Adipocyte-specific Nrf2 deletion negates nitro-oleic acid benefits on glucose tolerance in diet-induced obesity.

Obesity is commonly linked with white adipose tissue (WAT) dysfunction, setting off inflammation and oxidative stress, both key contributors to the cardiometabolic complications associated with obesity. To improve metabolic and cardiovascular health, countering these inflammatory and oxidative signaling processes is crucial. Offering potential in this context, the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) by nitro-fatty acids (NO2-FA) promote diverse anti-inflammatory signaling and counteract oxidative stress. Additionally, we previously highlighted that nitro-oleic acid (NO2-OA) preferentially accumulates in WAT and provides protection against already established high fat diet (HFD)-mediated impaired glucose tolerance. The precise mechanism accounting for these protective effects remained largely unexplored until now. Herein, we reveal that protective effects of improved glucose tolerance by NO2-OA is absent when Nrf2 is specifically ablated in adipocytes (ANKO mice). NO2-OA treatment did not alter body weight between ANKO and littermate controls (Nrf2fl/fl) mice on both the HFD and low-fat diet (LFD). As expected, at day 76 (before NO2-OA treatment) and notably at day 125 (daily treatment of 15 mg/kg NO2-OA for 48 days), both HFD-fed Nrf2fl/fl and ANKO mice exhibited increased fat mass and reduced lean mass compared to LFD controls. However, throughout the NO2-OA treatment, no distinction was observed between Nrf2fl/fl and ANKO in the HFD-fed mice as well as in the Nrf2fl/fl mice fed a LFD. Glucose tolerance tests revealed impaired glucose tolerance in HFD-fed Nrf2fl/fl and ANKO compared to LFD-fed Nrf2fl/fl mice. Notably, NO2-OA treatment improved glucose tolerance in HFD-fed Nrf2fl/fl but did not yield the same improvement in ANKO mice at days 15, 30, and 55 of treatment. Unraveling the pathways linked to NO2-OA's protective effects in obesity-mediated impairment in glucose tolerance is pivotal within the realm of precision medicine, crucially propelling future applications and refining novel drug-based strategies.

A FABP4-PPARγ signaling axis regulates human monocyte responses to electrophilic fatty acid nitroalkenes.

Nitro-fatty acids (NO2-FA) are electrophilic lipid mediators derived from unsaturated fatty acid nitration. These species are produced endogenously by metabolic and inflammatory reactions and mediate anti-oxidative and anti-inflammatory responses. NO2-FA have been postulated as partial agonists of the Peroxisome Proliferator-Activated Receptor gamma (PPARγ), which is predominantly expressed in adipocytes and myeloid cells. Herein, we explored molecular and cellular events associated with PPARγ activation by NO2-FA in monocytes and macrophages. NO2-FA induced the expression of two PPARγ reporter genes, Fatty Acid Binding Protein 4 (FABP4) and the scavenger receptor CD36, at early stages of monocyte differentiation into macrophages. These responses were inhibited by the specific PPARγ inhibitor GW9662. Attenuated NO2-FA effects on PPARγ signaling were observed once cells were differentiated into macrophages, with a significant but lower FABP4 upregulation, and no induction of CD36. Using in vitro and in silico approaches, we demonstrated that NO2-FA bind to FABP4. Furthermore, the inhibition of monocyte FA binding by FABP4 diminished NO2-FA-induced upregulation of reporter genes that are transcriptionally regulated by PPARγ, Keap1/Nrf2 and HSF1, indicating that FABP4 inhibition mitigates NO2-FA signaling actions. Overall, our results affirm that NO2-FA activate PPARγ in monocytes and upregulate FABP4 expression, thus promoting a positive amplification loop for the downstream signaling actions of this mediator.

Electrophilic nitro-fatty acids suppress allergic contact dermatitis in mice.

