Pharmacophore based approach to design inhibitors in Crustaceans: An insight into the molt inhibition response to the receptor guanylyl cyclase.

The first set of competitive inhibitors of molt inhibiting hormone (MIH) has been developed using the effective approaches such as Hip-Hop, virtual screening and manual alterations. Moreover, the conserved residues at 71 and 72 positions in the molt inhibiting hormone is known to be significant for selective inhibition of ecdysteroidogenesis; thus, the information from mutation and solution structure were used to generate common pharmacophore features. The geometry of the final six-feature pharmacophore was also found to be consistent with the homology-modeled MIH structures from various other decapod crustaceans. The Hypo-1, comprising six features hypothesis was carefully selected as a best pharmacophore model for virtual screening created on the basis of rank score and cluster processes. The hypothesis was validated and the database was virtually screened using this 3D query and the compounds were then manually altered to enhance the fit value. The hits obtained were further filtered for drug-likeness, which is expressed as physicochemical properties that contribute to favorable ADME/Tox profiles to eliminate the molecules exhibit toxicity and poor pharmacokinetics. In conclusion, the higher fit values of CI-1 (4.6), CI-4 (4.9) and CI-7 (4.2) in conjunction with better pharmacokinetic profile made these molecules practically helpful tool to increase production by accelerating molt in crustaceans. The use of feeding sub-therapeutic dosages of these growth enhancers can be very effectively implemented and certainly turn out to be a vital part of emerging nutritional strategies for economically important crustacean livestock.

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.

Inhibition of Hypoxic Pulmonary Vasoconstriction of Rats by Carbon Monoxide

Hypoxic pulmonary vasoconstriction (HPV), a unique response of pulmonary circulation, is critical to prevent hypoxemia under local hypoventilation. Hypoxic inhibition of K+ channel is known as an important O2-sensing mechanism in HPV. Carbon monoxide (CO) is suggested as a positive regulator of Ca2+-activated K+ channel (BK(Ca)), a stimulator of guanylate cyclase, and an O2-mimetic agent in heme moiety-dependent O2 sensing mechanisms. Here we compared the effects of CO on the HPV (Po2, 3%) in isolated pulmonary artery (HPV(PA)) and in blood-perfused/ventilated lungs (HPV(lung)) of rats. A pretreatment with CO (3%) abolished the HPV(PA) in a reversible manner. The inhibition of HPV(PA) was completely reversed by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor. In contrast, the HPV(lung) was only partly decreased by CO. Moreover, the partial inhibition of HPV(lung) by CO was affected neither by the pretreatment with ODQ nor by NO synthase inhibitor (L-NAME). The CO-induced inhibitions of HPV(PA) and HPV(lung) were commonly unaffected by tetraethylammonium (TEA, 2 mM), a blocker of BK(Ca). As a whole, CO inhibits HPV(PA) via activating guanylate cyclase. The inconsistent effects of ODQ on HPV(PA) and HPV(lung) suggest that ODQ may lose its sGC inhibitory action when applied to the blood-containing perfusate.

Methylene blue for vasoplegic syndrome treatment in heart surgery: fifteen years of questions, answers, doubts and certainties / Azul de metileno no tratamento da síndrome vasoplégica em cirurgia cardíaca: quinze anos de perguntas, respostas, dúvidas e certezas

