Hyperhomocysteinemia decreases intestinal motility leading to constipation

Elevated levels of plasma homocysteine (Hcy) called hyperhomocysteinemia (HHcy) have been implicated in inflammation and remodeling in intestinal vasculature, and HHcy is also known to aggravate the pathogenesis of inflammatory bowel disease (IBD). Interestingly, colon is the pivotal site that regulates Hcy levels in the plasma. We hypothesize that HHcy decreases intestinal motility through matrix metalloproteinase-9 (MMP-9)-induced intestinal remodeling leading to constipation. To verify this hypothesis, we used C57BL/6J or wild-type (WT), cystathionine β-synthase (CBS(+/-)), MMP-9(-/-), and MMP-9(-/-) + Hcy mice. Intestinal motility was assessed by barium meal studies and daily feces output. Plasma Hcy levels were measured by HPLC. Expression of ICAM-1, inducible nitric oxide synthase, MMP-9, and tissue inhibitors of MMPs was studied by Western blot and immunohistochemistry. Reactive oxygen species (ROS) including super oxide were measured by the Invitrogen molecular probe method. Tissue nitric oxide levels were assessed by a commercially available kit. Plasma Hcy levels in the treated MMP-9 group mice were comparable to CBS(+/-) mice. Barium meal studies suggest that intestinal motility is significantly decreased in CBS(+/-) mice compared with other groups. Fecal output-to-body weight ratio was significantly reduced in CBS(+/-) mice compared with other groups. There was significant upregulation of MMP-9, iNOS, and ICAM-1 expression in the colon from CBS(+/-) mice compared with WT mice. Levels of ROS, superoxide, and inducible nitric oxide were elevated in the CBS(+/-) mice compared with other groups. Results suggest that HHcy decreases intestinal motility due to MMP-9-induced intestinal remodeling leading to constipation.     

Calcitonin gene-related peptide released from endothelial progenitor cells inhibits the proliferation of rat vascular smooth muscle cells induced by angiotensin II

Remodeling by its very nature implies synthesis and degradation of extracellular matrix components (such as elastin, collagen, and connexins). Most of the vascular matrix metalloproteinase (MMP) are latent because of the presence of constitutive nitric oxide (NO). However, during oxidative stress peroxinitrite (ONOO-) activates the latent MMPs and instigates vascular remodeling. Interestingly, in mesenteric artery, homocysteine (Hcy) decreases the NO bio-availability, and folic acid (FA, an Hcy-lowering agent) mitigates the Hcy-mediated mesentery artery dysfunction. Dimethylarginine dimethylaminohydrolase-2 (DDAH-2) and endothelial nitric oxide synthase (eNOS) increases NO production. The hypothesis was that the Hcy decreased NO bio-availability, in part, activating MMP, decreasing elastin, DDAH-2, eNOS and increased vasomotor response by increasing connexin. To test this hypothesis,the authors used 12-week-old C57BJ/L6 wild type (WT) and hyperhomocysteinemic (HHcy)-cystathione beta synthase heterozygote knockout (CBS+/-) mice. Blood pressure measurements were made by radio-telemetry. WT and MMP-9 knockout mice were administered with Hcy (0.67 mg/ml in drinking water). Superior mesenteric artery and mesenteric arcade were analyzed with light and confocal microscopy. The protein expressions were measured by western blot analysis. The mRNA levels for MMP-9 were measured by RT-PCR. The data showed decreased DDAH-2 and eNOS expressions in mesentery in CBS-/+ mice compared with WT mice. Immuno-fluorescence and western blot results suggest increased MMP-9 and connexin-40 expression in mesenteric arcades of CBS-/+ mice compared with WT mice. The wall thickness of third-order mesenteric artery was increased in CBS-/+ mice compared to WT mice. Hcy treatment increased blood pressure in WT mice. Interestingly, in MMP-9 KO, Hcy did not increase blood pressure. These results may suggest that HHcy causes mesenteric artery remodeling and narrowing by activating MMP-9 and decreasing DDAH-2 and eNOS expressions, compromising the blood flow, instigating hypertension, and acute abdomen pain.     

