Cannabinoid receptor type 1 in the bed nucleus of the stria terminalis modulates cardiovascular responses to stress via local N-methyl-D-aspartate receptor/neuronal nitric oxide synthase/soluble guanylate cyclase/protein kinase G signaling.

BACKGROUND: Endocannabinoid neurotransmission in the bed nucleus of the stria terminalis is involved in the control of cardiovascular responses to stress. However, the local mechanisms involved is this regulation are not known. AIMS: The purpose of this study was to assess an interaction of bed nucleus of the stria terminalis endocannabinoid neurotransmission with local nitrergic signaling, as well as to investigate the involvement of local N-methyl-D-aspartate glutamate receptor and nitric oxide signaling in the control of cardiovascular responses to acute restraint stress by bed nucleus of the stria terminalis endocannabinoid neurotransmission in rats. METHODS: The first protocol evaluated the effect of intra-bed nucleus of the stria terminalis microinjection of the selective cannabinoid receptor type 1 receptor antagonist AM251 in nitrite/nitrate content in the bed nucleus of the stria terminalis following restraint stress. The other protocols evaluated the impact of local pretreatment with the selective N-methyl-D-aspartate glutamate receptor antagonist LY235959, the selective neuronal nitric oxide synthase inhibitor Nω-propyl-L-arginine, the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or the protein kinase G inhibitor KT5823 in restraint-evoked cardiovascular changes following bed nucleus of the stria terminalis treatment with AM251. RESULTS: Bilateral microinjection of AM251 into the bed nucleus of the stria terminalis increased local nitric oxide release during restraint stress. Bed nucleus of the stria terminalis treatment with the cannabinoid receptor type 1 receptor antagonist also enhanced the tachycardia caused by restraint stress, but without affecting arterial pressure increase and sympathetic-mediated cutaneous vasoconstriction. The facilitation of restraint-evoked tachycardia following bed nucleus of the stria terminalis treatment with the cannabinoid receptor type 1 receptor antagonist was completely inhibited by local pretreatment with LY235959, Nω-propyl-L-arginine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or KT5823. CONCLUSIONS: Our results provide evidence that bed nucleus of the stria terminalis endocannabinoid neurotransmission inhibits local N-methyl-D-aspartate/neuronal nitric oxide synthase/soluble guanylate cyclase/protein kinase G signaling, and this mechanism is involved in the control of the cardiovascular responses to stress.

Medial prefrontal cortex TRPV1 and CB1 receptors modulate cardiac baroreflex activity by regulating the NMDA receptor/nitric oxide pathway.

The ventral medial prefrontal cortex (vMPFC) facilitates the cardiac baroreflex response through N-methyl-D-aspartate (NMDA) receptor activation and nitric oxide (NO) formation by neuronal NO synthase (nNOS) and soluble guanylate cyclase (sGC) triggering. Glutamatergic transmission is modulated by the cannabinoid receptor type 1 (CB1) and transient receptor potential vanilloid type 1 (TRPV1) receptors, which may inhibit or stimulate glutamate release in the brain, respectively. Interestingly, vMPFC CB1 receptors decrease cardiac baroreflex responses, while TRPV1 channels facilitate them. Therefore, the hypothesis of the present study is that the vMPFC NMDA/NO pathway is regulated by both CB1 and TRPV1 receptors in the modulation of cardiac baroreflex activity. In order to test this assumption, we used male Wistar rats that had stainless steel guide cannulae bilaterally implanted in the vMPFC. Subsequently, a catheter was inserted into the femoral artery, for cardiovascular recordings, and into the femoral vein for assessing baroreflex activation. The increase in tachycardic and bradycardic responses observed after the microinjection of a CB1 receptors antagonist into the vMPFC was prevented by an NMDA antagonist as well as by the nNOS and sGC inhibition. NO extracellular scavenging also abolished these responses. These same pharmacological manipulations inhibited cardiac reflex enhancement induced by TRPV1 agonist injection into the area. Based on these results, we conclude that vMPFC CB1 and TRPV1 receptors inhibit or facilitate the cardiac baroreflex activity by stimulating or blocking the NMDA activation and NO synthesis.

Endothelial modulation of a nitric oxide donor complex-induced relaxation in normotensive and spontaneously hypertensive rats.

