iNOS Knockout Mice Research Articles

Absence of salt sensitivity in iNOS knockout mice

The ability of aminoguanidine to promote salt‐sensitive hypertension has led to the suggestion that the inducible form of nitric oxide synthase (iNOS, NOS‐2) contributes importantly to the regulation of salt balance and blood pressure (BP). Here, we tested the hypothesis that iNOS knockout mice (iNOS‐/‐) have increased salt sensitivity of BP relative to control animals (iNOS+/+). Male mice of either strain were placed on regular (0.5% NaCl, RS) or high (8% NaCl, HS) salt diet at 9 wks, and 24h diastolic (DBP) and systolic (SBP) BP were assessed by telemetry after 3 months. In iNOS+/+ mice, 3 months of HS was associated with a modest elevation of BP compared to the RS group (RS vs HS: DAP, 91 ± 4 vs 98 ± 6 mmHg, P<0.05, SAP, 124 ± 2 vs 128 ± 5 mmHg, NS), consistent with previous studies. In contrast, there was no evidence of this effect of HS in iNOS‐/‐ mice (RS vs HS: DAP: 95 ± 4 vs 92 ± 2 mmHg, NS, SAP: 126 ± 4 vs 124 ± 6 mmHg, NS). The BP of iNOS‐/‐ and iNOS+/+ mice on RS diet were not significantly different. The BP response to brief (4 day) increases (RS to HS) or decreases (HS to RS) in salt intake did not differ between the two strains. In summary, our results demonstrate that iNOS knockout mice are normotensive and do not exhibit an exaggerated BP response to high salt. We reject our original hypothesis and conclude that absence of iNOS does not lead to an obligatory increase of salt sensitivity or BP. Supported by the Canadian Institutes of Health Research.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Deficiency of iNOS Does Not Prevent Isoproterenol-induced Cardiac Hypertrophy in Mice

We investigated whether deficiency of inducible nitric oxide synthase (iNOS) could prevent isoproterenol-induced cardiac hypertrophy in iNOS knockout (KO) mice. Isoproterenol was continuously infused subcutaneously (15 mg/kg/day) using an osmotic minipump. Isoproterenol reduced body weight and fat mass in both iNOS KO and wild-type mice compared with saline-infused wild-type mice. Isoproterenol increased the heart weight in both iNOS KO and wild-type mice but there was no difference between iNOS KO and wild-type mice. Posterior wall thickness of left ventricle showed the same tendency with heart weight. Protein level of iNOS in the left ventricle was increased in isoproterenol-infused wild-type mice. The gene expression of interleukin-6 (IL-6) and transforming growth factor-beta (TGF-beta) in isoproterenol-infused wild-type was measured at 2, 4, 24, and 48-hour and isoproterenol increased both IL-6 (2, 4, 24, and 48-hour) and TGF-beta (4 and 24-hour). Isoproterenol infusion for 7 days increased the mRNA level of IL-6 and TGF-beta in iNOS KO mice, whereas the gene expression in wild-type mice was not increased. Phosphorylated form of extracellular signal-regulated kinases (pERK) was also increased by isoproterenol at 2 and 4-hour but was not increased at 7 days after infusion in wild-type mice. However, the increased pERK level in iNOS KO mice was maintained even at 7 days after isoproterenol infusion. These results suggest that deficiency of iNOS does not prevent isoproterenol-induced cardiac hypertrophy and may have potentially harmful effects on cardiac hypertrophy.

