Nitric Oxide-cyclic GMP Pathway Research Articles

Antidepressant- and anxiolytic-like actions of Cajanus cajan seed extract mediated through monoaminergic, nitric oxide-cyclic GMP and GABAergic pathways.

The seeds of Cajanus cajan (L) Millsp, are used in Traditional medicine for the treatment of anxiety and other neurological disorders. Hence, this study is designed to investigate the antidepressant- and anxiolytic-like properties of ethanol seed extract of Cajanus cajan (CC) in mice. CC (50, 100 or 200mg/kg, p.o.) was administered 1h before subjecting the animals to different behavioral models: forced swim test (FST) and tail suspension test (TST) (depressive-like behaviour), open field test (OFT), elevated plus maze (EPM), light-dark test (LDT) and hole-board test (HBT) for anxiety-like behaviour. To ascertain the pharmacodynamic of CC mice were pretreated with monoaminergic, nitrergic and GABAergic receptors antagonists. As well as molecular docking analysis of about 19 flavonoids present in CC on GABAA, α2 adrenoceptors and 5-HT2A receptors. CC (50, 100 or 200mg/kg, p.o.) treatment significantly reduced immobile time in both FST and TST when compared with vehicle-treated control. However, the pretreatment of mice with prazosin/yohimbine (α1/2 adrenoceptor antagonists, respectively), WAY100635 (5-HT1A receptor antagonist), ketanserin (5-HT2A receptor antagonist), sulpiride (dopamine D2 receptor antagonist), L-NG-Nitro arginine methyl ester (L-NAME), or methylene blue reversed the antidepressant-like effect of CC. In anxiety model, CC produced significant (p<0.05) increase in open arms exploration and head dipping behavior which was reversed by flumazenil (benzodiazepine receptor antagonist) in the EPM. Docking analysis showed significant binding affinity of orientin, vitexin, pinostrobin and quercetin with 5HT2A, α2-adrenoceptor and GABAA receptors. Findings from this study showed that C.cajan seeds extract exerts antidepressant-like effect through participation of monoaminergic systems (5-HT2 receptor, α1/α2-adrenoceptors, and dopamine D2-receptors), nitric oxide-cyclic GMP pathway and anxiolytic-like effect via GABAA benzodiazepine receptors. Moreso, presence of flavonoids with significant binding energies with monoaminergic and GABAergic systems support the potential of the extract in the management of mixed anxiety-depressive illness.

Xylazole inhibits NO-cGMP pathway in fetal rat nerve cells.

BackgroundXylazole (Xyl) is a veterinary anesthetic that is structurally and functionally similar to xylazine. However, the effects of Xyl in vitro remain unknown.ObjectivesThis study aimed to investigate the anesthetic mechanism of Xyl using fetal rat nerve cells treated with Xyl.MethodsFetal rat nerve cells cultured for seven days were treated with 10, 20, 30, and 40 μg/ mL Xyl for 0, 5, 10, 15, 20, 25, 30, 45, 60, 90, and 120 min. Variations of amino acid neurotransmitters (AANTs), Nitric oxide-Cyclic GMP (NO-cGMP) signaling pathway, and ATPase were evaluated.ResultsXyl decreased the levels of cGMP and NO in nerve cells. Furthermore, Xyl affected the AANT content and Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity in nerve cells. These findings suggested that Xyl inhibited the NO-cGMP signaling pathway in nerve cells in vitro.ConclusionsThis study provided new evidence that the anesthetic and analgesic effects of Xyl are related to the inhibition of the NO-cGMP signaling pathway.