Modulation of iNOS activity in age-related cardiac dysfunction

Based on the role of inducible nitric oxide synthase (iNOS) in heart failure, we hypothesized that the elevated expression of iNOS compared to young mice in the myocardium contributes to the age-related decline of left ventricular (LV) function. Cardiac iNOS mRNA and protein expression was singularly identified in old, wild type (WT) male mice (I6-month) and not in young WT male mice (6-month). Characterized with in vivo pressure-volume loops analysis, an age-related LV dysfunction was found in the old WT mice. The LV dysfunction of the aged mice was modified to that of the younger mice by the specific iNOS inhibitors, aminoguanidine (AMG, 10 mg/Kg, i.v. or infusion, n = 15) and S-methyl-isothiourea (MITU, 3 mg/Kg, i.v. n = 7), and declined with L-arginine (10 mg/Kg, i.v. n = 7). All three drugs had no effects on the LV function of young WT mice or old iNOS knockout (KO) mice. The NOx and cGMP levels were significantly higher only in the old WT mice (n = 6) and cGMP levels decreased to normal with AMG administration. In conclusion, these results suggested that the iNOS/NO/cGMP pathway may contribute to ventricular dysfunction during the aging process and that inhibition of iNOS activity significantly improved heart function in aged mice.     

Peroxisome proliferator ameliorates endothelial dysfunction in a murine model of hyperhomocysteinemia

To test the hypothesis that endothelial dysfunction in hyperhomocysteinemia was due to increased levels of nitrotyrosine and matrix metalloproteinase (MMP) activity in response to antagonism of peroxisome proliferator-activated receptor-alpha (PPAR-alpha), cystathionine beta-synthase (CBS) -/+ mice were bred, tail tissue was analyzed for genotype by PCR, and tail vein blood was analyzed for homocysteine (Hcy) by spectrofluorometry. To induce PPAR-alpha, mice were administered 8 microg/ml of ciprofibrate (CF) and grouped: 1) wild type (WT), 2) WT + CF, 3) CBS, 4) CBS + CF (n = 6 in each group). In these four groups of mice, plasma Hcy was 3.0 +/- 0.2, 2.5 +/- 1.2, 15.2 +/- 2.6 (P < 0.05 compared with WT), 11.0 +/- 2.9 micromol/l. Mouse urinary protein was 110 +/- 11, 86 +/- 6, 179 +/- 13, 127 +/- 9 microg.day(-1). kg(-1) by Bio-Rad dye binding assay. Aortic nitrotyrosine was 0.099 +/- 0.012, 0.024 +/- 0.004, 0.132 +/- 0.024 (P < 0.01 compared with WT), 0.05 +/- 0.01 (scan unit) by Western analysis. MMP-2 activity was 0.053 +/- 0.010, 0.024 +/- 0.002, 0.039 +/- 0.009, 0.017 +/- 0.006 (scan unit) by zymography. MMP-9 was specifically induced in CBS -/+ mice and inhibited by CF treatment. Systolic blood pressure (SPB) was 90 +/- 2, 88 +/- 16, 104 +/- 8 (P < 0.05 compared with WT), 96 +/- 3 mmHg. Aortic wall stress [(SPB. radius(2)/wall thickness)/2(radius + wall thickness)] was 10.2 +/- 1.9, 9.7 +/- 0.2, 16.6 +/- 0.8 (P < 0.05 compared with WT), 13.1 +/- 2.1 dyn/cm(2). The results suggest that Hcy increased aortic wall stress by increasing nitrotyrosine and MMP-9 activity.     

MMP‐13 deletion decreases profibrogenic molecules and attenuates N‐nitrosodimethylamine‐induced liver injury and fibrosis in mice

Connective tissue growth factor (CTGF) is involved in inflammation, pathogenesis and progression of liver fibrosis. Matrix metalloproteinase‐13 (MMP‐13) cleaves CTGF and releases several fragments, which are more potent than the parent molecule to induce fibrosis. The current study was aimed to elucidate the significance of MMP‐13 and CTGF and their downstream effects in liver injury and fibrosis. Hepatic fibrosis was induced using intraperitoneal injections of N‐nitrosodimethylamine (NDMA) in doses of 10 μg/g body weight on three consecutive days of each week over a period of 4 weeks in both wild‐type (WT) and MMP‐13 knockout mice. Administration of NDMA resulted in marked elevation of AST, ALT, TGF‐β1 and hyaluronic acid in the serum and activation of stellate cells, massive necrosis, deposition of collagen fibres and increase in total collagen in the liver of WT mice with a significant decrease in MMP‐13 knockout mice. Protein and mRNA levels of CTGF, TGF‐β1, α‐SMA and type I collagen and the levels of MMP‐2, MMP‐9 and cleaved products of CTGF were markedly increased in NDMA‐treated WT mice compared to the MMP‐13 knockout mice. Blocking of MMP‐13 with CL‐82198 in hepatic stellate cell cultures resulted in marked decrease of the staining intensity of CTGF as well as protein levels of full‐length CTGF and its C‐terminal fragments and active TGF‐β1. The data demonstrate that MMP‐13 and CTGF play a crucial role in modulation of fibrogenic mediators and promote hepatic fibrogenesis. Furthermore, the study suggests that blocking of MMP‐13 and CTGF has potential therapeutic implications to arrest liver fibrosis.     

