Selective Neuronal Nitric Oxide Synthase Inhibitor Research Articles

Influence of NO Synthase Inhibitors on Serotonin Release in the Medial Prefrontal Cortex during the Formation and Generalization of the Conditioned Fear Response in Rats

Serotonergic and nitrergic systems of the medial prefrontal cortex (mPFC) are involved in the control of fear generalization, but their local interaction during this function has been little studied. The aim of the work was to study the effect of blockade of endogenous nitrergic signals on serotonin release in the mPFC during the acquisition of a conditioned fear response (CFR – a fear model) and on the dynamics of its generalization. In male Sprague-Dawley rats using intracranial microdialysis in vivo and high-performance liquid chromatography with electrochemical detection, we found that the intra-mPFC infusion through the dialysis probe of the NO synthase inhibitor N-ω-nitro-L-arginine (NA, 0.5 mM) and selective neuronal NO synthase inhibitor N-ω-propyl-L-arginine (NPLA, 2mM) decreased the basal level of extracellular serotonin in the mPFC and reduced its rise, caused by the CFR acquisition (a paired presentation of a conditioned cue (CS+) and inescapable footshock). The intra-mPFC infusion of NA and NPLA increased animals’ freezing to a differential cue (CS–) not associated with footshock, during the first test, carried out 70 minutes after the CFR acquisition, but reduced it during repeated testing a day after the infusion, without changing freezing of the same animals to the potentially dangerous CS+. The data obtained indicate the involvement of endogenous NO in the activation of serotonin release in the mPFC, caused by the CFR acquisition. In addition, they show that the blockade of endogenous nitrergic signals of the mPFC, which enhances the initial generalization of the fear reaction, contributes to the extinction of the generalized fear, possibly due to inhibition of the serotonin release in the mPFC.

Analgesic and anti-inflammatory effects of modafinil in a mouse model of neuropathic pain: A role for nitrergic and serotonergic pathways

ABSTRACT Objectives To evaluate the effects of modafinil on neuropathic pain induced by sciatic nerve cuffing in mice, and possible contribution of nitrergic/inflammatory and serotonergic systems. Methods Neuropathic pain was induced by applying a polyethylene cuff around the left sciatic nerve. Seven days later, mice received modafinil (50, 100, and 200 mg/kg; intraperitoneal [i.p.]) and morphine (10 mg/kg, i.p.) as control. Mice also received pretreatments of the nonselective nitric oxide (NO) synthase (NOS) inhibitor L-NAME, the selective neuronal NOS inhibitor 7-nitroindazole, the selective inducible NOS inhibitor aminoguanidine, and the selective serotonin reuptake inhibitor citalopram before modafinil (100 mg/kg). von Frey test was used to evaluate mechanical allodynia. Additionally, sciatic nerves were collected for histopathological analysis. Tissue levels of NO metabolites, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were assessed. Results Animals whose sciatic nerves were cuffed had a significantly (P<0.001) decreased paw withdrawal threshold (PWT) compared with the sham-operated group. Modafinil (100 mg/kg) and morphine significantly reversed PWT (P<0.001). Pretreatments with L-NAME, 7-nitroindazole, aminoguanidine, and citalopram in different groups markedly reversed analgesic effects of modafinil. Tissue homogenates of Cuffed sciatic nerves showed significantly higher levels of NO metabolites, TNF-α and IL-6 (P<0.001). Modafinil lowered NO metabolites, TNF-α, and IL-6 levels (P<0.001). Histopathology illustrated marked axonal degeneration and shrinkage in the cuffed sciatic nerve, which were improved in the modafinil-treated group. Conclusions Modafinil exerts analgesic and neuroprotective effects in cuff-induced neuropathic mice via possible involvement of the nitrergic/inflammatory and serotonergic systems.

The synthetic peptide PnPP-19 potentiates erectile function via nNOS and iNOS

PnPP-19 peptide has a primary sequence design based on molecular modeling studies of PnTx2-6 toxin. It comprises the amino acid residues that are potentially significant for the pharmacological action of PnTx2-6. Ex vivo and in vivo experiments in normotensive, hypertensive, or diabetic murine models have shown a significant improvement in penile erection after administration of PnPP-19. Given the potential use of PnPP-19 in pharmaceutical formulations to treat erectile dysfunction and the lack of information concerning its mode of action, the present work investigates its activities on the nitrergic system. PnPP-19 induced a significant increase in nitric oxide (NO) and cGMP levels in corpus cavernosum (cc). These effects were inhibited by l-NAME, a non-selective inhibitor of nitric oxide synthase (NOS); were partially inhibited by 7- Nitroindazole, a selective inhibitor of neuronal NOS (nNOS); and were abolished by L-NIL, a selective inhibitor of inducible NOS (iNOS). This potentiating effect was not affected by atropine. PnPP-19 also led to changes in mRNA levels, protein expression and phosphorylation at specific sites of NOS, in cc. Assays using cavernous tissue from knockout mice to endothelial NOS (eNOS), nNOS or iNOS showed that PnPP-19 potentiates relaxation only in eNOS-knockout mice, which suggests an essential role for nNOS. Surprisingly, iNOS enhanced the potentiation of erectile function evoked by PnPP-19. Our results demonstrate that this new synthetic peptide potentiates erectile function via nitric oxide activation and reinforce its role as a new pharmacological tool for the treatment of erectile dysfunction.