OBJECTIVE: There is strong evidence that methylene blue (MB), an inhibitor of guanylate cyclase, is an excellent therapeutic option for vasoplegic syndrome (VS) treatment in heart surgery. The aim of this article is to review the MB's therapeutic function in the vasoplegic syndrome treatment. METHODS: Fifteen years of literature review. RESULTS: 1) Heparin and ACE inhibitors are risk factors; 2) In the recommended doses it is safe (the lethal dose is 40 mg/kg); 3) The use of MB does not cause endothelial dysfunction; 4) The MB effect appears in cases of nitric oxide (NO) up-regulation; 5) MB is not a vasoconstrictor, by blocking of the GMPc system it releases the AMPc system, facilitating the norepinephrine vasoconstrictor effect; 6) The most used dosage is 2 mg/kg as IV bolus followed by the same continuous infusion because plasmatic concentrations strongly decays in the first 40 minutes; 7) There is a possible "window of opportunity" for the MB's effectiveness. CONCLUSIONS: Although there are no definitive multicentric studies, the MB used to treat heart surgery VS, at the present time, is the best, safest and cheapest option, being a Brazilian contribution for the heart surgery

OBJETIVO: Existem fortes evidências de que o azul de metileno (AM), um inibidor da guanilato ciclase, é uma excelente opção terapêutica para o tratamento da síndrome vasoplégica (SV) em cirurgia cardíaca. O objetivo deste artigo é rever o papel terapêutico do AM no tratamento da SV. MÉTODOS: Revisão da literatura em período de 15 anos. RESULTADOS: 1) A heparina e inibidores da ECA são fatores de risco; 2) Nas doses preconizadas é droga segura (a dose letal é de 40 mg/kg); 3) O AM não causa disfunção endotelial; 4) O efeito do AM só aparece em caso de supra-regulação de óxido nítrico (NO); 5) O AM não é um vasoconstritor, pelo bloqueio do sistema GMPc ele "libera" o sistema AMPc, facilitando o efeito vasoconstritor da noradrenalina; 6) A dosagem mais utilizada é 2 mg/kg em bolus endovenosa, seguida de infusão contínua, pois a concentração plasmática decai acentuadamente nos primeiros 40 minutos; 7) Existe possível "janela de oportunidade" para efetividade do AM. CONCLUSÃO: Embora não existam estudos multicêntricos definitivos, a utilização do AM no tratamento da SV em cirurgia cardíaca é, na atualidade, a melhor, mais segura e barata opção, sendo contribuição brasileira

Cholinergic-NO-cGMP mediation of sildenafil-induced antinociception.

Acetylcholine and cholinomimetic agents with predominant muscarinic action are known to increase the concentration of cGMP by activation of nitric oxide signaling pathway in the nociceptive conditions. The present study was aimed to investigate the NO-cGMP-PDE5 pathway in nociceptive conditions in the experimental animals. Nociceptive threshold was assessed by acetic acid-induced writhing assay (chemonociception) or carrageenan-induced hyperalgesia. Sildenafil [1-5 mg/kg, ip, 50-200 microg/paw, intraplantar (ipl)] produced dose dependent antinociception in both the tested models. Coadministration of acetylcholine (50 mcg/paw, ipl) or cholinomimetic agent, neostigmine (0.1 mcg/kg, ip and 25 ng/paw, ipl) augmented the peripheral antinociceptive effect of sildenafil. This effect was sensitive to blockade by L-NAME (20 mg/kg, ip, 100 microg/paw, ipl), a non-selective NOS inhibitor and methylene blue (1 mg/kg, ip), a guanylate cyclase inhibitor, which per se had little or no effect in both the models of nociception. Further, the per se analgesic effect of acetylcholine and neostigmine was blocked by both L-NAME and methylene blue in the models of nociception, suggesting the activation of NO-cGMP pathway. Also, both L-NAME and methylene blue blocked the per se analgesic effect of sildenafil. These results indicate the peripheral accumulation of cGMP may be responsible for antinociceptive effect, and a possible interaction between cholinergic agents and PDE5 system in models of nociception.