Antinociceptive effect of tadalafil in various pain models: Involvement of opioid receptors and nitric oxide cyclic GMP pathway

Nitric oxide has been proven to play an important role in nociception, accordingly, its promoters, phosphodiesterase inhibitors have been investigated as pain response modulators. Aiming to evaluate the central antinociceptive effect of tadalafil, a phosphodiesterase 5 inhibitor, and to determine its EC50, tail flick and hot plate tests were employed. On the other hand, tadalafil antinociceptive peripheral effect was assessed through acetic acid-induced writhing model. Formalin test was used to appraise both non-inflammatory and inflammatory pain responses. In order to elaborate the involvement of opioid receptors and nitric oxide/cyclic guanosine monophosphate/potassium-ATP pathway in tadalafil-induced analgesia, mice were pretreated with naloxone, l-nitro-arginine-methyl-ester (l-NAME), methylene blue, and glibenclamide.The results illustrated that tadalafil had a significant antinociceptive effect in the tail flick, hot plate, acetic acid-induced writhing and formalin tests indicating the involvement of peripheral and central analgesic mechanisms. Moreover, tadalafil mechanism of action involved several receptors and mediators, specifically NO/cGMP pathway and opioid receptors. In the formalin test, naloxone significantly blocked the effect of tadalafil in the first phase and partially in the second phase which is an inflammatory pain-dependent aspect. l-NAME, methylene blue and glibenclamide partially blocked the effect of tadalafil in the first phase and enhanced its effect in the second phase which is related to nitric oxide role in the inflammatory process.As a conclusion, tadalafil possesses a potential analgesic effect via the involvement of opioid and nitric oxide pathways.

Correction: Mangiferin Prevents Guinea Pig Tracheal Contraction via Activation of the Nitric Oxide-Cyclic GMP Pathway

Correction: Mangiferin Prevents Guinea Pig Tracheal Contraction via Activation of the Nitric Oxide-Cyclic GMP Pathway

Mangiferin Prevents Guinea Pig Tracheal Contraction via Activation of the Nitric Oxide-Cyclic GMP Pathway

Previous studies have described the antispasmodic effect of mangiferin, a natural glucoside xanthone (2-C-β-Dgluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) that is present in mango trees and other plants, but its mechanism of action remains unknown. The aim of this study was to examine the potential contribution of the nitric oxide-cyclic GMP pathway to the antispasmodic effect of mangiferin on isolated tracheal rings preparations. The functional effect of mangiferin on allergic and non-allergic contraction of guinea pig tracheal rings was assessed in conventional organ baths. Cultured tracheal rings were exposed to mangiferin or vehicle, and nitric oxide synthase (NOS) 3 and cyclic GMP (cGMP) levels were quantified using western blotting and enzyme immunoassays, respectively. Mangiferin (0.1–10 µM) inhibited tracheal contractions induced by distinct stimuli, such as allergen, histamine, 5-hydroxytryptamine or carbachol, in a concentration-dependent manner. Mangiferin also caused marked relaxation of tracheal rings that were precontracted by carbachol, suggesting that it has both anti-contraction and relaxant properties that are prevented by removing the epithelium. The effect of mangiferin was inhibited by the nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) (100 µM), and the soluble guanylate cyclase inhibitor, 1H-[1], [2], [4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM), but not the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (SQ22536) (100 µM). The antispasmodic effect of mangiferin was also sensitive to K+ channel blockers, such as tetraethylammonium (TEA), glibenclamide and apamin. Furthermore, mangiferin inhibited Ca2+-induced contractions in K+ (60 mM)-depolarised tracheal rings preparations. In addition, mangiferin increased NOS3 protein levels and cGMP intracellular levels in cultured tracheal rings. Finally, mangiferin-induced increase in cGMP levels was abrogated by co-incubation with either ODQ or L-NAME. These data suggest that the antispasmodic effect of mangiferin is mediated by epithelium-nitric oxide- and cGMP-dependent mechanisms.

Phosphodiesterase-9 (PDE9) inhibition with BAY 73-6691 increases corpus cavernosum relaxations mediated by nitric oxide–cyclic GMP pathway in mice

