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Deinococcus radiodurans (Drad) is the most radioresistant organism known. Although mechanisms that underlie the extreme radioresistance of Drad are incompletely defined, resistance to UV irradiation-induced killing was found to be greatly attenuated in an NO synthase (NOS) knockout strain of Drad (Δnos). We now show that endogenous NO production is also critical for protection of Drad against γ-irradiation (3000 Gy), a result of accelerated growth recovery, not protection against killing. NO-donor treatment rescued radiosensitization in Δnos Drad but did not influence radiosensitivity in wild type Drad. To discover molecular mechanisms by which endogenous NO confers radioresistance, metabolite profiling studies were performed. Untargeted LC-MS-based metabolite profiling in Drad quantified relative abundances of 1425 molecules and levels of 294 of these were altered by >5-fold (p < 0.01). Unexpectedly, these studies identified a dramatic perturbation in carotenoid biosynthetic intermediates in Δnos Drad, including a reciprocal switch in the pathway end-products from deoxydeinoxanthin to deinoxanthin. NO supplementation rescued these nos deletion-associated changes in carotenoid biosynthesis, and fully-restored radioresistance to wildtype levels. Because carotenoids were shown to be important contributors to radioprotection in Drad, our findings suggest that endogenously-produced NO serves to maintain a spectrum of carotenoids critical for Drad's ability to withstand radiation insult., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although all three nitric oxide (NO) synthases (nNOS, iNOS, and eNOS) are expressed in injured arteries, it remains to be elucidated the role of the NOSs in their entirety in the vascular lesion formation. We addressed this issue in mice deficient in all NOS genes. Vascular injury was induced by permanent ligation of a unilateral carotid artery in wild-type (WT), singly, and triply NOS(-/-) mice. Two weeks after the procedure, constrictive vascular remodeling and neointimal formation were recognized in the ligated arteries. While constrictive remodeling was noted in the nNOS(-/-) and iNOS(-/-) genotypes, it was most accelerated in the n/i/eNOS(-/-) genotype. While neointimal formation was evident in the eNOS(-/-) and nNOS(-/-) genotypes, it was also most aggravated in the n/i/eNOS(-/-) genotype. Those lesions were reversed by long-term treatment with isosorbide dinitrate, a NO donor. Finally, we examined the involvement of bone marrow-derived cells in the vascular lesion formation. Bone marrow from the WT, singly, or triply NOS(-/-) mice was transplanted into the WT mice, and then the carotid ligation was performed. Intriguingly, constrictive remodeling and neointimal formation were both similarly most exacerbated in the case of the n/i/eNOS(-/-) bone marrow transplantation. These results indicate that the complete disruption of all the NOS genes causes markedly accelerated vascular lesion formation caused by blood flow disruption in mice in vivo, demonstrating the crucial vasculoprotective role of the whole endogenous NOS system. Our findings also suggest that the NOS system in bone marrow-derived cells may be involved in this vasculoprotective mechanism., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: The objective of the study was to investigate the transgenerational effect of fetal vascular programming, Study Design: Homozygous NOS3 knockout and wild type controls (NOS3+/+WT) were cross-bred to obtain heterozygous offspring that developed in (KO-/-) mothers lacking a functional NOS3 (KOM) vs wild-type control mothers (KOP). The first-generation KOM(+/-) and KOP(+/-) female mice were then bred with WT(+/+) males to obtain a second generation (F2). F2 offspring were genotyped and WT(+/+)-F2 mice were then used for in vivo blood pressure and in vitro vascular reactivity studies., Results: WT-F2 mice born to KOM mothers (KOM-F2WT) had significantly higher systolic blood pressure, mean arterial pressure, and pulse pressure, compared with WT-F2 born to KOP mothers. Male KOM-F2WT offspring had significantly increased response to phenylephrine (PE), compared with male KOP-F2WT. Male offspring had increased contractile responses to PE when compared with female. Acetylcholine responses were decreased in female KOM-F2WT, compared with female KOP-F2WT, but the difference was not statistically significant, Conclusion: Our findings support a transgenerational effect of fetal programming on the vascular phenotype and suggest possible gender specific adaptation.

Objective: The purpose of this study was to investigate vascular reactivity in heterozygous and homozygous offspring with a genetic predisposition for hypertension after postnatal cross-fostering to mothers with the opposite genetic inheritance of the NOS3 knockout allele., Study Design: Homozygous NOS3 knockout (C57BL/6J-NOS3(-/-KO)) and wild-type mice (NOS3(+/+WT)) were bred to obtain heterozygous litters with a paternally derived (NOS3(+/-pat)) or maternally derived (NOS3(+/-mat)) knockout allele. After delivery, heterozygous and homozygous litters were cross-fostered to a mother with the opposite NOS3 gene status. Carotid arteries were placed in a wire myograph for isometric tension recording with the use of contractile and relaxant agents. Statistical analysis with 1-way analysis of variance and Neuman-Keuls post-hoc testing was performed., Results: Increased sensitivity to phenylephrine and absent relaxation to acetylcholine in NOS3(+/-mat) was reversed with cross-fostering, and vasorelaxation to isoproterenol was increased. Contraction to calcium was increased in the cross-fostered paternally derived and wild-type litters., Conclusion: Postnatal interventions may alter the adult vascular profile favorably that is the result of an abnormal intrauterine environment.

The balance of arginine metabolism via nitric oxide synthase (NOS) or arginase is an important determinant of the inflammatory response of murine macrophages and dendritic cells. Here we analyzed the expression of the isoform arginase I in human myeloid cells. Using healthy donors and patients with arginase I deficiency, we found that in human leukocytes arginase I is constitutively expressed only in granulocytes and is not modulated by a variety of proinflammatory and anti-inflammatory stimuli in vitro. We demonstrate that arginase I is localized in azurophil granules of neutrophils and constitutes a novel antimicrobial effector pathway, likely through arginine depletion in the phagolysosome. Our findings demonstrate important differences between murine and human leukocytes with respect to regulation and function of arginine metabolism via arginase.