Inducible Nitric Oxide Synthase Mediates Hippocampal Caspase-3 Activation in Pneumococcal Meningitis

Brain damage in bacterial meningitis is still a major problem. More knowledge about the triggers and mechanisms of neuronal damage in bacterial meningitis is needed to improve outcome in bacterial meningitis. The most common bacterial meningitis pathogen—Streptococcus pneumoniae—causes caspase activation and neuronal apoptosis in the hippocampus via its toxins and extensive inflammatory potential. Nitric oxide (NO)—produced by inducible nitric oxide synthase (iNOS)—is a major inflammatory mediator clearly upregulated in the cerebrospinal fluid during pneumococcal meningitis. However, its effects in bacterial meningitis are still controversial. This article demonstrates that genetic inactivation of iNOS results in a marked reduction of caspase-3-mediated neuronal damage in experimental murine pneumococcal meningitis. Protection of hippocampal neurons in iNOS knockout mice was not due to differences in intrathecal growth of S. pneumoniae and must therefore be attributed to differences of host inflammatory mediators. This indicates that NO plays an important role in hippocampal caspase-3 activation during pneumococcal meningitis.

Kainic Acid-induced Neuronal Death is Attenuated by Aminoguanidine but Aggravated by L-NAME in Mouse Hippocampus

Nitric oxide (NO) has both neuroprotective and neurotoxic effects depending on its concentration and the experimental model. We tested the effects of NG-nitro-L-arginine methyl ester (L-NAME), a nonselective nitric oxide synthase (NOS) inhibitor, and aminoguanidine, a selective inducible NOS (iNOS) inhibitor, on kainic acid (KA)-induced seizures and hippocampal CA3 neuronal death. L-NAME (50 mg/kg, i.p.) and/or aminoguanidine (200 mg/kg, i.p.) were administered 1 h prior to the intracerebroventricular (i.c.v.) injection of KA. Pretreatment with L-NAME significantly increased KA-induced CA3 neuronal death, iNOS expression, and activation of microglia. However, pretreatment with aminoguanidine significantly suppressed both the KA-induced and L-NAME-aggravated hippocampal CA3 neuronal death with concomitant decreases in iNOS expression and microglial activation. The protective effect of aminoguanidine was maintained for up to 2 weeks. Furthermore, iNOS knockout mice (iNOS(-/-)) were resistant to KA-induced neuronal death. The present study demonstrates that aminoguanidine attenuates KA-induced neuronal death, whereas L-NAME aggravates neuronal death, in the CA3 region of the hippocampus, suggesting that NOS isoforms play different roles in KA-induced excitotoxicity.

Gene targeting demonstrates that inducible nitric oxide synthase is not essential for resistance to oral candidiasis in mice, or for killing of Candida albicans by macrophages in vitro

Oral candidiasis is caused by opportunistic infections with the yeast Candida albicans. Previous studies have demonstrated important roles for innate immunity and T helper type 1-mediated inflammatory reactions in recovery from infection, with macrophages and neutrophils as key effector cells. Both effector cell types use the inducible isoform of nitric oxide synthase (iNOS) to generate candidacidal molecules, but it is not clear whether nitric oxide (NO) is an absolute requirement for candidacidal effector activity. In this study we directly investigated the role of iNOS-derived NO in resistance to murine experimental oral candidiasis, using iNOS knockout mice. Knockout mice were no more susceptible to oral candidiasis than wild-type controls. Bone marrow-derived macrophages from the knockout mice killed C. albicans yeasts efficiently in vitro, and were still able to produce nitrites in an iNOS-independent manner, albeit less efficiently than wild-type controls. There were no significant differences in local mucosal production of interleukins 6, 12, 17A, or 23, interferon-gamma, or transforming growth factor-beta 24 h after oral challenge with C. albicans. These data suggest that iNOS-derived NO is not required for resistance to oral candidiasis in vivo, and that bone marrow-derived macrophages may have iNOS-independent means of generating reactive nitrogen species.