BACKGROUND: Reactions between nitric oxide (NO), nitrite (NO2-), and unsaturated fatty acids give rise to electrophilic nitro-fatty acids (NO2 -FAs), such as nitro oleic acid (OA-NO2 ) and nitro linoleic acid (LNO2 ). Endogenous electrophilic fatty acids (EFAs) mediate anti-inflammatory responses by modulating metabolic and inflammatory signal transduction reactions. Hence, there is considerable interest in employing NO2 -FAs and other EFAs for the prevention and treatment of inflammatory disorders. Thus, we sought to determine whether OA-NO2 , an exemplary nitro-fatty acid, has the capacity to inhibit cutaneous inflammation. METHODS: We evaluated the effect of OA-NO2 on allergic contact dermatitis (ACD) using an established model of contact hypersensitivity in C57Bl/6 mice utilizing 2,4-dinitrofluorobenzene as the hapten. RESULTS: We found that subcutaneous (SC) OA-NO2 injections administered 18 h prior to sensitization and elicitation suppresses ACD in both preventative and therapeutic models. In vivo SC OA-NO2 significantly inhibits pathways that lead to inflammatory cell infiltration and the production of inflammatory cytokines in the skin. Moreover, OA-NO2 is capable of enhancing regulatory T-cell activity. Thus, OA-NO2 treatment results in anti-inflammatory effects capable of inhibiting ACD by inducing immunosuppressive responses. CONCLUSION: Overall, these results support the development of OA-NO2 as a promising therapeutic for ACD and provides new insights into the role of electrophilic fatty acids in the control of cutaneous immune responses potentially relevant to a broad range of allergic and inflammatory skin diseases.

Nitro-oleic acid targets transient receptor potential (TRP) channels in capsaicin sensitive afferent nerves of rat urinary bladder.

Nitro-oleic acid (9- and 10-nitro-octadeca-9-enoic acid, OA-NO(2)) is an electrophilic fatty acid nitroalkene derivative that modulates gene transcription and protein function via post-translational protein modification. Nitro-fatty acids are generated from unsaturated fatty acids by oxidative inflammatory reactions and acidic conditions in the presence of nitric oxide or nitrite. Nitroalkenes react with nucleophiles such as cysteine and histidine in a variety of susceptible proteins including transient receptor potential (TRP) channels in sensory neurons of the dorsal root and nodose ganglia. The present study revealed that OA-NO(2) activates TRP channels on afferent nerve terminals in the urinary bladder and thereby increases bladder activity. The TRPV1 agonist capsaicin (CAPS, 1 µM) and the TRPA1 agonist allyl isothiocyanate (AITC, 30 µM), elicited excitatory effects in bladder strips, increasing basal tone and amplitude of phasic bladder contractions (PBC). OA-NO(2) mimicked these effects in a concentration-dependent manner (1 µM-33 µM). The TRPA1 antagonist HC3-030031 (HC3, 30 µM) and the TRPV1 antagonist diaryl piperazine analog (DPA, 1 µM), reduced the effect of OA-NO(2) on phasic contraction amplitude and baseline tone. However, the non-selective TRP channel blocker, ruthenium red (30 µM) was a more effective inhibitor, reducing the effects of OA-NO(2) on basal tone by 75% and the effects on phasic amplitude by 85%. In bladder strips from CAPS-treated rats, the effect of OA-NO(2) on phasic contraction amplitude was reduced by 65% and the effect on basal tone was reduced by 60%. Pretreatment of bladder strips with a combination of neurokinin receptor antagonists (NK1 selective antagonist, CP 96345; NK2 selective antagonist, MEN 10,376; NK3 selective antagonist, SB 234,375, 1 µM each) reduced the effect of OA-NO(2) on basal tone, but not phasic contraction amplitude. These results indicate that nitroalkene fatty acid derivatives can activate TRP channels on CAPS-sensitive afferent nerve terminals, leading to increased bladder contractile activity. Nitrated fatty acids produced endogenously by the combination of fatty acids and oxides of nitrogen released from the urothelium and/or afferent nerves may play a role in modulating bladder activity.