The role of inducible nitric oxide synthase (iNOS) in the progression of fibrosis during nonalcoholic steatohepatitis remains to be elucidated. This study examined the role of iNOS in the progression of fibrosis during steatohepatitis by comparing iNOS knockout (iNOS(-/-)) and wild-type (iNOS(+/+)) mice that were fed a high-fat diet. Severe fatty metamorphosis developed in the liver of iNOS(+/+) and iNOS(-/-) mice. Fibrotic changes were marked in iNOS(-/-) mice. Gelatin zymography showed that pro MMP-2 and pro MMP-9 protein expressions were more highly induced in iNOS(+/+) mice than in iNOS(-/-) mice. Active forms of MMP-2 and MMP-9 were clearly present only in the liver tissue of iNOS(+/+) mice. In situ zymography showed strong gelatinolytic activities in the liver tissue of iNOS(+/+) mice, but only spotty activity in iNOS(-/-)mice. iNOS may attenuate the progression of liver fibrosis in steatohepatitis, in part by inducing MMP-2 and MMP-9 expression and augmenting their activity.

Nitric oxide (NO) is involved in many physiological processes and also causes pathological effects by inducing apoptosis. It can enhance or suppress apoptosis depending on its concentration and the cell type involved. In this report, we used cDNA microarray analysis to show that SNAP, an NO donor, strongly induces Bcl-2/adenovirus E1B 19kDa-interacting protein 3 (BNIP3) in macrophages. BNIP3 is a mitochondrial pro-apoptotic protein that contains a Bcl-2 homology 3 domain and a COOH-terminal transmembrane (TM) domain. Macrophages activated by LPS/IFN-gamma produce nitric oxide synthase 2 (NOS2) and release endogenous NO. Expression of BNIP3 was also induced in macrophages by LPS/IFN-gamma, and the induction was blocked by a NOS2 inhibitor, S-methyl-isothiourea. Peritoneal macrophages from NOS2-null mice failed to produce BNIP3 in response to LPS/IFN-gamma. We conclude that BNIP3 expression in macrophages is controlled by the intracellular level of nitric oxide. Overexpression of BNIP3 but not of BNIP3 deltaTM, a BNIP3 mutant without the TM domain and C-terminal tail, led to apoptosis of the cells. Promoter analysis showed that the region between -281 and -1 of the 5'-upstream enhancer region of murine BNIP3 was sufficient for NO-dependent expression of BNIP3.

It is well established that cytokines such as tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) are involved in physiological sleep regulation, yet their downstream somnogenic mechanisms remain largely uninvestigated. Nitric oxide (NO) is an effector molecule for some TNFalpha actions. Neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) gene knockout (KO) mice sleep differently than their respective controls. In this study, we tested the hypothesis that NO mediates TNFalpha-induced sleep using iNOS and nNOS KO mice and their corresponding wild-type controls. Systemic administration of TNFalpha increased non-rapid eye movement sleep (NREMS) in the two control strains and in the iNOS KO mice during the first 4 h post-injection but failed to increase NREMS in nNOS KO mice. Rapid eye movement sleep (REMS) was suppressed by TNFalpha in nNOS controls but not in the other strains examined. The results suggest that TNFalpha affects sleep, in part, through nNOS.

Endogenous nitric oxide (NO) is known to modulate post-ischemic inflammatory response in various organs. However, the role of nitric oxide synthase isoforms (NOS) in mediating pulmonary post-ischemic inflammatory response is poorly understood. We therefore studied post-ischemic endothelial adhesion molecule expression and leukocyte migration in endothelial NOS knockout (eNOS-KO) mice subjected to pulmonary ischemia and reperfusion in vivo. Under anesthesia and mechanical ventilation, the left pulmonary hilum in wild-type (WT) and eNOS-KO mice was clamped for 1 hour, followed by reperfusion for up to 24 hours. In WT mice, we observed a selective up-regulation of both eNOS mRNA and protein in lung tissue, while inducible NOS (iNOS) and neuronal NOS (nNOS) remained unchanged. Survival in eNOS-KO mice was reduced due to severe pulmonary edema, underlining an increased susceptibility to ischemia-reperfusion (I/R) injury. Interstitial tissue infiltration by CD18- and CD11a-positive white blood cells as well as lung tissue water content peaked at 5 hours of reperfusion and were found significantly higher than in WT mice. Enhanced leukocyte-endothelial interaction was associated with pronounced up-regulation of vascular cell adhesion molecule (VCAM) in eNOS-KO mice during post-ischemic reperfusion. We conclude that eNOS attenuates post-ischemic inflammatory injury to the lung most probably via inhibition of endothelial adhesion molecule expression.

In this study, the role of nitric oxide (NO) in neuronal destruction during acute-phase Trypanosoma cruzi infection was evaluated in male C57BL/6 (WT, wild-type) mice and knockout mice [inducible nitric oxide synthase (iNOS)(-/-) and interferon (IFN)(-/-)]. Selected animals were infected by intraperitoneal injection of 100 trypomastigote forms of the Y strain of T. cruzi. Others were injected intraperitoneally with an equal volume of saline solution and served as controls. Our findings support those of previous studies regarding myenteric denervation in acute-phase T. cruzi infection. In addition, we clearly demonstrate that, despite the fact that parasite nests and similar inflammatory infiltrate in the intestinal wall were more pronounced in infected iNOS(-/-) mice than in infected WT mice, the former presented no reduction in myenteric plexus neuron numbers. Neuronal nerve profile expression, as revealed by the general nerve marker PGP 9.5, was preserved in all knockout animals. Infected IFN(-/-) mice suffered no significant neuronal loss and there was no inflammatory infiltrate in the intestinal wall. On days 5 and 10 after infection, iNOS activity was greater in infected WT mice than in controls, whereas iNOS activity in infected knockout mice remained unchanged. These findings clearly demonstrate that neuronal damage does not occur in NO-impaired infected knockout mice, regardless of whether inflammatory infiltrate is present (iNOS(-/-)) or absent (IFN(-/-)). In conclusion, our observations strongly indicate that myenteric denervation in acute-phase T. cruzi infection is because of IFN-gamma-elicited NO production resulting from iNOS activation in the inflammatory foci along the intestinal wall.

