Altered reactivity of gastric fundus smooth muscle in the mouse with targeted disruption of the kinin B(1) receptor gene

Relaxing action of sodium nitroprusside (SNP) was significantly reduced in the stomach fundus of mice lacking the kinin B(1) receptor (B(1)(-/-)). Increased basal cGMP accumulation was correlated with attenuated SNP induced dose-dependent relaxation in B(1)(-/-) when compared with wild type (WT) control mice. These responses to SNP were completely blocked by the guanylate cyclase inhibitor ODQ(10 mu M). It was also found that Ca(2+)-dependent, constitutive nitric oxide synthase (cNOS) activity was unchanged but the Ca(2+)-independent inducible NOS (iNOS) activity was greater in B(1)(-/-) mice than in WT animals. Zaprinast (100 mu M), a specific phosphodiesterase inhibitor, increased the nitrergic relaxations and the accumulation of the basal as well as the SNP-stimulated cGMP in WT but not in B(1)(-/-) stomach fundus. From these findings it is concluded that the inhibited phosphodiesterase activity and high level of cGMP reduced the resting muscle tone, impairing the relaxant responses of the stomach in B(1)(-/-) mice. In addition, it can be suggested that functional B(2) receptor might be involved in the NO compensatory mechanism associated with the deficiency of kinin B(1) receptor in the gastric tissue of the transgenic mice. (C) 2009 Elsevier Inc. All rights reserved.

Role of a Novel Tetrodotoxin-Resistant Sodium Channel in the Nitrergic Relaxation of Corpus Cavernosum from the South American Rattlesnake Crotalus Durissus Terrificus

Introduction. Coitus in snakes may last up to 28 hours; however, the mechanisms involved are unknown. Aim. To evaluate the relevance of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-phosphodiesterase type 5 (PDE5) system in snake corpus cavernosum reactivity. Methods. Hemipenes were removed from anesthetized South American rattlesnakes (Crotalus durissus terrificus) and studied by light and scanning electronic microscopy. Isolated Crotalus corpora cavernosa (CCC) were dissected from the non-spiny region of the hemipenises, and tissue reactivity was assessed in organ baths. Main Outcome Measures. Cumulative concentration-response curves were constructed for acetylcholine (ACh), sodium nitroprusside (SNP), 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272), and tadalafil in CCC precontracted with phenylephrine. Relaxation induced by electrical field stimulation (EFS) was also done in the absence and presence of N omega nitro-L-arginine methyl ester (L-NAME; 100 mu M), 1H-[1, 2, 4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 mu M) and tetrodotoxin (TTX; 1 mu M). Results. The hemipenes consisted of two functionally concentric corpora cavernosa, one of them containing radiating bundles of smooth muscle fibers (confirmed by alpha-actin immunostaining). Endothelial and neural nitric oxide synthases were present in the endothelium and neural structures, respectively; whereas soluble guanylate cyclase and PDE5 were expressed in trabecular smooth muscle. ACh and SNP relaxed isolated CCC, with the relaxations being markedly reduced by L-NAME and ODQ, respectively. BAY 41-2272 and tadalafil caused sustained relaxations with potency (pEC(50)) values of 5.84 +/- 0.17 and 5.10 +/- 0.08 (N = 3-4), respectively. In precontracted CCC, EFS caused frequency-dependent relaxations that lasted three times longer than those in mammalian CC. Although these relaxations were almost abolished by either L-NAME or ODQ, they were unaffected by TTX. In contrast, EFS-induced relaxations in marmoset CC were abolished by TTX. Conclusions. Rattlesnake CC relaxation is mediated by the NO-cGMP-PDE5 pathway in a manner similar to mammals. The novel TTX-resistant Na channel identified here may be responsible for the slow response of smooth muscle following nerve stimulation and could explain the extraordinary duration of snake coitus. Capel RO, Monica FZ, Porto M, Barillas S, Muscara MN, Teixeira SA, Arruda AMM, Pissinatti, L, Pissinatti A, Schenka AA, Antunes E, Nahoum C, Cogo JC, de Oliveira MA, and De Nucci G. Role of a novel tetrodotoxin-resistant sodium channel in the nitrergic relaxation of corpus cavernosum from the South American rattlesnake Crotalus durissus terrificus. J Sex Med 2011;8:1616-1625.

