Oxadiazolo[4,3-a]quinoxalin-1-one Research Articles

In vivo regulation of renal expression of (pro)renin receptor by a low-sodium diet

Effects of low salt (LS) on (pro)renin receptor (PRR) expression are not well established. We hypothesized that LS enhances renal PRR expression via the cGMP-protein kinase G (PKG) signaling pathway. Sprague-Dawley rats were fed a normal-salt (NS) or LS diet associated with intrarenal cortical administration of vehicle (V), the nitric oxide (NO) synthase inhibitor nitro-l-arginine methyl ester (l-NAME), the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP), the cGMP analog 8-bromoguanosine (8-Br)-cGMP, the guanylyl cyclase inhibitor 1H-[1, 2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or a PKG inhibitor (PKGi) for 6 days via osmotic minipump. We evaluated the effects of each treatment on renal interstitial fluid (RIF) levels of nitrate/nitrite and cGMP and renal PRR expression. There were no significant changes in blood pressure with any of the treatments. Urinary sodium excretion was significantly lower in rats given a LS diet. Compared with NS + V, RIF nitrate/nitrite and cGMP levels increased in LS + V rats. In NS groups, RIF nitrate/nitrite and cGMP levels did not change with l-NAME, ODQ, or PKGi and increased in response to SNAP. 8-Br-cGMP increased RIF cGMP but not RIF nitrate/nitrite. In LS groups, RIF nitrate/nitrite decreased with l-NAME and did not change with ODQ or PKGi whereas RIF cGMP decreased with l-NAME, ODQ, and PKGi. PRR mRNA and protein increased in LS + V. In NS rats, PRR mRNA and protein increased in response to 8-Br-GMP and were not affected by any of other treatments. In LS rats, PRR mRNA and protein decreased significantly in response to l-NAME, ODQ, and PKGi. We conclude that LS intake enhances renal expression of PRR via cGMP-PKG signaling pathway.

S-Nitrosylation Inhibits Pannexin 1 Channel Function

S-nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1-1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1(C40A), Panx1(C346A), and Panx1(C426A)). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1(C40A/C346A)) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.

Cyclic GMP/protein kinase G type‐Iα (PKG‐Iα) signaling pathway promotes CREB phosphorylation and maintains higher c‐IAP1, livin, survivin, and Mcl‐1 expression and the inhibition of PKG‐Iα kinase activity synergizes with cisplatin in non‐small cell lung cancer cells

Previously, our laboratory showed that nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type-Iα (PKG-Iα) signaling pathway plays an important role in preventing spontaneous apoptosis and promoting cell proliferation in both normal cells (bone marrow stromal cells and vascular smooth muscle cells) and certain cancer cells (ovarian cancer cells). In the present study, we investigated the novel role of the cGMP/PKG-Iα pathway in preventing spontaneous apoptosis, promoting colony formation and regulating phosphorylation of cAMP response element binding (CREB) protein and protein expression of inhibitor of apoptosis proteins (IAPs) and anti-apoptotic Bcl-2-related proteins in NCI-H460 and A549 non-small cell lung cancer (NSCLC) cells. 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), which blocks endogenous NO-induced activation of cGMP/PKG-Iα, induced apoptosis and decreased colony formation. ODQ also decreased CREB ser133 phosphorylation and protein expression of c-IAP1, livin, and survivin. DT-2 (inhibitor of PKG-Iα kinase activity) increased apoptosis by twofold and decreased CREB ser133 phosphorylation and c-IAP1, livin, and survivin expression. Gene knockdown of PKG-Iα expression using small-interfering RNA increased apoptosis and decreased CREB ser133 phosphorylation, and c-IAP1, livin, survivin, and Mcl-1 expression. Inhibition of PKG-Iα kinase activity with DT-2 dramatically enhanced pro-apoptotic effects of the chemotherapeutic agent cisplatin. Combined treatment of DT-2 and cisplatin increased apoptosis compared with cisplatin or DT-2 alone, showing a synergistic effect. The data suggest that the PKG-Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at ser133 and protein expression of c-IAP1, livin, survivin, and Mcl-1, preventing spontaneous apoptosis and promoting colony formation in NSCLC cells, which may limit the effectiveness of chemotherapeutic agents like cisplatin.