Anticonvulsant Effects of Thalidomide on Pentylenetetrazole-Induced Seizure in Mice: A Role for Opioidergic and Nitrergic Transmissions

Although accumulating evidence indicates that the immunomodulatory medication thalidomide exerts anticonvulsant properties, the mechanisms underlying such effects of thalidomide are still unknown. Our previous preclinical study suggested that nitric oxide (NO) signaling may be involved in the anticonvulsant effects of thalidomide in a mouse model of clonic seizure. Additionally, several studies have shown a modulatory interaction between thalidomide and opioids in opioids intolerance, nociception and neuropathic pain. However, it is unclear whether opioidergic transmission or its interaction with NO signaling is involved in the anticonvulsant effects of thalidomide. Given the fact that both opioidergic and nitrergic transmissions have bimodal modulatory effects on seizure thresholds, in the present study we explored the involvement of these signaling pathways in the possible anticonvulsant effects of thalidomide on the pentylenetetrazole (PTZ)-induced clonic seizure in mice. Our data showed that acute administration of thalidomide (5-50 mg/kg, i.p., 30 min prior PTZ injection) dose-dependently elevated PTZ-induced clonic seizure thresholds. Acute administration of low doses (0.5-3 mg/kg, i.p., 60 min prior PTZ) of morphine exerted anticonvulsant effects (P < 0.001), whereas higher doses (15-60 mg/kg, 60 min prior PTZ) had proconvulsant effects (P < 0.01). Acute administration of a non-effective anticonvulsant dose of morphine (0.25 mg/kg) prior non-effective dose of thalidomide (5 mg/kg) exerted a robust (P < 0.01) anticonvulsant effect. Administration of a non-effective proconvulsant dose of morphine (7.5 mg/kg) prior thalidomide (5 mg/kg) didn’t affect clonic seizure thresholds. Acute administration of a non-effective dose of the opioid receptor antagonist naltrexone (1 mg/kg, i.p.) significantly prevented anticonvulsant effects of thalidomide (10 mg/kg, i.p.). Pretreatment with non-effective dose of the NO precursor L-arginine (60 mg/kg, i.p.) significantly (P < 0.01) reduced the anticonvulsant effects of combined low doses of morphine (0.25 mg/kg) and thalidomide (5 mg/kg). Conversely, pretreatment with non-effective doses of either non-selective (L-NAME, 5 mg/kg, i.p.) or selective neuronal (7-nitroindazole, 30 mg/kg, i.p.) NO synthase (NOS) inhibitors significantly augmented the anticonvulsant effects of combined low doses of thalidomide and morphine, whereas the inducible NOS inhibitor aminoguanidine (100 mg/kg, i.p.) did not exert such effect. Our results indicate that opioidergic transmission and its interaction with neuronal NO signaling may contribute to the anti-seizure activity of thalidomide in the mice PTZ model of clonic seizure.

Involvement of l‐arginine‐nitric oxide pathway in the antidepressant and memory promoting effects of morin in mice

l-Arginine-nitric oxide pathway has been reported to be involved in the mediation of the psychopharmacological effects of many psychotropic drugs. Previous studies have shown that morin, a psychotropic compound isolated from mulberry leaf produces functional psychopharmacological effects indicative of antidepressant, antipsychotic, anxiolytic and nootropic properties. However, the role of l-arginine-nitric oxide pathway in the psychotropic effects of morin has not been fully investigated, hence, the need for this study. Male Swiss mice were pretreated individually or in combination with nitric oxide precursor [l-arginine (750 mg/kg, i.p.)], competitive nonselective nitric oxide synthase (NOS) inhibitor [N(G)-nitro-l-arginine methyl ester (l-NAME, i.p) (50 mg/kg)] or selective neuronal NOS inhibitor [methylene blue (3.75 mg/kg, i.p)] prior to morin (100 mg/kg, i.p.) or saline (10 mL/kg, i.p.) treatment. Psychopharmacological activities were then evaluated 30 min later using open field, Y-maze, and forced swim tests. l-Arginine significantly reversed the effects of morin on locomotion, memory and depression in mice. The reduced motor activity and enhanced memory function produced by morin were significantly attenuated by methylene blue but augmented the antimobility activity of morin in the FST. Moreover, l-NAME potentiated the psychopharmacological effects of morin in the open field and forced swim tests but reduced its memory promoting effect. Meanwhile, morin supplementation reversed the effects of l-arginine on l-NAME-treated mice in all behavioral models. The results of this study suggest that l-arginine-nitric oxide pathway might play a role in the modulation of the antidepressant and memory promoting effects of morin in mice.