The role of eNOS, iNOS, and NF-kappaB in upregulation and activation of cyclooxygenase-2 and infarct size reduction by atorvastatin

Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases myocardial expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), inducible NOS (iNOS), and cyclooxygenase-2 (COX2) in the rat. Inhibiting COX2 abolished the ATV-induced IS limitation without affecting p-eNOS and iNOS expression. We investigated 1) whether 3-day ATV pretreatment limits IS in eNOS(-/-) and iNOS(-/-) mice and 2) whether COX2 expression and/or activation by ATV is eNOS, iNOS, and/or NF-kappaB dependent. Male C57BL/6 wild-type (WT), University of North Carolina eNOS(-/-) and iNOS(-/-) mice received ATV (10 mg.kg(-1).day(-1); ATV(+)) or water alone (ATV(-)) for 3 days. Mice underwent 30 min of coronary artery occlusion and 4 h of reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin switch, and electrophoretic mobility shift assay. As a result, ATV reduced IS only in the WT mice. ATV increased eNOS, p-eNOS, iNOS, and COX2 levels and activated NF-kappaB in WT mice. It also increased myocardial COX2 activity. In eNOS(-/-) mice, ATV increased COX2 expression but not COX2 activity or iNOS expression. NF-kappaB was not activated by ATV in the eNOS(-/-) mice. In the iNOS(-/-) mice, eNOS and p-eNOS levels were increased but not iNOS and COX2 levels; however, NF-kappaB was activated. In conclusion, both eNOS and iNOS are essential for the IS-limiting effect of ATV. The expression of COX2 by ATV is iNOS, but not eNOS or NF-kappaB, dependent. Activation of COX2 is dependent on iNOS.     

Deficiency of inducible nitric oxide synthase attenuates immobilization-induced skeletal muscle atrophy in mice

The present study examined the effects of inducible nitric oxide synthase (iNOS) deficiency on skeletal muscle atrophy in single leg-immobilized iNOS knockout (KO) and wild-type (WT) mice. The left leg was immobilized for 1 wk, and the right leg was used as the control. Muscle weight and contraction-stimulated glucose uptake were reduced by immobilization in WT mice, which was accompanied with increased iNOS expression in skeletal muscle. Deficiency of iNOS attenuated muscle weight loss and the reduction in contraction-stimulated glucose uptake by immobilization. Phosphorylation of Akt, mTOR, and p70S6K was reduced to a similar extent by immobilization in both WT and iNOS KO mice. Immobilization decreased FoxO1 phosphorylation and increased mRNA and protein levels of MuRF1 and atrogin-1 in WT mice, which were attenuated in iNOS KO mice. Aconitase and superoxide dismutase activities were reduced by immobilization in WT mice, and deficiency of iNOS normalized these enzyme activities. Increased nitrotyrosine and carbonylated protein levels by immobilization in WT mice were reversed in iNOS KO mice. Phosphorylation of ERK and p38 was increased by immobilization in WT mice, which was reduced in iNOS KO mice. Immobilization-induced muscle atrophy was also attenuated by an iNOS-specific inhibitor N(6)-(1-iminoethyl)-l-lysine, and this finding was accompanied by increased FoxO1 phosphorylation and reduced MuRF1 and atrogin-1 levels. These results suggest that deficiency of iNOS attenuates immobilization-induced skeletal muscle atrophy through reduced oxidative stress, and iNOS-induced oxidative stress may be required for immobilization-induced skeletal muscle atrophy.     