Role of new agents affecting NO/cGMP pathway on ovalbumin-sensitized guinea pig trachea

ABSTRACTAsthma is a chronic inflammatory disease in which cell components play important roles. We aimed to evaluate the effects of NO/cGMP cleavage at trachea preparations isolated from ovalbumin-sensitized guinea pigs in vitro. Trachea rings were exposed to 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-12), (±)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide (NOR-4), 2-(2-methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl) methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), and electrical field stimulation (EFS). cGMP levels in trachea tissues were also measured. The relaxation responses of NOC-12, NOR-4, T-0156, and EFS were significantly decreased at ovalbumin-sensitized group. Nitric oxide (NO) donors significantly decreased the relaxation responses in the presence of 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ). L-Nitro-Arginine Methyl Ester (L-NAME) significantly decreased the EFS relaxation responses in both groups (experimental group and control group), but this effect was reversed by L-Arginine addition. In the experimental group, cGMP levels after EFS, carbachol, NOC-12, NOR-4, and T-0156 exposure were significantly lower than control group. In both groups, cGMP levels after NO donors’ exposure were significantly lower in the presence of ODQ and the cGMP levels after EFS + L-NAME were significantly lower than EFS alone. These results may show the increased formation of NO because of the increased iNOS activity in airway sensitization leading to the inhibition of cNOS resulting in the decrease of endogen NO and decrease of activation of guanylyl cyclase.

Nebivolol-induced vasodilation of renal afferent arterioles involves β3-adrenergic receptor and nitric oxide synthase activation

Nebivolol is a β(1)-adrenergic blocker that also elicits renal vasodilation and increases the glomerular filtration rate (GFR). However, its direct actions on the renal microvasculature and vasodilator mechanism have not been established. We used the in vitro blood-perfused juxtamedullary nephron technique to determine the vasodilator effects of nebivolol and to test the hypothesis that nebivolol induces vasodilation of renal afferent arterioles via an nitric oxide synthase (NOS)/nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway and the afferent arteriolar vasodilation effect may be mediated through the release of NO by activation of NOS via a β(3)-adrenoceptor-dependent mechanism. Juxtamedullary nephrons were superfused with nebivolol either alone or combined with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or the NOS inhibitor N(ω)-nitro-l-arginine (l-NNA) or the β-blockers metoprolol (β(1)), butoxamine (β(2)), and SR59230A (β(3)). Nebivolol (100 μmol/l) markedly increased afferent and efferent arteriolar diameters by 18.9 ± 3.0 and 15.8 ± 1.8%. Pretreatment with l-NNA (1,000 μmol/l) or ODQ (10 μmol/l) decreased afferent vasodilator diameters and prevented the vasodilator effects of nebivolol (2.0 ± 0.2 and 2.4 ± 0.6%). Metoprolol did not elicit significant changes in afferent vasodilator diameters and did not prevent the effects of nebivolol to vasodilate afferent arterioles. However, treatment with SR59230A, but not butoxamine, markedly attenuated the vasodilation responses to nebivolol. Using a monoclonal antibody to β(3)-receptors revealed predominant immunostaining on vascular and glomerular endothelial cells. These data indicate that nebivolol vasodilates both afferent and efferent arterioles and that the afferent vasodilator effect is via a mechanism that is independent of β(1)-receptors but is predominantly mediated via a NOS/NO/sGC/cGMP-dependent mechanisms initiated by activation of endothelial β(3)-receptors.

