce entire mitochondrial proteins nitration, responsible for the damage of renal mitochondria in diabetes.

The red blood cell as a biosensor for monitoring oxidative imbalance in chronic obstructive pulmonary disease: an ex vivo and in vitro study.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity in Western countries. The increased oxidative stress, caused by the release of reactive oxygen and nitrogen species (ROS/RNS) from inflammatory airways cells, contributes to the pathogenesis of the disease. The aim of the present study was to evaluate (a) whether the oxidative imbalance can lead to specific alterations of red blood cells (RBCs) from stable COPD patients; (b) whether treatment with N-acetyl-cysteine (NAC), in widespread use as mucolytic agent in clinical practice, can counteract these effects; and (c) whether an in vitro model represented by the exposure of RBC to ROS/RNS could mimic the in vivo situation. The results obtained clearly indicated that the RBC integrity and function are similarly altered in COPD patients and in ROS/RNS in vitro-treated samples and that NAC administration was capable of counteracting RBC oxidative modifications both in vivo, as detected by clinical and laboratory evaluations, and in vitro. Altogether these results point to RBC oxidative modifications as valuable bioindicators in the clinical management of COPD and indicate that in vitro RBC exposure to ROS/RNS as a useful tool in experimental studies aimed at the comprehension of the pathogenic mechanisms of the redox-associated diseases.

L-beta,beta-dimethylcysteine attenuates the haemodynamic responses elicited by systemic injections of peroxynitrite in anaesthetized rats.

1 There is direct chemical evidence that L-beta,beta-dimethylcysteine (L-penicillamine (L-PEN)) is a scavenger of peroxynitrite. The aim of this study was to determine whether L-PEN attenuates the haemodynamic responses elicited by peroxynitrite in pentobarbital-anaesthetized rats. 2 Peroxynitrite (1-20 micromol kg(-1), i.v.) elicited dose-dependent reductions in mean arterial blood pressure (MAP) and mesenteric and hindquarter vascular resistances. 3 L-PEN (2 mmol kg(-1), i.v.) elicited relatively minor but significant increases in MAP and vascular resistances. The initial reductions in MAP and vascular resistances elicited by peroxynitrite were not diminished after administration of L-PEN whereas they were much shorter in duration. As such, the total reductions in MAP and vascular resistances were markedly reduced by L-PEN. 4 The finding that L-PEN (2 mmol kg(-1), i.v.) did not affect the hypotensive or vasodilator responses elicited of the ATP-dependent potassium-channel agonist, cromakalim (3-18 microg kg(-1), i.v.), suggests that this dose of L-PEN is not a nonselective inhibitor of vasodilation. 5 These findings suggest that L-PEN may effectively scavenge peroxynitrite in vivo and/or interfere with the mechanisms by which peroxynitrite elicits its vasodilator responses.

Biological time-dependent difference in effect of peroxynitrite demonstrated by the mouse hot plate pain model.

We previously demonstrated the rhythmic pattern of L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) cascade in nociceptive processes. The coupled production of excess NO and superoxide leads to the formation of an unstable intermediate peroxynitrite, which is primarily responsible for NO-mediated toxicity. In the present study, we evaluated the biological time-dependent effects of exogenously administered peroxynitrite on nociceptive processes and peroxynitrite-induced changes in the analgesic effect of morphine using the mouse hot-plate pain model. Experiments were performed at four different times of day (1, 7, 13, and 19 hours after lights on, i.e., HALO) in mice of both sexes synchronized to a 12 h:12 h light-dark cycle. Animals were injected intraperitoneally (i.p.) with saline or 10 mg/kg morphine 30 min before and 0.001 mg/kg peroxynitrite 30 sec before hot-plate testing, respectively. The analgesic effect of morphine exhibited significant biological time-dependent differences in the thermally-induced algesia; whereas, administration of peroxynitrite alone exhibited either significant algesic or analgesic effect, depending on the circadian time of its injection. Concomitant administration of peroxynitrite and morphine reduced morphine-induced analgesia at three of the four different study time points. In conclusion, peroxynitrite displayed nociceptive and antinociceptive when administered alone according to the circadian time of treatment, while it diminished analgesic activity when administered in combination with morphine at certain biological times.