Cushing's syndrome and systemic administration of glucocorticoids are associated with hypertension, but the underlying molecular mechanism is only partially understood. We have shown previously that dexamethasone downregulates the expression of the endothelial NO synthase (eNOS) gene in human endothelial cells and in the rat and that this may contribute to the blood pressure-raising effect of the steroid [Proc. Natl. Acad. Sci. USA 96 (1999) 13357]. In the current communication, we demonstrated that dexamethasone increased mean arterial blood pressure in wild-type C-57 Bl6 mice (eNOS+/+ mice), but had no effect on blood pressure in mice with a disrupted eNOS gene (eNOS-/- mice) derived from the same strain. The NOS inhibitor ethylisothiourea, used for control purposes, showed a hypertensive effect in eNOS+/+ mice, but no such effect in eNOS-/- mice. Serum NO2-/NO3- levels, an indicator of total body NO synthesis, decreased significantly when eNOS+/+ mice were treated with dexamethasone. eNOS-/- mice had lower serum NO2-/NO3- levels per se, which were not changed significantly by dexamethasone. Dexamethasone decreased the expression of eNOS in three major organs of the mouse investigated, namely the heart, the liver, and the kidney. We conclude that the expressional downregulation of eNOS and the ensuing reduction in vascular NO production contributes to the hypertension caused by glucocorticoids.

Nitric oxide (NO), produced by NO-synthase (NOS), serves as an important vasodilator and inhibitory neurotransmitter. Inducible NOS (iNOS) is expressed in response to cytokine stimulation and is therefore not ordinarily present in healthy tissue. However, iNOS has been identified in certain organs, including the penis. The development of mice deficient in the iNOS gene (iNOS -/-) has provided a useful tool for the study of iNOS function. Therefore, an in vitro examination of vascular and nerve-mediated responses of corpus cavernosum (CC) and vascular responses of aorta from iNOS -/- mice and their wild-type controls was undertaken. Tissues were mounted in organ baths for agonist- and/or electrical field stimulation (EFS)-induced responses under isometric tension. CC from iNOS -/- mice developed increased sensitivity to phenylephrine (PE) and an increased maximum EFS-induced noradrenergic contraction of approximately 31%. Following PE precontraction, maximum relaxation to acetylcholine was reduced by approximately 39%; conversely, there was a 23% increase in relaxation to the NO-donor sodium nitroprusside. EFS-induced non-adrenergic, non-cholinergic (NANC) nerve-mediated relaxation was unaltered compared to control. Agonist-induced responses of aorta did not significantly differ between iNOS -/- and control mice. These results suggest that iNOS-derived NO may play a role in modulating erectile function and confirm that iNOS does not play a significant role in macrovascular function under normal physiological conditions.

Background/aims: Inducible nitric oxide synthase (iNOS) is found to have antiviral activity. Its role is evaluated using a murine acute hepatitis B virus (HBV) infection model., Methods: pHBV3.6 plasmid containing HBV genome was injected into mice by hydrodynamics-based in vivo transfection. HBV antigenemia and serum HBV-DNA were detected by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction, respectively. HBV replication in liver was analyzed by Northern and Southern blot. Intrahepatic leukocytes were isolated and analyzed with flow cytometry., Results: After hydrodynamics injection of pHBV3.6, HBV genome was synthesized in the liver and HBV-DNA, as well as hepatitis B surface antigen and hepatitis B e antigen were secreted into the blood. Anti-HBV antibody responses developed afterward. A murine acute HBV infection model was established with hydrodynamics injection of non-transponase based HBV-DNA. Using this protocol in iNOS deficient or wild type B6 mice, the level of HBV transcript, replicative intermediate, and antigenemia were higher in iNOS(-/-) than in B6 mice. The intrahepatic leukocytes in iNOS(-/-) mice were also affected after transfection., Conclusions: Our data suggests that the iNOS expression not only affects the HBV clearance, but also modulates the infiltrating leukocytes response to HBV antigens.

Expression of adrenomedullin, discovered as a vasodilatory peptide, is markedly up-regulated under pathological conditions such as tissue ischemia and inflammation, which are associated with neovascularization. Here, we tested the hypothesis that overly expressed adrenomedullin may augment collateral flow to ischemic tissues. We induced hindlimb ischemia in wild-type mice and injected a naked plasmid expressing human adrenomedullin or an empty vector into the ischemic muscle, followed by in vivo electroporation. Adrenomedullin markedly enhanced blood flow recovery as determined by Laser Doppler imaging. The mice treated with an empty vector suffered frequent autoamputation of the ischemic toe, which was completely prevented by adrenomedullin. Anti-CD31 immunostaining revealed that adrenomedullin significantly increased capillary density. The angiogenic effect of adrenomedullin was abrogated in endothelial nitric oxide synthase (eNOS)-deficient mice. These results indicate that adrenomedullin may promote collateral growth in response to ischemia through activation of eNOS.

Although nitric oxide (NO) plays important roles in pathogenesis of various liver diseases, the role of NO in the in vivo mechanism of Fas-mediated fulminant hepatitis is not known well. The effect of anti-Fas antibody (Jo2) on the survival, liver function, and histology was analyzed in wild-type (WT) and inducible NO synthase (iNOS)-deficient (iNOS(-/-)) mice. Upon intravenous injection of a lethal dose of Jo2, WT mice died on fulminant hepatitis within 12h. Under identical conditions, however, iNOS(-/-) mice showed strong resistance to Jo2 and survived without revealing liver injury. In conclusion, these observations suggest that regulation of NO metabolism may have therapeutic potential in the treatment of patients with fulminant hepatitis.

Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by N(G)-nitro-L-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only approximately 55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis.

Background: Arginine is required for the detoxification of ammonia and the synthesis of proteins, nitric oxide, agmatine, creatine, and polyamines, and it may promote lymphocyte function. In suckling mammals, arginine is synthesized in the enterocytes of the small intestine, but this capacity is lost after weaning., Objective: We investigated the significance of intestinal arginine production for neonatal development in a murine model of chronic arginine deficiency., Design: Two lines of transgenic mice that express different levels of arginase I in their enterocytes were analyzed., Results: Both lines suffer from a selective but quantitatively different reduction in circulating arginine concentration. The degree of arginine deficiency correlated with the degree of retardation of hair and muscle growth and with the development of the lymphoid tissue, in particular Peyer's patches. Expression of arginase in all enterocytes was necessary to elicit this phenotype. Phenotypic abnormalities were reversed by daily injections of arginine but not of creatine. The expression level of the very arginine-rich skin protein trichohyalin was not affected in transgenic mice. Finally, nitric oxide synthase-deficient mice did not show any of the features of arginine deficiency., Conclusions: Enterocytes are important for maintaining arginine homeostasis in neonatal mice. Graded arginine deficiency causes graded impairment of skin, muscle, and lymphoid development. The effects of arginine deficiency are not mediated by impaired synthesis of creatine or by incomplete charging of arginyl-transfer RNA.

Short episodes of ischemia and reperfusion in various organs may protect the organ itself, and the heart both as an immediate and a delayed effect. The present study investigates whether a systemic protection of vascular function occurs during adaption to ischemia. Brain ischemia was induced by bilateral ligation of the internal carotid arteries in C57BL6 mice, and 24-36 hours later rings of the thoracic aorta were mounted to study in vitro relaxation and contraction, or proteins were extracted for immunoblotting for endothelial nitric oxide synthase (eNOS) or inducible NOS (iNOS). eNOS decreased, while iNOS increased in the aortic wall after carotid artery ligation. In vitro contraction to increasing concentrations of prostaglandin F(2alpha) (PGF(2alpha)) was attenuated, while relaxation to acetylcholine (ACh) was enhanced. The latter was abolished by the iNOS-inhibitor aminoguanidine. When brain ischemia was induced in iNOS deficient mice, an increase of aortic eNOS was found 24 hours later. The ischemia-induced attenuated relaxation to PGF(2alpha) and enhanced relaxation to ACh were abolished. Aortic rings from mice with severe atherosclerosis (apolipoprotein E and low density lipoprotein receptor double knockout (ApoE/LDLr KO) mice) and spontaneous ischemic events in the heart or brain in vivo were also studied. Spontaneous ischemic events in ApoE/LDLr KO animals did not influence iNOS and eNOS in the vessel wall. A reduced contraction to PGF(2alpha) was observed, but relaxation to ACh was unchanged. These findings suggest that induced brain ischemia as a model of delayed, remote preconditioning protects vessel reactivity, and this protection is mediated by iNOS.

Acute administration of cadmium (Cd) to rats results in hepatotoxicity. Recent reports indicate that Kupffer cells, the resident macrophages of the liver, participate in the manifestation of Cd-induced hepatotoxicity. Nitric oxide (NO) is a reactive nitrogen radical produced by activated Kupffer cells via the induction of inducible nitric oxide synthase (iNOS). Nitric oxide can combine with superoxide to form peroxynitrite, a molecule that may participate in the toxic mechanisms of hepatotoxins, such as acetaminophen and bacterial endotoxin. It has been speculated that Cd also may exert its hepatotoxicity, in part, via the production of NO by iNOS. Therefore, this study was undertaken to determine whether iNOS contributes to Cd-induced hepatotoxicity. Wild-type (WT) mice were administered selective iNOS inhibitors (AMT and 1400W) concurrently and 3 h after administration of a hepatotoxic dose of Cd (4.0 mg Cd/mg). Additionally, WT and iNOS-null (iNOS-KO) mice were dosed iv with saline or 2.0, 2.5, 3.0, 3.5 or 4.0 mg Cd/kg. Serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities were quantified to assess liver injury. Administration of iNOS inhibitors failed to prevent Cd-induced hepatotoxicity. Also, Cd caused a dose-dependent increase in liver injury in both WT and iNOS-KO mice. The liver injury produced by Cd in the iNOS-KO mice was not different from that in WT at any dose. These data indicate that iNOS does not appear to mediate Cd-induced hepatotoxicity.

In acute glomerulonephritis (GN), increased nitric oxide (NO) production occurs, suggesting a pathophysiological role for NO in the disease process. Although NO potentially could have both toxic as well as protective effects, its exact role in the pathophysiology of GN is unclear and may depend on the NOS isoform generating NO. The protective effects of NO such as prevention of leukocyte and platelet activation and adhesion have been attributed to NO generated by endothelial nitric oxide synthase (eNOS). Evidence for a beneficial role for eNOS includes the demonstration of reduced eNOS expression in experimental models of GN as well as human biopsy specimens that is mostly likely due to endothelial cell necrosis. Reduced NO production in GN also may occur through reaction of NO with superoxide anions or the myeloperoxidase (MPO)/hypochlorous acid (HOCL) system. Further evidence has been provided by the observation that in several experimental models of GN, glomerular injury is exacerbated following treatment with non-selective NO inhibitors. Finally, the development of GN is severely aggravated in mice lacking a functional gene for eNOS as compared to wild-type mice, providing direct support for a protective role of eNOS-derived NO in acute GN.

