Non-specific Nitric Oxide Synthase Inhibitor Research Articles

The galactosylation of Nω-nitro-L-arginine enhances its anti-nocifensive or anti-allodynic effects by targeting glia in healthy and neuropathic mice

This study has investigated whether the galactosyl ester prodrug of Nω-nitro-L-arginine (NAGAL), shows enhanced analgesic efficacy in healthy mice and in models of visceral and neuropathic pain: the writhing test and the spared nerve injury (SNI), respectively. NAGAL was compared to methyl ester pro-drug of Nω-nitro-l-arginine (L-NAME), a widely exploited non-specific nitric oxide synthase (NOS) inhibitor, for analgesic potential. The writhing test revealed that the ED50 value, along with the 95% confidence limit (CL) was 3.82 (1.77–6.04) mg/kg for NAGAL and, 36.75 (20.07–68.37) mg/kg for L-NAME. Notably, NAGAL elicited a greater anti-allodynic effect than L-NAME did in neuropathic mice. Biomolecular and morphological studies revealed that spared nerve injury increased the expressions of pro-inflammatory enzymes (caspase-1) and two glial cell biomarkers: integrin alpha M (ITGAM) and glial fibrillary acidic protein (GFAP) in the spinal cord. Finally, GLUT-3, an isoform of the hexose transporters capable to bind NAGAL and inducible NOS (iNOS), were found to be over-expressed in the activated astrocytes of the spinal cord of neuropathic mice. NAGAL administration normalized expression levels of these biomarkers. NAGAL showed a greater efficacy in inhibiting visceral pain and allodynia than L-NAME possibly by a greater cell permeation through the hexose transporter which is highly over-expressed by activated glia.

Validating a Model of Nitric Oxide-Ca 2+ Crosstalk in Cardiac Myocytes

Recently, we have developed a mathematical model of activation of constitutive nitric oxide synthase (NOS)-cGMP signaling pathway and its crosstalk with the excitation-contraction coupling in rat cardiac myocytes. This model describes a dynamic regulation of intracellular nitric oxide ([NO]i) formation by changes in intracellular calcium [Ca2+]i signals. In particular, a key prediction of the model is that the formation of NO is sensitive to the frequency and magnitude of [Ca2+]i signaling. To validate these predictions, we tested experimentally correlations between increases in pacing frequency and the level of intracellular [Ca2+]i and formation of [NO]i in neonatal rat cardiac myocytes which were loaded with membrane permeable fluorescent indicators DAF-FM and Fluo-4, respectively. As predicted by the model, increasing pacing frequency from 0.5 Hz to 2.0 Hz enhanced diastolic [Ca2+]i and [NO]i formation. NO production was markedly blocked by eliminating the extracellular Ca2+ with 2 mM EGTA or by increasing Ca2+ buffering with BAPTA-AM or by treatment with non-specific NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), confirming that pacing-frequency-dependent NO synthesis was Ca2+ and NOS dependent. Several of our model simulations compared the effects of cytosolic vs. dyadic Ca2+ signals in NOS signaling, predicting that cytosolic Ca2+ signals may be sufficient to initiate NOS activation. Using the computational model, we found that reduction of extracellular sodium [Na+]0 from 137 mM to 70 mM under condition of inhibited SR would induce a Ca2+signal with magnitude similar to pacing-induced cytosolic Ca2+ signals. This occurs due to influx of Ca2+ through the Na+/Ca2+ exchanger. Experimental testing of this new protocol confirmed a broad, slow Na+-depletion-induced Ca2+ signal. The Na+ depletion protocol was sufficient to enhance NO synthesis similar to 0.5 Hz pacing. These results confirm the model prediction that cytosolic Ca2+ signals regulate NOS activity in cardiac myocytes.

Angiotensin-(1-7) increases neuronal potassium current via a nitric oxide-dependent mechanism.

