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

BAY 41-2272 inhibits human neutrophil functions

BAY 41-2272 is a guanylyl cyclase (GC) stimulator derived from YC-1 (3-[(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole]). Previous studies by our group showed that BAY 41-2272 activates human monocytes via soluble guanylyl cyclase (sGC) and cGMP. In this study, we investigated the effect of BAY 41-2272 on human neutrophil function and found that 30 μM BAY 41-2272 inhibits neutrophil migration (1.82-fold lower than FMLP, P < 0.05 by one-way ANOVA followed by Tukey's test), oxidative burst (1.70-fold lower than PMA, P < 0.05 by one-way ANOVA followed by Tukey's test), and IL-8 cytokine production (1.80-fold lower than PMA, P < 0.05 by one-way ANOVA followed by Tukey's test). Our results suggest that these effects are independent of the sGC pathway but dependent instead on cGMP production, as the response induced by 30 μM BAY 41-2272 was 6.40-fold greater than that observed in our negative control (P < 0.05 by parametric t-test). 1H-[1, 2, 4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), which is an irreversible inhibitor of sGC, was unable to reverse the effects of BAY 41-2272 on human neutrophils, indicating that this drug acts independently of sGC. Our results confirm the immunomodulatory effect of BAY 41-2272 on human neutrophils.

Antidiarrheal, vasorelaxant, and neuropharmacological actions of the diterpene tilifodiolide.

Salvia tiliifolia is used in folk medicine as a relaxant agent and for the treatment of diarrhea and neurodegenerative diseases. Tilifodiolide (TFD) is a diterpene obtained from this plant. The purpose of this work was to evaluate the antidiarrheal, vasorelaxant, and neuropharmacological actions of TFD. These effects were selected based on the folk medicinal use of S. tiliifolia. The antidiarrheal activity of 1-50 mg/kg p.o. TFD was assessed with the castor oil related tests. The vasorelaxant effect of TFD (0.9-298 μM) was performed with smooth muscle tissues from rats, and its mechanism of action was evaluated using different inhibitors. The sedative, anxiolytic, and antidepressant effects of 1-100 mg/kg TFD were assessed. The possible mechanisms of action of the anxiolytic and antidepressant effects of TFD were evaluated using inhibitors. TFD exhibited antidiarrheal (ED50 = 10.62 mg/kg) and vasorelaxant (EC50 = 48 ± 3.51 μM) effects. The coadministration of TFD with N(ω)-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), reverted the vasorelaxant action showed by TFD alone. TFD exerted anxiolytic actions (ED50 = 20 mg/kg) in the cylinder exploratory test, whereas TFD (50 mg/kg) showed antidepressant actions in the tail suspension test by 44%. The pretreatment with 2 mg/kg flumazenil partially reverted the anxiolytic actions of TFD, whereas the pretreatment with 1 mg/kg yohimbine abolished the antidepressant effects of TFD. In summary, TFD exerted antidiarrheal activity by decreasing the intestinal fluid accumulation and vasorelaxant effects mediated by nitric oxide and cyclic guanosine monophosphate. TFD showed anxiolytic and antidepressant effects by the partial involvement of gamma-Aminobutyric acid (GABA) receptors and the possible participation of α2-adrenoreceptors, respectively.

Effects of Nitric Oxide on Slow Waves and Spontaneous Contraction of Guinea Pig Gastric Antral Circular Muscle

We examined the effects of nitric oxide (NO) donors, S-nitroso-L-cysteine (Cys-NO) and 3-morpholinosydnonimine hydrochloride (SIN-1), on slow waves and contractile activity in the circular muscle of guinea pig gastric antrum. In the presence of atropine and guanethidine, electrical field stimulation (EFS) reduced the amplitude of phasic contraction. The effect of EFS was significantly inhibited by both the NO synthase inhibitor Nω-nitro-L-arginine methyl ester and a soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Cys-NO and SIN-1 mimicked the effect of EFS on phasic contraction and reduced the amplitude of slow waves in a concentration-dependent manner, with no effect on frequency and resting membrane potential. Phasic contraction was more sensitive to NO donors than slow waves. The inhibitory effects of NO donors were antagonized by ODQ and mimicked by a membrane permeable cGMP analogue 8-bromo-cGMP. Several K+ channel blockers such as apamin, iberiotoxin, and glibenclamide had no effect on the inhibitory action of SIN-1. These results suggest that NO inhibits the phasic contraction and slow waves through cGMP-dependent mechanisms in guinea pig gastric antrum. The effect of NO is unlikely to be mediated by the activation of Ca2+-activated or ATP-sensitive K+ channels.