Nitro-oleic acid inhibits firing and activates TRPV1- and TRPA1-mediated inward currents in dorsal root ganglion neurons from adult male rats.

Nitro-oleic acid (OA-NO(2)), an electrophilic fatty acid by-product of nitric oxide and nitrite reactions, is present in normal and inflamed mammalian tissues at up to micromolar concentrations and exhibits anti-inflammatory signaling actions. The effects of OA-NO(2) on cultured dorsal root ganglion (DRG) neurons were examined using fura-2 Ca(2+) imaging and patch clamping. OA-NO(2) (3.5-35 microM) elicited Ca(2+) transients in 20 to 40% of DRG neurons, the majority (60-80%) of which also responded to allyl isothiocyanate (AITC; 1-50 microM), a TRPA1 agonist, and to capsaicin (CAPS; 0.5 microM), a TRPV1 agonist. The OA-NO(2)-evoked Ca(2+) transients were reduced by the TRPA1 antagonist 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl) acetamide (HC-030031; 5-50 microM) and the TRPV1 antagonist capsazepine (10 microM). Patch-clamp recording revealed that OA-NO(2) depolarized and induced inward currents in 62% of neurons. The effects of OA-NO(2) were elicited by concentrations >or=5 nM and were blocked by 10 mM dithiothreitol. Concentrations of OA-NO(2) >or=5 nM reduced action potential (AP) overshoot, increased AP duration, inhibited firing induced by depolarizing current pulses, and inhibited Na(+) currents. The effects of OA-NO(2) were not prevented or reversed by the NO-scavenger carboxy-2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide. A large percentage (46-57%) of OA-NO(2)-responsive neurons also responded to CAPS (0.5 microM) or AITC (0.5 microM). OA-NO(2) currents were reduced by TRPV1 (diarylpiperazine; 5 microM) or TRPA1 (HC-030031; 5 microM) antagonists. These data reveal that endogenous OA-NO(2) generated at sites of inflammation may initially activate transient receptor potential channels on nociceptive afferent nerves, contributing to the initiation of afferent nerve activity, and later suppresses afferent firing.

Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives.

Nitroalkene fatty acid derivatives manifest a strong electrophilic nature, are clinically detectable, and induce multiple transcriptionally regulated anti-inflammatory responses. At present, the characterization and quantification of endogenous electrophilic lipids are compromised by their Michael addition with protein and small-molecule nucleophilic targets. Herein, we report a trans-nitroalkylation reaction of nitro-fatty acids with beta-mercaptoethanol (BME) and apply this reaction to the unbiased identification and quantification of reaction with nucleophilic targets. Trans-nitroalkylation yields are maximal at pH 7 to 8 and occur with physiological concentrations of target nucleophiles. This reaction is also amenable to sensitive mass spectrometry-based quantification of electrophilic fatty acid-protein adducts upon electrophoretic resolution of proteins. In-gel trans-nitroalkylation reactions also permit the identification of protein targets without the bias and lack of sensitivity of current proteomic approaches. Using this approach, it was observed that fatty acid nitroalkenes are rapidly metabolized in vivo by a nitroalkene reductase activity and mitochondrial beta-oxidation, yielding a variety of electrophilic and nonelectrophilic products that could be structurally characterized upon BME-based trans-nitroalkylation reaction. This strategy was applied to the detection and quantification of fatty acid nitration in mitochondria in response to oxidative inflammatory conditions induced by myocardial ischemia-reoxygenation.

Sulfenic acid in human serum albumin.

Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.

Oxidant-mediated impairment of nitric oxide signaling in sickle cell disease--mechanisms and consequences.