Actions of angiotensin-(1-7) [Ang-(1-7)], a heptapeptide of the renin-angiotensin system, in the periphery are mediated, at least in part, by activation of nitric oxide (NO) synthase (NOS) and generation NO(·). Studies of the central nervous system have shown that NO(·) acts as a sympathoinhibitory molecule and thus may play a protective role in neurocardiovascular diseases associated with sympathoexcitation, such as hypertension and heart failure. However, the contribution of NO in the intraneuronal signaling pathway of Ang-(1-7) and the subsequent modulation of neuronal activity remains unclear. Here, we tested the hypothesis that neuronal NOS (nNOS)-derived NO(·) mediates changes in neuronal activity following Ang-(1-7) stimulation. For these studies, we used differentiated catecholaminergic (CATH.a) neurons, which we show express the Ang-(1-7) receptor (Mas R) and nNOS. Stimulation of CATH.a neurons with Ang-(1-7) (100 nM) increased intracellular NO levels, as measured by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence and confocal microscopy. This response was significantly attenuated in neurons pretreated with the Mas R antagonist (A-779), a nonspecific NOS inhibitor (nitro-L-arginine methyl ester), or an nNOS inhibitor (S-methyl-L-thiocitrulline, SMTC), but not by endothelial NOS (eNOS) or inhibitory NOS (iNOS) inhibition {L-N-5-(1-iminoethyl)ornithine (L-NIO) and 1400W, respectively}. To examine the effect of Ang-(1-7)-NO(·) signaling on neuronal activity, we recorded voltage-gated outward K(+) current (I(Kv)) in CATH.a neurons using the whole cell configuration of the patch-clamp technique. Ang-(1-7) significantly increased I(Kv), and this response was inhibited by A-779 or S-methyl-L-thiocitrulline, but not L-NIO or 1400W. These findings indicate that Ang-(1-7) is capable of increasing nNOS-derived NO(·) levels, which in turn, activates hyperpolarizing I(Kv) in catecholaminergic neurons.

Specific inhibition of nitric oxide synthases at different time points in a murine model of pulmonary sepsis

Excessive production of nitric oxide (NO) by NO synthase (NOS) and a subsequent oxidative stress reaction are thought to be critically involved in the pathophysiology of sepsis. Previous studies suggested that NO production by neuronal NOS (nNOS) and inducible NOS (iNOS) is implemented in the disease process at different time points after the injury. Here we tested the roles of selective pharmacological inhibition of nNOS and iNOS at different time points in a murine model of pulmonary sepsis. The injury was induced by intranasal administration of live Pseudomonas aeruginosa (3.2 × 10 7 colony-forming units) in C57BL/6 wild-type mice. The animals received no treatment (control) or treatment with a specific nNOS inhibitor (4 or 8 h), iNOS inhibitor (4 or 8 h), or non-specific NOS inhibitor (4 or 8 h). In controls, the injury was associated with excessive releases of pro-inflammatory cytokines in the plasma, enhanced tissue lipid peroxidation, and decreased survival. Non-specific NOS inhibition at either time point did not influence survival and was not further investigated. While nNOS inhibition at 4 h was associated with a trend toward improved survival and significantly reduced contents of lung nitrite/nitrate (NO x ) and liver malondialdehyde, the blockade of nNOS at 8 h had no effect on these parameters. In contrast, early iNOS inhibition was associated with a trend toward decreased survival and no effects on lung NO x and liver malondialdehyde contents, whereas later iNOS blockade was associated with decreased malondialdehyde content in liver homogenates. In conclusion, pulmonary sepsis in mice may be beneficially influenced by specific pharmacological nNOS inhibition at an earlier time point and iNOS inhibition at a later time points post-injury. Future investigations should identify the time changes of the expression and activation of NOS isoforms.

Biologically active constituents from the fruiting body of Taiwanofungus camphoratus

Five new benzenoids, benzocamphorins A–E (1–5), and 10 recently isolated triterpenoids, camphoratins A–J (16–25), together with 23 known compounds including seven benzenoids (6–12), three lignans (13–15), and 13 triterpenoids (26–38) were isolated from the fruiting body of Taiwanofungus camphoratus. Their structures were established by spectroscopic analysis. Selected compounds were examined for cytotoxic and anti-inflammatory activities. Compounds 9 and 21 showed moderate cytotoxicity against MCF-7 and Hep2 cell lines with ED50 values of 3.4 and 3.0μg/mL, respectively. Compounds 21, 25, 26, 29–31, 33, and 36 demonstrated potent anti-inflammatory activity by inhibiting lipopolysaccharide (LPS)-induced nitric oxide (NO) production with IC50 values of 2.5, 1.6, 3.6, 0.6, 4.1, 4.2, 2.5, and 1.5μM, respectively, which were better than those of the nonspecific nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine methyl ester (l-NAME) (IC50: 25.8μM). These results may substantiate the use of T. camphoratus in traditional Chinese medicine (TCM) for the treatment of inflammation and cancer-related diseases. The newly discovered compounds deserve further development as anti-inflammatory candidates.