Hypotensive and vasorelaxing effects of the new NO-donor [Ru(terpy)(bdq)NO+](3+) in spontaneously hypertensive rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Upregulation of muscarinic receptors by long-term nitric oxide inhibition in the rat ileum

1. The aim of the present study was to examine the effects of long-term nitric oxide (NO) blockade on contractions of the rat ileum induced by muscarinic agonists.2. Male Wistar rats received the NO synthesis inhibitor N (G) -nitro-l-arginine methyl ester (l-NAME; 20 mg/rat per day) in drinking water for 7, 15, 30 and 60 days. Concentration-responses curves to methacholine and carbachol were obtained and pEC(50) values were calculated. Saturation binding assays were performed in membranes prepared from rat ileum after 60 days of l-NAME treatment and the dissociation constant (K-D ) and maximal number of binding sites (B-max ) were determined by Scatchard analysis.3. The NO synthase activity of the ileum was markedly reduced in all l-NAME-treated groups. At 60 days after l-NAME treatment, a significant increase in the potency of methacholine (fourfold) and carbachol (threefold) was observed. In binding studies, we found a significant increase in B-max for [(3) H]-quinuclidinyl benzilate of approximately 57% in the l-NAME treated group without any significant change in K-D values. The contractile response to methacholine was not modified by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (3 mumol/L). No morphological alterations in the rat ileum were observed in l-NAME-treated rats.4. Our findings suggest that treatment with l-NAME for 60 days induces a marked increase in the potency of methacholine and carbachol, as well as an increase in receptor number in the rat ileum.

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.

Isolation, homology modeling and renal effects of a C-type natriuretic peptide from the venom of the Brazilian yellow scorpion (Tityus serrulatus)

Mammalian natriuretic peptides (NPs) have been extensively investigated for use as therapeutic agents in the treatment of cardiovascular diseases. Here, we describe the isolation, sequencing and tridimensional homology modeling of the first C-type natriuretic peptide isolated from scorpion venom. In addition, its effects on the renal function of rats and on the mRNA expression of natriuretic peptide receptors in the kidneys are delineated. Fractionation of Tityusserrulatus venom using chromatographic techniques yielded a peptide with a molecular mass of 2190.64Da, which exhibited the pattern of disulfide bridges that is characteristic of a C-type NP (TsNP, T. serrulatus Natriuretic Peptide). In the isolated perfused rat kidney assay, treatment with two concentrations of TsNP (0.03 and 0.1μg/mL) increased the perfusion pressure, glomerular filtration rate and urinary flow. After 60min of treatment at both concentrations, the percentages of sodium, potassium and chloride transport were decreased, and the urinary cGMP concentration was elevated. Natriuretic peptide receptor-A (NPR-A) mRNA expression was down regulated in the kidneys treated with both concentrations of TsNP, whereas NPR-B, NPR-C and CG-C mRNAs were up regulated at the 0.1μg/mL concentration. In conclusion, this work describes the isolation and modeling of the first natriuretic peptide isolated from scorpion venom. In addition, examinations of the renal actions of TsNP indicate that its effects may be related to the activation of NPR-B, NPR-C and GC-C. © 2013 Elsevier Ltd.

Efeito da apocinina na hipertensão, disfunção endotelial e hipertrofia ventricular esquerda em Ratos Espontaneamente Hipertensos (SHR)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modulação nitrérgica na regulação ocitocinérgica da secreção do peptídeo natriurético atrial em cardiomiócitos

Historicamente conhecida por suas ações sobre o sistema reprodutor, hoje se sabe que a ocitocina (OT) também pode contribuir para a regulação da homeostase cardiovascular e hidroeletrolítica. A OT é produzida nos núcleos supra-óptico e paraventricular do hipotálamo e liberada para o plasma a partir de terminais neurais da pituitária posterior, no entanto, muitos estudos identificaram locais extra-cerebrais de produção OT, incluindo o coração e o endotélio vascular. A ativação de seus receptores em células endoteliais, bem como em sistemas hipotalâmicos/hipofisários e cardíaco, pode resultar na produção de óxido nítrico (NO). O presente trabalho teve como objetivo verificar o papel do NO na regulação da secreção de peptídeo natriurético atrial (ANP) estimulada por OT em cultura primária de cardiomiócitos de embriões de camundongos. Para tal, corações de embriões de camundongos Balb C, com 19 a 21 dias de vida intra-uterina, foram isolados e cultivados para os ensaios com OT e demais substâncias interferentes na síntese de NO e GMPc seu segundo mensageiro. A adição de concentrações crescentes de OT (0.1, 1, 10 e 100 μM) induziu aumento proporcional na secreção de ANP e nitrato para o meio, confirmando a ação estimuladora da OT em cardiomiócitos. O bloqueio da liberação de ANP estimulada por OT (10 μM) foi observada após adição de Ornitina Vasotocina (CVI-OVT) (100 μM), um antagonista específico de OT. Este antagonista inibiu a secreção basal de ANP, quando adicionado individualmente, sugerindo que a OT pode atuar via mecanismo autócrino, tônico estimulatório sobre a secreção de ANP. Amplificação da secreção de ANP estimulada por OT (10 μM) foi observada após sua associação com L-NAME, um inibidor da sintase de óxido nítrico (NOS) (600 μM), e ODQ (100 μM), um inibidor da guanilato ciclase solúvel, sugerindo a ocorrência de feedback negativo nitrérgico na liberação de ANP estimulada por OT no cardiomiócito. Os resultados obtidos mostraram modulação nitrérgica inibidora sobre a secreção de ANP estimulada por OT.