Inhibition of inducible nitric oxide synthase reduces an acute peripheral motor neuropathy produced by dermal burn injury in mice

The systemic inflammatory response produced by a full-thickness dermal burn injury is associated with a peripheral motor neuropathy. We previously reported that a 20% body surface area (BSA) full-thickness dermal burn in C57BL6 mice produced structural and functional deficits in motor axons at a distance from the burn site. The etiology of the neuropathy, however, is not well characterized. Burn injury leads to an increase in production of a number of proinflammatory mediators, including nitric oxide (NO). We tested the hypothesis that dermal burn-induced motor neuropathy is mediated by increased production of NO. NO synthase (NOS) activity was inhibited following a 20% BSA full-thickness burn by injection of non-specific NOS inhibitor, nitro-L-arginine methyl ester or inducible NOS (iNOS) inhibitors, L-N6-(1-iminoethyl) lysine, and aminoguanidine. NOS inhibitors also prevented the reduction in ventral roots mean axon caliber and the decrease in a motor nerve conduction velocity (MCV) following burn. iNOS knockout mice prevented MCV decrease in the first 3 days post-burn, but iNOS knockout MCV was significantly reduced at 7-14 days post-burn. These results suggest that an increase in NO production generated by systemic inflammatory response pathways after burn injury contributes to the development of structural and functional deficits in peripheral motor axons.

Methylmalonate-induced seizures are attenuated in inducible nitric oxide synthase knockout mice

Methylmalonic acidemias consist of a group of inherited neurometabolic disorders caused by deficiency of methylmalonyl-CoA mutase activity clinically and biochemically characterized by neurological dysfunction, methylmalonic acid (MMA) accumulation, mitochondrial failure and increased reactive species production. Although previous studies have suggested that nitric oxide (NO) plays a role in the neurotoxicity of MMA, the involvement of NO-induced nitrosative damage from inducible nitric oxide synthase (iNOS) in MMA-induced seizures are poorly understood. In the present study, we showed a decrease of time spent convulsing induced by intracerebroventricular administration of MMA (2μmol/2μL; i.c.v.) in iNOS knockout (iNOS−/−) mice when compared with wild-type (iNOS+/+) littermates. Visual analysis of electroencephalographic recordings (EEG) showed that MMA injection induced the appearance of high-voltage synchronic spike activity in the ipsilateral cortex which spreads to the contralateral cortex while quantitative electroencephalographic analysis showed larger wave amplitude during MMA-induced seizures in wild-type mice when compared with iNOS knockout mice. We also report that administration of MMA increases NOx (NO2 plus NO3 content) and 3-nitrotyrosine (3-NT) levels in a greater extend in iNOS+/+ mice than in iNOS−/− mice, indicating that NO overproduction and NO-mediated damage to proteins are attenuated in iNOS knockout mice. In addition, the MMA-induced decrease in Na+, K+-ATPase activity, but not in succinate dehydrogenase (SDH) activity, was less pronounced in iNOS−/− when compared with iNOS+/+ mice. These results reinforce the assumption that metabolic collapse contributes for the secondary toxicity elicited by MMA and suggest that oxidative attack by NO derived from iNOS on selected target such as Na+, K+-ATPase enzyme might represent an important role in this excitotoxicity induced by MMA. Therefore, these results may be of value in understating the pathophysiology of the neurological features observed in patients with methylmalonic acidemia and in the development of new strategies for treatment of these patients.

Pioglitazone protects the myocardium against ischemia-reperfusion injury in eNOS and iNOS knockout mice