The role of cytokines in the control of tissue parasitism and pathogenesis of experimental Chagas' disease was investigated. Wild-type and different cytokine as well as inducible nitric oxide synthase (iNOS) knockout mice were infected with the Colombian strain of Trypanosoma cruzi, and the kinetics of tissue parasitism, inflammatory reaction, parasitemia, and mortality were determined. We demonstrate the pivotal role of the interleukin (IL)-12/interferon (IFN)-gamma/iNOS axis and the antagonistic effect of IL-4 in controlling heart tissue parasitism, inflammation, and host resistance to acute infection with T. cruzi. Further, the heart and central nervous system were shown the main sites of reactivation of T. cruzi infection in mice lacking functional genes for IFN-gamma and IL-12, respectively. Our results also show that in contrast to IFN-gamma knockout (KO) mice, splenocytes from IL-12 KO mice infected with T. cruzi produced low levels of IFN-gamma upon stimulation with antigen. Consistently, high levels of anti-T. cruzi IgG2a antibodies were detected in the sera from IL-12 KO, but not from IFN-gamma KO mice, infected with the Colombian strain of T. cruzi. Thus, our results suggest that the level of IFN-gamma deficiency is a major determinant of the site of reactivation of T. cruzi infection in immunocompromised host.

Objective: The purpose of this study was to test whether omitting the vasodilator nitric oxide that is derived from any 1 of the 3 isoforms of nitric oxide synthase results in hypertension during pregnancy., Study Design: We measured systolic blood pressure before, during, and after pregnancy using an automated tail cuff method in 3 mutant (gene knockout) mouse strains in which the gene for neuronal nitric oxide, inducible nitric oxide, or endothelial nitric oxide was disrupted by gene targeting., Results: In neuronal nitric oxide gene knockout mice (n = 10), blood pressure was 100 +/- 3 mm Hg, not significantly different from 101 +/- 3 mm Hg in matched wild-type control mice (n = 10). Pregnancy did not change blood pressure or heart rate in either group. In inducible nitric oxide gene knockout mice (n = 9), blood pressure was 110 +/- 3 mm Hg, the same as in the wild-type control mice (110 +/- 2 mm Hg; n = 14). Blood pressure was unaffected by pregnancy in either group of mice. However, heart rate was significantly less in knockout mice (647 +/- 11 beats/min vs 666 +/- 9 beats/min; P <.005); this difference persisted through pregnancy. In endothelial nitric oxide gene knockout mice (n = 8), blood pressure was higher before pregnancy (114 +/- 4 mm Hg vs 103 +/- 4 mm Hg; P <.05) than in wild-type control mice (n = 9), but this difference disappeared during pregnancy, returning only after delivery. Heart rates were not different before pregnancy and were unaffected by pregnancy., Conclusion: There was no apparent increase in systolic blood pressure in any of the 3 nitric oxide synthase gene knockout strains during pregnancy compared to the wild-type control mice. This suggests that, at least in the mouse, genetic deficiency of any 1 isoform of nitric oxide synthase does not result in pregnancy-induced hypertension.

The objective of this study was to assess the role of inducible nitric oxide synthase (iNOS) in ischemia- and reperfusion (I/R)-induced liver injury. We found that partial hepatic ischemia involving 70% of the liver resulted in a time-dependent increase in serum alanine aminotransferase (ALT) levels at 1-6 h following reperfusion. Liver injury at 1, 3, and 6 h post-ischemia was not due to the infiltration of neutrophils as assessed by tissue myeloperoxidase (MPO) activity and histopathology. iNOS-deficient mice subjected to the same duration of ischemia and reperfusion showed dramatic and significant increases in liver injury at 3 but not 6 h following reperfusion compared to their wild type controls. Paradoxically, iNOS mRNA expression was not detected in the livers of wild type mice at any point during the reperfusion period and pharmacological inhibition of iNOS using L-N(6)(iminoethyl)-lysine (L-NIL) did not exacerbate post-ischemic liver injury at any time post-reperfusion. These data suggest that iNOS deficiency produces unanticipated genetic alterations that renders these mice more sensitive to liver I/R-induced injury., (Copyright 2001 Academic Press.)

Nitric oxide (NO) can modulate numerous genes through several pathways, yet some genes may be modulated only in the presence of the inflammatory stimuli that upregulate the inducible nitric oxide synthase (iNOS) rather than by NO alone. Furthermore, the role of prior expression of iNOS in the modulation of genes by NO is unknown. We addressed these issues in hepatocytes harvested from iNOS-null (iNOS(-/-)) mice exposed to NO by treatment with NO donors or by infection with an adenovirus-expressing human iNOS (Ad-iNOS), rather than by stimulation with inflammatory cytokines. Differential display and gene array analyses performed on mRNA derived from iNOS(-/-) hepatocytes demonstrated that infection with Ad-iNOS, but not infection with a control adenovirus expressing the beta-galactosidase gene (Ad-LacZ), induced a gene fragment identical to cytochrome P450 2E1 (CYP2E1). Northern analysis performed with this fragment demonstrated that treatment of iNOS(-/-) hepatocytes with Ad-iNOS or with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), but not control treatment or infection with Ad-LacZ, resulted in increased expression of CYP2E1. Inhibition of soluble guanylyl cyclase partially blocked the induction of CYP2E1 mRNA by Ad-iNOS. Rat hepatocytes treated with SNAP also exhibited increased expression of CYP2E1 mRNA. Preliminary studies, however, suggest that the induction of CYP2E1 in the rat hepatocytes treated with cytokines was not reduced in the presence of a NOS inhibitor. Our results suggest that CYP2E1 can be induced solely by NO derived from iNOS, at least partly in a cyclic GMP-dependent manner and independently of inflammatory stimuli or of prior exposure to NO., (Copyright 2001 Academic Press.)