Inhalation of NO during myocardial ischemia reduces infarct size and improves cardiac function

Bioactive NO carriers in circulating blood formed during NO inhalation selectively distribute blood flow to areas in need, and may thus improve collateral perfusion to the area-at-risk in acute myocardial infarction (AMI). Here, we tested the hypothesis that NO inhalation during the ischemic phase of AMI may improve left ventricular function and reduce infarct size in rats. Following left anterior descending coronary artery (LAD) occlusion, rats received 50 ppm NO for 2 h of ischemia, during subsequent 3 h of reperfusion, or for 5 h of ischemia and reperfusion. Effects of inhaled NO were compared to those of intravenous nitrite as a putative carrier formed during NO inhalation. Downstream signaling via soluble guanylate cyclase was tested by inhibition with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). NO inhalation during myocardial ischemia increased left ventricular systolic pressure, contractility, relaxation, and cardiac output, and reduced myocardial infarction size and area-at-risk as compared to untreated controls. NO inhalation during the reperfusion phase caused a comparable protective effect. Combined inhalation during ischemia and reperfusion did not further improve left ventricular hemodynamics, but had an additive protective effect on the myocardial area-at-risk. NO inhalation increased circulating nitrite levels, and mimicking of this effect by intravenous nitrite infusion achieved similar protection as NO inhalation during myocardial ischemia, while ODQ blocked the protective NO effect. Inhalation of NO during myocardial ischemia improves left ventricular function and reduces infarct size by mechanisms that increase levels of circulating nitrite and involve soluble guanylate cyclase. NO inhalation may represent a promising early intervention in AMI.

Tetrahydrobiopterin Protects Soluble Guanylate Cyclase against Oxidative Inactivation

Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress.

Nitric oxide (NO) enhances pemetrexed cytotoxicity via NO‑cGMP signaling in lung adenocarcinoma cells in�vitro and in�vivo

Pemetrexed (PEM) is a novel, multitargeted, antifolate, antineoplastic agent for the treatment of non-small cell lung cancer and malignant pleural mesothelioma. Additional effects of nitric oxide (NO) donors on the chemosensitivity of cancers have been reported. However, the effects of an NO donor on PEM-induced cytotoxicity remain unknown. In this study, we investigated the effects of the NO donors, NOC-18 on the cytotoxicity in A549 cells in vitro and of nitroglycerin (GTN), on the tumor growth of Lewis lung carcinoma cells in a murine syngraft model treated with PEM. The effects of NO donors on the expression of proteins associated with PEM metabolism, including thymidylate synthase (TS), reduced folate carrier 1 (RFC1), folylpolyglutamate synthase (FPGS), γ-glutamyl hydrolase (GGH) and multidrug resistance-related protein (MRP)5, and the effects of cyclic guanosine mono-phosphate (cGMP) signaling on these proteins were examined in A549 cells. Treatment with 100 nM NOC-18 for 3 days significantly enhanced PEM-induced cytotoxicity and increased the expression of RFC1 and FPGS in A549 cells. Treatment with 10 nM 8-bromo-cGMP (8-Br-cGMP) for 3 days also increased the expression of RFC1 and FPGS in A549 cells. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µm) significantly reversed the increase in RFC1 and FPGS expression induced by 100 nM NOC-18 in A549 cells. Combination therapy with GTN and PEM significantly reduced tumor growth compared with PEM alone in the syngraft model. The enhanced antitumor effect of GTN plus PEM was significantly reversed by the concomitant addition of ODQ. These findings suggest that NO donors, such as NOC-18 and GTN, enhance the anticancer effects of PEM by increasing the RFC1 and FPGS expression and stimulating cGMP signaling pathways in cancer cells.