Suppression of nitric oxide synthesis by L-NAME reverses the beneficial effects of pioglitazone on scopolamine-induced memory impairment in mice

Pioglitazone, an agonist of peroxisome proliferator-activated receptor gamma (PPARγ), which is widely used in treatment of type 2 diabetes, has shown some therapeutic effect in Alzheimer's disease. In this study, effects of acute pioglitazone on acquisition, consolidation and retrieval of memory, and also the involvement of nitric oxide (NO) in the effects of pioglitazone on spatial recognition memory has been investigated in a two-trial recognition Y-maze test and passive avoidance in mice. Memory impairment was induced by scopolamine (1 mg/kg, i.p.). Pioglitazone (10 and 20 mg/kg, p.o.) was administrated prior to either acquisition, consolidation or retention trials, while L-NAME (N-nitro- l-arginine methyl ester), a non-specific NO synthase inhibitor, was administered (10 mg/kg, i.p.) 30 min before each trial. Results: 1) pioglitazone improved the acquisition of recognition spatial memory-impaired by scopolamine; L-NAME dramatically reversed improving effects of pioglitazone on memory acquisition; 2) pioglitazone did not change the consolidation of spatial memory, impaired by scopolamine; 3) pioglitazone improved the retrieval of spatial memory and L-NAME did not alter the beneficial effect of pioglitazone; 4) pioglitazone did not affect scopolamine-induced cognitive impairments in the passive avoidance test. The present study demonstrates the beneficial effect of acute pioglitazone administration on acquisition and retrieval of scopolamine-induced cognitive deficits. This effect was reversed only in acquisition phase by nitric oxide synthase inhibitor, L-NAME, therefore, it could be concluded that NO might be involved in the pioglitazone beneficial effect of spatial memory acquisition.

Nicotine activation of neuronal nitric oxide synthase and guanylyl cyclase in the medulla increases blood flow of the common carotid artery in cats

Individual activation of nicotinic acetylcholine receptor (nAChR) or nitric oxide (NO) synthase in the dorsal facial area (DFA) increases blood flow of common carotid artery (CCA) supplying intra- and extra-cranial tissues. We investigated whether the activation of nAChR initiated the activation of NO synthase and guanylyl cyclase to increase CCA blood flow in anesthetized cats. Microinjections of nicotine (a non-selective nAChR agonist), or choline (a selective α7-nAChR agonist) in the DFA produced increases in CCA blood flow ipsilaterally. These increases were significantly reduced by pretreatment with NG-nitro-arginine methyl ester ( l-NAME, a non-specific NO synthase inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NO synthase inhibitor) or methylene blue (MB, a guanylyl cyclase inhibitor) but not by that with N5-(1-iminoethyl)- l-ornithine ( l-NIO, a potent endothelial NO synthase inhibitor). Control microinjection with d-NAME (an isomer of l-NAME), artificial cerebrospinal fluid or DMSO (a solvent for 7-NI) did not affect resting CCA blood flow, nor did they affect nicotine- or choline-induced response. In conclusion, activation of nAChR, at least α7-nAChR, led to the activation of neuronal NO synthase and guanylyl cyclase in the DFA, which induced an increase in CCA blood flow.

Glucagon-Like Peptide-1 Increases Myocardial Glucose Uptake via p38α MAP Kinase–Mediated, Nitric Oxide–Dependent Mechanisms in Conscious Dogs With Dilated Cardiomyopathy

Background— We have shown that glucagon-like peptide-1 (GLP-1[7–36] amide) stimulates myocardial glucose uptake in dilated cardiomyopathy (DCM) independent of an insulinotropic effect. The cellular mechanisms of GLP-1–induced myocardial glucose uptake are unknown. Methods and Results— Myocardial substrates and glucoregulatory hormones were measured in conscious, chronically instrumented dogs at control (n=6), DCM (n=9) and DCM after treatment with a 48-hour infusion of GLP-1 (7–36) amide (n=9) or vehicle (n=6). GLP-1 receptors and cellular pathways implicated in myocardial glucose uptake were measured in sarcolemmal membranes harvested from the 4 groups. GLP-1 stimulated myocardial glucose uptake (DCM: 20±7 nmol/min/g; DCM+GLP-1: 61±12 nmol/min/g; P =0.001) independent of increased plasma insulin levels. The GLP-1 receptors were upregulated in the sarcolemmal membranes (control: 98±2 density units; DCM: 256±58 density units; P =0.046) and were expressed in their activated (65 kDa) form in DCM. The GLP-1–induced increases in myocardial glucose uptake did not involve adenylyl cyclase or Akt activation but was associated with marked increases in p38α MAP kinase activity (DCM+vehicle: 97±22 pmol ATP/mg/min; DCM+GLP-1: 170±36 pmol ATP/mg/min; P =0.051), induction of nitric oxide synthase 2 (DCM+vehicle: 151±13 density units; DCM+GLP-1: 306±12 density units; P =0.001), and GLUT-1 translocation (DCM+vehicle: 21±3% membrane bound; DCM+GLP-1: 39±3% membrane bound; P =0.005). The effects of GLP-1 on myocardial glucose uptake were blocked by pretreatment with the p38α MAP kinase inhibitor or the nonspecific nitric oxide synthase inhibitor nitro- l -arginine. Conclusions— GLP-1 stimulates myocardial glucose uptake through a non–Akt-1–dependent mechanism by activating cellular pathways that have been identified in mediating chronic hibernation and the late phase of ischemic preconditioning.