We hypothesized that endothelium modulates relaxation induced by a nitric oxide (NO) donor ruthenium complex (TERPY, [Ru(terpy)(bdq)NO]3+) in mesenteric arteries of normotensive and spontaneously hypertensive (SHR) rats in different ways. We analyzed the mechanism involved in TERPY-induced relaxation in the second and third branches of mesenteric arteries and investigated how endothelium contributes to the TERPY vasodilator effect on SHR blood vessels. TERPY induced concentration-dependent relaxation in endothelium-denuded (E-) and endothelium-intact (E+) mesenteric arteries of normotensive rats and SHR. Pretreatment with ODQ (which inhibits soluble guanylyl cyclase) or TEA (tetraethylammonium, which blocks potassium channels) significantly reduced the TERPY vasodilator effect on E- mesenteric arteries of normotensive rats and SHR. The presence of endothelium shifted the concentration-effect curves for TERPY in E+ mesenteric arteries of normotensive rats to the right. Conversely, the presence of endothelium shifted the concentration-effect curves for TERPY in the case of SHR E+ mesenteric arteries to the left, which suggested increased potency. L-NNA, a more selective endothelial NO synthase (eNOS) inhibitor, reduced TERPY potency in SHR. The presence of endothelium and notably of NOS contributed to the TERPY vasodilator action in SHR: TERPY promoted eNOS Ser1177 phosphorylation with consequent NO production and increased soluble guanylyl cyclase activity, which may have directly activated potassium channels.

The Nitric oxide/CGMP/KATP pathway mediates systemic and central antinociception induced by resistance exercise in rats.

Resistance exercise (RE) is characterized to increase strength, tone, mass, and/or muscular endurance and also for produces many beneficial effects, such as blood pressure and osteoporosis reduction, diabetes mellitus control, and analgesia. However, few studies have investigated endogenous mechanisms involved in the RE-induced analgesia. Thus, the aim of this study was evaluate the role of the NO/CGMP/KATP pathway in the antinociception induced by RE. Wistar rats were submitted to acute RE in a weight-lifting model. The nociceptive threshold was measured by mechanical nociceptive test (paw-withdrawal). To investigate the involvement of the NO/CGMP/KATP pathway the following nitric oxide synthase (NOS) non-specific and specific inhibitors were used: N-nitro-l-arginine (NOArg), Aminoguanidine, N5-(1-Iminoethyl)-l-ornithine dihydrocloride (l-NIO), Nω-Propyl-l-arginine (l-NPA); guanylyl cyclase inhibitor, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ); and KATP channel blocker, Glybenclamide; all administered subcutaneously, intrathecally and intracerebroventricularly. Plasma and cerebrospinal fluid (CSF) nitrite levels were determined by spectrophotometry. The RE protocol produced antinociception, which was significantly reversed by NOS specific and unspecific inhibitors, guanylyl cyclase inhibitor (ODQ) and KATP channel blocker (Glybenclamide). RE was also responsible for increasing nitrite levels in both plasma and CSF. These finding suggest that the NO/CGMP/KATP pathway participates in antinociception induced by RE.

The expression of contextual fear conditioning involves activation of a NMDA receptor-nitric oxide-cGMP pathway in the dorsal hippocampus of rats.

The dorsal portion of the hippocampus is a limbic structure that is involved in fear conditioning modulation in rats. Moreover, evidence shows that the local dorsal hippocampus glutamatergic system, nitric oxide (NO) and cGMP modulate behavioral responses during aversive situations. Therefore, the present study investigated the involvement of dorsal hippocampus NMDA receptors and the NO/cGMP pathway in contextual fear conditioning expression. Male Wistar rats were submitted to an aversive contextual conditioning session and 48 h later they were re-exposed to the aversive context in which freezing, cardiovascular responses (increase of both arterial pressure and heart rate) and decrease of tail temperature were recorded. The intra-dorsal hippocampus administration of the NMDA receptor antagonist AP7, prior to the re-exposure to the aversive context, attenuated fear-conditioned responses. The re-exposure to the context evoked an increase in NO concentration in the dorsal hippocampus of conditioned animals. Similar to AP7 administration, we observed a reduction of contextual fear conditioning after dorsal hippocampus administration of either the neuronal NO synthase inhibitor N-propyl-L-arginine, the NO scavenger c-PTIO or the guanylate cyclase inhibitor ODQ. Therefore, the present findings suggest the possible existence of a dorsal hippocampus NMDA/NO/cGMP pathway modulating the expression of contextual fear conditioning in rats.