Marjoram Relaxes Rat Thoracic Aorta Via a PI3-K/eNOS/cGMP Pathway.

Despite pharmacotherapeutic advances, cardiovascular disease (CVD) remains the primary cause of global mortality. Alternative approaches, such as herbal medicine, continue to be sought to reduce this burden. Origanum majorana is recognized for many medicinal values, yet its vasculoprotective effects remain poorly investigated. Here, we subjected rat thoracic aortae to increasing doses of an ethanolic extract of Origanum majorana (OME). OME induced relaxation in a dose-dependent manner in endothelium-intact rings. This relaxation was significantly blunted in denuded rings. N(ω)-nitro-l-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) significantly reduced the OME-induced vasorelaxation. Cyclic guanosine monophosphate (cGMP) levels were also increased by OME. Moreover, wortmannin or LY294002 significantly reduced OME-induced vasorelaxation. Blockers of ATP-sensitive or Ca2+-activated potassium channels such as glibenclamide or tetraethylamonium (TEA), respectively, did not significantly affect OME-induced relaxation. Similarly, verapamil, a Ca2+ channel blocker, indomethacin, a non-selective cyclooxygenase inhibitor, and pyrilamine, a H1 histamine receptor blocker, did not significantly modulate the observed relaxation. Taken together, our results show that OME induces vasorelaxation via an endothelium-dependent mechanism involving the phosphoinositide 3-kinase (PI3-K)/ endothelial nitric oxide (NO) synthase (eNOS)/cGMP pathway. Our findings further support the medicinal value of marjoram and provide a basis for its beneficial intake. Although consuming marjoram may have an antihypertensive effect, further studies are needed to better determine its effects in different vascular beds.

Endothelium-dependent and endothelium-independent effects of 1-nitro-2-propylbenzene on rat aorta.

A group of nitro compounds contains a benzene ring in a short aliphatic chain with the NO2 group, property that supposedly favors its vasodilator profile. In this study, we evaluated in isolated rat aorta the effects of 1-nitro-2-propylbenzene (NPB), a nitro compound containing the NO2 in the aromatic ring. In aorta precontracted with KCl, NPB (1-3000μm) induced full endothelium-independent relaxation. In endothelium-intact preparations, phenylephrine-induced contractions were fully relaxed by NPB, effect unaltered by N(ω)-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In the concentration range of 30-300μm, NPB slightly but significantly potentiated the phenylephrine-induced contraction. Such potentiation was unaltered by the thromboxane-prostanoid receptor antagonist seratrodast, but was abolished by endothelium removal or by preincubation of endothelium-intact preparations with L-NAME, ODQ or by ruthenium red and HC-030031, blockers of subtype 1 of ankyrin transient receptor potential (TRPA1 ) channels. Verapamil exacerbated the potentiating effect of NPB. The potentiating effect was undetectable in preparations precontracted by 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U-46619). Relaxation was reduced by ruthenium red while it was enhanced by HC-030031. In conclusion, NPB has vasodilator properties but with a mechanism of action distinct from its analogues. Contrary to other nitro compounds, its relaxing effects did not involve recruitment of the guanylyl cyclase pathway. NPB has also endothelium-dependent potentiating properties on phenylephrine-induced contractions, a phenomenon that putatively required a role of endothelial TRPA1 channels. The present findings reinforce the notion that the functional group NO2 in the aliphatic chain of these nitro compounds determines favorably their vasodilator properties.

