Non-selective Nitric Oxide Synthase Research Articles

Spatial Memory in the Progeny of Rats Subjected to Different Types of Experimental Preeclampsia.

Spatial memory was studied in 2-month-old offspring of rats subjected to different types of experimental preeclampsia (replacement of drinking water with 1.8% NaCl from day 1 to 21 of gestation or intraperitoneal administration of non-selective NO-synthase inhibitor L-NAME to pregnant rats in a daily dose of 25 mg/kg for 7 days on gestation days 14-20). Spatial memory was evaluated in an elevated 8-arm radial maze. Both types of experimental preeclampsia impaired spatial (long-term and short-term) memory and can be used in the development of drugs correcting negative effects of this pregnancy complication on memory.

Centrally administered TNF increases arterial blood pressure independently of nitric oxide synthase

IntroductionEmerging evidence indicates that increased levels of TNF in the brain are associated with hypertension. Nitric oxide synthase (NOS) is involved in the central control of the cardiovascular system, exerting both pro- and antihypertensive effects. TNF induces hypothalamic synthesis of nitric oxide. AimWe checked if acutely administered TNF into the cerebral ventricles affects arterial blood pressure, heart rate and baroreflex sensitivity, and whether TNF actions are dependent on NOS in normotensive rats. MethodsWe carried out hemodynamic measurements in 6 groups of freely moving, adult Sprague-Dawley male rats, intracerebroventricularly (ICV) infused with either: 1) saline (5μl/h); 2) TNF (200ng/5μl/h); 3) non-selective NO synthase inhibitor – l-NG-Nitroarginine Methyl Ester (l-NAME) (1mg/5μl/h); 4) TNF together with l-NAME (200ng and 1mg/5μl/h, respectively); 5) neuronal NO synthase inhibitor – 7-nitroindazole sodium salt (7-NI) (20μg/10μl/h); 6) or TNF together with 7-NI (200ng and 20μg/10μl/h, respectively). Mean arterial blood pressure (MABP), heart rate (HR) and spontaneous baroreflex sensitivity (sBRS) evaluated by the sequence method were analysed. ResultsICV infusion of TNF caused a significant increase in MABP accompanied by a transient increase in HR, and a decrease in sBRS. ICV infusion of l-NAME increased MABP, but it did not change HR, nor sBRS. ICV infusion of 7-NI did not affect MABP, nor HR, nor sBRS. TNF administered together with l-NAME increased MABP with a transient increase in HR without changes of sBRS. Similarly, ICV infusion of TNF with 7-NI increased MABP without changes in HR and sBRS. ConclusionsCentrally administered TNF increases MABP and HR and blunts sBRS. The pressor effect of TNF appears to be independent of NOS activity in the brain. Inhibition of nNOS restores sBRS in TNF treated rats.

Role of NO/cGMP signaling pathway in dexmedetomidine-induced reduction of neuropathic pain in rats

Objective To investigate the role of nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway in dexmedetomidine-induced reduction of neuropathic pain (NP) in the rats. Methods Forty-two healthy male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 7 groups (n=6 each) using a random number table: normal control group (C group); NP group; dexmedetomidine group (D group); dimethyl sulfoxide (DMSO) group; a non-selective nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) group (L-NAME group); inactive enantiomer D-NAME group (D-NAME group); a soluble guanylyl cyclase inhibitor 1H-[1, 2, 4] oxidazole[4, 3-a] quinoxalin-1-one (ODQ) group (ODQ group). NP was induced by chronic constriction injury to the sciatic nerve in anesthetized rats.On day 7 after chronic constriction injury, dexmedetomidine 1.5 μg/kg was injected intrathecally in group D, and in DMSO, L-NAME, D-NAME and ODQ groups, DMSO 10 μl, L-NAME 100 μg, D-NAME 100 μg and ODQ 10 μg were injected intrathecally, respectively, and 25 min later dexmedetomidine 1.5 μg/kg was injected intrathecally.The thermal paw withdrawal latency was measured immediately after intrathecal administration and at 30, 60, 90, 120, 150, 180 and 210 min after intrathecal administration, and the area under the curve of thermal paw withdrawal latency was calculated to reflect the thermal pain threshold. Results Compared with group C, the thermal pain threshold was significantly decreased in the other six groups (P 0.05). Conclusion The mechanism by which dexmedetomidine reduces NP is related to activation of NO/cGMP signaling pathway in the rats. Key words: Dexmedetomidine; Neuralgia; Nitric oxide; Cyclic GMP; Spinal cord

