Nitric oxide down-regulates caveolin-1 expression in rat brains during focal cerebral ischemia and reperfusion injury

As a signalling molecule of the integral membrane protein family, caveolin participates in cellular signal transduction via interaction with other signalling molecules. The nature of interaction between nitric oxide (NO) and caveolin in the brain, however, remains largely unknown. In this study we investigated the role(s) of NO in regulating caveolin-1 expression in rat ischemic brains with middle cerebral artery occlusion (MCAO). Exposure to 1 h ischemia induced the increases in neuronal nitric oxide synthase (nNOS) and NO concentration with concurrent down-regulation of caveolin-1 expression in the ischemic core of rat brains. Subsequent 24 h or more reperfusion time led to an increase in inducible NOS (iNOS) expression and NO production, as well as a decline of caveolin-1 protein at the core and penumbra of the ischemic brain. Afterwards, NOS inhibitors and an NO donor were utilized to clarify the link between NO production and caveolin-1 expression in the rats with 1 h ischemia plus 24 h reperfusion. N(G)-nitro-l-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), N(6)-(1-iminoethyl)-lysine (NIL, an iNOS inhibitor), and 7-nitroindazole (7-NI, a nNOS inhibitor) prevented the loss of caveolin-1 in the core and penumbra of the ischemic brain, whereas l-N(5)-(1-iminoethyl)-ornithine (L-NIO, an endothelial NOS inhibitor) showed less effect than the other NOS inhibitors. S-Nitroso-N-acetylpenicillamine (SNAP, a NO donor) down-regulated the expression of caveolin-1 protein in normal and ischemic brains. These results, when taken together, suggest that NO modulates the expression of caveolin-1 in the brain and that the loss of caveolin-1 is associated with NO production in the ischemic brain.     

Acute and conditioned hypoxic tolerance augmented by endothelial nitric oxide synthase inhibition in mice.

To identify a possible role for nitric oxide (NO) in acute hypoxic tolerance (HT) we measured hypoxic survival time (HST), effect of hypoxic conditioning (HC), and survival following hypoxic conditioning while blocking or mimicking the action of nitric oxide synthase (NOS). To inhibit NOS, CD-1 mice were given supplemental endogenous NOS inhibitor asymmetrical dimethylarginine (ADMA) or a synthetic NOS inhibitor N(omega)-nitro-L-arginine (L-NNA), both of which nonselectively inhibit three of the isoforms of NOS [inducible (iNOS), neuronal (nNOS), and endothelial NOS (eNOS)]. ADMA (10 mg/kg i.p.) or saline vehicle was given 5 min before HST testing. L-NNA was given orally at 1 g/l in drinking water with tap water as the control for 48 h before testing. Both ADMA and L-NNA significantly increased HST and augmented the HC effect on HST. Neither the nNOS selective inhibitor 7-nitroindazole (7-NI) nor the iNOS selective inhibitor N-{[3-(aminomethyl)phenyl]methyl}-enthanimidamide (1400W) had a statistically significant effect on HST or HT. The NO donor, 3-morpholinosydnoeimine, when given alone did not significantly decrease HT, but it did mitigate the increased HT effect of L-NNA. These data confirm that acute hypoxic conditioning increases HT and that NOS inhibition by endogenous (ADMA) and a synthetic NOS inhibitor (L-NNA) further increases HT, whereas iNOS and nNOS inhibition does not, suggesting that it is the inhibition of eNOS that mediates enhancement of HT.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