Endothelial nitric oxide synthase (eNOS) activation with subsequent inducible NOS (iNOS), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase-2 (COX2) activation is essential to statin inhibition of myocardial infarct size (IS). In the rat, the peroxisome proliferator-activated receptor-gamma agonist pioglitazone (Pio) limits IS, upregulates and activates cPLA2 and COX2, and increases myocardial 6-keto-PGF1alpha levels without activating eNOS and iNOS. We asked whether Pio also limits IS in eNOS-/- and iNOS-/- mice. Male C57BL/6 wild-type (WT), eNOS-/-, and iNOS-/- mice received 10 mg.kg(-1).day(-1) Pio (Pio+) or water alone (Pio-) for 3 days. Mice underwent 30 min coronary artery occlusion and 4 h reperfusion, or hearts were harvested and subjected to ELISA and immunoblotting. As a result, Pio reduced IS in the WT (15.4+/-1.4% vs. 39.0+/-1.1%; P<0.001), as well as in the eNOS-/- (32.0+/-1.6% vs. 44.2+/-1.9%; P<0.001) and iNOS-/- (18.0+/-1.2% vs. 45.5+/-2.3%; P<0.001) mice. The protective effect of Pio in eNOS-/- mice was smaller than in the WT (P<0.001) and iNOS-/- (P<0.001) mice. Pio increased myocardial Ser633 and Ser1177 phosphorylated eNOS levels in the WT and iNOS-/- mice. iNOS was undetectable in all six groups. Pio increased cPLA2, COX2, and PGI2 synthase levels in the WT, as well as in the eNOS-/- and iNOS-/-, mice. Pio increased the myocardial 6-keto-PGF1alpha levels and cPLA2 and COX2 activity in the WT, eNOS-/-, and iNOS-/- mice. In conclusion, the myocardial protective effect of Pio is iNOS independent and may be only partially dependent on eNOS. Because eNOS activity decreases with age, diabetes, and advanced atherosclerosis, this effect may be relevant in a clinical setting and should be further characterized.

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

2093 CMR reveals that cardiac-specific overexpression of the inducible form of nitric oxide synthase induces Left Ventricular Hypertrophy in wild-type mice after AAV-Mediated direct gene transfer

Introduction CMR has previously shown that left ventricular remodeling (LVR) resulting from reperfused myocardial infarction (MI) is dramatically reduced in iNOS knock-out mice [1]. However, previous studies using transgenic mice with cardiac-specific overexpression of iNOS have yielded disparate results. One study reported a benign phenotype while a second reported that iNOS overexpression resulted in cardiac fibrosis, hypertrophy and dilatation. One explanation is that transgenic mice may develop compensatory mechanisms to down-regulate iNOS overexpression. Another possiblity is that the phenotype of iNOS overexpression may be variable in its penetrance due to limited availability of a necessary co-factor (tetrahydrobiopterin).

Bone marrow stromal cells induce apoptosis of lymphoma cells in the presence of IFNγ and TNF by producing nitric oxide

Bone marrow stromal cells (BMSCs) have been shown to promote the growth and survival of a wide variety of tumors. However, in the present study, we found that BMSCs induced apoptosis of lymphoma cells in the presence of INFγ and TNF. IFNγ and TNF dramatically induced the expression of inducible nitric oxide synthase (iNOS) by BMSCs in culture, and BMSCs generated from iNOS knockout mice did not induce apoptosis of lymphoma cells in the presence of IFNγ and TNF. In addition, we found that IFNγ and TNF also increased IL-6 expression by BMSCs, and anti-IL-6 further increased the killing of tumor cells by BMSCs. Taken together, our findings indicate that BMSCs induce apoptosis of lymphoma cells in the presence of IFNγ and TNF, and that the proapoptotic effect of BMSCs is mediated by nitric oxide. Our findings suggest a possibility to harness this proapoptotic feature of BMSCs for the development of novel therapeutic strategy to eliminate tumor cells, especially tumor cells in bone marrow.