Phosphodiesterase-9 (PDE9) specifically hydrolyzes cyclic GMP, and was detected in human corpus cavernosum. However, no previous studies explored the selective PDE9 inhibition with BAY 73-6691 in corpus cavernosum relaxations. Therefore, this study aimed to characterize the PDE9 mRNA expression in mice corpus cavernosum, and investigate the effects of BAY 73-6691 in endothelium-dependent and -independent relaxations, along with the nitrergic corpus cavernosum relaxations. Male mice received daily gavage of BAY 73-6691 (or dimethylsulfoxide) at 3 mg kg(-1) per day for 21 days. Relaxant responses to acetylcholine (ACh), nitric oxide (NO) (as acidified sodium nitrite; NaNO2 solution), sildenafil and electrical-field stimulation (EFS) were obtained in corpus cavernosum in control and BAY 73-6691-treated mice. BAY 73-6691 was also added in vitro 30 min before construction of concentration-responses and frequency curves. PDE9A and PDE5 mRNA expression was detected in the mice corpus cavernosum in a similar manner. In vitro addition of BAY 73-6691 neither itself relaxed mice corpus cavernosum nor changed the NaNO2, sildenafil and EFS-induced relaxations. However, in mice treated chronically with BAY 73-6691, the potency (pEC50) values for ACh, NaNO2 and sildenafil were significantly greater compared with control group. The maximal responses (Emax) to NaNO2 and sildenafil were also significantly greater in BAY 73-6691-treated mice. BAY 73-6691 treatment also significantly increased the magnitude and duration of the nitrergic corpus cavernosum relaxations (8-32 Hz). In conclusion, murine corpus cavernosum expresses PDE9 mRNA. Prolonged PDE9 inhibition with BAY 73-6691 amplifies the NO-cGMP-mediated cavernosal responses, and may be of therapeutic value for erectile dysfunction.

Curcumin induces differentiation of embryonic stem cells through possible modulation of nitric oxide-cyclic GMP pathway

Curcumin, an active ingredient of dietary spice used in curry, has been shown to exhibit anti-oxidant, anti-inflammatory and anti-proliferative properties. Using EB directed differentiation protocol of H-9 human embryonic stem (ES) cells; we evaluated the effect of curcumin (0-20 μmol/L) in enhancing such differentiation. Our results using real time PCR, western blotting and immunostaining demonstrated that curcumin significantly increased the gene expression and protein levels of cardiac specific transcription factor NKx2.5, cardiac troponin I, myosin heavy chain, and endothelial nitric oxide synthase during ES cell differentiation. Furthermore, an NO donor enhanced the curcumin-mediated induction of NKx2.5 and other cardiac specific proteins. Incubation of cells with curcumin led to a dose dependent increase in intracellular nitrite to the same extent as giving an authentic NO donor. Functional assay for second messenger(s) cyclic AMP (cAMP) and cyclic GMP (cGMP) revealed that continuous presence of curcumin in differentiated cells induced a decrease in the baseline levels of cAMP but it significantly elevated baseline contents of cGMP. Curcumin addition to a cell free assay significantly suppressed cAMP and cGMP degradation in the extracts while long term treatment of intact cells with curcumin increased the rates of cAMP and cGMP degradation suggesting that this might be due to direct suppression of some cyclic nucleotide-degrading enzyme (phosphodiesterase) by curcumin. These studies demonstrate that polyphenol curcumin may be involved in differentiation of ES cells partly due to manipulation of nitric oxide signaling.

The Nitric Oxide-Cyclic GMP Pathway Regulates FoxO and Alters Dopaminergic Neuron Survival in Drosophila

Activation of the forkhead box transcription factor FoxO is suggested to be involved in dopaminergic (DA) neurodegeneration in a Drosophila model of Parkinson's disease (PD), in which a PD gene product LRRK2 activates FoxO through phosphorylation. In the current study that combines Drosophila genetics and biochemical analysis, we show that cyclic guanosine monophosphate (cGMP)-dependent kinase II (cGKII) also phosphorylates FoxO at the same residue as LRRK2, and Drosophila orthologues of cGKII and LRRK2, DG2/For and dLRRK, respectively, enhance the neurotoxic activity of FoxO in an additive manner. Biochemical assays using mammalian cGKII and FoxO1 reveal that cGKII enhances the transcriptional activity of FoxO1 through phosphorylation of the FoxO1 S319 site in the same manner as LRRK2. A Drosophila FoxO mutant resistant to phosphorylation by DG2 and dLRRK (dFoxO S259A corresponding to human FoxO1 S319A) suppressed the neurotoxicity and improved motor dysfunction caused by co-expression of FoxO and DG2. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) also increased FoxO's activity, whereas the administration of a NOS inhibitor L-NAME suppressed the loss of DA neurons in aged flies co-expressing FoxO and DG2. These results strongly suggest that the NO-FoxO axis contributes to DA neurodegeneration in LRRK2-linked PD.