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 mu g/kg, orally; 0.0006 mu g/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 delta-opioid receptors, and partially reversed by norbinaltorphimine, an antagonist of kappa-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 delta-opioid and kappa-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. Behavioural Pharmacology 23:14-24 (C) 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

INTERACTION BETWEEN THE CARBON MONOXIDE AND NITRIC OXIDE PATHWAYS IN THE LOCUS COERULEUS DURING FEVER

FAPESP [2010/50882-1]

Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule

Lessa LM, Carraro-Lacroix LR, Crajoinas RO, Bezerra CN, Dariolli R, Girardi AC, Fonteles MC, Malnic G. Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule. Am J Physiol Renal Physiol 303: F1399-F1408, 2012. First published September 5, 2012; doi: 10.1152/ajprenal.00385.2011.-We previously demonstrated that uroguanylin (UGN) significantly inhibits Na+/H+ exchanger (NHE)3-mediated bicarbonate reabsorption. In the present study, we aimed to elucidate the molecular mechanisms underlying the action of UGN on NHE3 in rat renal proximal tubules and in a proximal tubule cell line (LLC-PK1). The in vivo studies were performed by the stationary microperfusion technique, in which we measured H+ secretion in rat renal proximal segments, through a H+-sensitive microelectrode. UGN (1 mu M) significantly inhibited the net of proximal bicarbonate reabsorption. The inhibitory effect of UGN was completely abolished by either the protein kinase G (PKG) inhibitor KT5823 or by the protein kinase A (PKA) inhibitor H-89. The effects of UGN in vitro were found to be similar to those obtained by microperfusion. Indeed, we observed that incubation of LLC-PK1 cells with UGN induced an increase in the intracellular levels of cAMP and cGMP, as well as activation of both PKA and PKG. Furthermore, we found that UGN can increase the levels of NHE3 phosphorylation at the PKA consensus sites 552 and 605 in LLC-PK1 cells. Finally, treatment of LLC-PK1 cells with UGN reduced the amount of NHE3 at the cell surface. Overall, our data suggest that the inhibitory effect of UGN on NHE3 transport activity in proximal tubule is mediated by activation of both cGMP/PKG and cAMP/PKA signaling pathways which in turn leads to NHE3 phosphorylation and reduced NHE3 surface expression. Moreover, this study sheds light on mechanisms by which guanylin peptides

Hypotensive and vasorelaxing effects of the new NO-donor [Ru(terpy)(bdq)NO+](3+) in spontaneously hypertensive rats

Drugs that release nitric oxide (NO) usually have limitations due to their harmful effects. Sodium nitroprusside (SNP) induces a rapid hypotension that leads to reflex tachycardia, which could be an undesirable effect in patients with heart disease, a common feature of hypertension. The nitrosyl ruthenium complex [Ru(terpy)(bdq)NO+](3+) (TERPY) is a NO donor that is less potent than SNP in denuded aortic rings. This study evaluated the hypotension and vasorelaxation induced by this NO donor in Wistar (W) and spontaneously hypertensive rats (SHR) and compared to the results obtained with SNP. Differently from the hypotension induced by SNP, the action of TERPY was slow, long lasting and it did not lead to reflex tachycardia in both groups. The hypotension induced by the NO-donors was more potent in SHR than in W. TERPY induced relaxation with similar efficacy to SNP, although its potency is lower in both strains. The relaxation induced by TERPY is similar in W and SHR, but SNP is more potent and efficient in SHR. The relaxation induced by TERPY is partially dependent on guanylate cyclase in SHR aorta. The NO released from the NO donors measured with DAF-2 DA by confocal microscopy shows that TERPY releases similar amounts of NO in W and SHR, while SNP releases more NO in SHR aortic rings. (c) 2012 Elsevier Inc. All rights reserved.