Peroxynitrite induces formation of N( epsilon )-(carboxymethyl) lysine by the cleavage of Amadori product and generation of glucosone and glyoxal from glucose: novel pathways for protein modification by peroxynitrite.

Accumulation of advanced glycation end products (AGEs) on tissue proteins increases with pathogenesis of diabetic complications and atherosclerosis. Here we examined the effect of peroxynitrite (ONOO(-)) on the formation of N( epsilon )-(carboxymethyl)lysine (CML), a major AGE-structure. When glycated human serum albumin (HSA; Amadori-modified protein) was incubated with ONOO(-), CML formation was detected by both enzyme-linked immunosorbent assay and high-performance liquid chromatography (HPLC) and increased with increasing ONOO(-) concentrations. CML was also formed when glucose, preincubated with ONOO(-), was incubated with HSA but was completely inhibited by aminoguanidine, a trapping reagent for alpha-oxoaldehydes. For identifying the aldehydes that contributed to ONOO(-)-induced CML formation, glucose was incubated with ONOO(-) in the presence of 2,3-diaminonaphthalene. This experiment led to identification of glucosone and glyoxal by HPLC. Our results provide the first evidence that ONOO(-) can induce protein modification by oxidative cleavage of the Amadori product and also by generation of reactive alpha-oxoaldehydes from glucose.

Peroxynitrite-mediated alpha-tocopherol oxidation in low-density lipoprotein: a mechanistic approach.

Previous reports proposed that peroxynitrite (ONOO-) oxidizes alpha-tocopherol (alpha-TOH) through a two-electron concerted mechanism. In contrast, ONOO- oxidizes phenols via free radicals arising from peroxo bond homolysis. To understand the kinetics and mechanism of alpha-TOH and gamma-tocopherol (gamma-TOH) oxidation in low-density lipoprotein (LDL) (direct vs. radical), we exposed LDL to ONOO- added as a bolus or an infusion. Nitric oxide (.NO), ascorbate and CO2 were used as key biologically relevant modulators of ONOO- reactivity. Although approximately 80% alpha-TOH and gamma-TOH depletion occurred within 5 min of incubation of 0.8 microM LDL with a 60 microM bolus of ONOO-, an equimolar infusion of ONOO- over 60 min caused total consumption of both antioxidants. gamma-Tocopherol was preserved relative to alpha-TOH, probably due to gamma-tocopheroxyl radical recycling by alpha-TOH. alpha-TOH oxidation in LDL was first order in ONOO- with approximately 12% of ONOO- maximally available. Physiological concentrations of.NO and ascorbate spared both alpha-TOH and gamma-TOH through independent and additive mechanisms. High concentrations of.NO and ascorbate abolished alpha-TOH and gamma-TOH oxidation. Nitric oxide protection was more efficient for alpha-TOH in LDL than for ascorbate in solution, evidencing the kinetically highly favored reaction of lipid peroxyl radicals with.NO than with alpha-TOH as assessed by computer-assisted simulations. In addition, CO2 (1.2 mM) inhibited both alpha-TOH and lipid oxidation. These results demonstrate that ONOO- induces alpha-TOH oxidation in LDL through a one-electron free radical mechanism; thus the inhibitory actions of.NO and ascorbate may determine low alpha-tocopheryl quinone accumulation in tissues despite increased ONOO- generation.

Reduced creatine kinase activity in transgenic amyotrophic lateral sclerosis mice.