The peripheral L-arginine–nitric oxide–cyclic GMP pathway and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain in rats

Crotalphine, a 14 amino acid peptide first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces a peripheral long-lasting and opioid receptor-mediated antinociceptive effect in a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. In the present study, we further characterized the molecular mechanisms involved in this effect, determining the type of opioid receptor responsible for this effect and the involvement of the nitric oxide-cyclic GMP pathway and of K⁺ channels. Crotalphine (0.2 or 5 μg/kg, orally; 0.0006 μg/paw), administered on day 14 after nerve constriction, inhibited mechanical hyperalgesia and low-threshold mechanical allodynia. The effect of the peptide was antagonized by intraplantar administration of naltrindole, an antagonist of δ-opioid receptors, and partially reversed by norbinaltorphimine, an antagonist of κ-opioid receptors. The effect of crotalphine was also blocked by 7-nitroindazole, an inhibitor of the neuronal nitric oxide synthase; by 1H-(1,2,4) oxadiazolo[4,3-a]quinoxaline-1-one, an inhibitor of guanylate cyclase activation; and by glibenclamide, an ATP-sensitive K⁺ channel blocker. The results suggest that peripheral δ-opioid and κ-opioid receptors, the nitric oxide-cyclic GMP pathway, and ATP-sensitive K⁺ channels are involved in the antinociceptive effect of crotalphine. The present data point to the therapeutic potential of this peptide for the treatment of chronic neuropathic pain.

Involvement of nitric oxide-cGMP pathway in the anticonvulsant effects of lithium chloride on PTZ-induced seizure in mice

Lithium is still the mainstay in the treatment of affective disorders as a mood stabilizer. Lithium also shows some anticonvulsant properties. While the underlying mechanisms of action of lithium are not yet exactly understood, we used a model of clonic seizure induced by pentylenetetrazole (PTZ) in male NMRI mice to investigate whether the anticonvulsant effect of lithium is mediated via NO-cGMP pathway. Injection of a single effective dose of lithium chloride (25 mg/kg) intraperitoneally (i.p.) increased significantly the seizure threshold (P<0.01). The anticonvulsant properties of the effective dose of lithium were prevented by pre-treatment with the per se non-effective doses of L-ARG [the substrate for nitric oxide synthase; NOS] (30 and 50 mg/kg) or sildenafil [a phosphodiesterase 5 inhibitor] (10 and 20 mg/kg). L-NAME [a non-specific NOS inhibitor] (5, 15 and 30 mg/kg), 7-NI [a specific neural NOS inhibitor] (30 and 60 mg/kg) or MB [a guanylyl cyclase inhibitor] (0.5 and 1 mg/kg) augmented the anticonvulsant effect of a sub-effective dose of lithium (10 mg/kg, i.p.). Whereas several doses of aminoguanidine [an inducible NOS inhibitor] (20, 50 and 100 mg/kg) failed to alter the anticonvulsant effect of lithium. Our findings demonstrated that nitric oxide-cyclic GMP pathway could be involved in the anticonvulsant properties of the lithium chloride. In addition, the role of constitutive NOS versus inducible NOS is prominent in this phenomenon.