Late preconditioning induced by NO donors, adenosine A1 receptor agonists, and delta1-opioid receptor agonists is mediated by iNOS

Although ischemia-induced late preconditioning (PC) is known to be mediated by inducible nitric oxide (NO) synthase (iNOS), the role of this enzyme in pharmacologically induced late PC remains unclear. We tested whether targeted disruption of the iNOS gene abrogates late PC elicited by three structurally different NO donors [diethylenetriamine/NO (DETA/NO), nitroglycerin (NTG), and S-nitroso-N-acetyl-penicillamine (SNAP)], an adenosine A1 receptor agonist [2-chloro-N6-cyclopentyladenosine (CCPA)], and a delta1-opioid receptor agonist (TAN-670). The mice were subjected to a 30-min coronary occlusion followed by 24 h of reperfusion. In iNOS knockout (iNOS-/-) mice, infarct size was similar to wild-type (WT) controls, indicating that iNOS does not modulate infarct size in the absence of PC. Pretreatment of WT mice with DETA/NO, NTG, SNAP, TAN-670, or CCPA 24 h before coronary occlusion markedly reduced infarct size. In iNOS-/- mice, however, the late PC effect elicited by DETA/NO, NTG, SNAP, TAN-670, and CCPA was completely abrogated. Furthermore, in WT mice pretreated with TAN-670 or CCPA, the selective iNOS inhibitor 1400W also abolished the delayed PC properties of these drugs; 1400W had no effect in WT mice. These data demonstrate that iNOS plays an obligatory role in NO donor-induced, adenosine A1 receptor agonist-induced, and delta1-opioid receptor agonist-induced late PC, underscoring the critical role of this enzyme as a common mediator of cardiac adaptations to stress.     

Testosterone production in mice lacking inducible nitric oxide synthase expression is sensitive to restraint stress

Immobilization stress (IMO) induces a rapid increase in glucocorticoid secretion [in rodents, corticosterone CORT)] and this is associated with decreased circulating testosterone (T) levels. Nitric oxide (NO), a reactive free radical and neurotransmitter, has been reported to be produced at higher rates in tissues such as brain during stress. The biosynthesis of T is also known to be dramatically suppressed by NO. Specifically, the inducible isoform of nitric oxide synthase (iNOS) was directly implicated in this suppression. To assess the respective roles of CORT and NO in stress-mediated inhibition of T production, adult wild-type (WT) and inducible nitric oxide synthase knockout (iNOS(-/-)) male mice were evaluated. Animals of each genotype were assigned to either basal control or 3-h IMO groups. Basal plasma and testicular T levels were equivalent in both genotypes, whereas testicular weights of mutant mice were significantly higher compared with WT animals. Exposure to 3-h IMO increased plasma CORT and decreased T concentrations in mice of both genotypes. Testicular T levels were also affected by stress in WT and mutant males, being sharply reduced in both genotypes. However, the concentrations of nitrite and nitrate, the stable metabolites of NO measured in testicular extracts, did not differ between control and stressed WT and iNOS(-/-) mice. These results support the hypothesis that CORT, but not NO, is a plausible candidate to mediate rapid stress-induced suppression of Leydig cell steroidogenesis.     

Susceptibility to ozone-induced acute lung injury in iNOS-deficient mice

Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO]NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide.     

Homocysteine decreases blood flow to the brain due to vascular resistance in carotid artery

An elevated level of Homocysteine (Hcy) is a risk factor for vascular dementia and stroke. Cysthathionine β Synthase (CBS) gene is involved in the clearance of Hcy. Homozygous individuals for (CBS−/−) die early, but heterozygous for (CBS−/+) survive with high levels of Hcy. The γ-Amino Butyric Acid (GABA) presents in the central nervous system (CNS) and functions as an inhibitory neurotransmitter. Hcy competes with GABA at the GABA_A receptor and affects the CNS function. We hypothesize that Hcy causes a decrease in blood flow to the brain due to increase in vascular resistance (VR) because of arterial remodeling in the carotid artery (CA). Blood pressure and blood flow in CA of wild type (WT), CBS−/+, CBS−/+ GABA_A−/− double knockout, and GABA_A−/− were measured. CA was stained with trichrome, and the brain permeability was measured. Matrix Metalloproteinases (MMP-2 and MMP-9), tissue inhibitor of metalloproteinase (TIMP-3, TIMP-4), elastin, and collagen-III expression were measured by real-time polymerase chain reaction (RT-PCR). Results showed an increase in VR in CBS−/+/GABA_A−/−double knockout > CBS−/+/ > GABA_A−/− compared to WT mice. Increased MMP-2, MMP-9, collagen-III and TIMP-3 mRNA levels were found in GABA_A−/−, CBS−/+, CBS−/+/GABA_A double knockout compared to WT. The levels of TIMP-4 and elastin were decreased, whereas the levels of MMP-2, MMP-9 and TIMP-3 increased, which indirectly reflected the arterial resistance. These results suggested that Hcy caused arterial remodeling in part, by increase in collagen/elastin ratio thereby increasing VR leading to the decrease in CA blood flow.     