In addition to its mediation of vascular relaxation and neurotransmission, nitric oxide (*NO) potently modulates oxygen radical reactions and inflammatory signaling. This participation of *NO in free radical and oxidative reactions will yield secondary oxides of nitrogen that display frequently-undefined reactivities and unique signaling properties. In sickle cell disease (SCD) inflammatory-derived oxidative reactions impair *NO-dependent vascular function. A combination of clinical and knockout-transgenic SCD mouse studies show increased rates of xanthine oxidase-dependent superoxide (O2*-) production and reveal the presence of an oxidative and nitrative inflammatory milieu in the sickle cell vasculature, kidney and liver. Considering the critical role of endothelial *NO production in regulating endothelial adhesion molecule expression, platelet aggregation, and both basal and stress-mediated vasodilation, the O2*- mediated reduction in *NO bioavailability can significantly contribute to the vascular dysfunction and organ injury associated with SCD.

Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration.

Nitrotyrosine formation is a hallmark of vascular inflammation, with polymorphonuclear neutrophil-derived (PMN-derived) and monocyte-derived myeloperoxidase (MPO) being shown to catalyze this posttranslational protein modification via oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (NO(2)(*)). Herein, we show that MPO concentrates in the subendothelial matrix of vascular tissues by a transcytotic mechanism and serves as a catalyst of ECM protein tyrosine nitration. Purified MPO and MPO released by intraluminal degranulation of activated human PMNs avidly bound to aortic endothelial cell glycosaminoglycans in both cell monolayer and isolated vessel models. Cell-bound MPO rapidly transcytosed intact endothelium and colocalized abluminally with the ECM protein fibronectin. In the presence of the substrates hydrogen peroxide (H(2)O(2)) and NO(2)(-), cell and vessel wall-associated MPO catalyzed nitration of ECM protein tyrosine residues, with fibronectin identified as a major target protein. Both heparin and the low-molecular weight heparin enoxaparin significantly inhibited MPO binding and protein nitrotyrosine (NO(2)Tyr) formation in both cultured endothelial cells and rat aortic tissues. MPO(-/-) mice treated with intraperitoneal zymosan had lower hepatic NO(2)Tyr/tyrosine ratios than did zymosan-treated wild-type mice. These data indicate that MPO significantly contributes to NO(2)Tyr formation in vivo. Moreover, transcytosis of MPO, occurring independently of leukocyte emigration, confers specificity to nitration of vascular matrix proteins.

Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease.

Plasma xanthine oxidase (XO) activity was defined as a source of enhanced vascular superoxide (O(2)( *-)) and hydrogen peroxide (H(2)O(2)) production in both sickle cell disease (SCD) patients and knockout-transgenic SCD mice. There was a significant increase in the plasma XO activity of SCD patients that was similarly reflected in the SCD mouse model. Western blot and enzymatic analysis of liver tissue from SCD mice revealed decreased XO content. Hematoxylin and eosin staining of liver tissue of knockout-transgenic SCD mice indicated extensive hepatocellular injury that was accompanied by increased plasma content of the liver enzyme alanine aminotransferase. Immunocytochemical and enzymatic analysis of XO in thoracic aorta and liver tissue of SCD mice showed increased vessel wall and decreased liver XO, with XO concentrated on and in vascular luminal cells. Steady-state rates of vascular O(2)( *-) production, as indicated by coelenterazine chemiluminescence, were significantly increased, and nitric oxide (( *)NO)-dependent vasorelaxation of aortic ring segments was severely impaired in SCD mice, implying oxidative inactivation of ( *)NO. Pretreatment of aortic vessels with the superoxide dismutase mimetic manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin markedly decreased O(2)( small middle dot-) levels and significantly restored acetylcholine-dependent relaxation, whereas catalase had no effect. These data reveal that episodes of intrahepatic hypoxia-reoxygenation associated with SCD can induce the release of XO into the circulation from the liver. This circulating XO can then bind avidly to vessel luminal cells and impair vascular function by creating an oxidative milieu and catalytically consuming (*)NO via O(2)( small middle dot-)-dependent mechanisms.

Catalytic consumption of nitric oxide by 12/15- lipoxygenase: inhibition of monocyte soluble guanylate cyclase activation.