Involvement of nitric oxide-cyclic guanosine monophosphate pathway in the antidepressant-like effect of tropisetron and ondansetron in mice forced swimming test and tail suspension test

Antidepressant-like effects of 5-hydroxytryptamine subtype 3 (5-HT3) antagonists including tropisetron and ondansetron have been previously demonstrated in the literature. It was reported that stimulation of 5-HT3 receptors activate the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway, which is involved in regulation of behavioral and emotional functions. In our study, treating animals with tropisetron (5, 10, and 30mg/kg) and ondansetron (0.01 and 0.1µg/kg) significantly decreased the immobility time in forced swimming test (FST) and tail-suspension test (TST). Co-administration of subeffective doses of tropisetron (1mg/kg) and ondansetron (0.001µg/kg) with subeffective dose of l-NAME (10mg/kg, nonselective NO synthase (NOS) inhibitor) and 7-nitroindazole (25mg/kg, neural NOS inhibitor) exerted antidepressant-like effect in FST and TST, while aminoguanidine (50mg/kg, inducible NOS inhibitor) did not enhance the antidepressant-like effect of 5-HT3 antagonists. Besides, l-arginine (750mg/kg, NO precursor) and sildenafil (5mg/kg, phosphodiesterase inhibitor) suppressed the anti-immobility effect of 5-HT3 antagonists. None of the treatments altered the locomotor behavior of mice in open-field test. Also, hippocampal (but not cortical) nitrite level was significantly lower in tropisetron and ondansetron-treated mice compared with saline-injected mice. Also, co-administration of 7-nitroindazole with tropisetron or ondansetron caused a significant decrease in hippocampal nitrite levels. In conclusion, we suggest that antidepressant-like effect of tropisetron and ondansetron are partially mediated by modulation of NO-cGMP pathway.

Possible involvement of nitrergic and opioidergic systems in the modulatory effect of acute chloroquine treatment on pentylenetetrazol induced convulsions in mice.

Chloroquine has long been used for the treatment of malaria and rheumatological disorders. Accumulating evidence suggests potential use of chloroquine as a neuroprotective agent. Several studies have reported that endogenous opioids and nitric oxide (NO) system mediate the chloroquine effects. In the present study, the involvements of endogenous opioids and NO in the modulatory effects of chloroquine on pentylenetetrazol-induced seizures were assessed in mice. Chloroquine 5mg/kg significantly increased the seizure threshold, but this effect was reversed with naltrexone 1mg/kg. Acute co-administration of l-NAME (non-selective NO synthase (NOS) inhibitor, 5mg/kg) or 7-NI (selective neuronal NOS inhibitor, 40 mg/kg) with the effective dose of chloroquine completely inhibited its anticonvulsant effects. Acute single injection of a sub-effective dose of l-arginine (NO precursor, 60 mg/kg) with a sub-effective dose of chloroquine 2.5mg/kg increased the seizure threshold but administration of L-arginine 60 mg/kg with chloroquine 10mg/kg decreased the seizure threshold. Moreover, the combination of the lower doses of naltrexone (0.1mg/kg) and 7-NI (15 mg/kg) showed additive effects in blocking the chloroquine-induced anticonvulsant properties. Chloroquine 5mg/kg enhanced the hippocampal nitrite levels. Chloroquine at the dose of 20mg/kg decreased the seizure threshold. This effect was inhibited through L-NAME (5mg/kg), 7-NI (40 mg/kg) and naltrexone (1mg/kg) administration with this dose of chloroquine. In conclusion, NO signaling probably through neuronal NOS, but not inducible NOS could be involved in the opioid-dependent anticonvulsant effects of chloroquine in this model of seizures in mice. It seems that nitric oxide and opioid systems are involved in modulatory effect of chloroquine on seizures induced by pentylenetetrazol.