Resveratrol augments nitric oxide generation and causes store calcium release in chromaffin cells

The cardiovascular protecting effect of the grape fruit trans-resveratrol has been explained among other factors, through augmentation of nitric oxide (NO) production in cardiovascular tissues. Another effect of low resveratrol concentration is the inhibition of single-vesicle quantal release of catecholamine from bovine adrenal chromaffin cells, that was recently suggested to be an additional factor contributing to its beneficial cardiovascular effects. We have investigated here the effects of a low concentration of trans-resveratrol (1μM) on Ca2+ and NO signaling pathways in bovine chromaffin cells, in an attempt to understand the mechanism underlying its previously reported inhibitory effects on quantal secretion. In cells loaded with fura-2 acetoxymethyl ester (fura-2), we have found that 1μM resveratrol produces a transient elevation of the cytosolic Ca2+ concentration ([Ca2+]c). This Ca2+ transient was drastically reduced when the Ca2+ store was depleted by ryanodine and dantrolene; it was also inhibited by Nω-nitro-l-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Furthermore, the Ca2+ transient was mimicked by NO donor S-nitroso-N-acetyl-penicillamine (SNAP). Resveratrol also enhanced the production of nitrites and NO, and L-NAME blocked both responses; in contrast, augmentation by SNAP of nitrites and NO was unaffected by ODQ and was only partially inhibited by L-NAME. On the basis of these results, we are proposing that resveratrol is mitigating the catecholamine surge occurring during stress, through its ability to elicit mild local [Ca2+]c transients and enhanced NO production, that blocks the last steps of exocytosis.

Peroxynitrite has potent pulmonary vasodilator activity in the rat

Peroxynitrite (PN) worsens pathological conditions associated with oxidative stress. However, beneficial effects have also been reported. PN has been shown to demonstrate vasodilator as well as vasoconstrictor properties that are dependent upon the experimental conditions and the vascular bed studied. PN-induced vascular smooth muscle relaxation may involve the formation of nitric oxide (NO) donors. The present results show that PN has significant vasodilator activity in the pulmonary and systemic vascular beds, and that responses to PN were not attenuated by L-penicillamine (L-PEN), a PN scavenger, whereas responses to sodium nitroprusside (SNP) were decreased. PN had a small inhibitory effect on decreases in arterial pressure in response to the NO donors diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and S-nitrosoglutathione (GSNO). PN partially reversed hypoxic pulmonary vasoconstriction. PN responses were attenuated by the soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and responses to PN and the PN precursor, 3-morpholinosydnonimine (SIN-1), were different. These data show that PN has potent pulmonary vasodilator activity in the rat, and provide evidence that a PN interaction with S-nitrosothiols is not the major mechanism mediating the response. These data suggest that responses to PN are mediated by the activation of sGC, and that PN has a small inhibitory effect on NO responses.

Endothelial Dysfunction in the Human Umbilical Artery due to Preeclampsia Can Be Prevented by Sildenafil

Objectives. We aimed to determine the effects of sildenafil in human umbilical artery preparation taken from preeclamptic or normal pregnant women, also to investigate underlying mechanisms in these effects. Study design. Eighteen pregnant women with preeclampsia and 18 healthy pregnant women were involved. Relaxation responses of sildenafil in presence and absence of nitric oxide (NO) synthase inhibitor, N-[omega]-nitro-l-arginine methyl ester (l-NAME), and soluble guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ), were compared between the preeclampsia group and control group. Results. Sildenafil-induced relaxation responses were significantly attenuated in the presence of preeclampsia, l-NAME or ODQ, but not totally abolished. Interestingly, except with ODQ incubation, in all set of experiments maximal relaxation response was achieved by sildenafil. Conclusion. These data indicate that sildenafil might effect vascular responsiveness of human umbilical artery through the involvement of NO/cyclic guanosine monophosphate (cGMP)-dependent and -independent pathways. Further investigations are needed to clarify the exact mechanisms.

Pro-survival Effects of 17β-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via Differential Actions of cGMP-dependent Protein Kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.

The regulatory role of the NO/cGMP signal transduction cascade during larval attachment and metamorphosis of the barnacleBalanus (=Amphibalanus) amphitrite

The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement in a dose-dependent manner. In contrast, both the NO scavenger carboxy-PTIO and the NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream guanylyl cyclase (GC) activity using a GC-selective inhibitor ODQ could also significantly accelerate larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a crucial stage for the larval decision to attach and metamorphose. In summary, we concluded that NO regulates larval settlement via mediating downstream cGMP signaling.