Critical role of nitric oxide in the modulation of prepulse inhibition in Swiss mice.

RATIONALE: Nitric oxide (NO) modulates the dopamine uptake and release processes and appears to be implicated in dopamine-related pathologies, such as schizophrenia. However, it is unclear whether there is excess or deficient NO synthesis in schizophrenia pathophysiology. Analyses of the intracellular pathways downstream of NO system activation have identified the cyclic nucleotide cyclic guanosine monophosphate (cGMP) as a possible target for drug development. Defects in the sensorimotor gating of the neural mechanism underlying the integration and processing of sensory information have been detected across species through prepulse inhibition (PPI). OBJECTIVES: The aim of this study was to investigate the effects of NO/cGMP increase on sensorimotor gating modulation during dopamine hyperfunction. METHODS: Mice were treated with NO donors and subjected to the PPI test. Treatment with the NO donor sodium nitroprusside was preceded by pretreatment with a soluble guanylate cyclase (sGC) inhibitor. Additionally, the mice were treated with NO donors and phosphodiesterases inhibitors prior to amphetamine treatment. RESULTS: Pretreatment with the NO donors enhanced the PPI response and attenuated the amphetamine-disruptive effects on the PPI. The sGC inhibitor did not modify the sodium nitroprusside effects. Additionally, the cGMP increase induced by a specific phosphodiesterase inhibitor did not modify the amphetamine-disruptive effect. CONCLUSIONS: This study provides the first demonstration that an increase in NO can improve the PPI response and block the amphetamine-disruptive effects on the PPI response. Our data are consistent with recent clinical results. However, these effects do not appear to be related to an increase in cGMP levels, and further investigation is thus required.

Inibição da guanilato ciclase pelo azul de metileno no choque circulatório causado por pancreatite aguda necrosante: uma palavra de cuidado embasada em modelo suíno / Guanylate cyclase inhibition by methylene blue in circulatory shock caused by acute necrotizing pancreatitis: a word of caution based on a porcine model

OBJETIVO: estudar o uso terapêutico do bloqueio da guanilato ciclase pelo azul de metileno em um modelo experimental de pancreatite aguda grave em suínos. MÉTODOS: a pancreatite aguda necrotizante foi induzida em porcos anestesiados por infusão ductal pancreática retrógrada de 1ml/kg de taurocolato de sódio a 5% e 8U/kg de enteroquinase. Três grupos foram estudados (n=5): controle (C), pancreatite (PA), "bolus" de azul seguido por pancreatite (AM+PA). Os dados incluíram enzimas séricas e do líquido abdominal, variáveis hemodinâmicas, hemogasometria arterial, volume de líquido abdominal, marcadores inflamatórios plasmáticos, nitrito/nitrato e mieloperoxidase e malondialdeído plasmático. Aplicou-se a análise de variância seguida do pós-teste de Bonferroni (p<0,05). RESULTADOS: os valores de amilase e lipase foram três e dez vezes mais elevados no grupo PA. A atividade da mieloperoxidase foi 50% superior no grupo PA. Os dados hemodinâmicos indicaram choque hipovolêmico precoce seguido de choque cardiogênico. Observou-se grave translocação de líquidos para a cavidade peritoneal. A nitrito/nitrato plasmática permaneceu inalterada. O grupo AM+PA teve aumento de cinco vezes do mieloperoxidase em comparação com o grupo C. CONCLUSÕES: a utilização de azul de metileno em suínos com pancreatite não demonstrou efeitos significativos sobre variáveis hemodinâmicas e inflamatórias. Seu uso terapêutico na pancreatite necro-hemorrágica pode ser inadequado e extremo cuidado deve ser tomado dado o aumento da peroxidação lipídica evidenciado pelo aumento dos valores do malondialdeído.