The Effects Of L-Arginine And L-Name On Coronary Flow And Oxidative Stress In Isolated Rat Hearts

AbstractThe aim of this experimental study was to assess the effects of the acute administration of L-arginine alone and in combination with L-NAME (a non-selective NO synthase inhibitor) on the coronary flow and oxidative stress markers in isolated rat hearts. The experimental study was performed on hearts isolated from Wistar albino rats (n=12, male, 8 weeks old, body mass of 180-200 g). Retrograde perfusion of the isolated preparations was performed using a modified method according to the Langendorff technique with a gradual increase in the perfusion pressure (40–120 cmH2O). The following values were measured in the collected coronary effluents: coronary flow, released nitrites (NO production marker), superoxide anion radical and the index of lipid peroxidation (measured as thiobarbiturate reactive substances). The experimental protocol was performed under controlled conditions, followed by the administration of L-arginine alone (1 mmol) and L-arginine (1 mmol) + L-NAME (30 μmol). The results indicated that L-arginine did not significantly increase the coronary flow or the release of NO, TBARS and the superoxide anion radical. These effects were partially blocked by the joint administration of L-arginine + L-NAME, which indicated their competitive effect. Hence, the results of our study do not demonstrate significant effects of L-arginine administration on the coronary flow and oxidative stress markers in isolated rat hearts.

Abstract 12185: Complete Disruption of All Nitric Oxide Synthases Markedly Reduces Cerebral Infarct Size After Middle Cerebral Artery Occlusion in Mice

Background: The role of each nitric oxide synthase (NOS) isoform in cerebral infarction has been studied in individual NOS isoform-deficient mice. It has been reported that, in a model of middle cerebral artery occlusion (MCAO), neuronal and inducible NOSs exacerbate cerebral infarction, whereas endothelial NOS conversely alleviates it. Although the role of the whole NOSs system in cerebral infarction has been examined in pharmacological studies with non-selective NOS inhibitors, the results are quit inconsistent, possibly because of non-specificity of the agents. In this study, we addressed this point in mice in which all three NOS genes are completely disrupted. Method and Results: We newly generated triple NOSs -/- mice and wild-type littermates by crossbreeding single NOS -/- mice. Transient (1 hour) or permanent MCAO was performed in male triple NOSs -/- and wild-type mice at 8-12 weeks of age (n=8-11). There was no anatomical difference in the structure of cerebral arteries between the triple NOSs -/- and the wild-type mice. Reductions in cerebral blood flow during MCAO were also comparable between the two genotypes. However, cerebral infarct size at 24 hours after transient MCAO was markedly reduced in the triple NOSs -/- genotype as compared with the wild-type genotype ( P <0.05). Cerebral infarct size at 24 hours after permanent MCAO was also markedly smaller in the triple NOSs -/- than in the wild-type genotype ( P <0.05). In addition, neurological deficit was significantly less and the survival rate was significantly better in the triple NOSs -/- genotype compared with the wild-type genotype (each P <0.05). Importantly, 3-nitrotyrosine protein levels (a marker of peroxynitrite production) in the cerebral infarct region were significantly decreased in the triple NOSs -/- genotype as compared with the wild-type genotype ( P <0.05), suggesting an involvement of decreased oxidative stress. Conclusions: These results provide the first evidence that complete disruption of all NOS genes markedly reduces cerebral infarct size after MCAO in mice, demonstrating a novel injurious role of the entire NOSs system in the pathogenesis of cerebral infarction. Inhibition of the NOSs system may be a new therapeutic option in the treatment of cerebral infarction.

Studying the functional condition of rat epididymis mitochondria at nitric oxide (II) synthesis change

Aim. To study the levels of nitric oxide (II) metabolites, lactate, endogenic carnitine and mitochondrial oxidoreductase activity in epididymis tissue at nitric oxide low synthesis and, based on the obtained data, to assess the functional condition of rat epididymis mitochondria.
 Methods. 16 Wistar rats were allocated to two equal groups: The first group was administered L-NG-nitroarginine methyl ester (L-NAME), which is non-selective NO-synthase inhibitor, at a dose of 25 mg/kg for 7 days. The second group (the control group) got 0.9% sodium chloride solution administered as intraperitoneal injection for 7 days. Homogenate was obtained from epididymis tissue (head and tail), and mitochondria were isolated from it by differential centrifugation. Activities of mitochondrial enzymes (lactate dehydrogenase, succinate dehydrogenase and superoxide dismutase) were measured, as well as concentrations of NO metabolites, lactate, total protein and endogenous carnitine.
 Results. Activity of succinate dehydrogenase was significantly decreased by 55 and 68%, lactate dehydrogenase activity - by 78 and 92%, superoxide dismutase - by 16 and 43% in the mitochondria of epididymis head and tail tissue, respectively in the group receiving 25 mg/kg of L-NAME, compared to the control group. The concentrations of NO metabolites decreased by 18 and 30%, lactate levels increased by 43 and 35%, the share of bounded carnitine decreased by 25% in epididymis tail. Changes of carnitine concentrations in epididymis head were non-significant.
 Conclusion. In L-NAME-induced deficiency of NO in epididymis tissues mitochondria, statistically significant decrease in the activity of mitochondrial oxidoreductases and lactate accumulation was noticed, indicating the development of secondary mitochondrial dysfunction.

