Neuronal nitric oxide synthase is resistant to ethanol.

To test for a possible role of nitric oxide (NO) in the neurotoxicity of ethanol, we studied the effects of ethanol on the neuronal NO synthase (nNOS) both in vitro and in vivo. Ethanol, up to 200 mM, did not change the NOS activity in the cerebellar homogenate or the production of NO by the cultured cerebellar granule cells. The number of NADPH diaphorase-positive cells in the culture did not change after the exposure to 200 mM ethanol in vitro. The NOS activity in the various brain regions of mice remained similar to the controls after the acute (3 g/kg) and the chronic (33 g/kg/day, 3.5 days) administration of ethanol. N(omega)-nitro-L-arginine, a NOS inhibitor, did not affect the ethanol-withdrawal behavior. These results indicate that nNOS is resistant to ethanol at clinically relevant concentrations and that ethanol affects the NO-operated system in the brain through a pathway other than that of nNOS.     

Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements

Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.     

Neuronal nitric oxide synthase catalyzes the reduction of 7-ethoxyresorufin.

Nitric oxide synthase (NOS: EC 1.14.13.39) catalyzes L-arginine oxidation to generate nitric oxide (NO) and L-citrulline. Recently, 7-ethoxyresorufin (7-ER), a specific substrate of cytochrome P-4501A1, was used as a cytochrome P-450 inhibitor to study the mechanism underlying the vasodilatation caused by some drugs, and was suggested to inhibit nitric oxide-mediated relaxation. Herein we demonstrate that 7-ER inhibits NO synthesis by uncoupling neuronal nitric oxide synthase (nNOS). 7-ER is a noncompetitive inhibitor of nNOS with respect to L-arginine with a Ki value of 0.76 +/- 0.06 microM. The decrease in NO formation is inversely correlated with an increase in NADPH oxidation. 7-ER binds to nNOS with a Km value of 0.68 +/- 0.07 microM, as calculated from the nNOS-dependent NADPH oxidation in the absence of L-arginine. nNOS catalyzes the reduction of 7-ER at the expense of NADPH. The flavoprotein inhibitor, diphenyleneiodonium chloride (100 microM), completely inhibited nNOS-dependent 7-ER reduction. While nitro-L-arginine (1 mM) and N(G)-nitro-L-arginine methyl ester (1 mM), specific inhibitors of nNOS, and phenylisocyanide (0.1 mM), a specific heme iron ligand, did not affect the reduction of 7-ER. These results indicate that the reductase domain, but not the oxygenase domain, of nNOS is involved in the reduction of 7-ER. 7-ER uncouples nNOS, shunting electrons from the reductase domain to the oxygenase domain of the enzyme. As a consequence, NO synthesis is inhibited.     

Neuronal nitric oxide synthase is necessary for elimination of Giardia lamblia infections in mice

NO produced by inducible NO synthase (NOS2) is important for the control of numerous infections. In vitro, NO inhibits replication and differentiation of the intestinal protozoan parasite Giardia lamblia. However, the role of NO against this parasite has not been tested in vivo. IL-6-deficient mice fail to control Giardia infections, and these mice have reduced levels of NOS2 mRNA in the small intestine after infection compared with wild-type mice. However, NOS2 gene-targeted mice and wild-type mice treated with the NOS2 inhibitor N-iminoethyl-L-lysine eliminated parasites as well as control mice. In contrast, neuronal NOS (NOS1)-deficient mice and wild-type mice treated with the nonspecific NOS inhibitor NG-nitro-L-arginine methyl ester and the NOS1-specific inhibitor 7-nitroindazole all had delayed parasite clearance. Finally, Giardia infection increased gastrointestinal motility in wild-type mice, but not in SCID mice. Furthermore, treatment of wild-type mice with NG-nitro-L-arginine methyl ester or loperamide prevented both the increased motility and the elimination of parasites. Together, these data show that NOS1, but not NOS2, is necessary for clearance of Giardia infection. They also suggest that increased gastrointestinal motility contributes to elimination of the parasite and may also contribute to parasite-induced diarrhea. Importantly, this is the first example of NOS1 being involved in the elimination of an infection.     

Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopoiesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells.     

Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban

Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1^–/–) have increased or decreased contractility, but consistently have found a slowed rate of intracellular Ca²⁺ ([Ca²⁺]_i) decline and relengthening. Contraction and [Ca²⁺]_i decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1^–/– and wild-type (WT) mice. We also simultaneously measured Ca²⁺ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1^–/–, and PLB^–/– mice. NOS1^–/– trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-methyl-L-thiocitrulline (SMLT) in WT myocytes (i.e., decreased Ca²⁺ transient and cell shortening amplitudes and prolonged decline of [Ca²⁺]_i). Alternatively, NOS1 inhibition with SMLT in PLB^–/– myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB serine16 phosphorylation. Furthermore, there was a decreased SR Ca²⁺ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. β-Adrenergic stimulation restored the slowed [Ca²⁺]_i decline in NOS1^–/– myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca²⁺]_i decline, and this effect is absent in PLB^–/– myocytes. Thus NOS1 signaling modulates PLB serine16 phosphorylation, in part, via peroxynitrite. NOS1; peroxynitrite; force-frequency response     

Neuronal nitric oxide synthase: expression in rat parietal cells

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.     

Uterine artery remodeling and reproductive performance are impaired in endothelial nitric oxide synthase-deficient mice

The progressive rise in uterine blood flow during pregnancy is accompanied by outward hypertrophic remodeling of the uterine artery (UA). This process involves changes of the arterial smooth muscle cells and extracellular matrix. Acute increases in blood flow stimulate endothelial production of nitric oxide (NO). It remains to be established whether endothelial NO synthase (eNOS) is involved in pregnancy-related arterial remodeling. We tested the hypothesis that absence of eNOS results in a reduced remodeling capacity of the UA during pregnancy leading to a decline in neonatal outcome. UA of nonpregnant and pregnant wild-type (Nos3+/+) and eNOS-deficient (Nos3-/-) mice were collected and processed for standard morphometrical analyses. In addition, cross sections of UA were processed for cytological (smoothelin, smooth muscle alpha-actin) and proliferation (Ki-67) immunostaining. We compared the pregnancy-related changes longitudinally and, together with the data on pregnancy outcome, transversally by analysis of variance with Bonferroni correction. During pregnancy, the increases in radius and medial cross sectional area of Nos3-/- UA was significantly less than those of Nos3+/+ UA. Smooth muscle cell dedifferentiation and proliferation were impaired in gravid Nos3-/- mice as deduced from the lack of change in the expression of smoothelin and smooth muscle alpha-actin, and the reduced Ki-67 expression. Until 17 days of gestation, litter size did not differ between both genotypes, but at birth the number of viable newborn pups and their weights were smaller in Nos3-/- than in Nos3+/+ mice. We conclude that absence of eNOS adversely affects UA remodeling in pregnancy, which may explain the impaired pregnancy outcome observed in these mice.     

Mechanisms associated to impaired activity of cardiac P-type ATPases in endothelial nitric oxide synthase knockout mice

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS^?/?) mice hearts that Na⁺/K⁺- and Ca²⁺-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS^?/? mice. Na⁺/K⁺-ATPase activity from crude preparations of adult male eNOS^?/? mice hearts and kidneys was reduced compared with wild-type animals (32 %, p?<?0.05 and 16 %, p?<?0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na⁺/K⁺-ATPase α1 isoform was not significantly changed, there was an important downregulation of the less abundant α2 isoform in the heart (57 %, p?<?0.0001). In addition, although cardiac Ca²⁺-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca²⁺-ATPase 2 protein in eNOS^?/? mice was very high (290 % compared with wild-type animals, p?<?0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p?<?0.01 and 35 %, p?<?0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases.     