OBJECTIVE: To study the therapeutic application of guanylate cyclase inhibition by methylene blue in an experimental model of acute pancreatitis in pigs. METHODS: acute necrotizing pancreatitis was induced in anesthetized pigs by the retrograde infusion of 1 ml/kg of 5% sodium taurocholate and 8 U/kg enterokinase in the pancreatic duct. Three groups were studied (n = 5): control (C), pancreatitis (AP), and MB bolus followed by pancreatitis (MB+P). The data included serum and abdominal fluid enzymes, hemodynamic variables, arterial hemogasometry, abdominal fluid volume, inflammatory markers, plasma nitrite/nitrate (NOx), plasma myeloperoxidase (MPO) and plasma malondialdehyde (MDA). One- and two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni test (p < 0.05). RESULTS: amylase and lipase were three and 10-fold higher in the AP group. Myeloperoxidase activity was 50% higher in the AP group. The hemodynamic data indicated early hypovolemic shock followed by cardiogenic shock. Severe fluid translocation to the peritoneal cavity was observed. Plasma NOx remained unchanged. The MB+P group had a five-fold increase in MDA compared with the C group. CONCLUSION: preemptive application of MB in pigs with AP demonstrated no significant effects on hemodynamic and inflammatory variables. The use of MB is inadequate in cases of exponential NO release, and extreme caution must be exercised, given the increase in lipid peroxidation based on the malondialdehyde dosage.

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-ß-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 Ca²âº-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.

Antihypertensive and vasorelaxant effects of dihydrospinochalcone-A isolated from Lonchocarpus xuul Lundell by NO production: computational and ex vivo approaches.

Current work was conducted to evaluate the vasorelaxant effect of dihydrospinochalcone-A (1) and isocordoin (2), compounds type chalcone isolated from Lonchocarpus xuul, an endemic tree of the Yucatan Peninsula, Mexico. Compounds 1 and 2 were found to induce significant relaxant effect in a concentration-dependent manner on aortic rat rings pre-contracted with noradrenaline (NA, 0.1 µM). Compound 1 was the most active and its effect was endothelium-dependent (Emax=79.67% and EC50=21.46 µM with endothelium and Emax=23.58% and EC50=91.8 µM without endothelium, respectively). The functional mechanism of action for 1 was elucidated. Pre-incubation with L-NAME (unspecific nitric oxide synthase inhibitor), indomethacin (unspecific COX inhibitor), ODQ (soluble guanylyl cyclase inhibitor), atropine (cholinergic receptor antagonist), TEA (unspecific potassium channel blocker) reduced relaxations induced by 1. Oral administration of 50 mg/kg of compound 1 exhibited significant decrease in diastolic and systolic blood pressure in SHR rats. The heart rate was not modified. Compound 1 was docked with a crystal structure of eNOS. Dihydrospinochalcone-A showed calculated affinity with eNOS in the C1 binding pockets, near the catalytic site; Trp449, Trp447 and His373 through aromatic and π-π interactions, also His463 and Arg367 are the residues that make hydrogen bonds with the carbonyl and hydroxyl groups. In conclusion, dihydrospinochalcone-A induces a significant antihypertensive effect due to its direct vasorelaxant action on rat aorta rings, through NO/sCG/PKG pathway and potassium channel opening.

The ventral hippocampus NMDA receptor/nitric oxide/guanylate cyclase pathway modulates cardiovascular responses in rats.

The hippocampus is a limbic structure that is involved in the expression of defensive reactions and autonomic changes in rats. The injection of L-glutamate (L-glu) into the ventral hippocampus (VH) decreases blood pressure and heart rate in anesthetized rats. Activation of NMDA receptors in the VH increases the production of nitric oxide (NO), leading to guanylate cyclase activation. The hypothesis of the present study was that a local NMDA receptor-NO-guanylate cyclase interaction mediates the cardiovascular effects of microinjection of L-glu into the VH. Microinjection of increasing doses of L-glu (30, 60 and 200 nmol/200 nL) into the VH of conscious rats caused dose-related pressor and tachycardiac responses. The cardiovascular effects of L-glu were abolished by local pretreatment with: the glutamate receptor antagonist AP-7 (0.4 nmol); the selective neuronal NO synthase (nNOS) inhibitor N(ω)-Propyl-L-arginine (0.04 nmol); the NO scavenger C-PTIO (2 nmol) or the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolol [4,3-a]quinoxalin-1-one (2 nmol). Moreover, these cardiovascular responses were blocked by intravenous pretreatment with: the ganglionic blocker mecamylamine (2mg/Kg); the nonselective ß-adrenergic receptor antagonist propranolol (2mg/Kg); the ß1-adrenergic receptor selective antagonist atenolol (1mg/kg). However, pretreatment with the selective α1-adrenergic receptor antagonist prazosin (0,5mg/kg) caused only a small reduction in the pressor response, without affecting the L-glu evoked tachycardia. In conclusion, our results suggest that cardiovascular responses caused by L-glu microinjection into the VH are mediated by NMDA glutamate receptors and involve local nNOS and guanylate cyclase activation. Moreover, these cardiovascular responses are mainly mediated by cardiac sympathetic nervous system activation, with a small involvement of the vascular sympathetic nervous system.