Activation of nitric oxide‐cyclic GMP pathway (NO‐cGMP) with nitrite or sildenafil, but not BAY 41‐2272 (BAY), blunts acute pulmonary embolism (APE)‐induced increases in circulating matrix metalloproteinase‐9 (MMP‐9) and oxidative stress

ObjectivesOxidative stress is a major upregulator of MMP‐9 and both are induced by APE, thus, we comparatively assessed the effects of NO‐cGMP activators with and without antioxidant properties on APE‐induced increases in MMP‐9.MethodsHemodynamic and biochemical evaluations were performed in non‐embolized dogs treated with saline (N= 5), and in embolized dogs (intravenous injections of microspheres) that received saline (n=9), or nitrite (6.75 µmol/kg over 15 min followed by 0.28 µmol/kg/min; n=5), or sildenafil (0.25 mg/kg over 30 min; n=5), or BAY (0.03, 0.1, 0.3, and 1 mg/kg/h; n=5). Plasma thiobarbituric acid reactive substances (TBARS) concentrations and MMP‐2 and MMP‐9 levels were determined using a fluorometric and gelatin zymography methods, respectively.ResultsAPE increased pulmonary artery pressure by ~25 mmHg. The infusion of each drug (nitrite, BAY, or sildenafil) reversed this increase by ~40% (all P&lt;0.05) and similar effects were seen on the pulmonary vascular resistance. While both nitrite and sildenafil produced no systemic effects, the infusion of BAY produced systemic hypotension (P&lt;0.05). While nitrite and sildenafil blunted the increases in plasma MMP‐9 levels and TBARS (all P&lt;0.05), BAY produced no such effects.ConclusionThese findings are consistent with the idea that NO‐cGMP activators with antioxidant effects prevent the release of MMP‐9 during APE. Supported by FAPESP

Participation of the GABA/benzodiazepine receptor and the NO-cyclicGMP pathway in the “antinociceptive-like effects” of diazepam

The mechanism of action underlying the “analgesic activity” of diazepam remains unclear. In this study, the possible participation of the GABA/benzodiazepine receptor and the nitric oxide-cyclic GMP (NO-cGMP) pathway was assessed utilizing the pain-induced functional impairment model in the rat (PIFIR). Nociception was induced by an intra-articular injection of 15% uric acid. Diazepam (1 and 2 mg/kg, i.p.) reversed the dysfunction induced by uric acid. Flumazenil (10 mg/kg, i.p.), a GABA/benzodiazepine receptor antagonist, abolished the “antinociceptive-like effect” of diazepam (at 2 mg/kg). The “antinociceptive-like effect” of diazepam (at 2 mg/kg) was antagonized by the non-selective nitric oxide synthase (NOS) inhibitor, N ω- l-nitro-arginine methyl ester hydrochloride ( l-NAME, 5 mg/kg, s.c.) (but not by its inactive isomer), and by the selective neuronal NOS inhibitor, 7-nitroindazole (7-NI, 1 mg/kg, i.p). While, the NO precursor, l-arginine (125 mg/kg, s.c.), but not d-arginine (125 mg/kg, s.c.), increased the “antinociceptive-like effect” of a non-effective dose of diazepam (1 mg/kg). Methylene blue (10 mg/kg, i.p.), a guanylate cyclase inhibitor, also prevented the “antinociceptive-like effect” of diazepam (at 2 mg/kg). These results suggest that the GABA/benzodiazepine receptor and the NO-cGMP pathway participate in the “antinociceptive-like effect” of diazepam.

Sildenafil augments the beneficial hemodynamic and histopathological effects of amlodipine in nitric oxide-deficient hypertensive rats: Role of nitric oxide–cyclic GMP pathway

The association of erectile dysfunction (ED) with cardiovascular diseases is so common. This study was carried out to investigate possible impact of sildenafil; the prototype phosphodiesterase 5 inhibitor used for treatment of ED, on the beneficial hemodynamic and histopathological effects of the prototype third generation calcium antagonist, amlodipine, in nitric oxide (NO)-deficient hypertensive rats. Hypertension was induced by 4-weeks treatment with N ω-nitro- l-arginine-methyl ester ( l-NAME). Animals were allocated into five groups: normal control, hypertensive control, amlodipine-treated group, sildenafil-treated group and combined treatment group. Drug treatment was started 2 weeks after l-NAME and continued together with l-NAME to the end of the treatment period. Systolic blood pressure (SBP), plasma nitrate/nitrite (NO x ) and plasma cGMP levels were evaluated at the end of the treatment period. Aortic and renal structural alterations were also investigated. l-NAME treatment caused elevation of SBP, reduction in plasma NO x and cGMP levels as well as adverse histological alterations in the tissues studied. Amlodipine normalized SBP, restored plasma NO x and cGMP levels and ameliorated the adverse histological changes seen in NO-deficient rats. When combined with sildenafil, both hemodynamic and histopathological effects of amlodipine were augmented with an underlying enhanced elevation of both plasma NO x and cGMP levels to statistically higher values than amlodipine alone. These results show that sildenafil augments the beneficial hemodynamic and histopathological effects of amlodipine in NO-deficient hypertensive rats with a pivotal role being played by NO–cGMP pathway. Whether this pharmacodynamic interaction could exist in other models of hypertension that do not share such biochemical derangement warrants further investigations.