Creatine (Cr), the substrate of the creatine kinase (CK) isoenzymes, was shown to be neuroprotective in several models of neurodegeneration, including amyotrophic lateral sclerosis (ALS). In order to investigate the mechanism of this beneficial effect, we determined CK activities and mitochondrial respiration rates in tissues from G93A transgenic mice, which overexpress a mutant form of human superoxide dismutase associated with familial ALS (FALS). While respiration rates of mitochondria from G93A transgenic or wild-type control mice isolated from spinal cord showed no difference, a significant and dramatic loss of CK activity could be detected in these tissues. In homogenates from spinal cord of G93A transgenic mice, CK activity decreased to 49% and in mitochondrial fractions to 67% compared to CK activities in wild-type control mice. Feeding the G93A transgenic mice with 2% Cr, the same tissues showed no statistically significant increase of CK activity compared to regular fed G93A transgenic mice. Experiments with isolated mitochondria, however, showed that Cr and adenosine triphosphate (ATP) protected mitochondrial CK activity against peroxynitrite-induced inactivation, which may play a role in tissue damage in neurodegeneration. Our data provide evidence for oxidative damage to the CK system in ALS, which may contribute to impaired energy metabolism and neurodegeneration.

Reactive nitrogen species scavenging, rather than nitric oxide inhibition, protects from articular cartilage damage in rat zymosan-induced arthritis.

1. The contribution of nitric oxide (NO) and peroxynitrite (PN) to inflammation in a zymosan-induced (1 mg, intra-articular, i.art.) rat model of arthritis was assessed by histopathology and by measuring the glycosaminoglycan (GAG) content of the articular cartilage. 2. Progression of the chronic synovitis in zymosan-induced arthritis (ZYA) was associated with increased nitrite and nitrotyrosine (3-NT) levels in the joint exudates that paralleled a progressive loss of the GAG content. An increase in 3-NT was also observed after i.art. PN. 3. The nonselective nitric oxide synthase (NOS) inhibitor l-N(G)-nitroarginine methyl ester (25-75 mg x kg(-1)day(-1)) or the selective inducible NOS inhibitor aminoguanidine (50-100 mg x kg(-1)day(-1)) given 1 h before (prophylactic) or 3 days after (therapeutic) injection of the zymosan ameliorated the synovitis, but worsened the GAG loss, as measured at the end of the experiment (day 7). 4. The PN scavenger uric acid (100-250 mg x kg(-1) i.p. four times daily) given prophylactically until the end of the experiment (day 14), in a dose compatible with its PN scavenging activity, significantly decreased both the synovitis and the GAG loss. 5. In conclusion, PN formation is associated with cartilage damage in addition to proinflammatory activity in ZYA. NOS inhibitors and a PN scavenger were able to reduce the cellular infiltration, while displaying opposite effects on cartilage homeostasis either by enhancing or ameliorating the damage, respectively.

Spinal mitochondrial-derived peroxynitrite enhances neuroimmune activation during morphine hyperalgesia and antinociceptive tolerance.

Treatment of severe pain by morphine, the gold-standard opioid and a potent drug in our arsenal of analgesic medications, is limited by the eventual development of hyperalgesia and analgesic tolerance. We recently reported that systemic administration of a peroxynitrite (PN) decomposition catalyst (PNDC) or superoxide dismutase mimetic attenuates morphine hyperalgesia and antinociceptive tolerance and reduces PN-mediated mitochondrial nitroxidative stress in the spinal cord. These results suggest the potential involvement of spinal PN signaling in this setting; which was examined in the present study. PN removal with intrathecal delivery of manganese porphyrin-based dual-activity superoxide/PNDCs, MnTE-2-PyP(5+) and the more lipophilic MnTnHex-2-PyP(5+), blocked hyperalgesia and antinociceptive tolerance in rats. Noteworthy is that intrathecal MnTnHex-2-PyP(5+) prevented nitration and inactivation of mitochondrial manganese superoxide dismutase. Mitochondrial manganese superoxide dismutase inactivation enhances the superoxide-to-PN pathway by preventing the dismutation of superoxide to hydrogen peroxide, thus providing an important enzymatic source for PN formation. Additionally, intrathecal MnTnHex-2-PyP(5+) attenuated neuroimmune activation by preventing the activation of nuclear factor kappa B, extracellular-signal-regulated kinase and p38 mitogen activated protein kinases, and the enhanced levels of proinflammatory cytokines, interleukin (IL)-1ß and IL-6, while increasing anti-inflammatory cytokines, IL-4 and IL-10. The role of PN was further confirmed using intrathecal or oral delivery of the superoxide-sparing PNDC, SRI-110. These results suggest that mitochondrial-derived PN triggers the activation of several biochemical pathways engaged in the development of neuroinflammation in the spinal cord that are critical to morphine hyperalgesia and tolerance, further supporting the potential of targeting PN as an adjunct to opiates to maintain pain relief.