Involvement of nNOS/NO/sGC/cGMP signaling pathway in cocaine sensitization and in the associated hippocampal alterations: does phosphodiesterase 5 inhibition help to drug vulnerability?

RATIONALE: Repeated cocaine administration induces behavioral sensitization in about 50 % of treated animals. Nitric oxide could be involved in the acquisition and maintenance of behavioral cocaine effects, probably by activation of neuronal nitric oxide synthase (nNOS)/NO/soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) signaling pathway, since inhibition of the nNOS enzyme attenuates development of sensitization in rats. On the other hand, increased cGMP availability by phosphodiesterase 5 inhibitors has been correlated to the misuse and recreational use of these agents and also to the concomitant use with illicit drugs in humans. Hippocampus is an important brain region for conditioning to general context previously associated to drug availability, influencing drug-seeking behavior and sensitization. Moreover, cocaine and other drugs of abuse can affect the strength of glutamate synapses in this structure, lastly modifying neuronal activity in main regions of the reward circuitry. OBJECTIVE: The objective of this study is to determine whether the pharmacological manipulation of nNOS/NO/sGC/cGMP signaling pathway altered changes induced by repeated cocaine exposure. RESULTS: The present investigation showed a relationship between behavioral cocaine sensitization, reduced threshold to generate long-term potentiation (LTP) in hippocampal dentate gyrus, and increased nNOS activity in this structure. However, when nNOS or sGC were inhibited, the number of sensitized animals was reduced, and the threshold to generate LTP was increased. The opposite occurred when cGMP availability was increased. CONCLUSION: We demonstrate a key role of the nNOS activity and NO/sGC/cGMP signaling pathway in the development of cocaine sensitization and in the associated enhancement of hippocampal synaptic transmission.

The lignan (-)-cubebin inhibits vascular contraction and induces relaxation via nitric oxide activation in isolated rat aorta.

Cubebin, the most abundant lignan in Piper cubeba, has been described as having several effects as trypanocidal, antimycobacterial, antispasmodic, antimicrobial, anti-inflammatory, and analgesic. This study investigated the vasorelaxant effect produced by (-)-cubebin in isolated rat aortic rings pre-contracted with phenylephrine (Phe), and the possible mechanism involved in this event was evaluated. Endothelium-dependent relaxation was evoked by acetylcholine and (-)-cubebin in intact aortic rings, while endothelium-independent vasorelaxation was elicited by sodium nitroprusside and (-)-cubebin in denuded rings. Cumulative concentration-response curves for Phe (10(-10) -10(-5) M) were determined for endothelium-intact and endothelium-denuded aortic rings in either the presence or absence of (-)-cubebin. Dose-response curves were also constructed for pre-incubation of vascular rings with Nω-nitro-L-arginine methyl ester (L-NAME) (a non-specific nitric oxide synthase inhibitor), indomethacin (an unspecific cyclooxygenase inhibitor), and 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) (a guanylyl cyclase inhibitor). (-)-Cubebin was found to exert a vasorelaxant effect irrespective of the presence of endothelium, which was abolished by pretreatment with L-NAME and ODQ, but not with indomethacin. In addition, (-)-cubebin was able to reduce Phe contraction in the case of intact rings. These results suggest that (-)-cubebin promotes vasorelaxation via NO/cGMP pathway in rat aorta, without prostacyclin involvement.

Involvement of the nitric oxide/cyclic guanylate monophosphate pathway in the pilocarpine-induced seizure model in mice.