Nitric oxide (NO) has been implicated in various aspects of the atherogenic process and has been shown to possess both protective and cytotoxic properties. Recently, increased expression of inducible nitric oxide synthase (iNOS) has been detected in atherosclerotic lesions, although there is no consensus as to its pathogenetic significance [1,2]. In this longitudinal study we show that iNOS plays an important protective role in the atherogenic process. Indirect systolic blood pressure was measured by photoplethysmography in unanesthetized mice fed either a basal or high salt diet, and found to be significantly higher in iNOS-deficient mice than in wild type controls at three months of age (P=0.038 (basal diet) and P=0.0005 (high salt diet)). In addition, relative to controls, the iNOS-deficient mice had significantly elevated serum cholesterol levels at 3, 9 and 12 months of age (P=0.0017, 0.0001 and 0.0002 for the respective ages) as well as a significantly higher incidence of atherosclerotic plaques. These findings suggest that iNOS targeted mutant mice, historically used as an animal model to investigate the role of nitric oxide in the inflammatory response [3,4], may also serve as a model for the study of cholesterol homeostasis and atherogenesis.

Background: The obstructed kidney in unilateral ureteral obstruction (UUO) is characterized by renal atrophy and tissue loss, which is mediated by renal tubular apoptosis. We sought to determine whether NO is involved in renal tubular apoptosis in vitro and in vivo., Methods: Rat renal tubular epithelial cells (NRK-52E) were subjected to mechanical stretch, and apoptosis and cell size were analyzed by flow cytometry. Furthermore, we studied UUO in mice lacking the gene for inducible nitric oxide synthase (iNOS-/-) and their wild-type littermates. Tubular apoptosis and proliferation were detected by immunostaining. NOS activity and NOS expression were assessed by a citrulline assay and Western blot, respectively., Results: Stretching-induced apoptosis in NRK-52E, which was reduced when NO was increased; conversely, stretch-induced apoptosis was increased when a NOS inhibitor was added to the cells. Stretched cells are larger and more apoptotic than unstretched cells. In UUO, the obstructed kidney of iNOS-/- mice exhibited more apoptotic renal tubules than the wild-type mice through 14 days of UUO. The obstructed kidney of iNOS-/- mice at day 3 showed more proliferative tubules compared with wild type. The obstructed kidney of wild-type mice exhibited higher total NOS activity until day 7 after UUO compared with iNOS-/- mice. However, the obstructed kidney of day 14 wild-type mice exhibited significantly lower iNOS activity and protein compared with the day 0 kidney., Conclusion: These results suggest that mechanical stretch is related to renal tubular apoptosis and that NO plays a protective role in this system in UUO.

Cytokines may participate in islet destruction during the development of type 1 diabetes. Expression of inducible nitric oxide synthase (iNOS) and subsequent NO formation induced by IL-1 beta or (IL-1 beta + IFN-gamma) may impair islet function in rodent islets. Inhibition of iNOS or a deletion of the iNOS gene (iNOS -/- mice) protects against cytokine-induced beta-cell suppression, although cytokines might also induce NO-independent impairment. Presently, we exposed wild-type (wt, C57BL/6 x 129SvEv) and iNOS -/- islets to IL-1 beta (25 U/ml) and (IL-1 beta (25 U/ml) + IFN-gamma (1000 U/ml)) for 48 h. IL-1 beta and (IL-1 beta + IFN-gamma) induced a significant increase in NO formation in wt but not in iNOS -/- islets. Both IL-1 beta and (IL-1 beta + IFN-gamma) impaired glucose-stimulated insulin release and reduced the insulin content of wt islets, while (IL-1 beta + IFN-gamma) reduced glucose oxidation rates and cell viability. IL-1 beta exposure to iNOS -/- islets impaired glucose-stimulated insulin release, increased insulin accumulation and reduced the insulin content, without any increase in cell death. Exposure to (IL-1 beta + IFN-gamma) had no effect on iNOS -/- islets except reducing the insulin content. Our data suggest that IL-1 beta may inhibit glucose-stimulated insulin release by pathways that are not NO-dependent and not related to glucose metabolism or cell death., (Copyright 2001 Academic Press.)

We recently reported that nitrotyrosine and acetaminophen (APAP)-cysteine protein adducts colocalize in the hepatic centrilobular cells following a toxic dose of APAP to mice. Whereas APAP-adducts are formed by reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine, nitrotyrosine residues are formed by reaction of tyrosine with peroxynitrite. Peroxynitrite is formed from nitric oxide (NO) and superoxide. This manuscript examines APAP (300 mg/kg) hepatotoxicity in mice lacking inducible nitric oxide synthase activity (NOS2 null or knockout mice; C57BL/6-Nos2(tm1Lau)) and in the wildtype mice. In a time course the ALT levels in the exposed NOS2 null mice were approximately 50% of the wildtype mice; however, histological examination of liver sections indicated similar levels of centrilobular hepatic necrosis in both wild-type and NOS2 null mice. Serum nitrate plus nitrite levels (NO synthesis) were identical in saline-treated NOS2 null and wild-type mice (53 +/- 2 microM). APAP increased NO synthesis in wild-type mice only. The increases paralleled the increases in ALT levels with peak levels of serum nitrate plus nitrite at 6 h (168 +/- 27 microM). In wild-type mice hepatic tyrosine nitration was greatly increased relative to saline treated controls. Tyrosine nitration increased in NOS2 null mice also, but the increase was much less. APAP increased hepatic malonaldehyde levels (lipid peroxidation) in NOS2 null mice only. The results suggest the presence of multiple pathways to APAP-mediated hepatic necrosis, one via nitrotyrosine, as in the wild-type mice, and another that is not dependent upon inducible nitric oxide synthase activity, but which may involve increased superoxide., (Copyright 2001 Academic Press.)