Differential effects of peroxynitrite on contractile protein properties in fast- and slow-twitch skeletal muscle fibers of rat.

Oxidative modification of contractile proteins is thought to be a key factor in muscle weakness observed in many pathophysiological conditions. In particular, peroxynitrite (ONOO(-)), a potent short-lived oxidant, is a likely candidate responsible for this contractile dysfunction. In this study ONOO(-) or 3-morpholinosydnonimine (Sin-1, a ONOO(-) donor) was applied to rat skinned muscle fibers to characterize the effects on contractile properties. Both ONOO(-) and Sin-1 exposure markedly reduced maximum force in slow-twitch fibers but had much less effect in fast-twitch fibers. The rate of isometric force development was also reduced without change in the number of active cross bridges. Sin-1 exposure caused a disproportionately large decrease in Ca(2+) sensitivity, evidently due to coproduction of superoxide, as it was prevented by Tempol, a superoxide dismutase mimetic. The decline in maximum force with Sin-1 and ONOO(-) treatments could be partially reversed by DTT, provided it was applied before the fiber was activated. Reversal by DTT indicates that the decrease in maximum force was due at least in part to oxidation of cysteine residues. Ascorbate caused similar reversal, further suggesting that the cysteine residues had undergone S-nitrosylation. The reduction in Ca(2+) sensitivity, however, was not reversed by either DTT or ascorbate. Western blot analysis showed cross-linking of myosin heavy chain (MHC) I, appearing as larger protein complexes after ONOO(-) exposure. The findings suggest that ONOO(-) initially decreases maximum force primarily by oxidation of cysteine residues on the myosin heads, and that the accompanying decrease in Ca(2+) sensitivity is likely due to other, less reversible actions of hydroxyl or related radicals.

Is there a trigger role of peroxynitrite in the anti-arrhythmic effect of ischaemic preconditioning and peroxynitrite infusion?

This study has examined whether peroxynitrite (PN), generated during the preconditioning (PC) procedure or administered by brief intracoronary infusions, plays a trigger role in the anti-arrhythmic effects of preconditioning and peroxynitrite in anaesthetized dogs. To achieve this we infused the peroxynitrite scavenger uric acid (UA; 0.2 mg/kg/min, i.v.) over a 30 min period, just prior to a 25 min occlusion of the left anterior descending coronary artery, in preconditioned (UA+PC, n=8), peroxynitrite-treated (UA+PN, n=8) and in control (UAC; n=9) dogs. The effects were compared to those obtained from groups (PC, n=10; PN, n=10; C1, n=14) without uric acid administration. Severities of ischaemia (ST-segment elevation, inhomogeneity of electrical activation) and ventricular arrhythmias (VPBs, VT, VF), plasma nitrate/nitrite levels, as well as myocardial superoxide and nitrotyrosine productions were determined. Both preconditioning and the infusion of peroxynitrite increased nitrotyrosine formation which was abolished by the simultaneous administration of urate. Despite this, the protective effects of preconditioning (i.e. reductions in arrhythmias, superoxide and nitrotyrosine productions, as well as the increase in nitric oxide availability), occurring during the prolonged period of occlusion and reperfusion were still present. In contrast, urate completely abolished the protection resulted from peroxynitrite administration. This effect is most probably due to the fact that urate has already scavenged peroxynitrite during the infusion. Interestingly, urate itself, given prior to ischaemia and reperfusion, was also protective. We conclude that peroxynitrite in nanomolar concentrations can induce an anti-arrhythmic effect but peroxynitrite, generated during the preconditioning stimulus, is not necessary for the preconditioning-induced anti-arrhythmic protection.