Nitric oxide and glutamate are contributors of anti-seizure activity of rubidium chloride: A comparison with lithium

The neuro-protective effects of rubidium and lithium as alkali metals have been reported for different central nervous system dysfunctions including mania and depression. The aim of this study was evaluating as well as comparing the effects of rubidium chloride (RbCl) and lithium chloride (LiCl) on different seizures paradigms in mice and determining the involvement of NMDA receptors and nitrergic pathway.To assess the seizures threshold, animals received intravenous pentylenetetrazole (PTZ, 0.5%; 1 mL/min). Male NMRI mice (6–8 weeks) received intraperitoneal (i.p.) injections of different doses of RbCl and LiCl. Doses greater than 10 mg/kg of RbCl showed a significant anticonvulsant activity 60 min after administration; the anticonvulsant effects of LiCl was observed at the doses more than 5 mg/kg and after 30 min in PTZ-induced seizure threshold. But, RbCl (10, 20 mg/kg, i.p) or LiCl (5, 10 mg/kg, i.p) injection did not induce protection against maximal electroshock (MES) or intraperitoneal injection of PTZ lethal dose (80 mg/kg)-induced seizure models.Pre-treatment with L-NAME (non-selective nitric oxide synthase (NOS) inhibitor, 10 mg/kg; i.p.) and 7-nitroindazole (selective neuronal NOS inhibitor, 30 mg/kg; i.p.) enhanced the anticonvulsive effects of both RbCl (5 mg/kg, i.p.) and LiCl (1 mg/kg, i.p.) in PTZ-induced seizure threshold model. Injection of MK-801 (NMDA receptor antagonist, 0.05 mg/kg; i.p.) before RbCl (5 mg/kg, i.p.; P < 0.001) and LiCl (1 mg/kg, i.p.; P < 0.001) administration increased the anti-seizure activity. But, treatment with L-arginine (precursor of nitric oxide, 100 mg/kg; i.p.) decreased the seizure threshold of both RbCl (20 mg/kg, i.p.; P < 0.001) and LiCl (10 mg/kg, i.p.; P < 0.001). Measurement of nitrite levels in hippocampus of animals revealed a remarkable reduction after treatment with RbCl (20 mg/kg, i.p; P < 0.05) and LiCl (10 mg/kg, i.p; P < 0.01).To conclude, rubidium may protect central nervous system against seizures in PTZ-induced seizures threshold model through NMDA/nitrergic pathways with a similarity to lithium effects in mice.

Nitric oxide synthase inhibitors protect against brain and liver damage caused by acute malathion intoxication.

To investigate the effect of NG-nitro-l-arginine methyl ester (l-NAME), a non-selective nitric oxide synthase (NOS) inhibitor, and 7-nitroindazole (7-NI), a selective neuronal NOS inhibitor, on oxidative stress and tissue damage in brain and liver and on DNA damage of peripheral blood lymphocytes in malathion intoxicated rats. Malathion (150mg/kg) was given intraperitoneally (i.p.) along with l-NAME or 7-NI (10 or 20mg/kg, i.p.) and rats were euthanized 4h later. The lipid peroxidation product malondialdehyde (MDA), nitric oxide (nitrite), reduced glutathione (GSH) concentrations and paraoxonase-1 (PON-1) activity were measured in both brain and liver. Moreover, the activities of glutathione peroxidase (GPx) acetylcholinesterase (AChE), and butyrylcholinesterase (BChE), total antioxidant capacity (TAC), glucose concentrations were determined in brain. Liver enzyme determination, Comet assay, histopathological examination of brain and liver sections and inducible nitric oxide synthase (iNOS) immunohistochemistry were also performed. (i) Rats treated with only malathion exhibited increased nitric oxide and lipid peroxidation (malondialdehyde) accompanied with a decrease in GSH content, and PON-1 activity in brain and liver. Glutathione peroxidase activity, TAC, glucose concentrations, AChE and BChE activities were decreased in brain. There were also raised liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and increased DNA damage of peripheral blood lymphocytes (Comet assay). Malathion caused marked histopathological changes and increased the expression of iNOS in brain and liver tissues. (ii) In brain of malathion-intoxicated rats, l-NAME or 7-NI resulted in decreased nitrite and MDA contents while increasing TAC and PON1 activity. Reduced GSH and GPx activity showed an increase by l-NAME. AChE activity increased by 20mg/kg l-NAME and 10mg/kg 7-NI. AChE activity decreased by the higher dose of 7-NI while either dose of 7-NI resulted in decreased BChE activity. (iii) In liver of malathion-intoxicated rats, decreased MDA content was observed after l-NAME or 7-NI. Nitrite level was unchanged by l-NAME but increased after 7-NI which also resulted in decreased GSH concentration and PON1 activity. Either inhibitor resulted in decreased liver ALT activity. (iv) DNA damage of peripheral blood lymphocytes was markedly inhibited by l-NAME or 7-NI treatment. (v) iNOS expression in brain and liver decreased by l-NAME or 7-NI. (vi) More marked improvement of the histopathological alterations induced by malathion in brain and liver was observed after 7-NI compared with l-NAME. In malathion intoxicated rats, the neuronal NOS inhibitor 7-NI and to much less extent l-NAME were able to protect the brain and liver tissue integrity along with improvement in oxidative stress parameters. The decrease in DNA damage of peripheral blood lymphocytes by NOS inhibitors also suggests the involvement of nitric oxide in this process.