Changes in Gastric Smooth Muscle Cell Contraction during Pregnancy: Effect of Estrogen.

It is well known that pregnancy is associated with frequent gastrointestinal (GI) disorders and symptoms. Moreover, previous reports have shown that estrogen, which changes in levels during pregnancy, participates in the regulation of GI motility and is involved in the pathogenesis of various functional disorders in the stomach. The aim of the current study was to explore the changes in the expression of estrogen receptors (ERs) and examine the effect of estrogen on nitric oxide- (NO-) cyclic guanosine monophosphate (cGMP) pathway and thus relaxation in gastric smooth muscle cells (GSMC) during pregnancy. Single GSMC from early-pregnant and late-pregnant Sprague-Dawley rats were used. Protein and mRNA expression levels of ERs were measured via specifically designed enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), respectively. NO and cGMP levels were measured via specifically designed ELISA kits. Effect of estrogen on acetylcholine- (ACh-) induced contraction of single GSMC was measured via scanning micrometry in the presence or absence of the NO synthase inhibitor, N-nitro-L-arginine (L-NNA), or guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Estrogen increased both NO and cGMP levels and their levels were greater in early compared to late pregnancy. Expression of ERs was greater in early compared to late pregnancy. ACh induced greater contraction of GSMC in late pregnancy compared to early pregnancy. Estrogen inhibited ACh-induced contraction in both periods of pregnancy. Importantly, pretreatment of GSMC with either L-NNA or ODQ abolished estrogen inhibitory action on muscle contraction. In conclusion, GSMC contractile behavior undergoes drastic changes in response to estrogen during pregnancy and this might explain some of the pregnancy-associated gastric disorders.

Bupivacaine-induced contraction is attenuated by endothelial nitric oxide release modulated by activation of both stimulatory and inhibitory phosphorylation (Ser1177 and Thr495) of endothelial nitric oxide synthase

This study examined the mechanism associated with the endothelium-dependent attenuation of vasoconstriction induced by bupivacaine (BPV), with a particular focus on the upstream cellular signaling pathway of endothelial nitric oxide synthase (eNOS) phosphorylation induced by BPV in human umbilical vein endothelial cells (HUVECs). BPV concentration-response curves were investigated in the isolated rat aorta. The effects of Nω-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), methylene blue, calmidazolium, the Src kinase inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and the combination of L-arginine and L-NAME on BPV-induced contraction in endothelium-intact aorta preparations were examined. The effects of BPV alone and in combination with PP2 on the phosphorylation of eNOS (at Ser1177 or Thr495), caveolin-1 and Src kinase were examined in HUVECs. BPV-induced contraction was lower in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, methylene blue and calmidazolium increased BPV-induced contraction in endothelium-intact aortae, whereas PP2 alone and combined treatment with L-arginine and L-NAME inhibited BPV-induced contraction. Low-concentration BPV (30 µM) induced both stimulatory (Ser1177) and inhibitory (Thr495) phosphorylation of eNOS in HUVECs. However, high-concentration BPV (150 µM) induced only stimulatory (Ser1177) eNOS phosphorylation. Additionally, phosphorylation of Src kinase, caveolin-1 and inhibitory eNOS (Thr495) induced by low-concentration BPV was inhibited by PP2. These results suggest that contraction induced by low-concentration BPV is attenuated by endothelial nitric oxide release, which is modulated both stimulatory (Ser1177) and inhibitory eNOS phosphorylation (Thr495). BPV-induced phosphorylation of eNOS (Thr495) is indirectly mediated by an upstream cellular signaling pathway involving Src kinase (Tyr416) and caveolin-1 (Tyr14).