Nitric oxide (NO) has been implicated in the local regulation of bone metabolism. However, the contribution made by specific NO synthase (NOS) enzymes is unclear. Here we show that endothelial NOS gene knockout mice (eNOS-/-) have marked abnormalities in bone formation. Histomorphometric analysis of eNOS-/- femurs showed bone volume and bone formation rate was reduced by up to 45% (P: < 0.01) and 52% (P: < 0.01), respectively. These abnormalities were prevalent in young (6 to 9 weeks old) adults but by 12 to 18 weeks bone phenotype was restored toward wild-type. Dual energy X-ray absorptiometry analysis confirmed the age-related bone abnormalities revealing significant reductions in femoral (P: < 0.05) and spinal bone mineral densities (P: < 0.01) at 8 weeks that were normalized at 12 weeks. Reduction in bone formation and volume was not related to increased osteoclast numbers or activity but rather to dysfunctional osteoblasts. Osteoblast numbers and mineralizing activity were reduced in eNOS-/- mice. In vitro, osteoblasts from calvarial explants showed retarded proliferation and differentiation (alkaline phosphatase activity and mineral deposition) that could be restored by exogenous administration of a NO donor. These cells were also unresponsive to 17ss-estradiol and had an attenuated chemotactic response to transforming growth factor-beta. In conclusion, eNOS is involved in the postnatal regulation of bone mass and lack of eNOS gene results in reduced bone formation and volume and this is related to impaired osteoblast function.

The objective of this study was to determine what roles the endothelial cell and inducible isoforms of nitric oxide synthase (eNOS, iNOS) play in ischemia and reperfusion (I/R)-induced liver injury in vivo in mice genetically deficient in each isoform of NOS. We found that 45 min of partial (70%) liver ischemia and 5 h of reperfusion induced substantial liver injury as assessed by the release of large and significant amounts of the liver-specific enzyme alanine aminotransferase (ALT) into the serum of wild-type (wt) mice. The enhanced ALT levels were not due to increased recruitment of potentially damaging PMNs, which could mediate hepatocyte injury, as neither histopathological inspection nor quantitative MPO determinations revealed the presence of PMNs in the liver at this time point. In addition, we observed a significant enhancement in liver injury in eNOS-deficient but not iNOS-deficient mice subjected to liver I/R compared to postischemic wt mice. Taken together, these data suggest that eNOS- but not iNOS-derived NO plays an important role in limiting or downregulating I/R-induced liver injury in vivo following 5 h of reperfusion., (Copyright 2000 Academic Press.)

Mice sensitized with SCHISTOSOMA: mansoni eggs and IL-12 develop liver granulomas, on subsequent infection, which are smaller and less fibrotic than those in nonsensitized mice. The protective response is accompanied by a shift in the type-2 cytokine profile to one dominated by type-1 cytokines. The deviated response is associated with marked increases in inducible nitric oxide synthase (NOS-2) activity. Here, we demonstrate, by using NOS-2-deficient mice, that the anti-inflammatory and anti-fibrotic effects of the type-1 response are completely NOS-2-dependent. Strikingly, despite developing a polarized type-1 cytokine response that was similar in magnitude, the egg/IL-12-sensitized NOS-deficient mice developed granulomas 8 times larger than WT mice did. There was also no decrease in hepatic fibrosis in the sensitized mutant animals. Interferon-gamma-deficient mice failed to exhibit the exacerbated inflammatory response, despite displaying a marked deficiency in nitric oxide production. However, immune deviation was unsuccessful in the latter animals, which suggested that the increase in inflammation in NOS-deficient mice resulted from a polarized but nitric oxide-deficient type-1 response. These results reveal a beneficial role for NOS-2 in the regulation of inflammation and suggest that the ultimate success of Th2-to-Th1 immune deviation strategies will rely on the efficient activation of NOS-2 expression in downstream effector cells.

The deleterious effects of lipopolysaccharide (LPS) during endotoxic shock are associated with the secretion of tumor necrosis factor (TNF) and the production of nitric oxide (NO), both predominantly released by tissue macrophages. We analyzed the mechanism by which LPS induces apoptosis in bone marrow-derived macrophages (BMDM). LPS-induced apoptosis reached a plateau at about 6 hours of stimulation, whereas the production of NO by the inducible NO-synthase (iNOS) required between 12 and 24 hours. Furthermore, LPS-induced early apoptosis was only moderately reduced in the presence of an inhibitor of iNOS or when using macrophages from iNOS -/-mice. In contrast, early apoptosis was paralleled by the rapid secretion of TNF and was almost absent in macrophages from mice deficient for one (p55) or both (p55 and p75) TNF-receptors. During the late phase of apoptosis (12-24 hours) NO significantly contributed to the death of macrophages even in the absence of TNF-receptor signaling. NO-mediated cell death, but not apoptosis induced by TNF, correlated with the induction of p53 and Bax genes. Thus, LPS-induced apoptosis results from 2 independent mechanisms: first and predominantly, through the autocrine secretion of TNF-alpha (early apoptotic events), and second, through the production of NO (late phase of apoptosis). (Blood. 2000;95:3823-3831)

Study Objectives: Inducible nitric oxide synthase (iNOS) is upregulated in a number of inflammatory lung conditions, and exhaled nitric oxide (NO) concentration is increased. However, previous studies in children with cystic fibrosis (CF) have shown that exhaled NO is reduced. The purpose of this investigation was to study exhaled NO concentration in adults with CF, and to investigate the effect of CF genotype and respiratory tract infection on this measurement., Design: Exhaled and nasal NO levels were measured in 54 adult CF subjects and 37 healthy nonsmoking age-matched subjects using a chemiluminesence analyzer. Spirometry (FEV(1) and FVC), CF genotype, and bacterial colonization were also recorded., Setting: This study was conducted at a national CF center., Results: The mean age of patients was 26.9 years, and the mean FEV(1) was 50.5% predicted (range, 17 to 104%). Nasal NO in the CF patients (mean, 520 parts per billion [ppb]; confidence interval [CI], 452 to 588) was significantly lower (p < 0.001) than in control subjects (987 ppb; CI, 959 to 1,015). Exhaled NO was significantly lower (p < 0. 001) in CF patients (5.0 ppb; CI, 4.1 to 6.1) than in control subjects (7.3 ppb; CI, 6.8 to 7.8). FEV(1) did not correlate with nasal or exhaled NO. No association was observed between genotype and NO values or colonization with Pseudomonas aeruginosa., Conclusions: Despite the airway inflammation that is characteristic of CF, both nasal and exhaled NO were reduced. There was no association with genotype or infection status. As NO has bacteriostatic effects and may augment mucociliary clearance, this observation may be of clinical importance.