A study of diaphyseal nutrient foramina in human tibia

To investigate the effect of NG-nitro-l-arginine methyl ester (l-NAME), a non-selective nitric oxide synthase (NOS) inhibitor, and 7-nitroindazole (7-NI), a selective neuronal NOS inhibitor, on oxidative stress and tissue damage in brain and liver and on DNA damage of peripheral blood lymphocytes in malathion intoxicated rats.Malathion (150 mg/kg) was given intraperitoneally (i.p.) along with l-NAME or 7-NI (10 or 20 mg/kg, i.p.) and rats were euthanized 4 h later. The lipid peroxidation product malondialdehyde (MDA), nitric oxide (nitrite), reduced glutathione (GSH) concentrations and paraoxonase-1 (PON-1) activity were measured in both brain and liver. Moreover, the activities of glutathione peroxidase (GPx) acetylcholinesterase (AChE), and butyrylcholinesterase (BChE), total antioxidant capacity (TAC), glucose concentrations were determined in brain. Liver enzyme determination, Comet assay, histopathological examination of brain and liver sections and inducible nitric oxide synthase (iNOS) immunohistochemistry were also performed.(i) Rats treated with only malathion exhibited increased nitric oxide and lipid peroxidation (malondialdehyde) accompanied with a decrease in GSH content, and PON-1 activity in brain and liver. Glutathione peroxidase activity, TAC, glucose concentrations, AChE and BChE activities were decreased in brain. There were also raised liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and increased DNA damage of peripheral blood lymphocytes (Comet assay). Malathion caused marked histopathological changes and increased the expression of iNOS in brain and liver tissues. (ii) In brain of malathion-intoxicated rats, l-NAME or 7-NI resulted in decreased nitrite and MDA contents while increasing TAC and PON1 activity. Reduced GSH and GPx activity showed an increase by l-NAME. AChE activity increased by 20 mg/kg l-NAME and 10 mg/kg 7-NI. AChE activity decreased by the higher dose of 7-NI while either dose of 7-NI resulted in decreased BChE activity. (iii) In liver of malathion-intoxicated rats, decreased MDA content was observed after l-NAME or 7-NI. Nitrite level was unchanged by l-NAME but increased after 7-NI which also resulted in decreased GSH concentration and PON1 activity. Either inhibitor resulted in decreased liver ALT activity. (iv) DNA damage of peripheral blood lymphocytes was markedly inhibited by l-NAME or 7-NI treatment. (v) iNOS expression in brain and liver decreased by l-NAME or 7-NI. (vi) More marked improvement of the histopathological alterations induced by malathion in brain and liver was observed after 7-NI compared with l-NAME.In malathion intoxicated rats, the neuronal NOS inhibitor 7-NI and to much less extent l-NAME were able to protect the brain and liver tissue integrity along with improvement in oxidative stress parameters. The decrease in DNA damage of peripheral blood lymphocytes by NOS inhibitors also suggests the involvement of nitric oxide in this process.