The present study was designed to investigate the involvement of the nitric oxide (NO)/cyclic guanylate monophosphate pathway in pilocarpine-induced seizures in mice. Male Swiss mice (26-32 g) were used as the in vivo model. The following pharmacological tools were utilized: the non-selective NO synthase (NOS) inhibitor L-NAME (10 mg/kg, i.p.), a preferential inducible NOS (iNOS) inhibitor aminoguanidine (25 mg/kg, i.p.), a highly specific iNOS inhibitor 1400W (2.5 mg/kg, i.p.), the NO donor L-arginine (150 mg/kg, i.p.), and the soluble guanylyl cyclase inhibitor ODQ (10 mg/kg, i.p.). The animals were divided into groups (n = 8) and pretreated for 30 min before receiving pilocarpine (400 mg/kg, i.p.), while the control group received only pilocarpine. They were observed for 60 min to measure initial seizure latency, latency till death, and mortality. An administration of L-NAME or ODQ delayed the onset of initial seizure, increased latency till death, and produced a 25% survival rate. Aminoguanidine increased the initial seizure and latency until death, and administration of 1400W did not have an effect. Incremental increases of NO by L-arginine were capable of decreasing the seizure and death latency. These results support the idea that the constitutive NOS, probably neuronal NOS, followed by soluble guanylyl cyclase activation is involved in the convulsive responses caused by pilocarpine administration.

Nitric oxide generated by the compound RuBPY promotes the vascular smooth cell membrane hyperpolarization.

The cis-[Ru(bpy)(2)(py)NO(2)](PF(6)) specie (RuBPY) has been used as nitric oxide (NO) delivery agent. It is an NO reservoir and it is thermodynamically stable in aqueous solution. This study aimed to evaluate the NO specie generated by RuBPY as compared to NO released from sodium nitroprusside (SNP) and to study the cellular mechanisms specially focusing the activation of soluble guanylyl-cyclase (sGC), K(+) channels and the cell membrane hyperpolarization, which are the main targets for NO-inducing vascular relaxation. NO generated by RuBPY and the vascular smooth muscle cell (VSMC) membrane potential were measured by confocal microscopy. The cellular mechanisms of aorta relaxation were investigated using K(+) channel blockers and sGC inhibitor. NO released from RuBPY was higher than NO released from SNP. RuBPY released only radicalar NO(0) and SNP released both NO(-) and NO(0). The concentration-effect curves for RuBPY-induced relaxation was shifted to the right by inhibition of sGC with ODQ and by the non-selective blockade of K(+) channels with TEA. The simultaneous combination of ODQ and TEA abolished the vasorelaxation induced by RuBPY. The membrane potential measured by the sensitive dye 4-Di-ANNEPS demonstrated that RuBPY induces cell membrane hyperpolarization. Taken together, our results indicate that the large amount of NO(0) specie generated by RuBPY induces vasorelaxation due to activation of sGC, K(+) channels sensitive to TEA, and cell membrane hyperpolarization. These results indicate that NO(0) generated from RuBPY can also directly activate the K(+) channels in an independent way of sGC.

Guanylate cyclase inhibition by methylene blue in circulatory shock caused by acute necrotizing pancreatitis: a word of caution based on a porcine model.

OBJECTIVE: To study the therapeutic application of guanylate cyclase inhibition by methylene blue in an experimental model of acute pancreatitis in pigs. METHODS: acute necrotizing pancreatitis was induced in anesthetized pigs by the retrograde infusion of 1 ml/kg of 5% sodium taurocholate and 8 U/kg enterokinase in the pancreatic duct. Three groups were studied (n = 5): control (C), pancreatitis (AP), and MB bolus followed by pancreatitis (MB+P). The data included serum and abdominal fluid enzymes, hemodynamic variables, arterial hemogasometry, abdominal fluid volume, inflammatory markers, plasma nitrite/nitrate (NOx), plasma myeloperoxidase (MPO) and plasma malondialdehyde (MDA). One- and two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni test (p < 0.05). RESULTS: amylase and lipase were three and 10-fold higher in the AP group. Myeloperoxidase activity was 50% higher in the AP group. The hemodynamic data indicated early hypovolemic shock followed by cardiogenic shock. Severe fluid translocation to the peritoneal cavity was observed. Plasma NOx remained unchanged. The MB+P group had a five-fold increase in MDA compared with the C group. CONCLUSION: preemptive application of MB in pigs with AP demonstrated no significant effects on hemodynamic and inflammatory variables. The use of MB is inadequate in cases of exponential NO release, and extreme caution must be exercised, given the increase in lipid peroxidation based on the malondialdehyde dosage.