A COX/5-LOX Inhibitor Licofelone Revealed Anticonvulsant Properties Through iNOS Diminution in Mice.

Licofelone is a COX/5-LOX inhibitor, which recently was approved as an effective treatment for osteoarthritis. Beside its analgesic and anti-inflammatory effects, some reports show neuro-protective properties for this agent in central nervous system. Several lines of evidence declare the involvement of COX or LOX isoenzymes in epileptic disorders. To set the foundation for future research into the neurobiology of licofelone as a potential therapeutic agent, we studied the effect of licofelone in an animal model of epilepsy. Although different neurotransmitters and neuro-modulators like nitric oxide were introduced as suggested targets of licofelone, the underlying mechanisms of central effects of this drug are not still fully understood. We have utilized pentylenetetrazole-induced clonic seizure model to investigate the behavioral consequences of licofelone administration and its possible mechanisms in seizure susceptibility. Licofelone revealed anticonvulsant properties at the dose of 10mg/kg (i.p) or higher in mice. Pre-treatment with NO (nitric oxide) donor, L-arginine, reversed this anticonvulsant effects dose dependently. L-NAME, as a non-selective nitric oxide synthase (NOS) inhibitor, potentiated the anticonvulsant effects of licofelone. A neuronal NOS inhibitor, 7-NI did not affect seizure threshold alone or in combination with licofelone. Using non-effective doses of selective inhibitors of inducible NOS, aminoguanidine or 1400W, significantly increased the seizure threshold when were accompanied by licofelone in low doses. These data support the involvement of NO as an important role player in the central neuro-protective properties of licofelone. Furthermore, it implies that down regulation of iNOS seems crucial for anticonvulsant properties of this COX/5-LOX inhibitor in seizure susceptibility.

Enhanced nitric oxide bioavailability in coronary arteries prevents the onset of heart failure in rats with myocardial infarction

AimThe endothelium, mainly via nitric oxide (NO) release, adjusts the coronary flow. Cardiac function is closely linked to blood flow; thus, we tested the hypothesis that NO modulation in coronary arteries could be differentially adjusted after myocardial infarction (MI) in the presence or absence of heart failure (HF). Methods and resultsFour weeks after coronary occlusion, the infarcted rats were subdivided into rats without (MI) or with HF signs according to haemodynamic parameters. The septal coronary arteries were subsequently used to perform functional and molecular experiments. Acetylcholine (ACh)-induced relaxation was decreased in the coronary arteries following HF, whereas it was enhanced in the arteries of the MI compared with those of SHAM-operated (SO) rats. The relaxation induced by the NO donor was similar among the groups. NO production, which was evaluated by 4,5-diaminofluorescein diacetate, was reduced in the coronary arteries of the HF group and increased in the arteries with MI after ACh-induced stimulation. HF coronary arteries exhibited oxidative stress, which was evaluated via ethidium bromide-positive nuclei, whereas it was decreased in MI. To evaluate the mechanisms involved in the enhanced ACh-induced relaxation in the arteries following MI, certain septal coronary arteries were pre-incubated with L-NAME (a nonselective NO synthase (NOS) inhibitor), 7-NI (a selective neuronal NOS (nNOS) inhibitor) or LY294002 (a PI3-kinase inhibitor). L-NAME and LY294002 reduced ACh-induced relaxation in the MI and SO rats; however, these effects were greater in the MI arteries. 7-NI reduced only the ACh-relaxation in MI. In addition, the eNOS, nNOS, Akt, and superoxide dismutase isoform protein expressions were greater in the coronary arteries of the MI than in those of the SO groups. ConclusionOur data suggested that endothelial function was closely related to cardiac function after coronary occlusion. The coronary arteries from the HF rats exhibited reduced NO bioavailability, whereas the MI rats exhibited increased NO bioavailability because of increased eNOS/nNOS/PI3-kinase/Akt pathway and a reduction in ROS generation. These results suggest that enhanced NO modulation can prevent the onset of HF.