12/15-Lipoxygenase (LOX) activity is elevated in vascular diseases associated with impaired nitric oxide (( small middle dot)NO) bioactivity, such as hypertension and atherosclerosis. In this study, primary porcine monocytes expressing 12/15-LOX, rat A10 smooth muscle cells transfected with murine 12/15-LOX, and purified porcine 12/15-LOX all consumed *NO in the presence of lipid substrate. Suppression of LOX diene conjugation by *NO was also found, although the lipid product profile was unchanged. *NO consumption by porcine monocytes was inhibited by the LOX inhibitor, eicosatetraynoic acid. Rates of arachidonate (AA)- or linoleate (LA)-dependent *NO depletion by porcine monocytes (2.68 +/- 0.03 nmol x min(-1) x 10(6) cells(-1) and 1.5 +/- 0.25 nmol x min(-1) x 10(6) cells(-1), respectively) were several-fold greater than rates of *NO generation by cytokine-activated macrophages (0.1-0.2 nmol x min(-1) x 10(6) cells(-1)) and LA-dependent *NO consumption by primary porcine monocytes inhibited *NO activation of soluble guanylate cyclase. These data indicate that catalytic *NO consumption by 12/15-LOX modulates monocyte *NO signaling and suggest that LOXs may contribute to vascular dysfunction not only by the bioactivity of their lipid products, but also by serving as catalytic sinks for *NO in the vasculature.

Halogenated 2,5-pyrrolidinediones: synthesis, bacterial mutagenicity in Ames tester strain TA-100 and semi-empirical molecular orbital calculations.

The chloroimide 3,3-dichloro-4-(dichloromethylene)-2,5-pyrrolidinedione, a tetrachloroitaconimide, is the principal mutagen produced by chlorination of simulated poultry chiller water. It is the second most potent mutagenic disinfection by-product of chlorination ever reported. Six of seven new synthetic analogs of this compound are direct-acting mutagens in Ames tester strain TA-100. Computed energies of the lowest unoccupied molecular orbital (E(LUMO)) and of the radical anion stability (DeltaH(f)(rad)-DeltaH(f)) from MNDO-PM3 for the chloroimides show a quantitative correlation with the Ames TA-100 bacterial mutagenicity values. The molar mutagenicities of these direct acting mutagenic imides having an exocyclic double bond fit the same linear correlation (lnM(m) vs. E(LUMO); lnM(m) vs. DeltaH(f)(rad)--DeltaH(f)) as the chlorinated 2(5H)-furanones, including the potent mutagen MX, 3-chloro-4-(dichloro-methyl)-5-hydroxy-2(5H)-furanone, a by-product of water chlorination and paper bleaching with chlorine. Mutagenicity data for related haloimides having endocyclic double bonds are also given. For the same number of chlorine atoms, the imides with endocyclic double bonds have significantly higher Ames mutagenicity compared to their structural analogs with exocyclic double bonds, but do not follow the same E(LUMO) or DeltaH(f)(rad)-DeltaH(f) correlation as the exocyclic chloroimides and the chlorinated 2(5H)-furanones.

Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin.