The 2-nitrate-1,3-dibuthoxypropan, a new nitric oxide donor, induces vasorelaxation in mesenteric arteries of the rat.

The reduced availability of nitric oxide (NO) is associated with cardiovascular diseases. Therefore, NO donors such as organic nitrates are useful for the treatment of these disorders. The 2-nitrate-1,3-dibuthoxypropan (NDBP) is an organic nitrate synthesized from glycerin, which the pharmacological effects have not been investigated. In this study we evaluated the vasorelaxant effect induced by NDBP in superior mesenteric artery from rats. In phenylephrine pre-contracted artery rings, NDBP (10(-8)-10(-4)M) elicited concentration-dependent and endothelium-independent relaxation, which were attenuated by hydroxocobalamin-HDX (30 µM), a NO extracellular scavenger, and 1-H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one-ODQ (10 µM), an inhibitor of soluble guanylyl cyclase (sGC). In addition, the NDBP-induced relaxation was reduced by non-selective K(+) channels blocker KCl (20 mM) or selective K(+) channels blockers such as tetraethylammonium-TEA (B(KCa), 1 mM), charybdotoxin-ChTX (B(KCa), 100 nM), glibenclamide (K(ATP), 1µM) and 4-aminopyridine-4-AP (K(V), 1mM). In preparations with ODQ (10 µM) plus TEA (1 mM), the response was virtually abolished. In rat smooth muscle cells culture, NDBP (10(-6)-10(-4)M) caused concentration-dependent increases in NO levels. These findings suggest that NDBP causes vasorelaxation through NO generation and activation of the sCG/cGMP/PKG pathway.

Guanylate cyclase inhibition by methylene blue as an option in the treatment of vasoplegia after a severe burn. A medical hypothesis.

Today it is known that severe burns can be accompanied by the phenomenon of vasoplegic syndrome (VS), which is manifested by persistent and diffuse vasodilation, hypotension and low vascular resistance, resulting in circulatory and respiratory failure. The decrease in systemic vascular resistance observed in VS is associated with excessive production of nitric oxide (NO). In the last 2 decades, studies have reported promising results from the administration of an NO competitor, methylene blue (MB), which is an inhibitor of the soluble guanylate cyclase (sGC), in the treatment of refractory cases of vasoplegia. This medical hypothesis rationale is focused on the tripod of burns/vasoplegia catecholamine resistant/methylene blue. This article has 3 main objectives: 1) to study the guanylate cyclase inhibition by MB in burns; 2) to suggest MB as a viable, safe and useful co-adjuvant therapeutic tool of fluid resuscitation, and; 3) to suggest MB as burns hypotensive vasoplegia amine-resistant treatment.

Immunohistochemical and functional characterization of nitric oxide signaling pathway in isolated aorta from Crotalus durissus terrificus.

We characterized the nitric oxide (NO)-cyclic GMP-phosphodiesterase-5 (PDE5) pathway in Crotalus durissus terrificus aorta. Concentration responses curves to acetylcholine (ACh), sodium nitroprusside (SNP), BAY41-2272 (soluble guanylyl cyclase [sGC] stimulator), BAY60-2770 (sGC activator) and tadalafil (PDE5 inhibitor) were constructed in phenylephrine (10 µM)-precontracted tissues with intact (E(+)) or denuded (E(-)) endothelium. ACh (0.0001-10 µM) and SNP (0.0001-10 µM) relaxed aorta, which were reduced by the NO synthase (L-NAME,100 µM) or the sGC inhibitors (ODQ, 10 µM). Tadalafil (0.0001-10 µM) relaxed E(+) rings with potency (pEC(50)) and maximal response (E(max)) values of 7.34±0.02 and 105±8%, respectively. E(-) or ODQ treatment significantly (P<0.05) reduced tadalafil relaxations (66±18% and 71±7%, respectively). BAY41-2272 (0.0001-300 nM) produced concentration-dependent relaxations in E(+) rings, which were reduced by addition of either ODQ or L-NAME (16.0- and 5.2-fold rightward shifts, respectively). The relaxation of BAY60-2770 was markedly potentiated by ODQ and L-NAME (41.0- and 9.7-fold leftward shifts, respectively), whereas in E(-) the pEC(50) values were shifted by 7-fold to the right. Immunohistochemistry, followed validation by transcriptomic analysis, revealed the presence of eNOS in endothelium, whereas nNOS was observed only in perivascular nerves. sGC and PDE5 were expressed in smooth muscle. Thus, NO-sGC-PDE5 pathway is evolutionarily present in Crotalus sp. vessels, and has a remarkable degree of functional similarity to mammalian vessels.