Hypercapnic Acidosis Reduces Oxidative Reactions in Endotoxin-induced Lung Injury

Hypercapnic acidosis frequently occurs when patients with acute lung injury are initially ventilated with low tidal volume "protective" strategies. Hypercapnic acidosis per se, in the absence of any change in tidal volume or airway pressure, is protective when instituted before the onset of injury. However, the mechanisms by which hypercapnic acidosis confers this protection are incompletely understood, in particular, the effects on pulmonary oxidative reactions, which are potent mediators of tissue damage, have not been previously examined in vivo. After anesthesia, tracheostomy, and the intratracheal instillation of endotoxin to establish lung injury, rats were mechanically ventilated for 6 h in normocapnia (21% O2, 0% CO2). Rats were then randomized to either normocapnic (21% O2, 0% CO2) or hypercapnic (21% O2, 5% CO2) ventilation and a nonspecific nitric oxide synthase inhibitor (N-monomethyl-L-arginine) or vehicle. Dihydrorhodamine was administered intravenously, and the lungs were removed for determination of the oxidative formation of rhodamine by spectrofluorimetry after 20 min. Thus, rats were randomly assigned to either: normocapnia-endotoxin (n = 12), normocapnia-endotoxin-N-monomethyl-L-arginine (n = 9), hypercapnia-endotoxin (n = 11), or hypercapnia-endotoxin-N-monomethyl-L-arginine (n = 10). Hypercapnic acidosis significantly reduced the pulmonary oxidative reactions in the inflamed lung compared with normocapnia. Nitric oxide synthase blockade did not alter endotoxin-induced oxidative reactions. Hypercapnic acidosis reduced oxidative reactions in the acutely injured lung in vivo, within minutes of onset and was not reliant on nitric oxide-dependent peroxynitrite production. This rapid onset antioxidant action is a previously undescribed mechanism by which hypercapnic acidosis could act, even when acute lung injury is well established.

014 Atorvastatin protects human myocardium from lethal ischaemia-reperfusion injury by activating the risk pathway

Background Previous animal studies have demonstrated that acute treatment with atorvastatin at reperfusion reduces myocardial infarct size by up-regulating the Akt and Erk1/2 components of the Reperfusion Injury Salvage Kinase (RISK) pathway. Whether this cardioprotective strategy applies to human cardiac tissue is unknown. Objective To determine whether atorvastatin, administered at reperfusion, protects human atrial tissue from ischaemia-reperfusion injury (IRI) via activation of the RISK pathway. Methods Local UCLH/UCL Ethical Committee approval was granted. Human atrial trabeculae were isolated from right atrial appendage tissue harvested from consenting adult patients undergoing elective cardiac surgery. The atrial trabeculae were subjected to 90 min of hypoxia followed by 120 min reoxygenation as simulated ischaemia-reperfusion injury. Following this, recovery of contractile function was determined and compared to baseline. Atrial trabeculae were randomised to the following groups: 1. Control (N=14); 2. Hypoxic preconditioning positive control (N=4); 3. Atorvastatin (25 μM) at reperfusion (N=9); 4. Atorvastatin plus UO126 (10 μM), a MEK1/2-Erk1/2 inhibitor (N=4); 5. Atorvastatin plus LY294002 (15 μM), a PI3K-Akt inhibitor (N=4); 6. Atorvastatin plus L-NAME (100 μM), a non-specific nitric oxide synthase inhibitor (N=7); 7. Atorvastatin plus 1400W (5μM), a specific inducible nitric oxide synthase inhibitor (N=5); 8. Inhibitor-only controls (N=18). Results Control atrial trabeculae recovered 37.5±1.6% of baseline contractile function following simulated IRI. Treatment with atorvastatin 25 μM at reperfusion significantly improved the recovery of contractile function (61.1±3.8%, p Conclusions Atorvastatin, administered at reperfusion, protects human atrial muscle from simulated IRI via activation of the Akt and Erk1/2 components of the RISK pathway and nitric oxide synthase.