Activation of the NMDA receptor–neuronal nitric oxide synthase pathway within the ventral bed nucleus of the stria terminalis mediates the negative affective component of pain

Pain consists of sensory and affective components. Although the neuronal mechanisms underlying the sensory component of pain have been studied extensively, those underlying its affective component are only beginning to be elucidated. Previously, we showed the pivotal role of the ventral part of the bed nucleus of the stria terminalis (vBNST) in the negative affective component of pain. Here, we examined the role of glutamate-nitric oxide (NO) signaling in the affective component of pain in rats using a conditioned place aversion (CPA) test. Intra-vBNST injection of either CNQX (an AMPA receptor antagonist) or MK-801 (an NMDA receptor antagonist) dose-dependently attenuated intraplantar formalin-induced CPA (F-CPA) without reducing nociceptive behaviors. In vivo microdialysis showed that extracellular oxidative NO metabolites (NOx) levels were significantly increased by intraplantar formalin injection. Intra-vBNST injection of NPLA (a selective neuronal NO synthase (nNOS) inhibitor), c-PTIO (a NO scavenger), or ZL006 (a postsynaptic density-95 (PSD-95)-nNOS interaction inhibitor) dose-dependently suppressed F-CPA without attenuating nociceptive behaviors. Intra-vBNST injection of NOR3 (a NO donor) produced CPA in a dose-dependent manner in the absence of noxious stimulation. Furthermore, whole-cell patch-clamp electrophysiology in the vBNST slices revealed that NOR3 induced depolarization of hyperpolarization-activated cation current (Ih)-positive vBNST neurons, which was blocked by the NO scavenger. These results suggest that activation of glutamatergic transmission and subsequent nNOS-derived NO production within the vBNST mediate the negative affective component of pain and that NO-evoked excitation of Ih-positive vBNST neurons may be among the cellular mechanisms underlying pain-induced aversion.

Endothelial nitric oxide synthase in rat brain is downregulated by sub-chronic antidepressant treatment.

Nitric oxide (NO) is a neurotransmitter that may be related to major depressive disorder (MDD) because the selective neuronal NO synthase (NOS) inhibitor, 7-nitroindazole, induces a dose-dependent antidepressant-like effect. NO modulates major neurotransmitters involved in the neurobiology of MDD, such as norepinephrine, serotonin, dopamine, and glutamate. In this study, we investigated the effects of antidepressants as NO modulators in acute and sub-chronic treatments. Rats were injected with the SSRI paroxetine (PAR, 10mg/kg), the SNRI milnacipran (MIL, 30mg/kg), or the NaSSA mirtazapine (MIR, 10mg/kg) for acute (1h) or sub-chronic (3weeks) treatment prior to analysis of nine brain regions (frontal cortex, temporal cortex, striatum, thalamus, hippocampus, midbrain, pons, cerebellum, and olfactory bulb). The mRNA expression levels of three NOS subtypes (neuronal: nNOS, inducible: iNOS, and endothelial: eNOS) were analyzed using real-time PCR with Taqman probes. Acute MIR treatment significantly increased nNOS mRNA expression in the hippocampus, midbrain, cerebellum and olfactory bulb, and iNOS mRNA expression in the frontal cortex and midbrain. Acute PAR and MIR treatments significantly increased eNOS mRNA expression in most brain regions. Conversely, sub-chronic treatment with all types of antidepressants resulted in significant decreases of eNOS mRNA expression in most brain regions. Sub-chronic treatment with the three types of antidepressants consistently decreased eNOS mRNA expression levels in the rat brain. These effects may be associated with the involvement of the NO system in the mechanism of action of antidepressants.

Effects of oleuropein on pentylenetetrazol-induced seizures in mice: involvement of opioidergic and nitrergic systems.

Oleuropein, a well-known olive polyphenol, has been shown to mediate neuroprotection in Alzheimer's disease and cerebral ischemia. We investigated the effects of oleuropein on pentylenetetrazole (PTZ)-induced seizures in male NMRI mice, with diazepam as the standard drug. We also examined the possible involvement of opioidergic/nitrergic pathways in the probable effects of oleuropein. Intraperitoneal (i.p.) administration of different doses of oleuropein (10, 20 and 30mg/kg) significantly increased the seizure threshold 60min prior to induction of seizure, in a dose-dependent manner. Administration of naltrexone (10mg/kg, i.p.), an opioid receptor antagonist, completely reversed the anticonvulsant effects of oleuropein (10mg/kg). On the other hand, the anticonvulsant effect of oleuropein (10mg/kg) was blocked by a non-effective dose of nonspecific inhibitor of nitric oxide synthase (NOS), L-NAME (1 and 10mg/kg, i.p) and a selective inhibitor of neuronal NOS, 7-nitroindazole (30mg/kg, i.p.). However, the nitric oxide precursor, L-arginine (30 and 60mg/kg, i.p.) potentiated the anticonvulsant activity of oleuropein (10mg/kg). A selective inducible NOS inhibitor, aminoguanidine (100mg/kg, i.p.) did not change the anticonvulsant activity of oleuropein. It seems that the opioidergic system and constitutive neuronal NOS may be involved in the anticonvulsant properties of oleuropein.