The role of nitric oxide, superoxide and peroxynitrite in the anti-arrhythmic effects of preconditioning and peroxynitrite infusion in anaesthetized dogs.

BACKGROUND AND PURPOSE: Both ischaemia preconditioning (PC) and the intracoronary infusion of peroxynitrite (PN) suppress ischaemia and reperfusion (I/R)-induced arrhythmias and the generation of nitrotyrosine (NT, a marker of PN). However, it is still unclear whether this latter effect is due to a reduction in nitric oxide (NO) or superoxide (O(2)(-)) production. EXPERIMENTAL APPROACH: Dogs anaesthetized with chloralose and urethane were infused, twice for 5 min, with either saline (control) or 100 nM PN, or subjected to similar periods of occlusion (PC), 5 min prior to a 25 min occlusion and reperfusion of the left anterior descending coronary artery. Severities of ischaemia and ventricular arrhythmias, as well as changes in the coronary sinus nitrate/nitrite (NOx) levels were assessed throughout the experiment. The production of myocardial NOx, O(2)(-) and NT was determined following reperfusion. KEY RESULTS: Both PC and PN markedly suppressed the I/R-induced ventricular arrhythmias, compared to the controls, and increased NOx levels during coronary artery occlusion. Reperfusion induced almost the same increases in NOx levels in all groups, but superoxide production and, consequently, the generation of NT were significantly less in PC- and PN-treated dogs than in controls. CONCLUSIONS AND IMPLICATIONS: Since both PC and the administration of PN enhanced NOx levels during I/R, the attenuation of endogenous PN formation in these dogs is primarily due to a reduction in the amount of O(2) produced. Thus, the anti-arrhythmic effect of PC and PN can almost certainly be attributed to the preservation of NO availability during myocardial ischaemia.

Renal hemodynamic and excretory responses to intra-arterial infusion of peroxynitrite in anesthetized rats.

Peroxynitrite (ONOO(-)) is formed endogenously by the reaction of nitric oxide (NO) and superoxide (O(2)(-)). To examine the hypothesis that OONO(-) cause renal vasodilation at low concentrations but cause vasoconstriction at higher concentrations, we examined renal responses to intra-arterial infusion of incremental doses of OONO(-) (10, 20, and 40 microg.kg(-1).min(-1); 45 min each) in anesthetized rats. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined by PAH and inulin clearance. In control rats (n = 6), low dose (10 microg.kg(-1).min(-1)) of OONO(-) increased RBF by 10 +/- 3% and GFR by 15 +/- 5%. The higher doses (20 and 40 microg.kg(-1).min(-1)) mostly reversed these responses which were -7 +/- 4 and -27 +/- 7% (P < 0.05) in RBF and -0.1 +/- 4.8 and -14 +/- 12% in GFR, respectively. There were no appreciable changes in urine flow (V) and sodium excretion (U(Na)V) during OONO(-) infusion. However, in rats pretreated with NO synthase (NOS) inhibitor, l-NAME (50 microg.kg(-1).min(-1); n = 5), these doses of ONOO(-) significantly reduced RBF (-26 +/- 7, -27 +/- 6, and -44 +/- 3%) and GFR (-21 +/- 6, -25 +/- 8, and -32 +/- 12%) with variable increases in V or U(Na)V. Long-term infusion of OONO(-) (10 microg.kg(-1).min(-1) for 75 min) in another set of control rats (n = 5) also showed similar vasodilator and hyperfiltration responses. These data indicate that ONOO(-) acts as an oxidant at high concentration but provides renoprotective function at low concentration that depends on intact NOS activity.

Regulation of cGMP-dependent protein kinase-mediated vasodilation by hypoxia-induced reactive species in ovine fetal pulmonary veins.