Effects of a New Glutamic Acid Derivative on Myocardial Contractility of Stressed Animals under Conditions of Nitric Oxide Synthesis Blockade.

Glufimet (glutamic acid derivative) in a dose of 28.7 mg/kg limited the reduction of the cardiac functional reserve in animals subjected to 24-h stress under conditions of nonselective NO synthase blockade with L-NAME (10 mg/kg). Adrenoreactivity and increased afterload tests showed that the increment of myocardial contraction/relaxation rates, left-ventricular pressure, and HR were significantly higher in glufimet-treated stressed animals with NO synthesis blockade than in animals which received no glufimet. The efficiency of glufimet was higher than that of phenibut (the reference drug).

Elevated level of nitric oxide mediates the anti-depressant effect of rubidium chloride in mice

Rubidium has been used to treat psychiatric conditions including depression. We examined the antidepressant activity of rubidium chloride (RbCl) in male mice and the possible interference of nitric oxide (NO) in this effect. Mouse forced swimming test (FST) and tail suspension test (TST) were used to evaluate the antidepressant-like effect of RbCl. These drugs were used in this study: NG-l-arginine methyl ester (l-NAME), a non-selective nitric oxide synthase (NOS) inhibitor, 7-Nitroindazole and aminoguanidine, selective neuronal and inducible NOS inhibitors, respectively, and l-arginine, an NO precursor. We studied the changes of serum and hippocampus nitrite level after different treatments. RbCl (30mg/kg), when administered 60min before the tests, significantly reduced the immobility time. Non-effective doses of l-NAME (10mg/kg) and aminoguanidine (50mg/kg), co-administered with the effective dose of RbCl (30mg/kg), reversed the anti-immobility effect of RbCl, while 7-NI (25mg/kg) could not prevent the diminishing effect of RbCl on immobility time. Moreover, co-administration of non-effective doses of l-arginine (750mg/kg) and RbCl (10mg/kg) decreased the immobility time. None of the mentioned treatments altered the locomotor activity of mice in open-field test. Nitrite level was significantly increased in serum and hippocampus of animals after RbCl (30mg/kg) administration and this nitrite level elevation was reversed by non-effective dose of l-NAME and aminoguanidine, but not 7-NI. Our data for the first time reveal the role of NO pathway in the antidepressant-like activity of RbCl, concluding that this effect results from elevation of NO through involvement of iNOS in mice.

PP.27.05

Objective: β-adrenoreceptor overstimulation with isoproterenol (ISO) induces endothelial dysfunction and hyperreactivity to phenylephrine (PHE). Statins, HMG-CoA reductase inhibitors, has pleiotropic effects that improve endothelial function. The aim of this study is to evaluate the effect of pretreatment with simvastatin (SIMV) on the hyperreactivity to PHE in the aorta of ISO-treated mice. Design and method: C57BL6/J mice were treated with SIMV (5 μg/g/day; gavage) or vehicle (VHC). From day 7, both groups received VHC (soybean oil; sc) or ISO (15 μg/g/kg; sc) for more 7 days. After treatment, the experimental groups (CT, ISO, SIMV and SIMV + ISO) were killed and heart, tibia and thoracic aorta were collected. The analysis of cardiac hypertrophy (CH) was performed using the ratio of heart weight / length of the tibia. PHE-induced contraction (10–10–10–5 M) in the presence or absence of non-selective nitric oxide (NO) synthase inhibitor (L-NAME, 100 μM) were evaluated in aortic rings. The fluorescent dye hydroethidine was used to evaluate the in situ production of reactive oxygen species (ROS) and endothelial NO synthase (eNOS) dimerization by Western Blot. Results are analyzed by Two-way ANOVA; p < 0.05 *vs CT, #vs. SINV. Results: ISO (26 ± 3% *) and SIMV + ISO (20 ± 2% #) presented CH when compared to controls. The maximum response (Rmax) to PHE was reduced in SIMV aortas when compared to CT. However, this response was higher in ISO and SIMV + ISO compared to their respective control. L-NAME increased Rmax to PHE in all groups. The magnitude of this response was greater in SIMV and reduced in ISO and SIMV + ISO. ROS bioavailability increased in ISO and SIMV + ISO, while did not change in SIMV. The ratio of eNOS dimer to monomer was significantly lower in aortas from ISO and SIMV + ISO groups. Conclusions: SIMV, despite increasing the NO bioavailability, was not able to reverse the reduction of nitrergic modulation, oxidative stress and eNOS uncoupled, which result in PHE hyperreactivity in aortas from ISO-treated mice.