Mechanisms underlying the vasorelaxant effect of trans-4-methoxy-β-nitrostyrene in the rat mesenteric resistance arteries

Mechanisms underlying the vasorelaxant effects of the synthetic nitro compound, trans-4-methoxy-β-nitrostyrene (T4MN) were studied in isolated small resistance arteries from spontaneously hypertensive rats (SHRs). T4MN caused vasorelaxation in endothelium-intact third-order branches of the mesenteric artery pre-contracted with noradrenaline (NA). This effect was unchanged by indomethacin and atropine but was significantly reduced by endothelium removal, L-NAME, LY294002, glybenclamide, TEA, apamin, TRAM 34, or by the association of apamin and TRAM 34. Pretreatment with the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the T4MN-induced relaxation in endothelium-intact, but not in denuded preparations. Incubation of small resistance arteries with T4MN increased nitric oxide (NO) production, an effect that was blocked by L-NAME. In Ca2+-free medium, T4MN inhibits the contractions induced by (i) NA, (ii) exogenous calcium through receptor- or voltage-operated Ca2+ channels and (iii) those evoked by Ca2+ influx through stores-operated Ca2+ channels activated by thapsigargin-induced Ca2+ store depletion. In contrast, T4MN was inert against the transient contraction induced by caffeine in Ca2+-free medium. In conclusion, T4MN induced effective vasorelaxant effects in isolated small resistance arteries from SHRs. This vasorelaxation seems to be mediated partly by an endothelium-dependent mechanism involving activation of Akt/eNOS/NO pathway and partly by an endothelium-independent mechanism through activation of sGC/cGMP/PKG pathway in vascular smooth muscle, leading to inhibition of Ca2+ influx from the extracellular milieu and IP3-sensitive intracellular Ca2+ release as well as activation of potassium channels.

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one Attenuates Oxidative Trauma and Recuperate Beam Walk and Adhesive Removal Behavior in MPTP Parkinsonian Mice Model

Recent researches have suggested 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase inhibitor may attenuate motor impairments in Parkinson's disease (PD). The antiparkinsonian activity of ODQ were studied on motor abnormalities induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to provide a better understanding of this drug group. The objective of the present study is to evaluate the effect of ODQ on behavioral parameters such as Beam walk test, Adhesive removal test and to assess the biochemical changes due to ODQ against MPTP induced PD mice model. Eighteen C57BL/6J male mice were included in the study, divided into three groups of 6 each. Group A mice were treated with vehicle (Normal saline). Group B mice were subjected to MPTP sub acute protocol. Group C mice were treated with MPTP as according to sub acute protocol and administered with ODQ subcutaneous injection after final MPTP dose. Behavioral tests like Beam walk test, Adhesive removal test, along with Biochemical correlation were done using standard methods. Narrow beam walk and adhesive removal behavior were significantly reversed, and Superoxide dismutase (SOD) levels were enhanced in ODQ treated group compared to MPTP intoxicated mice group. Soluble guanylate cyclase inhibitor ODQ, could be a potential treatment for maintaining the balance of antioxidant and oxidant biochemical environment during oxidative stress which may be helpful for treating PD, targeting one or more factors of its multiple etiological factors.

Effect of progesterone on nitric oxide/cyclic guanosine monophosphate signaling and contraction in gastric smooth muscle cells.

Previous studies have shown that progesterone could inhibit muscle contraction in various sites of the gastrointestinal tract. The underlying mechanisms responsible for these inhibitory effects of progesterone are not fully known. The aim of the current study was to investigate the effect of progesterone on the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway and muscle contraction in the stomach. Single gastric smooth muscle cells from female Sprague-Dawley rats were used. The expression of progesterone receptor (PR) mRNA was analyzed by reverse transcription polymerase chain reaction. NO and cGMP levels were measured via specific ELISAs. Acetylcholine (ACh)-induced contraction of single gastric muscle cells preincubated with progesterone was measured via scanning micrometry in the presence or absence of the NO synthase inhibitor, Nω-Nitro-L-arginine (L-NNA), or guanylyl cyclase inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and expressed as percent shortening from resting cell length. PR expression was detected in the stomach muscle cells. Progesterone inhibited ACh-induced gastric muscle cell contraction. Furthermore, progesterone increased NO and cGMP levels in single gastric muscle cells. Most notably, pre-incubation of muscle cells with either L-NNA or ODQ abolished the inhibitory action of progesterone on muscle contraction. These present observations suggest that progesterone promotes muscle cell relaxation in the stomach potentially via the NO/cGMP pathway.