大鼠脑缺血再灌注后海马神经细胞NOS表达与细胞凋亡及复方丹参的保护作用

目的探讨大鼠局灶性脑缺血再灌注后海马神经细胞一氧化氮合酶(NOS)的表达与神经细胞凋亡的关系及中药复方丹参的保护作用。方法采用大脑中动脉内栓线阻断法(MCAO)造成局灶性脑缺血再灌注模型。用原位细胞凋亡检测方法观察海马神经细胞凋亡;用免疫组织化学方法检测大鼠海马神经细胞(nNOS、iNOS)的表达并做图像分析。结果与假手术对照组比较,脑缺血再灌注2h后缺血侧海马CA1、CA3区神经细胞nNOS、iNOS表达升高,并出现神经细胞凋亡,随着再灌注时间的延长,神经细胞iNOS的表达明显增强,凋亡神经细胞数逐渐增多,至24h达高峰,但神经细胞nNOS的表达并未见明显增强。复方丹参保护组神经细胞nNOS、iNOS的表达和凋亡神经细胞数明显低于缺血再灌组(P<0.01)。结论脑缺血再灌注后缺血侧海马CA1、CA3区神经细胞nNOS的表达增强,iNOS的表达显著升高,使NO的形成增加,这可能是介导脑缺血再灌注后神经细胞凋亡的机制之一。复方丹参具有下调神经细胞nNOS、iNOS的表达,减少NO的生成,抑制细胞凋亡,减轻缺血再灌注对大鼠海马损伤的作用。     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

Amyloid-beta (25-35) increases activity of neuronal NO-synthase in rat brain

Nitric oxide (NO) is a free radical with multiple functions in the nervous system. NO plays an important role in the mechanisms of neurodegenerative diseases including Alzheimer's disease. The main source of NO in the brain is an enzymatic activity of nitric oxide synthase (NOS). The aim of the present study was to analyze the expression and activity of both neuronal (nNOS) and inducible (iNOS) isoenzymes in the cerebral cortex and hippocampus of rats after intracerebroventricular administration of amyloid-beta (A beta) peptide fragment A beta(25-35). NADPHd histochemistry as well as immunohistochemistry were also used to investigate nNOS and iNOS expression in rat brain. The data presented here show that A beta(25-35) did not influence levels of nNOS or iNOS mRNA or protein expression in both structures studied. A beta(25-35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity. A beta(25-35) decreased the number of NADPHd-expressing neurons in the neocortex, but it did not significantly influence the number NADPHd-positive cells in the hippocampus. The peptide had no effect on the number of nNOS containing cells. We hypothesize that increased synthesis of NO induced by A beta(25-35) is related to qualitative alterations of nNOS molecule, but not to changes in NOS protein expression.     

The participation of brain NO synthase in blood pressure control of adult spontaneously hypertensive rats

Increased blood pressure (BP) in genetic hypertension is usually caused by high activity of sympathetic nervous system (SNS) which is enhanced by central angiotensin II but lowered by central nitric oxide (NO). We have therefore evaluated NO synthase (NOS) activity as well as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) protein expression in brainstem and midbrain of adult spontaneously hypertensive rats (SHR) characterized by enhanced sympathetic vasoconstriction. We also studied possible participation of brain NO in antihypertensive effects of chronic captopril treatment of adult SHR. NOS activity was increased in midbrain of SHR compared to Wistar-Kyoto (WKY) rats. This could be ascribed to enhanced iNOS expression, whereas nNOS expression was unchanged and eNOS expression was reduced in this brain region. In contrast, no significant changes of NOS activity were found in brainstem of SHR in which nNOS and iNOS expression was unchanged, but eNOS expression was increased. Chronic captopril administration lowered BP of adult SHR mainly by attenuation of sympathetic tone, whereas the reduction of angiotensin II-dependent vasoconstriction and the decrease of residual BP (amelioration of structural remodeling of resistance vessels) were less important. This treatment did not affect significantly either NOS activity or expression of any NOS isoform in the two brain regions. Our data do not support the hypothesis that altered brain NO formation contributes to sympathetic hyperactivity and high BP of adult SHR with established hypertension.     