The effect of acute aripiprazole treatment on chemically and electrically induced seizures in mice: The role of nitric oxide.

Aripiprazole is an antipsychotic drug which acts through dopamine and serotonin receptors. Aripiprazole was noted to have antiseizure effects in a study on mice, while it induced seizures in a few human case reports. Dopaminergic and serotonergic systems relate to nitric oxide, and aripiprazole also has effects on dopamine and serotonin receptors. This study investigated the effects of aripiprazole on seizures and the potential role of nitric oxide in the process. The following three models were examined to explore the role of aripiprazole on seizures in mice: 1 - pentylenetetrazole administered intravenously, 2 - pentylenetetrazole administered intraperitoneally, and 3 - electroshock. Aripiprazole administration delayed clonic seizure in intravenous and intraperitoneal pentylenetetrazole models. In the electroshock-induced seizure model, tonic seizure and mortality protection percent were increased after aripiprazole administration. In intraperitoneal administration of pentylenetetrazole, aripiprazole effects on clonic seizure latency were significantly decreased when l-NAME - a nonselective nitric oxide synthase (NOS) inhibitor, 7-nitroindazole - a selective neuronal NOS (nNOS) inhibitor, or aminoguanidine - a selective inducible NOS (iNOS) inhibitor was injected before aripiprazole administration. In the intravenous pentylenetetrazole method, administration of l-NAME or aminoguanidine inhibited aripiprazole effects on clonic seizure threshold. Aminoguanidine or l-NAME administration decreased aripiprazole-induced protection against tonic seizures and death in the electroshock model. In both intravenous and intraperitoneal seizure models, aripiprazole and l-arginine coadministration delayed the onset of clonic seizures. Moreover, it increased protection against tonic seizures and death in intraperitoneal pentylenetetrazole and electroshock models. In conclusion, the release of nitric oxide via iNOS or nNOS may be involved in anticonvulsant properties of aripiprazole.

The Effects of Sub-Chronic Treatment with Aripiprazole on Pentylenetetrazole- and Electroshock-Induced Seizures in Mice: The Role of Nitric Oxide

Almost all antipsychotics have been associated with a risk of epileptic seizure provocation. Aripiprazole is a novel atypical antipsychotic. The risk of seizures with aripiprazole is reported to be the lowest among atypical agents. In this study, we investigated the effect of aripiprazole on seizure of mice in sub-chronic treatments. We also examined the interaction of nitric oxide (NO) with aripiprazole in seizure experiments. Mice received aripiprazole for 6 days and then on the 7th day aripiprazole was injected 60 min before intraperitoneal pentylenetetrazole or electroshock. <smlcap>L</smlcap>-NAME (non-selective NO synthase (NOS) inhibitor), 7-nitroindazole (neuronal NOS selective inhibitor), aminoguanidine (inducible NOS selective inhibitors) or <smlcap>L</smlcap>-arginine (NO donor), all were injected 5 min before aripiprazole in separate groups. The results of both seizure models demonstrated anti-epileptic properties of aripiprazole in sub-chronic administrations. Co-administration of aripiprazole and selective and non-selective NOS inhibitors prevented the anticonvulsant effect of aripiprazole. While <smlcap>L</smlcap>-arginine and aripiprazole co-administration increased the clonic seizure threshold and protection against tonic seizure and death, these effects were not significant. The current results indicated that aripiprazole has anticonvulsant effects probably through the release of NO.

GLP-1 Receptor Agonist Exenatide Increases Capillary Perfusion Independent of Nitric Oxide in Healthy Overweight Men.