Endothelial cells influence the sodium nitroprusside mediated inhibition of platelet aggregation by an as yet unkown pathway

The clinical use of Sodium nitroprusside (SNP) may be associated with an alteration of platelet function. The main focus of this study was the effect of SNP on platelet aggregation in the absence or presence of endothelial cells.Methods: Platelets were incubated with different concentrations of SNP with and without endothelial cells. Platelet aggregation was induced by ADP.Results: Platelet aggregation was significantly inhibited by all concentrations of SNP. Endothelial cells significantly increased this inhibitory effect of SNP. Time course studies showed an inverse correlation of incubation time to platelet aggregation inhibition in the absence of endothelial cells, and a direct correlation in the presence of endothelial cells. Blocking platelet and endothelial cell guanylate cyclase with 1 H-(1,2,4)-oxadiazolo(4,3-a) quinoxalin-1-one (ODQ), or pretreatment of the endothelial cells with cyclooxygenase – inhibitors, had no influence on the increased inhibitory effect of the endothelial cells. Cyanide reversed the inhibitory effect of SNP completely.Conclusion: Endothelial cells play an important role in the SNP mediated inhibition of platelet aggregation. The effect is reversible only by cyanide, not by blocking classical NO signal transduction.

Paclitaxel-Induced Endothelial Dysfunction in Living Rats Is Prevented by Nicorandil via Reduction of Oxidative Stress

Paclitaxel-eluting stents dramatically reduce rates of in-stent restenosis; however, paclitaxel is known to lead to endothelial dysfunction. Protective effects of nicorandil on paclitaxel-induced endothelial dysfunction by examining flow-mediated dilation (FMD) were investigated in anesthetized rats. After 7-day osmotic infusion of paclitaxel (5 mg/kg per day), FMD was measured by high-resolution ultrasound in the femoral artery of living rats. Paclitaxel significantly reduced FMD (21.6% ± 3.2% to 7.1% ± 1.7%); this reduction was prevented by co-treatment with nicorandil (15 mg/kg per day), while paclitaxel did not affect nitroglycerin-induced vasodilation. Diazoxide and tempol, but not isosorbide dinitrate, had an effect similar to nicorandil in preventing paclitaxel-induced decrease in FMD. Nicorandil significantly prevented paclitaxel-induced reduction in acetylcholine-induced vasodilation. On the underling mechanisms, paclitaxel increased reactive oxygen species (ROS) production (dihydrorhodamine 123, DCF fluorescence intensity) and NADPH oxidase (p47phox, gp91phox mRNA) in arteries and human coronary artery endothelial cells (HCAECs), while paclitaxel reduced nitric oxide (NO) release (DAF-2 fluorescence intensity), but not endothelial NO synthase (eNOS) phosphorylation in HCAECs. Nicorandil prevented the increased ROS production in arteries and HCAECs, which was 5-hydroxydecanoate (5-HD)-sensitive but 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)-resistant, without significant effect on the reduced NO release. In conclusion, nicorandil prevents paclitaxel-induced endothelial dysfunction, which may be brought by improved NO bioavailability due to the reduction of oxidative stress via KATP channel activation.

An anxiolytic-like effect of hyperbaric oxygen in the mouse light/dark exploration test

AimsWe studied whether hyperbaric oxygen (HBO2) treatment, which is known to increase production of nitric oxide (NO) in the brain, might also produce an NO-dependent anxiolytic-like behavioral response. Main methodsMale NIH Swiss mice (20–25g) were subjected to a 60-min HBO2 treatment at different absolute atmospheres, and anxiety was assessed using the light/dark exploration test at different time intervals following the cessation of HBO2 treatment. To ascertain the underlying mechanism of action, other groups of mice were pretreated with the NO synthase inhibitor NG-monomethyl-l-arginine acetate, the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO), the soluble guanylyl cyclase-inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or the benzodiazepine antagonist flumazenil to determine their influence on the HBO2-induced anxiolytic-like effect. Key findingsA 60-min HBO2 treatment at 3.0 absolute atmospheres increased the time spent by mice in the light compartment that lasted up to 90min following the end of HBO2 treatment. This anxiolytic effect of HBO2 was significantly reduced by pretreatment with L-NMMA, carboxy-PTIO, ODQ and flumazenil. SignificanceBased on these findings, we conclude that a 60-min HBO2 treatment is capable of inducing an anxiolytic effect that possibly involves NO, cyclic GMP and the benzodiazepine binding site.