BACKGROUND: Altered endothelial cell nitric oxide (NO(*)) production in atherosclerosis may be due to a reduction of intracellular tetrahydrobiopterin, which is a critical cofactor for NO synthase (NOS). In addition, previous literature suggests that inactivation of NO(*) by increased vascular production superoxide (O(2)(*-)) also reduces NO(*) bioactivity in several disease states. We sought to determine whether these 2 seemingly disparate mechanisms were related. METHODS AND RESULTS: Endothelium-dependent vasodilation was abnormal in aortas of apoE-deficient (apoE(-/-)) mice, whereas vascular superoxide production (assessed by 5 micromol/L lucigenin) was markedly increased. Treatment with either liposome-entrapped superoxide dismutase or sepiapterin, a precursor to tetrahydrobiopterin, improved endothelium-dependent vasodilation in aortas from apoE(-/-) mice. Hydrogen peroxide had no effect on the decay of tetrahydrobiopterin, as monitored spectrophotometrically. In contrast, superoxide modestly and peroxynitrite strikingly increased the decay of tetrahydrobiopterin over 500 seconds. Luminol chemiluminescence, inhibitable by the peroxynitrite scavengers ebselen and uric acid, was markedly increased in apoE(-/-) aortic rings. In vessels from apoE(-/-) mice, uric acid improved endothelium-dependent relaxation while having no effect in vessels from control mice. Treatment of normal aortas with exogenous peroxynitrite dramatically increased vascular O(2)(*-) production, seemingly from eNOS, because this effect was absent in vessels lacking endothelium, was blocked by NOS inhibition, and did not occur in vessels from mice lacking eNOS. CONCLUSIONS: Reactive oxygen species may alter endothelium-dependent vascular relaxation not only by the interaction of O(2)(*-) with NO(*) but also through interactions between peroxynitrite and tetrahydrobiopterin. Peroxynitrite oxidation of tetrahydrobiopterin may represent a pathogenic cause of "uncoupling" of NO synthase.

Interactions between nitric oxide and lipid oxidation pathways: implications for vascular disease.

Nitric oxide ((.)NO) signaling pathways and lipid oxidation reactions are of central importance in both the maintenance of vascular homeostasis and the progression of vascular disease. Because both of these pathways involve free radical species that can also react together at extremely fast rates, convergent interactions between these pathways are expected. Biochemical and cell biology studies have defined multiple interactions of (.)NO with oxidizing lipids that could lead to either vascular protection or potentiation of inflammatory vascular injury. For example, low levels of (.)NO generated by endothelial nitric oxide synthase can terminate propagating lipid radicals and inhibit lipoxygenases, reactions that would be protective. Alternatively, if generated at elevated levels, for example, after inducible nitric oxide synthase expression in inflammation, (.)NO can be converted to prooxidant species, such as peroxynitrite (ONOO(-)) and nitrogen dioxide ((.)NO(2)), that can potentiate inflammatory injury to vascular cells. Finally, both enzymatic and nonenzymatic lipid oxidation reactions can influence (.)NO bioactivity by directly scavenging (.)NO or altering the induction and catalytic activity of nitric oxide synthase enzymes. In this review, we summarize the biochemical interactions between (.)NO and lipid oxidation reactions and discuss the recognized and potential roles of these reactions in the vasculature.

Reaction of peroxynitrite with Mn-superoxide dismutase. Role of the metal center in decomposition kinetics and nitration.

Manganese superoxide dismutase (Mn-SOD), a critical mitochondrial antioxidant enzyme, becomes inactivated and nitrated in vitro and potentially in vivo by peroxynitrite. Since peroxynitrite readily reacts with transition metal centers, we assessed the role of the manganese ion in the reaction between peroxynitrite and Mn-SOD. Peroxynitrite reacts with human recombinant and Escherichia coli Mn-SOD with a second order rate constant of 1.0 +/- 0.2 x 10(5) and 1.4 +/- 0.2 x 10(5) m(-)1 s(-)1 at pH 7.47 and 37 degrees C, respectively. The E. coli apoenzyme, obtained by removing the manganese ion from the active site, presents a rate constant <10(4) m(-)1 s(-)1 for the reaction with peroxynitrite, whereas that of the manganese-reconstituted apoenzyme (apo/Mn) was comparable to that of the holoenzyme. Peroxynitrite-dependent nitration of 4-hydroxyphenylacetic acid was increased 21% by Mn-SOD. The apo/Mn also promoted nitration, but the apo and the zinc-substituted apoenzyme (apo/Zn) enzymes did not. The extent of tyrosine nitration in the enzyme was also affected by the presence and nature (i.e. manganese or zinc) of the metal center in the active site. For comparative purposes, we also studied the reaction of peroxynitrite with low molecular weight complexes of manganese and zinc with tetrakis-(4-benzoic acid) porphyrin (tbap). Mn(tbap) reacts with peroxynitrite with a rate constant of 6.8 +/- 0.1 x 10(4) m(-)1 s(-)1 and maximally increases nitration yields by 350%. Zn(tbap), on the other hand, affords protection against nitration. Our results indicate that the manganese ion in Mn-SOD plays an important role in the decomposition kinetics of peroxynitrite and in peroxynitrite-dependent nitration of self and remote tyrosine residues.