Developmental exposure to polychlorinated biphenyls or methylmercury, but not to its combination, impairs the glutamate–nitric oxide–cyclic GMP pathway and learning in 3-month-old rats

Prenatal exposure to polychlorinated biphenyls (PCBs) or methylmercury (MeHg) contaminated food may affect brain development, leading to long-term alterations in cognitive function. Both types of contaminants, PCBs and MeHg, are often found together contaminating food, especially fish in some polluted areas. Exposure to combinations of neurotoxicants may exert different effects on the developing nervous system than exposure to individual contaminants. Developmental exposure (during pregnancy and lactation) to PCB126 or PCB153 impairs learning ability when the rats are 3 months old. Impairment of learning seems to be a consequence of impairment of the function of the glutamate–nitric oxide (NO)–cGMP pathway in brain in vivo. The aims of the present work were 1) to assess whether perinatal exposure to MeHg also affects the function of the glutamate–NO–cGMP pathway in brain in vivo analyzed by in vivo brain microdialysis and/or the ability to learn the Y maze task when the rats are 3 months old, and 2) to assess whether perinatal exposure to combinations of MeHg with PCB153 or PCB126 potentiates, decreases or does not modify the effects of the individual neurotoxicants. Perinatal exposure to PCB126, PCB153 or MeHg impaired the function of the glutamate–NO–cGMP pathway in cerebellum and learning ability. However, co-exposure to PCB126+MeHg or PCB153+MeHg inhibits the impairment of the pathway or learning ability. These results support that the function of this pathway modulates learning of the Y maze task. Moreover, they show that co-exposure to these PCBs and MeHg does not exacerbate, but reduces the effects on the ability to learn this task.

Effect of Glutamine Synthesis Inhibition with Methionine Sulfoximine on the Nitric Oxide–Cyclic GMP Pathway in the Rat Striatum Treated Acutely with Ammonia: A Microdialysis Study

Ammonia neurotoxicity is associated with overactivation of N-methyl-D-aspartate (NMDA) receptors leading to enhanced nitric oxide and cyclic GMP synthesis and to accumulation of reactive oxygen and nitrogen species. Ammonia is detoxified in the brain via synthesis of glutamine, which if accumulated in excess contributes to astrocytic swelling, mitochondrial dysfunction and cerebral edema. This study was aimed at testing the hypothesis that the activity of the NMDA/NO/cGMP pathway is controlled by the ammonia-induced production of Gln in the brain. Ammonium chloride (final concentration 5 mM), infused for 40 min to the rat striatum via a microdialysis probe, caused a significant increase in Gln (by 40%), NO oxidation products (nitrite+nitrate=NOx) (by 35%) and cGMP (by 50%) concentration in the microdialysate. A Gln synthetase inhibitor, methionine sulfoximine (MSO, 5 mM), added directly to the microdialysate, completely prevented ammonia-mediated production of Gln, and paradoxically, it increased ammonia-mediated production of NOx and cGMP by 230% and 250%, respectively. Of note, MSO given alone significantly reduced basal Gln concentration in the rat striatum, had no effect on the basal NOx concentration, and attenuated basal concentration of cGMP in the microdialysate by 50%. The results of the present study suggest that Gln, at physiological concentrations, may ameliorate excessive activation of the NO-cGMP pathway by neurotoxic concentrations of ammonia. However, in view of potential direct interference of MSO with the pathway, exogenously added Gln and less toxic modulators of Gln content and/or transport will have to be employed in further studies on the underlying mechanisms.