Activation of PAR‐2 Elicits NO‐Dependent and CGRP‐Independent Dilation of the Dural Artery

The goal of this study was to determine the vascular effects of protease-activated receptor-2 (PAR-2) activation in the rat cranial vasculature. The role of PAR-2 in pain and inflammatory conditions has been established but the information available on its effects and receptor distribution in the trigeminal vascular axis is limited. We studied the dilatory function and expression of PAR-2 in the neuro-vascular circuit, critical in migraine pathogenesis. We also investigated the interaction of PAR-2 with calcitonin gene-related peptide (CGRP) and dural mast cells. We used an improved model of intravital microscopy on the closed cranial window in rats to study the vascular effects of PAR-2 activating peptides (PAR-2 APs; SLIGRL-NH(2), 2-Furoyl-LIGRLO-NH(2)) in the dural vasculature. Measurement of immunoreactive CGRP in skull halves and in trigeminal nucleus caudalis was done by using an enzyme-linked immunosorbent assay. We also analyzed the presence of PAR-2 in different migraine relevant tissues by quantitative real-time PCR and Western blot analysis. PAR-2 APs and trypsin induced a dose-dependent increase in dural artery diameter. The topical application of a nonspecific nitric oxide synthase (NOS) inhibitor, L-N(G)-Nitroarginine methyl ester, attenuated SLIGRL-NH(2) responses. Olcegepant, a CGRP receptor antagonist, did not a have significant effect on the SLIGRL-NH(2) responses, though exogenous CGRP responses were completely blocked. There was no significant release of CGRP from skull halves incubated with SLIGRL-NH(2) as compared with those incubated with the corresponding negative peptide. Chronic mast cell degranulation did not change the vascular effects of PAR-2 APs. mRNA and protein expression of PAR-2 were found throughout trigeminovasuclar axis. PAR-2 activation leads to vasodilation of dural arteries and these responses are partially mediated by nitric oxide. As PAR-2 is present throughout trigeminovasuclar axis, it may have a role in migraine pathogenesis, independent of CGRP and mast cell mediated mechanism.

Agmatine enhances the anticonvulsant effect of lithium chloride on pentylenetetrazole-induced seizures in mice: Involvement of L-arginine/nitric oxide pathway

After nearly 60 years, lithium is still the mainstay in the treatment of mood disorders. In addition to its antimanic and antidepressant effects, lithium also has anticonvulsant properties. Similar to lithium, agmatine plays a protective role in the central nervous system against seizures and has been reported to enhance the effect of different antiepileptic agents. Moreover, both agmatine and lithium have modulatory effects on the L-arginine/nitric oxide pathway. This study was designed to investigate: (1) whether agmatine and lithium exert a synergistic effect against clonic seizures induced by pentylenetetrazole and (2) whether or not this synergistic effect is mediated through inhibition of the L-arginine/nitric oxide pathway. In our study, acute administration of a single potent dose of lithium chloride (30 mg/kg ip) increased seizure threshold, whereas pretreatment with a low and independently noneffective dose of agmatine (3 mg/kg) potentiated a subeffective dose of lithium (10 mg/kg). N G-L-arginine methyl ester (L-NAME, nonspecific nitric oxide synthase inhibitor) at 1 and 5 mg/kg and 7-nitroindazole (7-NI, preferential neuronal nitric oxide synthase inhibitor) at 15 and 30 mg/kg augmented the anticonvulsant effect of the noneffective combination of lithium (10 mg/kg ip) and agmatine (1 mg/kg), whereas several doses (20 and 40 mg/kg) of aminoguanidine (inducible nitric oxide synthase inhibitor) failed to alter the seizure threshold of the same combination. Furthermore, pretreatment with independently noneffective doses (30 and 60 mg/kg) of L-arginine (substrate for nitric oxide synthase) inhibited the potentiating effect of agmatine (3 mg/kg) on lithium (10 mg/kg). Our findings demonstrate that agmatine and lithium chloride have synergistic anticonvulsant properties that may be mediated through the L-arginine/nitric oxide pathway. In addition, the role of constitutive nitric oxide synthase versus inducible nitric oxide synthase is prominent in this phenomenon.