Red Ginseng Saponin Fraction A Isolated from Korean Red Ginseng by Ultrafiltration on the Porcine Coronary Artery

Red ginseng saponin fraction-A (RGSF-A) contains a high percentage of panaxadiol saponins that were isolated from Korean red ginseng by ultrafiltration. The aim of this study was to elucidate the effects of RGSF-A on the porcine distal left anterior descending (LAD) coronary artery. The relaxant responses to RGSF-A were examined during contractions induced by 100 nM U46619 (9,11-dideoxy-9a,11a-methanoepoxy-prostaglandin F2a), a stable analogue of thromboxane A2. RGSF-A dose-dependently induced biphasic (fast- and slow-) relaxation in the distal LAD coronary artery in the presence of an intact endothelium. The fast-relaxation was quickly achieved in a minute, and then the slow-relaxation was slowly developed and sustained for more than thirty minutes after the administration of RGSF-A. The slow-relaxation had a tendency to be bigger than the fast-relaxation. Fast relaxation induced by RGSF-A was almost blocked by Nω-Nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor. However slow-relaxation induced by RGSF-A was only partially inhibited by L-NAME and ODQ. In the endothelium-removed ring, RGSF-A evoked only slow-relaxation to a certain extent. These data suggest that RGSF-A induced both endothelium dependent fast- and slow-relaxation and endothelium independent slow-relaxation in the porcine distal LAD coronary artery. The endothelium dependent fast-relaxation is mediated by the nitric oxide (NO)-cGMP pathway, and the endothelium dependent slow-relaxation is at least partially mediated by the NO-cGMP pathway. However, the endothelium-independent slow-relaxation remains to be elucidated.

Tadalafil analgesia in experimental arthritis involves suppression of intra‐articular TNF release

We investigated the effect of the phosphodiesterase-5 inhibitor, tadalafil, on the acute hypernociception in rat models of arthritis. Rats were treated with either an intra-articular injection of zymosan (1 mg) or surgical transection of the anterior cruciate ligament (as an osteoarthritis model). Controls received saline intra-articular or sham operation respectively. Joint pain was evaluated using the articular incapacitation test measured over 6 h following zymosan or between 4 and 7 days after anterior cruciate ligament transection. Cell counts, tumour necrosis factor-α (TNF-α), interleukin-1 (IL-1), and the chemokine, cytokine-induced neutrophil chemoattractant-1 (CINC-1) were measured in joint exudates 6 h after zymosan. Groups received tadalafil (0.02-0.5 mg·kg⁻¹ per os) or saline 2 h after intra-articular zymosan. Other groups received the µ-opioid receptor antagonist naloxone or the cGMP inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) before tadalafil. Tadalafil dose-dependently inhibited hypernociception in zymosan and osteoarthritis models. In zymosan-induced arthritis, tadalafil significantly decreased cell influx and TNF-α release but did not alter IL-1 or CINC-1 levels. Pretreatment with ODQ but not with naloxone prevented the anti-inflammatory effects of tadalafil. Therapeutic oral administration of tadalafil provided analgesia mediated by guanylyl cyclase and was independent of the release of endogenous opioids. This effect of tadalafil was associated with a decrease in neutrophil influx and TNF-α release in inflamed joints.