Toxin Tx2‐6 from the spider “Phoneutria nigriventer” improves the impaired erectile function in DOCA‐Salt hypertensive rats

Accidents involving the venom of spider P. nigriventer are characterized by different symptoms including priapism. The aim of this study is to investigate the role of the toxin Tx2-6, a peptide extracted from the venom of P. nigriventer, in the penile erection. Erectile function was measured in urethane anesthetized Wistar rats (230–250g) by continuous monitoring mean arterial pressure (MAP) and intracavernosal pressure (ICP) during electrical stimulation of the major pelvic ganglion. Voltage-response curves (0.5–3.0V, 12Hz, 0.1ms, 30s each step) were performed before and (15 min) after subcutaneous injection of Tx2-6 (12 μg/Kg). The erectile response induced by ganglionic stimulation was significantly potentiated after injection of Tx2-6. Incubation with Tx2-6 (0,1 and 0,01μg/mL) caused a significant release of NO from rat isolated strips of corpus cavernosum, detected through a NO indicator (DAF-FM) using confocal microscopy. Subcutaneous injection of Tx2-6 significantly improved the impaired erectile response induced by ganglionic stimulation in DOCA-salt hypertensive compared to sham-operated rats. The results in this study indicate that Tx2-6 is probably the component of the venom responsible for the priapism observed in poisoning accidents with the P. nigriventer spider, and that Tx2-6 induces and or potentiates penile erection by facilitating the nitric oxide-cyclic GMP pathway. Furthermore, our results suggest that Tx2-6 is a pharmacological tool with a great potential to treat erectile dysfunction. Financial support: CNPq, CAPES, NIH (HL-74167)

Activation of soluble guanylate cyclase by nitric oxide is increased in lymphocytes from both rats chronically exposed to 2,5-hexanedione and workers chronically exposed to n-hexane

Although occupational exposure to n-hexane induces neurotoxic effects in the central and peripheral nervous systems, the mechanisms of its neurotoxicity remain unclear. n-Hexane is metabolized to 2,5-hexanedione (2,5-HD), which is the neurotoxic agent and the indicator chosen for the biological monitoring of exposed workers. It has been previously reported that chronic exposure to 2,5-HD impairs the glutamate–nitric oxide–cyclic GMP pathway at the level of activation of soluble guanylate cyclase (sGC) enzyme by nitric oxide (NO), both in cultured neurons and in the cerebellum of rats in vivo. The aim of this study was to assess whether the activation of sGC by NO is also altered in lymphocytes from rats treated with 2,5-HD and/or workers chronically exposed to n-hexane. Lymphocytes were isolated from male Wistar rats treated with 2,5-HD in drinking water, and from blood samples from shoe-factory workers environmentally and chronically exposed to n-hexane. Urine samples were also collected from workers at the end of the shift in order to measure the urinary levels of 2,5-HD. Activation of sGC by NO was significantly higher (p<0.05) in lymphocytes from rats treated with 2,5-HD than in control rats. In isolated lymphocytes from exposed workers the activation of sGC by NO also increases (p<0.05) in contrast to the controls. The results presented here indicate that the activation of lymphocytes could be an indicator of the toxicity produced by being exposed to n-hexane, since the effects observed in workers chronically exposed to n-hexane are similar to those found in rats chronically treated with 2,5-HD in drinking water.