The insulinotropic gut-derived hormone glucagon-like peptide-1 (GLP-1) increases capillary perfusion via a nitric oxide-dependent mechanism in rodents. This improves skeletal muscle glucose use and cardiac function. In humans, the effect of clinically used GLP-1 receptor agonists (GLP-1RAs) on capillary density is unknown. We aimed to assess the effects of the GLP-1RA exenatide on capillary density as well as the involvement of nitric oxide in humans. We included 10 healthy overweight men (age, 20-27 years; body mass index, 26-31 kg/m(2)). Measurements were performed during intravenous infusion of placebo (saline 0.9%), exenatide, and a combination of exenatide and the nonselective nitric oxide-synthase inhibitor L-N(G)-monomethyl arginine. Capillary videomicroscopy was performed, and baseline and postocclusive (peak) capillary densities were counted. Compared with placebo, exenatide increased baseline and peak capillary density by 20.1% and 8.3%, respectively (both P=0.016). Concomitant L-N(G)-monomethyl arginine infusion did not alter the effects of exenatide. Vasomotion was assessed using laser Doppler fluxmetry. Exenatide nonsignificantly reduced the neurogenic domain of vasomotion measurements (R=-5.6%; P=0.092), which was strongly and inversely associated with capillary perfusion (R=-0.928; P=0.036). Glucose levels were reduced during exenatide infusion, whereas levels of insulin were unchanged. Acute exenatide infusion increases capillary perfusion via nitric oxide-independent pathways in healthy overweight men, suggesting direct actions of this GLP-1RA on microvascular perfusion or interaction with vasoactive factors.

Oxidative stress impairs NO‐dependent cutaneous vasodilation but not NO‐dependent sweating in young adults during high intensity exercise

Our recent work showed that nitric oxide (NO)‐dependent sweating and cutaneous vasodilation are diminished in young males during intermittent exercise at high (700 W) relative to moderate (400 W) rates of metabolic heat production. We tested the hypothesis that this impairment results from increased oxidative stress during high intensity exercise. On separate days, nine young (24 ± 2 years) healthy males performed two 30 min bouts of semi‐recumbent cycling in the heat (35°C, 20% RH) at a rate of metabolic heat production of i) 500 W or ii) 700 W. The first and second exercise bouts were followed by 20 and 40 min of recovery, respectively. Forearm local sweat rate (LSR) and cutaneous vascular conductance (CVC) were measured at 4 skin sites that were continuously perfused via intradermal microdialysis with: 1) lactated Ringer's solution (Control), 2) 10 mM ascorbate (ASC, antioxidant), 3) 10 mM L‐NG‐Nitroarginine methyl ester (L‐NAME, non‐selective NO synthase inhibitor), or 4) 10 mM ASC + 10 mM L‐NAME. During moderate intensity exercise (i.e., 500 W), L‐NAME and ASC + L‐NAME reduced LSR and CVC compared to the Control site (all P &lt; 0.05), while no effect of ASC was observed (all P &gt; 0.05). Conversely, high intensity exercise performed at 700 W did not result in differences in LSR between treatment sites (P = 0.85). However, compared to Control, CVC was elevated at the ASC (all P &lt; 0.05) while it was reduced at the L‐NAME (all P &lt; 0.05) and ASC + L‐NAME (all P &lt; 0.05) sites. Altogether these results suggest that oxidative stress impairs NO‐dependent cutaneous vasodilation but not LSR during intense exercise. Support: Natural Sciences and Engineering Research Council of Canada.

A primary role for kinin B1 receptor in inflammation, organ damage, and lethal thrombosis in a rat model of septic shock in diabetes.

Diabetes mellitus and septic shock increase the incidence of mortality by thrombosis. Although kinin B1 receptor (B1R) is involved in both pathologies, its role in platelet function and thrombosis remains unknown. This study investigates the expression, the inflammatory, and pro-thrombotic effects of B1R in a model of septic shock in diabetic rats. Sprague-Dawley rats were made diabetic with streptozotocin (STZ) (65 mg/kg, i.p.). Four days later, control and STZ-diabetic rats were injected with lipopolysaccharide (LPS) (2 mg/kg, i.p.) or the vehicle. B1R antagonist (SSR240612, 10 mg/kg by gavage) was given either acutely (12 and 24 h prior to endpoint analysis) or daily for up to 7 days. Moreover, a 7-day treatment was given either with cyclooxygenase (COX)-2 inhibitor (niflumic acid, 5 mg/kg, i.p.), non-selective COX-1 and COX-2 inhibitor (indomethacin, 10 mg/kg, i.p.), non-selective nitric oxide synthase (NOS) inhibitor (L-NAME, 50 mg/kg by gavage), iNOS inhibitor (1400W, 5 mg/kg, i.p.), or heparin (100 IU/kg, s.c.). The following endpoints were measured: edema and vascular permeability (Evans blue dye), B1R expression (qRT-PCR, western blot, flow cytometry), aggregation in platelet-rich plasma (optical aggregometry), and organ damage (histology). Rats treated with STZ, LPS, and STZ plus LPS showed significant increases in edema and vascular permeability (heart, kidney, lung, and liver) and increased expression of B1R in heart and kidney (mRNA) and platelets (protein). Lethal septic shock induced by LPS was enhanced in STZ-diabetic rats and was associated with lung and kidney damage, including platelet micro-aggregate formation. SSR240612 prevented all these abnormalities as well as STZ-induced hyperglycemia and LPS-induced hyperthermia. Similarly to SSR240612, blockade of iNOS and COX-2 improved survival. Data provide the first evidence that kinin B1R plays a primary role in lethal thrombosis in a rat model of septic shock in diabetes. Pharmacological rescue was made possible with B1R antagonism or by inhibition of iNOS and COX-2, which may act as downstream mechanisms.