Atropine and ODQ antagonize tetanic fade induced by L-arginine in cats

Although it has been demonstrated that nitric oxide (NO) released from sodium nitrite induces tetanic fade in the cat neuromuscular preparations, the effect of L-arginine on tetanic fade and its origin induced by NO have not been studied in these preparations. Furthermore, atropine reduces tetanic fade induced by several cholinergic and anticholinergic drugs in these preparations, whose mechanism is suggested to be mediated by the interaction of acetylcholine with inhibitory presynaptic muscarinic receptors. The present study was conducted in cats to determine the effects of L-arginine alone or after pretreatment with atropine or 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ) on neuromuscular preparations indirectly stimulated at high frequency. Drugs were injected into the middle genicular artery. L-arginine (2 mg/kg) and S-nitroso-N-acetylpenicillamine (SNAP; 16 µg/kg) induced tetanic fade. The Nw-nitro-L-arginine (L-NOARG; 2 mg/kg) alone did not produce any effect, but reduced the tetanic fade induced by L-arginine. D-arginine (2 mg/kg) did not induce changes in tetanic fade. The tetanic fade induced by L-arginine or SNAP was reduced by previous injection of atropine (1.0 µg/kg) or ODQ (15 µg/kg). ODQ alone did not change tetanic fade. The data suggest that the NO-synthase-GC pathway participates in the L-arginine-induced tetanic fade in cat neuromuscular preparations. The tetanic fade induced by L-arginine probably depends on the action of NO at the presynaptic level. NO may stimulate guanylate cyclase increasing acetylcholine release and thereby stimulating presynaptic muscarinic receptors

Effects of methylene blue and indomethacin on sodium nitroprusside-induced relaxations of the guinea pig tracheal smooth muscle

The aim of the present investigation was to evaluate the potential modulatory effect of the guinea pig tracheal smooth muscle intrinsic tone on the relaxant responses to sodium nitroprusside and the effects of methylene blue on this response. Paired tracheal chains were mounted for isotonic contractions under 500 mg of tension in Krebs-Henseleit solution. The intrinsic modulatory tone was inhibited by indomethacin, in a concentration that did not have any effect over carbachol induced contractions. Sodium nitroprusside-induced relaxations were the same in the absence or presence of the modulatory tone. Methylene blue inhibited 50 per cent sodium nitroprusside-induced relaxations, in the presence or absence of the intrinsic system. This suggests that sodium nitroprusside-induced relaxations are mediated through guanylyl-cylase activation and that these are not under the modulation of the intrinsic prostaglandinergic tone.

Effects of methylene blue on the development of intrinsic contractile responses of the guinea pig tracheal smooth muscle

The aim of the present study was to investigate the effects of methylene bue, a guanylyl cyclase inhibitor, on the development of intrinsic contractile responses of the guinea pig tracheal smooth muscle. Paired tracheal chains were mounted for isotonic contractions under 500 mg of tension in Krebs-Henseleit solution. The intrinsic contractile tone modulated carbachol-induced contractions and was inhibited by indomethacin, suggesting the involvement of cyclooxygenase products on this tone. Methylene blue (5x10(-5)M) irreversibly inhibited the intrinsic contractile responses. considering that methylene blue prevents any endogenous production of cGMP, it would be expected to enhance the contractions. However, since methylene blue has effects over nitric oxide synthase and nitric oxide itself, we suggest that guanylyl cyclase activations is not important for the development of the intrinsic tone.

Inhibición de la vía del óxido nítrico en el shock séptico refractario / Inhibition of nitric oxide way in the refractary septic shock

To assess the acute effects of methylene blue infusion, an inhibitor of nitric oxyde synthesis, on hemodynamic parameters in patients with refractory septic shock. Fourteen patients admitted to intensive care units with septic shock of diverse etiologies and unable to maintain median arterial pressures over 60 mm Hg with the use of at least 2 vasoactive drugs, were studied. All received a 1 mg/kg bolus of methylene blue. Hemodinamic parameters were measured before and 30, 60, 120 and 180 min after the bolus. Systolic and diastolic blood pressure and systemic vascular resistance increased in all patients. There were no significant changes in cardiac output, oxygen consumption or extraction. Methylene blue has an acute pressor effect in patients with septic shock