Nitric oxide production contributes to Bacillus anthracis edema toxin-associated arterial hypotension and lethality: ex vivo and in vivo studies in the rat.

We showed previously that Bacillus anthracis edema toxin (ET), comprised of protective antigen (PA) and edema factor (EF), inhibits phenylephrine (PE)-induced contraction in rat aortic rings and these effects are diminished in endothelial-denuded rings. Therefore, employing rat aortic ring and in vivo models, we tested the hypothesis that nitric oxide (NO) contributes to ET's arterial effects. Compared with rings challenged with PA alone, ET (PA + EF) reduced PE-stimulated maximal contractile force (MCF) and increased the PE concentration producing 50% MCF (EC50) (P < 0.0001). Compared with placebo, l-nitro-arginine methyl-ester (l-NAME), an NO synthase (NOS) inhibitor, reduced ET's effects on MCF and EC50 in patterns that approached or were significant (P = 0.06 and 0.03, respectively). In animals challenged with 24-h ET infusions, l-NAME (0.5 or 1.0 mg·kg(-1)·h(-1)) coadministration increased survival to 17 of 28 animals (60.7%) compared with 4 of 27 (14.8%) given placebo (P = 0.01). Animals receiving l-NAME but no ET all survived. Compared with PBS challenge, ET increased NO levels at 24 h and l-NAME decreased these increases (P < 0.0001). ET infusion decreased mean arterial blood pressure (MAP) in placebo and l-NAME-treated animals (P < 0.0001) but l-NAME reduced decreases in MAP with ET from 9 to 24 h (P = 0.03 for the time interaction). S-methyl-l-thiocitrulline, a selective neuronal NOS inhibitor, had effects in rings and, at a high dose in vivo models, comparable to l-NAME, whereas N'-[3-(aminomethyl)benzyl]-acetimidamide, a selective inducible NOS inhibitor, did not. NO production contributes to ET's arterial relaxant, hypotensive, and lethal effects in the rat.

Atorvastatin attenuates the antinociceptive tolerance of morphine via nitric oxide dependent pathway in male mice.

The development of morphine-induced antinociceptive tolerance limits its therapeutic efficacy in pain management. Atorvastatin, or competitive inhibitor of 3-hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase, is mainstay agent in hypercholesterolemia treatment. Beyond the cholesterol-lowering activity, exploration of neuroprotective properties of this statin indicates its potential benefit in central nervous disorders. The aim of the present study was to assess the effects of atorvastatin in development and expression of morphine-induced analgesic tolerance in male mice and probable involvement of nitric oxide. Chronic and acute treatment with atorvastatin 10 and 20mg/kg, respectively, could alleviate morphine tolerance in development and expression phases. Chronic co-administration of nitric oxide synthase (NOS) inhibitors including L-NAME (non selective NOS inhibitor; 2mg/kg), aminoguanidine (selective inducible NOS inhibitor; 50mg/kg) and 7-NI (selective neuronal NOS inhibitor; 15mg/kg) with atorvastatin blocked the protective effect of atorvastatin in tolerance reversal. Moreover, reversing the atorvastatin effect was also observed in acute simultaneous treatment of L-NAME (5mg/kg) and aminoguanidine (100mg/kg) with atorvastatin. Co-treatment of guanylyl cyclase inhibitor, ODQ (chronic dose: 10mg/kg and acute dose: 20mg/kg) was associated with prevention of atorvastatin anti-tolerance properties. Our results revealed that the atorvastatin modulating role in morphine antinociceptive tolerance is mediated at least in part via nitric oxide in animal pain models of hot plate and tail flick.

Cardiovascular drugs and triazole based kinase inhibitors as a new strategies for the treatment of Alzheimer disease

Atherosclerosis and stroke (including cerebrovascular atheroand arteriosclerosis with neurological consequences such as Alzheimer disease (AD)) are two leading causes of ageassociated disability, dementia, and death. Five million people suffer from AD only in the United States and this figure will balloon to 16 million persons, mostly the elderly, by the year 2040. Conventional wisdom for the last 20 years has decreed that AD is a "neurodegenerative" disorder caused primarily by the abnormal deposition of a brain tissue protein called amyloid beta. Neurodegenerative disorders are characterized by an entire loss of cognitive function and inappropriate death of nerve cells in the brain areas that control such functions as memory and language. The goal of this review is to determine the fact that there is a tight relationship between atherosclerosis and stroke-induced vascular lesions in AD pathology. We have also analyzed current status of the potential possibility of the therapeutics effects of such cardiovascular drugs as atorvastatin, simvastatin, and candesartan, as well as nitric oxide synthase (NOS) inhibitors 7-nitroindazole (a selective inhibitor of neuronal NOS), aminoguanidine (a selective inhibitor of inducible NOS), 1-nitroimidazole ornithine (a selective inhibitor of endothelial NOS), and purine-triazole based kinase inhibitors as a completely new treatment strategy in animal models that mimics cholesterol induced AD and/or AD-like pathology, which deserves special attention.