Map kinase and PKC signaling pathways modulate NGF-mediated apoE transcription

The present study assessed the mechanisms by which nerve growth factor (NGF) increased the level of apolipoprotein E (apoE) in PC12 cells. NGF (50ng/mL) significantly increased apoE protein levels following 72h of treatment. Similarly NGF increased luciferase activity in cells transfected with a luciferase reporter construct containing a 500bp fragment of the apoE promoter, indicating NGF-induced apoE expression is regulated, at least in part, at the level of transcription. The non-selective nitric oxide synthase (NOS) inhibitor Nɷ-nitro-L-arginine methylester (L-NAME; 20mM) did not attenuate the NGF-mediated increase in luciferase activity, while the inducible NOS inhibitor s-methylisothiourea (S-MIU; 2mM) partially attenuated this action of NGF. Inhibition of MAP kinase activation with 50μM U0126 or pre-treatment with the PKC inhibitor bisindolylmaleimide 1 (BIS-1; 10μM) prevented the NGF-mediated activation of the apoE promoter. Pre-treatment with the phospholipase C (PLC) inhibitor U73122 (5μM) partially inhibited the NGF-induced increase in luciferase activity while the Akt inhibitor LY294002 (10μM) had no effect. These data suggest NGF-induced apoE transcription requires MAP kinase and PKC activation and that these TrkA signaling pathways may be modulated by NO.

Ginsenoside Re inhibits pacemaker potentials via adenosine triphosphate-sensitive potassium channels and the cyclic guanosine monophosphate/nitric oxide-dependent pathway in cultured interstitial cells of Cajal from mouse small intestine

BackgroundGinseng belongs to the genus Panax. Its main active ingredients are the ginsenosides. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the gastrointestinal (GI) tract. To understand the effects of ginsenoside Re (GRe) on GI motility, the authors investigated its effects on the pacemaker activity of ICCs of the murine small intestine. MethodsInterstitial cells of Cajal were dissociated from mouse small intestines by enzymatic digestion. The whole-cell patch clamp configuration was used to record pacemaker potentials in cultured ICCs. Changes in cyclic guanosine monophosphate (cGMP) content induced by GRe were investigated. ResultsGinsenoside Re (20–40μM) decreased the amplitude and frequency of ICC pacemaker activity in a concentration-dependent manner. This action was blocked by guanosine 5′-[β-thio]diphosphate [a guanosine-5'-triphosphate (GTP)-binding protein inhibitor] and by glibenclamide [an adenosine triphosphate (ATP)-sensitive K+ channel blocker]. To study the GRe-induced signaling pathway in ICCs, the effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) and RP-8-CPT-cGMPS (a protein kinase G inhibitor) were examined. Both inhibitors blocked the inhibitory effect of GRe on ICC pacemaker activity. L-NG-nitroarginine methyl ester (100μM), which is a nonselective nitric oxide synthase (NOS) inhibitor, blocked the effects of GRe on ICC pacemaker activity and GRe-stimulated cGMP production in ICCs. ConclusionIn cultured murine ICCs, GRe inhibits the pacemaker activity of ICCs via the ATP-sensitive potassium (K+) channel and the cGMP/NO-dependent pathway. Ginsenoside Re may be a basis for developing novel spasmolytic agents to prevent or alleviate GI motility dysfunction.