NO production by cNOS and iNOS reflects blood pressure changes in LPS-challenged mice

Increased nitric oxide (NO) production is the cause of hypotension and shock during sepsis. In the present experiments, we have measured the contribution of endothelial (e) and inducible (i) nitric oxide synthase (NOS) to systemic NO production in mice under baseline conditions and upon LPS treatment (100 microg/10 g ip LPS). NO synthesis was measured by the rate of conversion of l-[guanidino-15N2]arginine to l-[ureido-15N]citrulline, and the contribution of the specific NOS isoforms was evaluated by comparing NO production in eNOS-deficient [(-/-)] and iNOS(-/-) mice with that in wild-type (WT) mice. Under baseline conditions, NO production was similar in WT and iNOS(-/-) mice but lower in eNOS(-/-) mice [WT: 1.2 +/- 0.2; iNOS(-/-): 1.2 +/- 0.2; eNOS(-/-): 0.6 +/- 0.3 nmol. 10 g body wt-1. min-1]. In response to the challenge with LPS (5 h), systemic NO production increased in WT and eNOS(-/-) mice but fell in iNOS(-/-) mice [WT: 2.7 +/- 0.3; eNOS(-/-): 2.2 +/- 0.6; iNOS(-/-): 0.7 +/- 0.1 nmol. 10 g body wt-1. min-1]. After 5 h of LPS treatment, blood pressure had dropped 14 mmHg in WT but not in iNOS(-/-) mice. The present findings provide firm evidence that, upon treatment with bacterial LPS, the increase of NO production is solely dependent on iNOS, whereas that mediated by cNOS is reduced. Furthermore, the data show that the LPS-induced blood pressure response is dependent on iNOS.     

Homocysteine decreases blood flow to the brain due to vascular resistance in carotid artery

An elevated level of Homocysteine (Hcy) is a risk factor for vascular dementia and stroke. Cysthathionine β Synthase (CBS) gene is involved in the clearance of Hcy. Homozygous individuals for (CBS−/−) die early, but heterozygous for (CBS−/+) survive with high levels of Hcy. The γ-Amino Butyric Acid (GABA) presents in the central nervous system (CNS) and functions as an inhibitory neurotransmitter. Hcy competes with GABA at the GABA_A receptor and affects the CNS function. We hypothesize that Hcy causes a decrease in blood flow to the brain due to increase in vascular resistance (VR) because of arterial remodeling in the carotid artery (CA). Blood pressure and blood flow in CA of wild type (WT), CBS−/+, CBS−/+ GABA_A−/− double knockout, and GABA_A−/− were measured. CA was stained with trichrome, and the brain permeability was measured. Matrix Metalloproteinases (MMP-2 and MMP-9), tissue inhibitor of metalloproteinase (TIMP-3, TIMP-4), elastin, and collagen-III expression were measured by real-time polymerase chain reaction (RT-PCR). Results showed an increase in VR in CBS−/+/GABA_A−/−double knockout > CBS−/+/ > GABA_A−/− compared to WT mice. Increased MMP-2, MMP-9, collagen-III and TIMP-3 mRNA levels were found in GABA_A−/−, CBS−/+, CBS−/+/GABA_A double knockout compared to WT. The levels of TIMP-4 and elastin were decreased, whereas the levels of MMP-2, MMP-9 and TIMP-3 increased, which indirectly reflected the arterial resistance. These results suggested that Hcy caused arterial remodeling in part, by increase in collagen/elastin ratio thereby increasing VR leading to the decrease in CA blood flow.     