Rewarding properties of sildenafil citrate in mice: role of the nitric oxide-cyclic GMP pathway

Sildenafil citrate is widely prescribed for erectile dysfunction and acts by inhibiting phosphodiesterase type-5, resulting in accumulation of cyclic-guanosine monophosphate (cGMP) via activation of nitric oxide synthase (NOS). The nitric oxide (NO) system is relevant to the rewarding effects of various drugs of abuse. Several epidemiologic studies indicate that sildenafil is abused in a recreational fashion. In the present study, the rewarding properties of sildenafil and probable involvement of the NO-cGMP pathway were investigated in adult male NMRI mice. The ability of sildenafil citrate (1-40 mg/kg) to produce conditioned place preference (CPP) was studied in an unbiased CPP paradigm. The effects of NO precursor L-arginine, nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), and the inhibitor of guanylyl cyclase methylene blue (MB) on sildenafil-induced CPP were assessed. Mice that received sildenafil (20 and 40 mg/kg) in one environment during conditioning phase displayed a preference for this environment. Both L-NAME (5 mg/kg) and MB (1 mg/kg) in combination with sildenafil (20 mg/kg) suppressed the acquisition of sildenafil-induced place preference. Lower and per se noneffective dose of sildenafil (10 mg/kg) and L-arginine (60 mg/kg), when coadministered, exerted a significant place conditioning. Sildenafil shows rewarding properties that may involve the NO-cGMP pathway.

Peripheral and central activation of nitric oxide-cyclic GMP pathway by sildenafil

Experimental studies have indicated the importance of cAMP and cGMP in modulation of peripheral sensory neurons leading to hyperalgesic response. The concentration of both depends upon the activity of phosphodiesterase, which is responsible for their degradation. The aim of the present study was to evaluate the effect of the PDE-5 inhibitor sildenafil on central or peripheral administration in formalin-induced hyperalgesia in rats. Sildenafil dose-dependently and significantly attenuated both the early and late phase of formalin-induced hyperalgesia on central administration. However, sildenafil on peripheral administration inhibited only the late phase of formalin-induced hyperalgesia in rats. The anti-nociceptive effect of sildenafil was blocked by L-NAME, a non-selective NOS inhibitor, and methylene blue (MB), a guanylate cyclase inhibitor, but sildenafil itself had little or no effect on the first phase of the formalin test in rats. The results from the present study indicates that sildenafil, besides peripheral actions, has a central anti-nociceptive effect, which may be due to activation of the NO-cGMP pathway, as this effect was blocked by L-NAME and MB. PDE-5 inhibitors could be considered as a new class of anti-nociceptive agents for future drug development.

Involvement of the nitric oxide–cyclic GMP pathway and neuronal nitric oxide synthase in ATP‐induced Ca2+ signalling in cochlear inner hair cells

We recently demonstrated that extracellular adenosine 5'-triphosphate (ATP) induced nitric oxide (NO) production in the inner hair cells (IHCs) of the guinea pig cochlea, which inhibited the ATP-induced increase in the intracellular Ca(2+) concentrations ([Ca(2+)](i)) by a feedback mechanism [Shen, J., Harada, N. & Yamashita, T. (2003) Neurosci. Lett., 337, 135-138]. We herein investigated the role of the NO-cGMP pathway and neuronal NO synthase (nNOS) in the ATP-induced Ca(2+) signalling in IHCs using the Ca(2+)-sensitive dye fura-2 and the NO-sensitive dye DAF-2. Fura-2 fluorescence-quenching experiments with Mn(2+) showed that ATP triggered a Mn(2+) influx. L-N(G)-nitroarginine methyl ester (L-NAME), a nonspecific NOS inhibitor, accelerated the ATP-induced Mn(2+) influx while S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, suppressed it. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase, and KT5823, an inhibitor of cGMP-dependent protein kinase, enhanced the ATP-induced [Ca(2+)](i) increase. 8-Bromoguanosine-cGMP, a membrane-permeant analogue of cGMP mimicked the effects of SNAP. Moreover, the effects of 7-nitroindazole, a selective nNOS inhibitor, mimicked the effects of L-NAME regarding both the enhancement of the ATP-induced Ca(2+) response and the attenuation of NO production. Immunofluorescent staining of nNOS using a single IHC revealed that nNOS was distributed throughout the IHCs, but enriched in the apical region of the IHCs as shown by intense staining. In conclusion, the ATP-induced Ca(2+) influx may be the principal source for nNOS activity, which may interact with P2X receptors in the apical region of IHCs. Thereafter, NO can be produced and conversely inhibits the Ca(2+) influx via the NO-cGMP-PKG pathway by a feedback mechanism.