Imatinib Mesylate (Gleevec) as Protein-tyrosine Kinase Inhibitor Elicits Smooth Muscle Relaxation in Isolated Human Prostatic Tissue

To evaluate the mechanism of action of imatinib mesylate (Gleevec), a protein tyrosine kinase inhibitor on the human prostate with benign prostatic hyperplasia. Prostate samples were obtained from 16 patients with benign prostatic hyperplasia (mean age 68.3 ± 1.9 years), who had undergone transurethral prostatectomy. In tissue bath studies, cumulative concentration-response curves were constructed for imatinib after precontraction with 120 mM KCl. Imatinib-induced relaxation was quantitated in tissues treated with l-N(G)-Nitroarginine Methyl Ester (l-NAME) (an inhibitor of nitric oxide synthase) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (a soluble guanylyl cyclase inhibitor). Two K+ channel blockers (adenosine triphosphate [K(ATP)] and Large-conductance Ca(2+)-activated K(+) channels [BK(Ca2+)] channels) were also evaluated as antagonists of imatinib-induced relaxation and repeated in the presence of the α-adrenergic receptor blocker alfuzosin. An electrical field stimulation (1-20 Hz, 5 ms, 5 seconds, 60 V)-induced contractile response was performed on strips incubated with imatinib (10(-3) M). KCl-induced contractions in human prostatic tissue were significantly inhibited by imatinib (maximal response 84.9 ± 4.5%) and were attenuated by l-NAME (42%, P < .001) and ODQ (43%, P < .001). This relaxant effect was also suppressed by glibenclamide (adenosine triphosphate-sensitive K+ channel blocker, 41%, P < .001) and tetraethylammonium (BK(Ca2+) channel blocker, 24%, P < .05). Imatinib induced prostatic smooth muscle relaxation in vitro. This effect was suppressed by l-NAME and ODQ, showing a dependence on the nitric oxide-cyclic guanosine monophosphate pathway and modulated by the K(ATP) and BK(Ca2+) K+ channels. Our findings suggest that imatinib can augment relaxation of human prostatic tissues by way of a novel ligand-protein tyrosine kinase signaling pathway.

Mechanisms involved in the nitric oxide-induced vasorelaxation in porcine prostatic small arteries

Benign prostatic hypertrophy has been known to be related with glandular ischemia processes, and nitric oxide (NO) is a potent vasodilator agent. Therefore, the current study investigates the mechanisms underlying the NO-induced vasorelaxation in pig prostatic small arteries. In microvascular myographs, relaxation to electrical field stimulation (EFS), or to exogenous (S)-nitroso-N-acetylpenicillamine (SNAP) and acetylcholine (ACh), was observed on noradrenaline-precontracted prostatic small arterial rings under non-adrenergic and non-cholinergic (NANC) conditions. EFS (1-16Hz) and exogenous SNAP (0.1-30μM) evoked frequency- and concentration-dependent relaxation, respectively. Tetrodotoxin, a neuronal voltage-gated Na(+) channel blocker, abolished the EFS-evoked relaxation. ACh (1nM-10μM) induced concentration-dependent relaxation, which was reduced by the NO synthase inhibitor N(G)-nitro-L: -arginine (L: -NOARG). L: -NOARG also reduced the EFS-elicited relaxation but failed to modify the response to SNAP. 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and iberiotoxin (IbTX), blockers of soluble guanylyl cyclase and large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, respectively, reduced EFS-, SNAP-, and ACh-induced relaxation. The combination of ODQ with IbTX did not produce further inhibition of the responses to either SNAP or ACh, compared with ODQ alone. Blockade of cyclooxygenases and intermediate and small conductance Ca(2+)-activated, ATP-dependent, and voltage-gated K(+) channels did not change the EFS and SNAP responses. In conclusion, our results suggest that NO and non-NO non-prostanoid factor(s) derived from NANC nerves are involved in the vasodilatation of pig prostatic small arteries. NO produces relaxation through soluble guanylyl cyclase activation-dependent BK(Ca) channel opening and through guanylyl cyclase-independent mechanisms. The vasodilatation elicited by NO could be useful to prevent prostatic ischemia.