Alterations of NO synthase isoforms in brain and kidney of rats with genetic and salt hypertension

Both brain and peripheral nitric oxide (NO) play a role in the control of blood pressure and circulatory homeostasis. Central NO production seems to counteract angiotensin II-induced enhancement of sympathetic tone. The aim of our study was to evaluate NO synthase (NOS) activity and protein expression of its three isoforms--neuronal (nNOS), endothelial NOS (eNOS) and inducible (iNOS)--in two brain regions involved in blood pressure control (diencephalon and brainstem) as well as in the kidney of young adult rats with either genetic (12-week-old SHR) or salt-induced hypertension (8-week-old Dahl rats). We have demonstrated reduced nNOS and iNOS expression in brainstem of both hypertensive models. In SHR this abnormality was accompanied by attenuated NOS activity and was corrected by chronic captopril treatment which prevented the development of genetic hypertension. In salt hypertensive Dahl rats nNOS and iNOS expression was also decreased in the diencephalon where neural structures important for salt hypertension development are located. As far as peripheral NOS activity and expression is concerned, renal eNOS expression was considerably reduced in both genetic and salt-induced hypertension. In conclusions, we disclosed similar changes of NO system in the brainstem (but not in the diencephalon) of rats with genetic and salt-induced hypertension. Decreased nNOS expression was associated with increased blood pressure due to enhanced sympathetic tone.     

Differential and constitutive expression of neuronal, inducible, and endothelial nitric oxide synthase mRNAs and proteins in pathologically normal human tissues.

Nitric oxide (NO) is produced by NO synthases (nNOS, iNOS, and eNOS) expressed in various human tissues and depending on the amount of NO produced in each tissue, the physiological function of NO is determined. However, due to the difficulty in obtaining normal human tissues, little is known about the basal levels of each of the three NOS mRNAsand proteins expressed constitutively in various human tissues. Results of the present study indicate that the basal levels of each of the three NOS mRNAs and proteins expressed in various regions of brain and peripheral tissues are different both in their sizes and in their contents. In Northern blot analysis, two different-sized mRNAs were found for each NOS isozymes: for the nNOS, approximately 12 and <12 kb mRNAs; for the iNOS, 4.2 and 4.5 kb mRNAs; for the eNOS, 4.2 and 4.4 kb mRNAs. In the Western blot, several different-sized NOS proteins were detected ( approximately 160, approximately 140, and approximately 130 kDa for nNOS; approximately 130 kDa for iNOS and eNOS) with tissue-specific expression patterns. These differential expression patterns of NOS mRNAs and proteins were caused by alternative splicing in the open-reading frame, and 5'- and/or 3'-untranslated regions of NOS mRNAs. These results suggest that regulation for differential expression of the three NOS genes in various human tissues may occur by alternative splicing of the NOS mRNAs in tissue-specific patterns.     

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.     

Effects of 1400W, a potent selective inducible NOS inhibitor, on histamine- and leukotriene D4-induced relaxation of isolated guinea pig nasal mucosa.

To clarify the role of inducible nitric oxide synthase (iNOS) in the relaxation of nasal vasculature, the effects of a potent selective iNOS inhibitor, N-[(3-aminomethyl)benzyl]acetamidine (1400W), on histamine- and leukotriene D4 (LTD4)-induced relaxations of isolated nasal septal mucosae were examined in naive guinea pigs. In addition to eNOS and nNOS, Western blots demonstrated a distinct expression of iNOS in nasal mucosal tissues of naive guinea pigs. In isolated nasal septal mucosae precontracted with norepinephrine (3 x 10(-5)M), both histamine (10(-7)-10(-3)M) and LTD4 (10(-10)-10(-7)M) exhibited relaxations, which were inhibited by a NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; 10(-4)M). The inhibitory effect of L-NMMA was reversed by L-arginine (10(-3)M), indicating that the relaxations induced by histamine and LTD4 are mediated by NO. Furthermore, both the histamine- and LTD4-induced relaxations were also significantly attenuated by 1400W (10(-5)M). These findings suggest an involvement of NO generated by iNOS in agonist-induced relaxation of nasal mucosal vasculature in naive guinea pigs.     