Neuronal nitric oxide synthase proteolysis limits the involvement of nitric oxide in kainate-induced neurotoxicity in hippocampal neurons

In this work, we investigated the role of nitric oxide (NO) in neurotoxicity triggered by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation in cultured hippocampal neurons. In the presence of cyclothiazide (CTZ), short-term exposures to kainate (KA; 5 and 15 min, followed by 24-h recovery) decreased cell viability. Both NBQX and d-AP-5 decreased the neurotoxicity caused by KA plus CTZ. Long-term exposures to KA plus CTZ (24 h) resulted in increased toxicity. In short-, but not in long-term exposures, the presence of NO synthase (NOS) inhibitors (l-NAME and 7-NI) decreased the toxicity induced by KA plus CTZ. We also found that KA plus CTZ (15-min exposure) significantly increased cGMP levels. Furthermore, short-term exposures lead to decreased intracellular ATP levels, which was prevented by NBQX, d-AP-5 and NOS inhibitors. Immunoblot analysis revealed that KA induced neuronal NOS (nNOS) proteolysis, gradually lowering the levels of nNOS according to the time of exposure. Calpain, but not caspase-3 inhibitors, prevented this effect. Overall, these results show that NO is involved in the neurotoxicity caused by activation of non-desensitizing AMPA receptors, although to a limited extent, since AMPA receptor activation triggers mechanisms that lead to nNOS proteolysis by calpains, preventing a further contribution of NO to the neurotoxic process.     

Decreased viability of nitric oxide synthase double knockout mice

Nitric oxide acts as an important intracellular messenger in a variety of systems, including reproduction. Previous studies have shown the importance of nitric oxide in embryo development. NO is produced from l-arginine by the enzyme, nitric oxide synthase (NOS), which has three isoforms: endothelial (NOS3), neural (NOS1), and inducible (NOS2). We hypothesize that, because of the importance of NOS in development, at least two NOS isoforms are required in order for normal embryo development to occur. Through the generation of NOS3/NOS2, NOS3/NOS1, and NOS2/NOS1 double knockout mice, we found that while litter size remains unchanged, the expected number of generated double knockout mice varies significantly from what would be predicted by Mendelian genetics. Estrous cycles were similar for both DKO and the wild-type mice, and both groups were deemed fertile by their ability to mate with wild-type (CD-1) mice. Together, these results lead us to conclude that the lack of two NOS isoforms leads to a decreased viability in mice because of a developmental problem in the double knockout embryo.     

Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase.

BACKGROUND: Nitric oxide (NO) has been implicated as a mediator of penile erection, because the neuronal isoform of NO synthase (NOS) is localized to the penile innervation and NOS inhibitors selectively block erections. NO can also be formed by two other NOS isoforms derived from distinct genes, inducible NOS (iNOS) and endothelial NOS (eNOS). To clarify the source of NO in penile function, we have examined mice with targeted deletion of the nNOS gene (nNOS- mice). MATERIALS AND METHODS: Mating behavior, electrophysiologically induced penile erection, isolated erectile tissue isometric tension, and eNOS localization by immunohistochemistry and Western blot were performed on nNOS- mice and wild-type controls. RESULTS: Both intact animal penile erections and isolated erectile tissue function are maintained in nNOS mice, in agreement with demonstrated normal sexual behaviors, but is stereospecifically blocked by the NOS inhibitor, L-nitroarginine methyl ester (L-NAME). eNOS is abundantly present in endothelium of penile vasculature and sinusoidal endothelium within the corpora cavemosa, with levels that are significantly higher in nNOS- mice than in wild-type controls. CONCLUSIONS: eNOS mediates NO-dependent penile erection in nNOS- animals and normal penile erection. These data clarify the role of nitric oxide in penile erection and may have implications for therapeutic agents with selective effects on NOS isoforms.     

Inhibition of neuronal nitric oxide synthase increases aggressive behavior in mice.