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 involvement of neuronal nitric oxide synthase in the anti-epileptic action of curcumin on pentylenetetrazol-kindled rats

In this study, it was investigated whether a NO signaling pathway is involved in the anti-epileptic effect of curcumin on pentylenetetrazol (PTZ)-kindled rats. PTZ-kindled rats received different doses of curcumin that were administered intraperitoneally for 24 days. Either a non-selective inhibitor of nitric oxide synthase (NOS) (N-nitro-L-arginine methyl ester (L-NAME)), a selective inhibitor of neuronal NOS (7-Nitroindazole (7-NI)), a selective inhibitor of inducible NOS (aminoguanidine (AG)), or a NO precursor (L-arginine (L-ARG)) was administered chronically to evaluate the role of NO in curcumin's anti-seizure effect. A chronic administration of curcumin (200 mg/kg) was most effective for decreasing the mean frequency of epileptiform discharge. Furthermore, a pretreatment with L-NAME or 7-NI augmented the anti-epileptic effect of curcumin. In contrast, AG failed to significantly alter the anti-epileptic effect of curcumin. A pretreatment with L-ARG temporally reversed the anti-epileptic effect of curcumin in the early stage, but in the late stage, it potentiated curcumin's anti-epileptic effect. These findings suggest that the L-arginine-nitric oxide pathway may be involved in the anti-epileptic properties of curcumin, and that the role of nNOS (and not iNOS) is prominent in this neuroprotective feature.

Antidepressant action via the nitric oxide system: A pilot study in an acute depressive model induced by arginin.

Nitric oxide (NO) may be a neurotransmitter related to major depressive disorder (MDD) because the selective neuronal NO synthase (NOS) inhibitor, 7-nitroindazole, induces dose-dependent antidepressant-like effects. However, its role in MDD is not yet known. The purpose of our study was to determine if antidepressants improve depression via the NO pathway using an acute depressive rat model induced by l-arginine (AR). Three types of antidepressants were examined, fluoxetine (FLX, 10mg/kg), milnacipran (MIL, 30mg/kg), and mirtazapine (MIR, 10mg/kg), in a depressive model that used AR (750mg/kg) pretreatment. mRNA expression levels of three NOS subtypes were analyzed by real-time PCR, as well as serum NO levels. Significant increases in iNOS mRNA expression levels were found in brain regions after AR treatment, although the eNOS gene tended to decrease with AR injection. After antidepressant treatment, there were no mRNA expression changes in either nNOS or iNOS. However, eNOS mRNA expression significantly increased with FLX (cerebellum, P=0.011; hippocampus, P=0.011; midbrain, P=0.011; pons, P=0.013; striatum, P=0.011; and thalamus, P<0.001). There was a statistically significant increase in serum NO levels with MIL treatment (P=0.011). We conclude that changes in eNOS mRNA levels in the brain with FLX treatment, and amount of serum NO with MIL treatment may be related to antidepressant effects of both agents, but further experiments are needed to confirm involvement of the NO system in MDD.

P2 purinergic receptor activation of neuronal nitric oxide synthase and guanylyl cyclase in the dorsal facial area of the medulla increases blood flow in the common carotid arteries of cats

In the dorsal facial area (DFA) of the medulla, an activation of either P2 purinergic receptor or nitric oxide synthase (NOS) results in the release of glutamate, leading to an increase in blood flow of the common carotid artery (CCA). It is not known whether activation of the P2 receptor by ATP may mediate activation of NOS/guanylyl cyclase to cause glutamate release and/or whether l-Arg (nitric oxide (NO) precursor) may also cause ATP release from any other neuron, to cause an increase in CCA flow. We demonstrated that microinjections of P2 receptor agonists (ATP, α,β-methylene ATP) or NO precursor (l-arginine) into the DFA increased CCA blood flow. The P2-induced CCA blood flow increase was dose-dependently reduced by pretreatment with NG-nitro-arginine methyl ester (l-NAME, a non-specific NOS inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NOS inhibitor) or methylene blue (MB, a guanylyl cyclase inhibitor) but not by that with d-NAME (an isomer of l-NAME) or N5-(1-iminoethyl)-l-ornithine (l-NIO, a potent endothelial NOS inhibitor). Involvement of glutamate release in these responses were substantiated by microdialysis studies, in which perfusions of ATP into the DFA increased the glutamate concentration in dialysates, but co-perfusion of ATP with l-NAME or 7-NI did not. Nevertheless, the arginine-induced CCA blood flow increase was abolished by combined pretreatment of l-NAME and MB, but not affected by pretreatment with a selective P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS). In conclusion, ATP activation of the P2 receptor in the DFA induced activation of neuronal NOS/guanylyl cyclase, which causes glutamate release leading to an increase in CCA blood flow. However, arginine activation of neuronal NOS/guanylyl cyclase, which also caused glutamate release and CCA blood flow increase, did not induce activation of P2 receptors. These findings provide important information for drug design and/or developing therapeutic strategies for the diseases associated with CCA blood flow that supplies intra- and extra-cranial tissues.