Nitric oxide depresses connexin 43 after myocardial infarction in mice

Heart failure (HF) is a major cause of death and morbidity. Connexin 43 (Cx43) content is reduced in the failing myocardium, but regulating factors have not been identified. In HF, inducible nitric oxide synthase (iNOS)-induced high levels of nitric oxide (NO) cause apoptosis and cardiac dysfunction. However, a direct iNOS-Cx43 link has not been demonstrated. We investigated this relationship in mice after myocardial infarction. Effects of myocardial infarction were evaluated 2 weeks after coronary artery ligation in wild-type C57BL/6 (WT) and iNOS(-/-) knockout mice. Myocardial Cx43 and Cx45 content were assessed by immunofluorescence confocal imaging and western blotting. Cardiac function was evaluated in anaesthetized mice using a micro pressure-tipped catheter inserted into the left ventricle. Despite similar infarct size, deficiency in iNOS resulted in significantly lower plasma nitrate/nitrite levels, better haemodynamic performance and lower mortality 2 weeks after coronary ligation. Myocardial Cx43, but not Cx45, content was lower in WT mice following ligation. The reduction in Cx43 was less in iNOS(-/-) compared with WT mice. To assess the direct effect of NO on Cx43 expression, cultured neonatal mouse cardiomyocytes were employed. Incubation with the NO donor, S-nitroso-N-acetylpenicillamine, elicited a dose-dependent decrease in Cx43 content in cultured neonatal cardiomyocytes. Increased NO production from iNOS depressed cardiac performance and contributed to the decreased myocardial Cx43 content 2 weeks after myocardial infarction.

Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis

Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression.

Additional lack of iNOS attenuates diastolic dysfunction in aged ET-1 transgenic miceThis article is one of a selection of papers published in the special issue (part 1 of 2) on Forefronts in Endothelin.

Endothelin-1 (ET-1) exhibits potent proinflammatory and profibrotic properties. Moreover, inflammation is a potent stimulus for inducible NO synthase (iNOS), which has been shown to contribute to cardiac injury. We thus hypothesized that ET-1-induced cardiac injury is attenuated by concomitant lack of iNOS. We established crossbred animals of ET-1 transgenic mice (ET+/+) and iNOS knockout mice (iNOS-/-). At 13 months of age, mice were allocated according to their genotype to one of 4 study groups: wild type (WT) controls (n=8); ET-1 transgenic (ET+/+) mice (n=10); iNOS knockout (iNOS-/-) mice (n=7); and crossbred (ET+/+ iNOS-/-) mice (n=15). Left ventricular function was determined in vivo by using a tip catheter. Animals were subsequently euthanized and hearts were harvested for weight assessment and histologic evaluation. No cardiac hypertrophy was present, as evidenced by similar mean cardiac weight and myocyte diameter in all groups. Cardiac perivascular fibrosis was significantly increased in ET+/+ and iNOS-/- groups versus WT, whereas ET+/+ iNOS-/- mice did not differ from WT. Regarding left ventricular function, plasma B-type natriuretic peptide was elevated in ET+/+ and iNOS-/- mice, but again in crossbred animals this effect was blunted. Heart catheterization revealed a significantly increased stiffness constant in both ET-overexpressing groups versus WT, but this increase was significantly attenuated in the ET+/+iNOS-/- group versus the ET+/+ group. Parameters indicating systolic heart failure (EF, cardiac output), however, were not different between all study groups. Our study demonstrates that ET transgenic mice develop left ventricular stiffening with subsequent diastolic dysfunction in a slow, age-dependent manner. Additional knock out of iNOS significantly attenuates cardiac injury. We thus conclude that ET-1-induced cardiac injury is at least partially mediated by iNOS.

Cardiac Fibrogenesis Following Infarction in Mice With Deletion of Inducible Nitric Oxide Synthase