Abstract 4105: Nitric-oxide-mediated inhibition of Na+/H+ exchange (NHE) is a novel mechanism of bile acid induced damage: Relevance to esophageal carcinogenesis

Abstract Background: Barrett's esophagus (BE) is a premalignant condition that is associated with gastroesophageal reflux disease (GERD). Gastric acid and bile acids are two components of refluxate that induce proliferation, DNA damage and oxidative stress. In combination, the effects of gastric acid and bile acids are amplified and synergistic. The major goal of this study was to evaluate the effect of bile acids alone or in combination with acid on intracellular pH (pHi). A mechanism responsible for bile acid-induced pHi alterations was examined. Methods: CP-A cells (non-dysplastic Barrett's esophagus cells) were treated with medium acidified to pH 5.5 and/or 0.5mM bile acid cocktail in the presence/absence of inhibitors of nitric oxide synthase (NOS) or NHE (sodium-hydrogen exchanger). pHi was measured by BCECF microfluorimetry in perfused cells, nitric oxide by DAF-FM fluorescence and DNA damage by the comet assay. Western blot analysis was used to detect the expression of phosphorylated (activated) eNOS, iNOS and NHE1. Results: A dose-dependent decrease in pHi was observed in CP-A cells exposed to bile acids. Bile acid-induced intracellular acidification was abolished when NO production was inhibited by a nonspecific NOS inhibitor L-NAME or by thea specific iNOS inhibitor 1400W. In contrast the NHE inhibitor DMA, significantly decreased the bile acid-mediated pHi response. Exposure of the cells to acidified medium (pH5.5) containing a 0.5mM bile acid cocktail, markedly decreased pHi pHi to a value below extracellular pH. Importantly, medium acidified to pH5.5 containing 0.5mM bile acids induced a similar decrease in pHi as medium acidified to pH 4.5 without bile acids. This The same treatment resulteds in a 250% increase in acid-mediated DNA damage compared to medium acidified to pH5.5 in CP-A cells. Conclusion: In summary, a new mechanism of bile acid induced damage was identified. Bile acids alone or in combination with acidic pH significantly decrease intracellular pH in esophageal cells. The results suggest the effect ofresponse tpo bile acids is the consequence of NOS activation and that leads to NHE inhibition. The mMarked increase in DNA damage after suggests exposure to bile acids in combination with acid may play a crucial role in esophageal carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4105.

Endothelial nitric oxide synthase activity involves in the protective effect of ascorbic acid against penicillin-induced epileptiform activity

Ascorbic acid and nitric oxide are known to play important roles in epilepsy. The aim of present study was to identify the involvement of nitric oxide (NO) in the anticonvulsant effects of ascorbic acid on penicillin-induced epileptiform activity in rats. Intracortical injection of penicillin (500, International Units (IU)) into the left sensorimotor cortex induced epileptiform activity within 2–5 min. Thirty minutes after penicillin injection, nitric oxide synthase (NOS) inhibitor, N G-nitro- l-arginine methyl ester ( l-NAME, 100 mg/kg), neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI, 40 mg/kg), NO substrate, l-arginine (500 mg/kg) were administered with the most effective dose of ascorbic acid (100 mg/kg) intraperitoneally (i.p.). The administration of l-arginine significantly decreased the frequency of epileptiform activity while administration of l-NAME did not influence the mean frequency of epileptiform activity. Injection of 7-NI decreased the mean frequency of epileptiform activity but did not influence amplitude. Ascorbic acid decreased both the mean frequency and amplitude of penicillin-induced epileptiform activity in rats. The application of l-NAME partially and temporarily reversed the anticonvulsant effects of ascorbic acid. The results support the hypothesis of neuro-protective role for NO and ascorbic acid. The protective effect of ascorbic acid against epileptiform activity was partially and temporarily reversed by nonspecific nitric oxide synthase inhibitor l-NAME, but not selective neuronal nitric oxide synthase inhibitor 7-NI, indicating that ascorbic acid needs endothelial-NOS/NO route to decrease penicillin-induced epileptiform activity.

Role of inducible nitric oxide synthase on the development of virus-associated asthma exacerbation which is dependent on Th1 and Th17 cell responses

Asthma is characterized by airway inflammation induced by immune dysfunction to inhaled antigens. Although respiratory viral infections are the most common cause of asthma exacerbation, immunologic mechanisms underlying virus-associated asthma exacerbation are controversial. Clinical evidence indicates that nitric oxide (NO) levels in exhaled air are increased in exacerbated asthma patients compared to stable patients. Here, we evaluated the immunologic mechanisms and the role of NO synthases (NOSs) in the development of virus-associated asthma exacerbation. A murine model of virus-associated asthma exacerbation was established using intranasal challenge with ovalbumin (OVA) plus dsRNA for 4 weeks in mice sensitized with OVA plus dsRNA. Lung infiltration of inflammatory cells, especially neutrophils, was increased by repeated challenge with OVA plus dsRNA, as compared to OVA alone. The neutrophilic inflammation enhanced by dsRNA was partly abolished in the absence of IFN-gamma or IL-17 gene expression, whereas unaffected in the absence of IL-13. In terms of the roles of NOSs, dsRNA-enhanced neutrophilic inflammation was significantly decreased in inducible NOS (iNOS)-deficient mice compared to wild type controls; in addition, this phenotype was inhibited by treatment with a non-specific NOS inhibitor (L-NAME) or an specific inhibitor (1400 W), but not with a specific endothelial NOS inhibitor (AP-CAV peptide). Taken together, these findings suggest that iNOS pathway is important in the development of virus-associated exacerbation of neutrophilic inflammation, which is dependent on both Th1 and Th17 cell responses.