We previously reported that hypoxia attenuates cGMP-dependent protein kinase (PKG)-mediated relaxation in pulmonary vessels (Am J Physiol Lung Cell Mol Physiol 279: L611-L618, 2003). To determine whether hypoxia-induced reactive oxygen and nitrogen species (ROS and RNS, respectively) may be involved in the downregulation of PKG-mediated relaxation, ovine fetal intrapulmonary veins were exposed to 4 h of normoxia or hypoxia, with or without scavengers of ROS [N-acetylcysteine (NAC)] or peroxynitrite (quercetin and Trolox) and preconstricted with endothelin-1. Hypoxia decreased the relaxation response to 8-bromo-cGMP, PKG protein expression, and kinase activity and increased tyrosine nitration in PKG. However, ROS and RNS scavengers prevented these changes. To determine whether increased PKG nitration diminishes PKG activity, pulmonary vein smooth muscle cells (PVSMC) were exposed to shorter-term (30 min) hypoxia, which increased PKG nitration and decreased PKG activity but did not alter PKG protein expression. Increased dihydro-2,7-dichlorofluorescein diacetate (DCFH(2)-DA) fluorescence in PVSMC after 4 h or 30 min of hypoxia was not observed in the presence of NAC, quercetin, or Trolox, suggesting increased ROS and RNS production. Increased PKG nitration and the associated decrease in PKG activity in PVSMC after 30 min of hypoxia were also reversed on reoxygenation. The consequences of PKG nitration were assessed by exposure of purified PKG-Ialpha to peroxynitrite, which caused increased 3-nitrotyrosine immunoreactivity and inhibition of kinase activity. Our data suggest that, after 30 min of hypoxia, reversible covalent modification of PKG by hypoxia-induced reactive species may be an important mechanism by which the relaxation response to cGMP is regulated. However, after 4 h of hypoxia, PKG nitration and decreased PKG expression are involved.

Plasmodium berghei resists killing by reactive oxygen species.

Reactive oxygen species (ROS) are widely believed to kill malarial parasites. C57BL/6 mice injected with P. berghei inocula incubated with supraphysiological doses of NO (< or =150 microM) or with peroxynitrite (220 microM), however, exhibited parasitemia similar to that seen with those given control inocula, and there was no difference in disease development. Only treatment of inocula with NO doses nearing saturation (> or =1.2 mM) resulted in no detectable parasitemia in the recipients; flow cytometric analysis with a vital dye (hydroethidine) indicated that 1.5 mM NO lysed the erythrocytes rather than killing the parasites. The hemoglobin level in the inocula was about 8 muM; the hemoglobin was mainly oxyhemoglobin (oxyHb) (96%), which was converted to methemoglobin (>95%) after treatment with 150 microM NO. The concentrations of 150 microM of NO and 220 microM of peroxynitrite were far in excess of the hemoglobin concentration (approximately 8 microM), and yet no parasite killing was detected. We therefore conclude that hemoglobin protects Plasmodium parasites from ROS, but the parasite likely possesses intrinsic defense mechanisms against ROS.

Prostacyclin in the cardiovascular system: new aspects and open questions.

Several indications exist that prostacyclin (PGI(2)) release in the cardiovascular system might be affected by cyclooxygenase (COX)-2-specific inhibitors. This could reflect an inhibition of PGI(2) synthesis in the endothelium although in these cells mainly COX-1 is expressed. Inflammation and stress induce COX-2 in smooth muscle cells which could have happened in patients with cardiac diseases. Herein, we show that also cardiomyocytes contain PGI(2) synthase in intercalated discs as a third source of PGI(2) in the cardiovascular system. Another aim of this study was to explain the finding that PGI(2) synthase in lipopolysaccharide (LPS)-treated smooth muscle cells, in contrast to endothelial cells, is resistant to nitration and inhibition by peroxynitrite. By using redox cyclers, the nitration occurred and confirmed our previous hypothesis that a high peroxidative activity of such cells keeps peroxynitrite below the effective levels of 50 nM. Considering enhanced oxidative stress in aged vessels, we postulated and verified that endothelial dysfunction in aged vessels is due to nitration and inhibition of PGI(2) synthase. Such data underline the role of PGI(2) as a potent mediator for regaining and maintaining the normal resting state of cells in a COX-2 dependent fashion.