Nitric oxide (NO) radicals generated by endothelial nitric oxide synthase (eNOS) are involved in the regulation of vascular tone. In addition, NO radicals derived from eNOS inhibit platelet aggregation and leukocyte adhesion to the endothelium and, thus, may have anti-inflammatory effects. To study the role of eNOS in renal inflammation, the development of accelerated anti-glomerular basement membrane (GBM) glomerulonephritis was examined in mice lacking a functional gene for eNOS and compared with wild-type (WT) C57BL/B6j mice. WT C57BL/6j mice (n = 12) and eNOS knockout (-/-) mice (n = 12) were immunized intraperitoneally with sheep IgG (0.2 mg in complete Freund's adjuvant). At day 6.5 after immunization, mice received a single i.v. injection of sheep anti-mouse GBM (1 mg in 200 microl PBS). Mice were sacrificed at day 1 and 10 after induction of the disease. All WT mice survived until day 10, whereas 1 eNOS-/- mouse died and 2 more became moribund, requiring sacrifice. At day 10, eNOS-/- mice had higher levels of blood urea nitrogen than WT mice (P < 0.02), although proteinuria was comparable. Immunofluorescence microscopy documented similar IgG deposition in both WT and eNOS-/- mice, but eNOS-/- mice had more extensive glomerular staining for fibrin at day 10 (P < 0.007). At day 10, light microscopy demonstrated that eNOS-/- mice had more severe glomerular thrombosis (P < 0.003) and influx of neutrophils (P < 0. 006), but similar degrees of overall glomerular endocapillary hypercellularity and crescent formation. In conclusion, accelerated anti-GBM glomerulonephritis is severely aggravated in eNOS-/- mice, especially with respect to glomerular capillary thrombosis and neutrophil infiltration. These results indicate that NO radicals generated by eNOS play a protective role during renal inflammation.

Background: Inducible nitric oxide synthase (iNOS) is enhanced during acute rejection. Pharmacologic inhibition of nitric oxide synthase (NOS) activity has had variable effects on graft survival in a number of animal models. To further characterize the requirement and effects of iNOS during acute allograft rejection, we examined rejection responses of mice completely deficient of iNOS., Methods: Heterotopic cardiac allografts were performed using wild-type and iNOS deficient mice (iNOS[-/-]) as recipients. Graft survival was determined by abdominal palpation. At days 3 and 7 following transplantation, grafts were harvested and analyzed histologically. Cytokine messenger RNA (mRNA) expression was measured by ribonuclease protection assay., Results: Mean survival time of cardiac allografts did not differ between wild-type (18 +/- 3 days) and iNOS(-/-) recipients (16 +/- 2 days). At 3 days, findings of moderate acute rejection were seen in both recipients groups, although modestly reduced in iNOS(-/ -) mice. By 7 days, allografts in both groups demonstrated severe rejection. Within grafts at day 3, there was a 3-fold reduction in IL-1beta expression and a 4-fold reduction in IL-1RA in iNOS(-/-) recipients (p = 0.03 andp = 0.04, respectively) compared to wild-type recipients. Expression of other proinflammatory cytokines was detected in the grafts from both recipients, but was not significantly different. Finally, rejection responses to iNOS(-/-) cardiac allografts were nearly identical to wild-type allografts., Conclusions: Rejection of cardiac allografts by iNOS(-/-) mice occurs in a similar fashion to wild-type recipients, with extensive inflammation and proinflammatory cytokine production. While iNOS may play a role in cytokine induction by macrophages, these studies suggest that iNOS is not required for efficient cardiac graft rejection.

Intranasal infection of mice by Vesicular Stomatitis Virus (VSV) often leads to breakdown of the blood-brain barrier (BBB). The role of Interleukin 12 (IL-12) and nitric oxide synthase (NOS) was examined here. Wild-type (WT), NOS-1 knockout (KO), and NOS-3 KO mice were infected with VSV and treated with either IL-12 or medium. IL-12 treatment of uninfected hosts did not result in pathology. In contrast with WT and NOS-1 KO mice, where extensive gross and ultrastructural correlation of BBB breakdown were evident following infection, in NOS-3 KO mice, integrity of the BBB was observed. Thus NOS-3 activity in astrocytes, endothelial cells, or ependymal cells may play an essential role in regulating the BBB., (Copyright 1999 Academic Press.)

The purpose of the present study was to test the hypothesis that restenosis is associated with decreased constitutive nitric oxide synthase activity. Male miniswine with moderately elevated serum cholesterol levels underwent cardiac catheterization and oversized balloon injury to the right and left circumflex coronary arteries, followed 2 weeks later by repeat injury on the same coronary segments. After 4 weeks, the coronary arteries were either immediately frozen in liquid nitrogen or pressure-perfusion fixed and prepared for histologic examination. Constitutive nitric oxide synthase activity was quantified using a fibroblast reporter cell method, while constitutive nitric oxide synthase protein was compared between balloon-injured and non-balloon-injured arteries using Western blot analysis. Immunohistochemical studies were performed using a specific antibody against constitutive nitric oxide synthase protein. Following balloon injury, there was decreased constitutive nitric oxide synthase activity in balloon-injured coronary arteries, compared to distal non-balloon-injured segments from the same artery. Histological examination demonstrated an intact endothelium. Specific antibody staining revealed that there was less constitutive nitric oxide synthase protein reactivity by immunohistochemical analysis. Western analysis confirmed less constitutive nitric oxide synthase protein. The data are consistent with the hypothesis that restenosis is associated with decreased endothelial cell nitric oxide production. The data suggest this is secondary to a decreased amount of constitutive nitric oxide synthase enzyme in the endothelium. A deficiency in constitutive nitric oxide synthase enzyme may contribute to the impaired second messenger and paracrine functions of the endothelium observed during restenosis following balloon injury, including abnormal vasomotion, extracellular matrix formation, and platelet aggregation.