Studies have shown that the absence of inducible nitric oxide synthase (iNOS) improves cardiac function and survival after myocardial infarction (MI). The responsible mechanisms, however, remain uncertain. Cardiac iNOS is significantly increased after MI, which is colocalized with fibrous tissue formation. Herein, we tested our hypothesis that iNOS is involved in the development of cardiac fibrosis. Wild-type and iNOS-knockout mice were subjected to MI by left coronary artery ligation. At week 1, 2, 3, and 4 post-MI, we addressed cardiac expression of profibrogenic mediator, growth of collagen-producing cells, collagen synthesis, and degradation. In the infarcted myocardium of wild-type and iNOS-knockout mice, transforming growth factor (TGF)-beta1 expression was significantly increased, particularly in the early stage; myofibroblasts appeared and became abundant for over 4 weeks; matrix metalloproteinase-1 expression was low, whereas tissue inhibitor of matrix metalloproteinase-1 was significantly elevated; type-I collagen mRNA was significantly increased and collagen was continuously accumulated. In the noninfarcted myocardium, TGF-beta1 and type-I collagen mRNA levels as well as collagen volume were also elevated, but less evident than infarcted myocardium. However, there was no significant difference in cardiac TGF-beta1 expression, myofibroblast population, collagen synthesis/degradation, and collagen volume between wild-type and iNOS-knockout mice with MI. The current study suggests that iNOS-induced nitric oxide production may not mediate cardiac fibrosis after MI. Thus, other mechanisms are involved in nitrosative stress-induced cardiac dysfunction after MI.

Estradiol abolishes reduction in cell death by the opioid agonist Met5-enkephalin after oxygen glucose deprivation in isolated cardiomyocytes from both sexes

There is evidence for differences in the response to the treatment of cardiovascular disease in men and women. In addition, there are conflicting results regarding the effectiveness of pharmacologically induced protection or ischemic preconditioning in females. We investigated whether the ability of Met(5)-enkephalin (ME) to reduce cell death after oxygen-glucose deprivation (OGD) is influenced by the presence of 17beta-estradiol (E(2)) in a nitric oxide (NO)- and estrogen receptor-dependent manner. On postnatal day 7 to 8, murine cardiomyocytes from wild-type or inducible NO synthase (iNOS) knockout mice were separated by sex, isolated by collagenase digestion, cultured for 24 h, and subjected to 90 min OGD and 180 min reoxygenation at 37 degrees C (n = 4 to 5 replicates). Cell cultures were incubated in E(2) for 15 min or 24 h before OGD. ME was used to increase cell survival. Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. Data are presented as means +/- SE. As a result, in both sexes, ME-induced cell survival was lost in the presence of E(2), and the ability of ME to improve cell survival was restored after treatment with the estrogen receptor antagonist ICI-182780. Furthermore, iNOS was necessary for ME to increase cell survival following OGD in vitro. We conclude that ME-induced reduction in cell death is abolished by E(2) in a sex-independent manner via activation of estrogen receptors, and this interaction is dependent on iNOS.

Inducible Nitric Oxide Synthase (iNOS) is Not Required for IL-2–induced Hypotension and Vascular Leak Syndrome in Mice

Dose limiting side effects of interleukin-2 (IL-2) include severe hypotension and vascular leak syndrome (VLS). Previous studies have shown that nitric oxide (NO) synthesis is strongly induced after IL-2 treatment of C3H/HeN mice (180,000 IU b.i.d. for 5 d). We employed knockout mice (on C57BL/6 background) to test the role of the inducible NO synthase (iNOS) in mediating IL-2 toxicity. In contrast to C3H/HeN mice, which developed hypotension and VLS after 10 doses of only 180,000 IU IL-2, C57BL/6 mice were far more resistant requiring increased doses of 800,000 IU IL-2 (b.i.d., 5 d) to induce hypotension and VLS. Serum interferon-gamma levels were significantly more elevated by IL-2 treatment in C3H/HeN mice than in C57BL/6, correlating with the severity of hypotension and VLS. Urinary excretion of NO metabolites was markedly reduced in iNOS knockout mice (C57BL/6 iNOS) after IL-2 treatment. A surprising finding was that these mice still developed profound hypotension and VLS. Similar findings were observed after administration of a iNOS specific inhibitor, L-N[6]-(1-iminoethyl)lysine (L-NIL). In contrast, a general NOS inhibitor, N-monomethyl-L-arginine, prevented both hypotension and vascular leak. The superoxide dismutase mimetic, M40403, reversed IL-2-induced hypotension but not VLS in knockout mice. Thus, peroxynitrite-mediated mechanisms are likely responsible for hypotension, whereas NO-induced changes in vascular permeability result in VLS. The iNOS enzyme is not necessary for pathogenesis of IL-2-induced cardiovascular toxicity. These results imply that other NOS isoforms, such as endothelial NOS, may play a major role in the development of IL-2-induced cardiovascular toxicity.