Hypercapnia induces injury to alveolar epithelial cells via a nitric oxide-dependent pathway.

Ventilator strategies allowing for increases in carbon dioxide (CO(2)) tensions (hypercapnia) are being emphasized to ameliorate the consequences of inflammatory-mediated lung injury. Inflammatory responses lead to the generation of reactive species including superoxide (O(2)(-)), nitric oxide (.NO), and their product peroxynitrite (ONOO(-)). The reaction of CO(2) and ONOO(-) can yield the nitrosoperoxocarbonate adduct ONOOCO(2)(-), a more potent nitrating species than ONOO(-). Based on these premises, monolayers of fetal rat alveolar epithelial cells were utilized to investigate whether hypercapnia would modify pathways of.NO production and reactivity that impact pulmonary metabolism and function. Stimulated cells exposed to 15% CO(2) (hypercapnia) revealed a significant increase in.NO production and nitric oxide synthase (NOS) activity. Cell 3-nitrotyrosine content as measured by both HPLC and immunofluorescence staining also increased when exposed to these same conditions. Hypercapnia significantly enhanced cell injury as evidenced by impairment of monolayer barrier function and increased induction of apoptosis. These results were attenuated by the NOS inhibitor N-monomethyl-L-arginine. Our studies reveal that hypercapnia modifies.NO-dependent pathways to amplify cell injury. These results affirm the underlying role of.NO in tissue inflammatory reactions and reveal the impact of hypercapnia on inflammatory reactions and its potential detrimental influences.

Catalytic consumption of nitric oxide by prostaglandin H synthase-1 regulates platelet function.

Nitric oxide (( small middle dot)NO) plays a central role in vascular homeostasis via regulation of smooth muscle relaxation and platelet aggregation. Although mechanisms for ( small middle dot)NO formation are well known, removal pathways are less well characterized, particularly in cells that respond to ( small middle dot)NO through activation of soluble guanylate cyclase. Herein, we report that ( small middle dot)NO is catalytically consumed by prostaglandin H synthase-1 (PGHS-1) through acting as a reducing peroxidase substrate. With purified ovine PGHS-1, ( small middle dot)NO consumption requires peroxide (LOOH or H(2)O(2)), with a K(m)( (app)) for 15(S)hydroperoxyeicosatetraenoic acid (HPETE) of 3. 27 +/- 0.35 microm. During this, 2 mol ( small middle dot)NO are consumed per mol HPETE, and loss of HPETE hydroperoxy group occurs with retention of the conjugated diene spectrum. Hydroperoxide-stimulated ( small middle dot)NO consumption requires heme incorporation, is not inhibited by indomethacin, and is further stimulated by the reducing peroxidase substrate, phenol. PGHS-1-dependent ( small middle dot)NO consumption also occurs during arachidonate, thrombin, or activation of platelets (1-2 microm.min(-1) for typical plasma platelet concentrations) and prevents ( small middle dot)NO stimulation of platelet soluble guanylate cyclase. Platelet sensitivity to ( small middle dot)NO as an inhibitor of aggregation is greater using a platelet-activating stimulus () that does not cause ( small middle dot)NO consumption, indicating that this mechanism overcomes the anti-aggregatory effects of ( small middle dot)NO. Catalytic consumption of ( small middle dot)NO during eicosanoid synthesis thus represents both a novel proaggregatory function for PGHS-1 and a regulated mechanism for vascular ( small middle dot)NO removal.