Enhancement of the anti‐inflammatory and anti‐arthritic effects of theophylline by a low dose of a nitric oxide donor or non‐specific nitric oxide synthase inhibitor

Although there are many new specific phosphodiesterase inhibitors with anti-inflammatory activity, none have yet reached the market because of their low therapeutic efficacy. Our study was aimed to evaluate the anti-inflammatory and anti-arthritic effect of an established phosphodiesterase inhibitor, theophylline, and to investigate the effect of the nitric oxide (NO) donor, sodium nitroprusside (SNP) or NO synthase inhibitor, L-N(G)-monomethyl arginine (L-NMMA) on its actions. The effects of theophylline alone and combined with SNP or L-NMMA on the pathogenesis of adjuvant-induced arthritis in rats were evaluated. Prophylactic or therapeutic doses of theophylline significantly ameliorated the pathogenesis of adjuvant arthritis in rats as evidenced by a significant decrease in the arthritis index, hind paws volume, ankle joint diameter, fever, body weight loss and hyperalgesia in a dose-dependent manner. Inflammatory cellular infiltrate in synovium of ankle joint and pannus formation were also markedly inhibited. Interleukin-10 (IL-10) levels were significantly increased in arthritic rats given theophylline alone or in combination with either SNP or L-NMMA. Co-administration of a low dose of SNP or L-NMMA enhanced significantly the anti-inflammatory and anti-arthritic effect of theophylline. In contrast, a high dose of SNP counteracted the anti-inflammatory and anti-arthritic effects of theophylline. These findings confirm the anti-inflammatory and anti-arthritic activities of theophylline and suggest a new approach to enhance the anti-inflammatory and anti-arthritic effects of theophylline would be to administer it in combination with a low dose of a NO donor or a non-specific NO synthase inhibitor.

Direct effect of cigarette smoke on human pulmonary artery tension

The effect of chronic cigarette smoke on pulmonary artery (PA) tension has been studied extensively; nevertheless, the direct effect of cigarette smoke is poorly understood. We investigated the direct effect of cigarette smoke extract (CSE) on PA tension in non-smokers, smokers, and COPD patients in vitro. PA samples from 35 patients who underwent lung resection were examined by measuring isometric tension in response to increasing serotonin concentrations. CSE dose dependently inhibited the response to serotonin in smokers and COPD patients, and to a lesser extent in non-smokers. CSE-induced relaxation was similarly inhibited by the nonspecific nitric oxide synthase (NOS) inhibitor l-NOARG and the specific inducible NOS (iNOS) inhibitor l-NIL, mainly in non-smokers and smokers, and to a lesser extent in COPD patients. Immunostaining of iNOS in PA samples was greater for smokers and COPD patients compared with non-smokers, which explains the lesser effect of CSE on PA tension in non-smokers. Moreover, CSE induced the release of nitrite via iNOS in human PA smooth muscle cells. In conclusion, CSE inhibition of serotonin-induced PA contraction was mediated mainly by iNOS in non-smokers, smokers, and COPD patients, but in different ways, which may be explained by differential iNOS expression in the PA of these patients.

Influence of L-arginine on the nitric oxide concentration and level of oxidative stress during ischemia-reperfusion injury in a rat model

This study aimed at evaluating whether supplementation of L-arginine in the course of ischemia and reperfusion syndrome after acute, experimental ischemia of the hind legs of the rat, could influence nitric oxide (NO) concentration and selected biochemical parameters concentration related to the oxidative stress. The study included 64 male Wistar rats. The animals were divided into four groups: Group I = control, Group II = placebo, Group III = L-arginine (500 mg/kg body mass/day for 5 days, p.o.), Group IV = L-arginine and nonspecific nitric oxide synthase (NOS) inhibitor - N(G)-Nitro-L-arginine-methyl ester (L-NAME) (75 micromol/ rat/day for 5 days, p.o.). Each group was further divided into subgroups: 1 = animals not subjected to ischemia and reperfusion, 2 = animals underwent 4-hour ischemia and subsequent reperfusion. Animals from Subgroup 2 were anesthetized and submitted to acute tourniquet ischemia of the hind limb. Blood samples were collected from all anesthetized rats by puncturing the right ventricle to assess total antioxidant status, lipids' peroxide concentration and nitric oxide concentration before ischemia and at 4th hour of ischemia and at 30th, 60th or 120th minute of reperfusion. We found that administration of L-arginine to rats resulted in significant increase of NO, level of total antioxidant status (TAS), and decrease of lipid' peroxide concentration when compared to the control and placebo groups. All these laboratory changes were progressing along with lengthening of reperfusion time. Simultaneous application of L-NAME led to reversal of phenomena caused by L-arginine. The present results suggest that L-arginine may protect tissues and organs against ischemia-reperfusion injury. The potential therapeutic role of L-arginine administration in prevention and treatment of ischemia-reperfusion syndrome consequences needs further investigation in humans.