Nitric oxide synthase modulates the antihyperalgesic effect of the NMDA receptor antagonist MK-801 on Carrageenan-induced inflammatory pain in rats.

The N-methyl-D-aspartate (NMDA) receptor, an ionotropic glutamate receptor, may play a significant role in the development and maintenance of an inflammatory pain. Activation of NMDA receptors may cause nitric oxide (NO) release through activation of NO synthase (NOS). MK-801, a noncompetitive NMDA receptor antagonist is commonly used as a neuropharmacological tool. The interaction between MK-801 and NOS in the inflammatory pain has not been evaluated before. We investigated whether MK-801 affects inflammatory pain and whether NOS modulates the effect of MK-801. Carrageenan-induced hyperalgesia was evaluated by measuring the withdrawal response to mechanical stimuli, using an electronic version of the von Frey anesthesiometer in Wistar rats. MK-801 given subcutaneously (0.5-20 μg/kg) or intraplantarly (0.1 and 0.15 μg/paw) significantly reduced mechanical hyperalgesia. Intraplantarly given MK-801 exerted a local antihyperalgesic effect, because when applied to the contralateral side it did not reduce mechanical sensitivity in the ipsilateral side. N-nitro-L-arginine methyl ester hydrochloride (5 and 10 mg/kg), a non-selective NOS inhibitor, significantly reduced the effects of MK-801. N-ω-Propyl-L-arginine hydrochloride (0.5-2 mg/kg), a selective inhibitor of neuronal NOS, increased the antihyperalgesic effect of MK-801, whereas S-methylisothiourea (5-15 μg/kg), a selective inhibitor of inducible NOS, lowered the antihyperalgesic effect of MK-801. Importantly, each NOS inhibitor given alone did not affect carrageenan-induced hyperalgesia. In conclusion, MK-801 is effective against inflammatory pain and its antihyperalgesic effect is modulated in a different ways by NOS, being enhanced by a neuronal NOS inhibitor but reduced by an inducible NOS inhibitor.

Intermedin in Paraventricular Nucleus Attenuates Sympathetic Activity and Blood Pressure via Nitric Oxide in Hypertensive Rats

Intermedin (IMD) is a member of calcitonin/calcitonin gene-related peptide family, which shares the receptor system consisting of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs). This study investigated the effects of IMD in paraventricular nucleus (PVN) on renal sympathetic nerve activity and mean arterial pressure and its downstream mechanism in hypertension. Rats were subjected to 2-kidney 1-clip (2K1C) surgery to induce renovascular hypertension or sham operation. Acute experiments were performed 4 weeks later under anesthesia. IMD mRNA and protein were downregulated in 2K1C rats. Bilateral PVN microinjection of IMD caused greater decreases in renal sympathetic nerve activity and mean arterial pressure in 2K1C rats than in sham-operated rats, which were prevented by pretreatment with adrenomedullin receptor antagonist AM22-52 or nonselective nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester, and attenuated by selective neuronal NO synthase inhibitor N(ω)-propyl-l-arginine hydrochloride or endothelial NO synthase inhibitor N(5)-(1-iminoethyl)-l-ornithine dihydrochloride. AM22-52 increased renal sympathetic nerve activity and mean arterial pressure in 2K1C rats but not in sham-operated rats, whereas calcitonin/calcitonin gene-related peptide receptor antagonist calcitonin/calcitonin gene-related peptide 8-37 had no significant effect. CRLR and RAMP3 mRNA, as well as CRLR, RAMP2, and RAMP3 protein expressions, in the PVN were increased in 2K1C rats. Microinjection of IMD into the PVN increased the NO metabolites (NOx) level in the PVN in 2K1C rats, which was prevented by AM22-52. Chronic PVN infusion of IMD reduced, but AM22-52 increased, blood pressure in conscious 2K1C rats. These results indicate that IMD in the PVN inhibits sympathetic activity and attenuates hypertension in 2K1C rats, which are mediated by adrenomedullin receptors (CRLR/RAMP2 or CRLR/RAMP3) and its downstream NO.