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen‐induced retinopathy: beneficial effect of the absence of AQP4

Hypoxia‐dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin‐4 (AQP4) is involved in the hypoxia‐dependent VEGF upregulation in the retina of a mouse model of oxygen‐induced retinopathy (OIR). The expression levels of VEGF, the hypoxia‐inducible factor‐1α (HIF‐1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF‐1 binding site (HBS) in the VEGF gene promoter, the binding of HIF‐1α to the HBS, the retinal vascularization and function have been determined in the retina of wild‐type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF‐1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF‐1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF‐1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF‐1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF‐induced retinal damage in response to hypoxia.     

Ascorbate inhibits iNOS expression and preserves vasoconstrictor responsiveness in skeletal muscle of septic mice

Inducible nitric oxide synthase (iNOS) expression in blood vessels contributes to the vascular hyporeactivity characteristic of sepsis. Our previous work demonstrated in vitro that ascorbate inhibits iNOS expression in lipopolysaccharide- and interferon-gamma-stimulated skeletal muscle endothelial cells (ECs) through an antioxidant mechanism. The present study evaluated in vivo the hypothesis that administration of ascorbate decreases oxidative stress, prevents endothelial iNOS expression, and improves vascular reactivity in septic skeletal muscle. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Plasma nitrite and nitrate (NOx) levels were elevated by 6 h after CLP. Prior ascorbate bolus injection (200 mg/kg body wt iv) blocked the elevation of plasma NOx and abolished the expression of iNOS protein and activity in the septic skeletal muscle. We also demonstrated that iNOS mRNA determined by RT-PCR was induced in the microvascular ECs of the muscle at 3 h after CLP. This induction was attenuated by prior ascorbate administration. Ascorbate inhibition of iNOS expression was associated with decreased oxidant levels in the septic muscle. Moreover, ascorbate administration restored partially the baseline arterial pressure and preserved completely the microvascular constriction and arterial pressure responses to norepinephrine in CLP mice. These results suggest that early administration of ascorbate may be a valuable adjunct treatment of sepsis.     

Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS

Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.     

Mitochondrial mechanism of oxidative stress and systemic hypertension in hyperhomocysteinemia

Formation of homocysteine (Hcy) is the constitutive process of gene methylation. Hcy is primarily synthesized by de-methylation of methionine, in which s-adenosyl-methionine (SAM) is converted to s-adenosyl-homocysteine (SAH) by methyltransferase (MT). SAH is then hydrolyzed to Hcy and adenosine by SAH-hydrolase (SAHH). The accumulation of Hcy leads to increased cellular oxidative stress in which mitochondrial thioredoxin, and peroxiredoxin are decreased and NADH oxidase activity is increased. In this process, Ca2+-dependent mitochondrial nitric oxide synthase (mtNOS) and calpain are induced which lead to cytoskeletal de-arrangement and cellular remodeling. This process generates peroxinitrite and nitrotyrosine in contractile proteins which causes vascular dysfunction. Chronic exposure to Hcy instigates endothelial and vascular dysfunction and increases vascular resistance causing systemic hypertension. To compensate, the heart increases its load which creates adverse cardiac remodeling in which the elastin/collagen ratio is reduced, causing cardiac stiffness and diastolic heart failure in hyperhomocysteinemia.     

Mice lacking inducible nitric oxide synthase develop exacerbated hepatic inflammatory responses induced by Plasmodium berghei NK65 infection

Infection of mice with Plasmodium berghei NK65 represents a well-recognized malaria model in which infection is accompanied by an intense hepatic inflammatory response. Enzyme-inducible nitric oxide synthase is an important regulator of inflammation and leukocyte recruitment in microvessels, but these functions have yet to be evaluated in experimental malaria. In this study, we assessed the involvement of inducible nitric oxide synthase in inflammatory responses to murine experimental malaria induced by P. berghei NK65. We observed that wild type (WT) and nitric oxide synthase (iNOS)-deficient mice (iNOS^−/−) mice showed similar levels of parasitemia following P. berghei NK65 infection, although infected iNOS^−/− mice presented early mortality. Inducible nitric oxide synthase deficiency led to increased leukocyte rolling and adhesion to the liver in iNOS^−/− mice relative to the WT animals, as observed via intravital microscopy. Infected iNOS^−/− mice also exhibited increased hepatic leukocyte migration and subsequent liver damage, which was associated with high serum levels of the cytokines TNF-α, IL-6 and IL-10. Our data suggest potential role for the iNOS enzyme as a regulator of hepatic inflammatory response induced by P. berghei NK65-infection, and its absence leads to exacerbated inflammation and sequential associated-hepatic damage in the animals.