NO donors-relaxation is impaired in aorta from hypertensive rats due to a reduced involvement of K(+) channels and sarcoplasmic reticulum Ca(2+)-ATPase.

AIMS: To examine the vasodilatation induce by the NO donors, [Ru(terpy)(bdq)NO](3+) (TERPY) and sodium nitroprusside (SNP), and to compare their effects in aortic rings from hypertensive 2K-1C and normotensive 2K rats. MAIN METHODS: Vascular reactivity was performed in aortic rings pre-contracted with phenylephrine (Phe 100nM). We have analyzed the maximal relaxation (Emax) and potency (pD(2)) of NO donors. KEY FINDINGS: Potency of SNP was greater than TERPY in both arterial groups. The vasodilatation induced by TERPY was greater in 2K than in 2K-1C, and it was inhibited by sGC inhibitor ODQ in 2K and in 2K-1C aortic rings. ODQ did not alter the efficacy to SNP, but it reduced its potency in 2K and 2K-1C. The blockade of K(+) channels reduced the potency of TERPY only in aortic rings of 2K. On the other hand, the potency of SNP was reduced in both 2K and 2K-1C. The combination of ODQ and TEA reduced the relaxation induced by TERPY and SNP in 2K and reduced the efficacy to SNP in 2K-1C aortic rings but it had no additional effect on the TERPY relaxation in 2K-1C aortas. The production of cGMP induced by TERPY was greater than that produced by SNP, which was similarly increased in 2K and 2K-1C. Sarcoplasmic reticulum Ca-ATPase inhibition only impaired the relaxation induced by SNP in 2K aortic rings. SIGNIFICANCE: Taken together, our results provide evidences that in this model of hypertension, impaired K(+) channels activation by TERPY and SERCA activation by SNP may contribute to decreased vasodilatation.

Ketamine activates the L-arginine/Nitric oxide/cyclic guanosine monophosphate pathway to induce peripheral antinociception in rats.

BACKGROUND: The involvement of the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway in antinociception has been implicated as a molecular mechanism of antinociception produced by several antinociceptive agents, including µ-, κ-, or δ-opioid receptor agonists, nonsteroidal analgesics, cholinergic agonist, and α2C adrenoceptor agonist. In this study, we investigated whether ketamine, a dissociative anesthetic N-methyl-D-aspartate receptor antagonist, was also capable of activating the L-arginine/NO/cGMP pathway and eliciting peripheral antinociception. METHODS: The rat paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E2. All drugs were locally administered into the right hindpaw of male Wistar rats. RESULTS: Ketamine (10, 20, 40, 80 µg/paw) elicited a local antinociceptive effect that was antagonized by the nonselective NOS inhibitor L-NOARG (12, 18, and 24 µg/paw) and by the selective neuronal NOS inhibitor L-NPA (12, 18, and 24 µg/paw). In another experiment, we used the inhibitors L-NIO and L-NIL (24 µg/paw) to selectively inhibit endothelial and inducible NOS, respectively. These 2 drugs were ineffective at blocking the effects of the peripheral ketamine injection. In addition, the level of nitrite in the homogenized paw indicated that exogenous ketamine is able to induce NO release. The soluble guanylyl cyclase inhibitor ODQ (25, 50, and 100 µg/paw) blocked the action of ketamine, and the cGMP-phosphodiesterase inhibitor zaprinast (50 µg/paw) enhanced the antinociceptive effects of low-dose ketamine (10 µg/paw). CONCLUSIONS: Our results suggest that ketamine stimulates the L-arginine/NO/cyclic GMP pathway via neuronal NO synthase to induce peripheral antinociceptive effects.