The role of nitric oxide in the inhibitory effect of ghrelin against penicillin-induced epileptiform activity in rat

Ghrelin, a gastric peptide with key action on food intake, has been recently recognized as a potential antiepileptic agent. In the present study, we investigated the involvement of nitric oxide in the effect of ghrelin on penicillin-induced epileptiform activity in rat. Thirty minutes after penicillin injection, ghrelin, at doses of 0.5, 1, 2 microg, was administered intracerebroventricularly (i.c.v.). Ghrelin, at a dose of 1 microg, significantly decreased the mean frequency of epileptiform activity without changing the amplitude whereas other doses of ghrelin (0.5 and 2 microg) did not alter either the mean of frequency or amplitude of epileptiform activity. The effects of systemic administration of nitric oxide synthase (NOS) inhibitors, non-selective N(G)-nitro-l-arginine methyl ester (l-NAME), selective neuronal NOS inhibitor, 7-nitroindazole (7-NI) and NO substrate, l-arginine on the anticonvulsive effects of ghrelin were investigated. The administration of l-NAME (60 mg/kg, i.p.), 15 min before ghrelin (1microg) application, reversed the anti-epileptiform effects of ghrelin whereas 7-NI (40 mg/kg, i.p.) did not influence it. The present study provides electrophysiological evidence that the intracerebroventricular injection of ghrelin has an inhibitory effect against epileptiform activity in the penicillin model of epilepsy. The anti-epileptiform activity of ghrelin was reversed by nonspecific nitric oxide synthase inhibitor l-NAME, but not selective neuronal nitric oxide synthase inhibitor 7-NI, indicating that ghrelin requires activation of endothelial-NOS/NO route in the brain.

Direct evidence of nitric oxide release from neuronal nitric oxide synthase activation in the left ventricle as a result of cervical vagus nerve stimulation

Information regarding vagal innervation in the cardiac ventricle is limited and the direct effect of vagal stimulation on ventricular myocardial function is controversial. We have recently provided indirect evidence that the anti-fibrillatory effect of vagus nerve stimulation on the ventricle is mediated by nitric oxide (NO). The aim of this study was to provide direct evidence for the release of nitric oxide in the cardiac ventricle during stimulation of the efferent parasympathetic fibres of the cervical vagus nerve. The isolated innervated rabbit heart was employed with the use of the NO fluorescent indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) during stimulation of the cervical vagus nerves and acetylcholine perfusion in the absence and presence of the non-specific NO synthase inhibitor NG-nito-L-arginine (L-NNA) and the neuronal NO synthase selective inhibitor 1-(2-trifluormethylphenyl)imidazole (TRIM). Using the novel fluorescence method in the beating heart, we have shown that NO-dependent fluorescence is increased by 0.92 +/- 0.26, 1.20 +/- 0.30 and 1.91 +/- 0.27% (during low, medium and high frequency, respectively) in the ventricle in a stimulation frequency-dependent manner during vagus nerve stimulation, with comparable increases seen during separate stimulation of the left and right cervical vagus nerves. Background fluorescence is reduced during perfusion with L-NNA and the increase in fluorescence during high frequency vagal stimulation is inhibited during perfusion with both L-NNA (1.97 +/- 0.35% increase before L-NNA, 0.00 +/- 0.02% during L-NNA) and TRIM (1.78 +/- 0.18% increase before TRIM, -0.11 +/- 0.08% during TRIM). Perfusion with 0.1 microM acetylcholine increased NO fluorescence by 0.76 +/- 0.09% which was blocked by L-NNA (change of 0.00 +/- 0.03%) but not TRIM (increase of 0.82 +/- 0.21%). Activation of cardiac parasympathetic efferent nerve fibres by stimulation of the cervical vagus is associated with NO production and release in the ventricle of the rabbit, via the neuronal isoform of nitric oxide synthase.