高压氧对急性脑创伤后皮层一氧化氮合酶mRNA表达的影响

目的:拟观察高压氧(HBO)治疗对急性创伤性颅脑损伤后皮层NOSmRNA表达的影响,探讨HBO治疗急性脑损伤的机理。方法:采用自由落体法打击模型制备SD大鼠急性脑创伤,伤后1 h、12 h采用0.25 MPaHBO治疗,伤后6 h、24 h取样皮层,应用半定量逆转录聚合酶链反应(RT-PCR)观察神经元型一氧化氮合酶(nNOS)、内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)mRNA表达量变化。结果:0.25MPaHBO治疗各时间组nNOS、eNOS和iNOSmRNA较急性颅脑损伤各时间组显著下降(P<0.01),且HBO治疗24 h组较6 h组下降更明显(P<0.05,P<0.01),0.25 MPa常氧高氮各时间组与急性颅脑损伤各时间组NOSmRNA表达量无统计学意义。结论:HBO治疗可以下调nNOSmRNA、iNOSmRNA和eNOSmRNA的表达量,可能为HBO治疗脑创伤的机理之一。     

Effect of subchronic acrylamide exposure on the expression of neuronal and inducible nitric oxide synthase in rat brain

Acrylamide (ACR) is a known industrial neurotoxic chemical. Evidence suggests that ACR neurotoxic effect is related to brain neurotransmission disturbances. Since nitric oxide (NO) acts as a neurotransmission modulator and is produced by nitric oxide synthase (NOS), the neuronal NOS (nNOS) and inducible NOS (iNOS) expression pattern were determined in rat cerebral cortex and striatum after subchronic exposure to ACR. Using immunocytochemistry, the neuronal count of nNOS or optical density of iNOS from sections at three coronal levels, bregma 1.0, -0.4, and -2.3 mm, were compared between ACR-treated and control rats. At all three levels, nNOS expressions were uniformly decreased in most of the neocortical subregions following the treatment of ACR. At bregma level 1.0 mm, total numbers of nNOS expressing neurons were significantly decreased to 58.7% and 64.7% of the control in the cortex and striatum of ACR-treated rats, respectively. However, at the bregma level -2.3 mm, ACR treatment did not produce a significant difference in the numbers of nNOS expressing neurons both in the cortex and striatum. Contrary to nNOS, iNOS expressions were consistently increased to approximately 32% in the neocortex and 25% in the striatum, following the subchronic ACR treatment. These data suggest that subchronic ACR exposure involves compensatory mechanism on nNOS and iNOS expression to maintain the homeostasis of NO at the rostral part of the neocortex and the striatum. However, in the caudal brain, increased iNOS expression did not suppress nNOS expression. Therefore, the present study is consistent with the hypothesis that ACR toxicity is mediated through the disturbance to the NO signaling pathway and exhibits a rostrocaudal difference through the differential expressions of nNOS and iNOS in the neocortex and the striatum.     

Isoflurane induces second window of preconditioning through upregulation of inducible nitric oxide synthase in rat heart

The second window of preconditioning (SWOP) induced by inhalation of volatile anesthetics has been documented in the rat heart and is triggered by nitric oxide synthase (NOS), but involvement of NOS in the mediator phase of isoflurane-induced SWOP has not been demonstrated. We tested the hypothesis that isoflurane-induced SWOP is mediated through upregulation of inducible NOS (iNOS). Rats inhaled 0.75 minimum alveolar concentration (MAC) isoflurane, 1.5 MAC isoflurane, or O2 for 2 h. After 24, 48, 72, and 96 h, the isolated heart was perfused with buffer and subjected to 30 min of ischemia followed by 2 h of reperfusion. Inhalation of 0.75 and 1.5 MAC isoflurane significantly limited infarct size after ischemia-reperfusion 24-72 h after isoflurane inhalation. The maximum effect was obtained 48 h after inhalation of 1.5 MAC isoflurane. Postischemic left ventricular function was improved only 48 h after inhalation of 1.5 MAC isoflurane. iNOS expression and activity in the heart were increased 24-72 h after inhalation of 1.5 MAC isoflurane; this increase was less pronounced after inhalation of 0.75 MAC isoflurane. A selective iNOS inhibitor, 1400W (10 microM), abolished iNOS activation and cardioprotection induced 48 h after inhalation of 1.5 MAC isoflurane. These results suggest that isoflurane inhalation induces SWOP after 24-72 h through overexpression and activation of iNOS in the rat heart.     

Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.     

Secretagogue-stimulated pancreatic secretion is differentially regulated by constitutive NOS isoforms in mice

Nitric oxide (NO) and NO synthase (NOS) play controversial roles in pancreatic secretion. NOS inhibition reduces CCK-stimulated in vivo pancreatic secretion, but it is unclear which NOS isoform is responsible, because NOS inhibitors lack specificity and three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Mice having individual NOS gene deletions were used to clarify the NOS species and cellular interactions influencing pancreatic secretion. In vivo secretion was performed in anesthetized mice by collecting extraduodenal pancreatic duct juice and measuring protein output. Nonselective NOS blockade was induced with N(omega)-nitro-L-arginine (L-NNA; 10 mg/kg). In vivo pancreatic secretion was maximal at 160 pmol.kg(-1).h(-1) CCK octapeptide (CCK-8) and was reduced by NOS blockade (45%) and eNOS deletion (44%). Secretion was unaffected by iNOS deletion but was increased by nNOS deletion (91%). To determine whether the influence of NOS on secretion involved nonacinar events, in vitro CCK-8-stimulated secretion of amylase from isolated acini was studied and found to be unaltered by NOS blockade and eNOS deletion. Influence of NOS on in vivo secretion was further examined with carbachol. Protein secretion, which was maximal at 100 nmol.kg(-1).h(-1) carbachol, was reduced by NOS blockade and eNOS deletion but unaffected by nNOS deletion. NOS blockade by L-NNA had no effect on carbachol-stimulated amylase secretion in vitro. Thus constitutive NOS isoforms can exert opposite effects on in vivo pancreatic secretion. eNOS likely plays a dominant role, because eNOS deletion mimics NOS blockade by inhibiting CCK-8 and carbachol-stimulated secretion, whereas nNOS deletion augments CCK-8 but not carbachol-stimulated secretion.     

Down-Regulation of Neuronal Nitric Oxide Synthase by Nitric Oxide After Oxygen-Glucose Deprivation in Rat Forebrain Slices

Abstract : The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor ( Z )-1-[(2-aminoethyl)- N -(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Neuronal NOS-mediated nitration and inactivation of manganese superoxide dismutase in brain after experimental and human brain injury

Manganese superoxide dismutase (MnSOD) provides the first line of defense against superoxide generated in mitochondria. SOD competes with nitric oxide for reaction with superoxide and prevents generation of peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. Thus, sufficient amounts of catalytically competent MnSOD are required to prevent mitochondrial damage. Increased nitrotyrosine immunoreactivity has been reported after traumatic brain injury (TBI); however, the specific protein targets containing modified tyrosine residues and functional consequence of this modification have not been identified. In this study, we show that MnSOD is a target of tyrosine nitration that is associated with a decrease in its enzymatic activity after TBI in mice. Similar findings were obtained in temporal lobe cortical samples obtained from TBI cases versus control patients who died of causes not related to CNS trauma. Increased nitrotyrosine immunoreactivity was detected at 2 h and 24 h versus 72 h after experimental TBI and co-localized with the neuronal marker NeuN. Inhibition and/or genetic deficiency of neuronal nitric oxide synthase (nNOS) but not endothelial nitric oxide synthase (eNOS) attenuated MnSOD nitration after TBI. At 24 h after TBI, there was predominantly polymorphonuclear leukocytes accumulation in mouse brain whereas macrophages were the predominant inflammatory cell type at 72 h after injury. However, a selective inhibitor or genetic deficiency of inducible nitric oxide synthase (iNOS) failed to affect MnSOD nitration. Nitration of MnSOD is a likely consequence of peroxynitrite within the intracellular milieu of neurons after TBI. Nitration and inactivation of MnSOD could lead to self-amplification of oxidative stress in the brain progressively enhancing peroxynitrite production and secondary damage.