BACKGROUND: Mice with targeted disruption of the gene for the neuronal isoform of nitric oxide synthase (nNOS) display exaggerated aggression. Behavioral studies of mice with targeted gene deletions suffer from the criticism that the gene product is missing not only during the assessment period but also throughout development when critical processes, including activation of compensatory mechanisms, may be affected. To address this criticism, we have assessed aggressive behavior in mice treated with a specific pharmacological inhibitor of nNOS. MATERIALS AND METHODS: Aggressive behavior, as well as brain citrulline levels, were monitored in adult male mice after treatment with a specific nNOS inhibitor, 7-nitroindazole (7-NI) (50 mg/kg i.p.), which is known to reduce NOS activity in brain homogenates by > 90%. As controls, animals were treated with a related indazole, 3-indazolinone (3-I) (50 mg/kg i.p.) that does not affect nNOS or with on oil vehicle. RESULTS: Mice treated with 7-NI displayed substantially increased aggression as compared with oil- or 3-I-injected animals when tested in two different models of aggression. Drug treatment did not affect nonspecific locomotor activities or body temperature. Immunohistochemical staining for citrulline in the brain revealed a dramatic reduction in 7-NI-treated animals. CONCLUSIONS: 7-NI augmented aggression in WT mice to levels displayed by nNOS- mice, strongly implying that nNOS is a major mediator of aggression. NOS inhibitors may have therapeutic roles in inflammatory, cardiovascular, and neurologic diseases. The substantial aggressive behavior soon after administration of an nNOS inhibitor raises concerns about adverse behavioral sequelae of such pharmacological agents.     

PGAP1 knock-out mice show otocephaly and male infertility

A palmitate linked to the inositol in glycosylphosphatidylinositol (GPI) is removed in the endoplasmic reticulum immediately after the conjugation of GPI with proteins in most cells. Previously, we identified PGAP1 (post GPI attachment to proteins 1) as a GPI inositoldeacylase that removes the palmitate from inositol. A defect in PGAP1 caused a delay in the transport of GPI-anchored proteins (GPI-APs) from the endoplasmic reticulum to the cell surface in Chinese hamster ovary cells, although the cell-surface expression of GPI-APs in the steady state was normal. Nevertheless, in most cells, GPI-APs undergo deacylation. To elucidate the biological significance of PGAP1 in vivo, we established PGAP1 knock-out mice. Most PGAP1 knock-out mice showed otocephaly, a developmental defect, and died right after birth. However, some survived with growth retardation. Male knock-out mice showed severely reduced fertility despite the capability of ejaculation. Their spermatozoa were normal in number, motility, and ability to ascend the uterus, but were unable to go into the oviduct. In vitro, PGAP1-deficient spermatozoa showed weak attachment to the zona pellucida and a severely diminished rate of fertilization. Therefore, an extra acyl chain in GPI anchors caused severe deleterious effects to development and sperm function.     

Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis

Increasing evidence suggests that depression may be associated with a lack of hippocampal neurogenesis. It is well established that neuronal nitric oxide synthase (nNOS)-derived NO exerts a negative control on the hippocampal neurogenesis. Using genetic and pharmacological methods, we investigated the roles of nNOS in depression induced by chronic mild stress (CMS) in mice. Hippocampal nNOS over-expression was first observed 4 days and remained elevated 21 and 56 days after exposure to CMS. The mice exposed to CMS exhibited behavioral changes typical of depression, and impaired neurogenesis in the hippocampus. The CMS-induced behavioral despair and hippocampal neurogenesis impairment were prevented and reversed in the null mutant mice lacking nNOS gene (nNOS−/−) and in the mice receiving nNOS inhibitor. Disrupting hippocampal neurogenesis blocked the antidepressant effect of nNOS inhibition. Moreover, nNOS−/− mice exhibited antidepressant-like properties. Our findings suggest that nNOS over-expression in the hippocampus is essential for chronic stress-induced depression and inhibiting nNOS signaling in brain may represent a novel approach for the treatment of depressive disorders.