Inhibition of reactive oxygen species attenuates aneurysm formation in a murine model

Reactive oxygen species (ROS) are increased in human abdominal aortic aneurysms (AAA). NADPH oxidases are the predominant source of superoxide anion (O 2 −) in the vasculature. Inducible nitric oxide synthase (iNOS) produces a significant amount of nitric oxide (NO) during inflammatory processes. We hypothesized that ROS produced by NADPH oxidases and iNOS played an important role in aneurysm formation. We examined this hypothesis using selective blockade of NADPH oxidases and iNOS in a murine model of AAA. Mice, including C57BL/6, iNOS knockout (iNOS −/−) mice, and its background matched control (C57BL/6), underwent AAA induction by periaortic application of CaCl 2. Aortic diameter was measured at aneurysm induction and harvest. Beginning 1 week prior to aneurysm induction and continuing to aortic harvest 6 weeks later, one group of the C57BL/6 mice were treated with orally administered apocynin (NADPH oxidase inhibitor). Control mice were given water. The mean diameter and change in diameter of each group were compared with concurrent controls. Aortic levels of the NO metabolite, NO x (NO 2 and NO 3), were significantly increased in CaCl 2-treated wild type mice. INOS −/− mice were partly resistant to aneurysm induction. This was associated with reduced expression of matrix metalloproteinase (MMP)-2 and MMP-9 and decreased production of NO x in the aortic tissues. Inhibition of NADPH oxidase by apocynin also blocked aneurysm formation. In conclusion, both iNOS deficiency and NADPH oxidase inhibition suppressed aneurysm formation in association with decreased NO x levels. These studies suggest that both NADPH oxidase and iNOS pathways contribute to ROS production and AAA development.

Effect of Inducible Nitric Oxide Synthase on Cerebral Blood Flow after Experimental Traumatic Brain Injury in Mice

Inducible nitric oxide synthase (iNOS) has been suggested to play a complex role in the response to central nervous system insults such as traumatic brain injury (TBI) and cerebral ischemia. In the current study, we quantified maps of regional cerebral blood flow (CBF) using an arterial spin-labeling magnetic resonance imaging (MRI) technique, at 24 and 72 h after experimental TBI in iNOS knockout (KO) and wild-type (WT) mice. Our hypothesis was that iNOS would contribute to the level of CBF at 72 h after experimental TBI in mice. Comparing anatomical brain regions of interest (ROIs) at 24-h post controlled cortical impact (CCI), there were significant reductions in CBF in the hemisphere, cortex, and contusion-rich area of the cortex of injured animals versus naive, regardless of genotype. Regional assessment of CBF at 72 h after injury demonstrated that recovery of CBF was reduced in the ipsilateral hippocampus, thalamus, and amygdala/piriform cortex in iNOS KO versus WT mice by 26%, 15%, and 21%, respectively; this attenuated recovery was restricted to structures outside the contusion. These regions with reduced CBF in iNOS KO mice represented ROIs where CBF in the WT was either numerically or statistically greater than that seen in respective WT naive, suggesting a contribution of iNOS to delayed posttraumatic hyperemia. However, pixel analysis denoted that the contribution of iNOS to CBF at 72 h was not limited to hyperemia flows. In conclusion, iNOS plays a role in the recovery of CBF after CCI in mice. Questions remain if this effect represents a homeostatic component of CBF recovery, pathologic vasodilatation linked to inflammation, or NO-mediated facilitation of angiogenesis.