Role of nitric oxide and endocannabinoids in synaptic plasticity in the perirhinal cortex and in visual recognition memory

Introduction and aims of the research Nitric oxide (NO) and endocannabinoids (eCBs) are major retrograde messengers, involved in synaptic plasticity (long-term potentiation, LTP, and long-term depression, LTD) in many brain areas (including hippocampus and neocortex), as well as in learning and memory processes. NO is synthesized by NO synthase (NOS) in response to increased cytosolic Ca2+ and mainly exerts its functions through soluble guanylate cyclase (sGC) and cGMP production. The main target of cGMP is the cGMP-dependent protein kinase (PKG). Activity-dependent release of eCBs in the CNS leads to the activation of the Gαi/o-coupled cannabinoid receptor 1 (CB1) at both glutamatergic and inhibitory synapses. The perirhinal cortex (Prh) is a multimodal associative cortex of the temporal lobe, critically involved in visual recognition memory. LTD is proposed to be the cellular correlate underlying this form of memory. Cholinergic neurotransmission has been shown to play a critical role in both visual recognition memory and LTD in Prh. Moreover, visual recognition memory is one of the main cognitive functions impaired in the early stages of Alzheimer’s disease. The main aim of my research was to investigate the role of NO and ECBs in synaptic plasticity in rat Prh and in visual recognition memory. Part of this research was dedicated to the study of synaptic transmission and plasticity in a murine model (Tg2576) of Alzheimer’s disease. Methods Field potential recordings. Extracellular field potential recordings were carried out in horizontal Prh slices from Sprague-Dawley or Dark Agouti juvenile (p21-35) rats. LTD was induced with a single train of 3000 pulses delivered at 5 Hz (10 min), or via bath application of carbachol (Cch; 50 μM) for 10 min. LTP was induced by theta-burst stimulation (TBS). In addition, input/output curves and 5Hz-LTD were carried out in Prh slices from 3 month-old Tg2576 mice and littermate controls. Behavioural experiments. The spontaneous novel object exploration task was performed in intra-Prh bilaterally cannulated adult Dark Agouti rats. Drugs or vehicle (saline) were directly infused into the Prh 15 min before training to verify the role of nNOS and CB1 in visual recognition memory acquisition. Object recognition memory was tested at 20 min and 24h after the end of the training phase. Results Electrophysiological experiments in Prh slices from juvenile rats showed that 5Hz-LTD is due to the activation of the NOS/sGC/PKG pathway, whereas Cch-LTD relies on NOS/sGC but not PKG activation. By contrast, NO does not appear to be involved in LTP in this preparation. Furthermore, I found that eCBs are involved in LTP induction, but not in basal synaptic transmission, 5Hz-LTD and Cch-LTD. Behavioural experiments demonstrated that the blockade of nNOS impairs rat visual recognition memory tested at 24 hours, but not at 20 min; however, the blockade of CB1 did not affect visual recognition memory acquisition tested at both time points specified. In three month-old Tg2576 mice, deficits in basal synaptic transmission and 5Hz-LTD were observed compared to littermate controls. Conclusions The results obtained in Prh slices from juvenile rats indicate that NO and CB1 play a role in the induction of LTD and LTP, respectively. These results are confirmed by the observation that nNOS, but not CB1, is involved in visual recognition memory acquisition. The preliminary results obtained in the murine model of Alzheimer’s disease indicate that deficits in synaptic transmission and plasticity occur very early in Prh; further investigations are required to characterize the molecular mechanisms underlying these deficits.

Impaired vascular function in normoglycemic mice prone to autoimmune diabetes: role of nitric oxide

Type 1 diabetes is an immuno-inflammatory condition which increases the risk of cardiovascular disease, particularly in young adults. This study investigated whether vascular function is altered in mice prone to autoimmune diabetes and whether the nitric oxide (NO)-cyclic GMP axis is involved. Aortic rings suspended in organ chambers and precontracted with phenylephrine were exposed to cumulative concentrations of acetylcholine. To investigate the role of NO, some experiments were performed in the presence of either 1400W (N-(3-aminomethyl)benzyl-acetamidine hydrochloride), a selective inhibitor of the iNOS-isoform, L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride), an inhibitor of all three NOS-isoforms, or ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a selective inhibitor of guanylate cyclase. Moreover, contractility to phenylephrine, big endothelin-1, and endothelin-1 was assessed and histological analysis and iNOS immunohistochemistry were performed. Endothelium-dependent relaxation was reduced in prediabetic NOD mice (78+/-4 vs. 88+/-2%, respectively, P<0.05 vs. control) despite normal plasma glucose levels (n.s. vs. control). Preincubation with 1400W further attenuated responses in prediabetic (P<0.05 vs. untreated) but not in diabetic or in control mice. In contrast, basal NO bioactivity remained unaffected until the onset of diabetes in NOD mice. Contractile responses to big endothelin-1 and endothelin-1 were reduced in prediabetic animals (P<0.05 vs. control), whereas in diabetic mice only responses to big endothelin-1 were decreased (P<0.05 vs. control). These data demonstrate that endothelium-dependent and -independent vascular function in NOD mice is abnormal already in prediabetes in the absence of structural injury. Early proinflammatory activation due to iNOS in diabetes-prone NOD mice appears to be one of the mechanisms contributing to impaired vasoreactivity.

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments.

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.