Perivascular adipose tissue protects against the vascular dysfunction induced by acute ethanol intake: Role of hydrogen peroxide

AimWe investigated the consequences of acute ethanol intake on the anti-contractile effect of perivascular adipose tissue (PVAT). MethodsThe effects of a single dose of ethanol (1 g/kg; p.o. gavage) on the vascular function were assessed within 30 min in male Wistar rats. ResultsEthanol decreased the relaxation induced by acetylcholine and increased the contraction induced by phenylephrine in endothelium-intact, but not in endothelium-denuded aortas without PVAT. The vascular dysfunction induced by ethanol was not observed in aortic rings with PVAT. Nω-Nitro-l-arginine methyl ester (L-NAME), NG-nitro-l-arginine (L-NNA) and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), but not tiron or tempol, increased the contraction induced by phenylephrine in endothelium-intact aortas with PVAT from control and ethanol-treated rats. Catalase increased phenylephrine-induced contraction in aortas with PVAT from ethanol-treated rats, but not from control rats. Conversely, inhibition of catalase with aminotriazole decreased phenylephrine-induced contraction in aortas from ethanol-treated rats. Treatment with ethanol increased hydrogen peroxide (H2O2) levels and decreased catalase activity in aortas with PVAT. Ethanol increased superoxide anion (O2−) generation in aortas with or without PVAT. Superoxide dismutase (SOD) activity was not affected by ethanol intake. In situ quantification of H2O2 using 2′7′dichlorodihydrofluorescein diacetate (DCFH-DA) revealed increased levels of H2O2 in periaortic PVAT from ethanol-treated rats. However, in situ evaluation of nitric oxide (NO) in both aorta and PVAT showed no differences between groups. ConclusionsOur study provides novel evidence that the periaortic PVAT protects against the vascular dysfunction induced by acute ethanol intake through a mechanism that involves increased generation of H2O2.

Activation of Large Conductance, Calcium-Activated Potassium Channels by Nitric Oxide Mediates Apelin-Induced Relaxation of Isolated Rat Coronary Arteries.

Apelin increases coronary blood flow, cardiac contractility, and cardiac output. Based on these favorable hemodynamic effects, apelin and apelin-like analogs are being developed for treating heart failure and related disorders; however, the molecular mechanisms underlying apelin-induced coronary vasodilation are unknown. This study aimed to elucidate the signaling pathways by which apelin causes smooth muscle relaxation in coronary arteries. Receptors for apelin (APJ receptors) were expressed in coronary arteries, as determined by Western blot and polymerase chain reaction analyses. Immunofluorescence imaging studies identified APJ receptors on endothelial and smooth muscle cells. In isolated endothelial cells, apelin caused an increase in 4,5-diaminofluorescein fluorescence that was abolished by nitro-l-arginine (NLA) and F13A (H-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Ala-OH), an APJ receptor antagonist, consistent with increased nitric oxide (NO) production. In arterial rings, apelin caused endothelium-dependent relaxations that were abolished by NLA, F13A, and iberiotoxin. Neither oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) nor DT-2, a protein kinase G inhibitor, had any effect on apelin-induced relaxations, and apelin itself had no effect on intracellular cGMP accumulation in coronary arteries. Patch-clamp studies in isolated smooth muscle cells demonstrated that the NO donors, diethyl amine NONOate and sodium nitroprusside, caused increases in large conductance, calcium-activated potassium channel (BKCa) currents, which were inhibited by iberiotoxin but not ODQ. Thus, apelin causes endothelium-dependent relaxation of coronary arteries by stimulating endothelial APJ receptors and releasing NO, which acts in a cGMP-independent manner and increases BKCa activity in the underlying smooth muscle cells. These results provide a mechanistic basis for apelin-induced coronary vasodilation and may provide guidance for the future development of novel apelin-like therapeutic agents.

Vasodilator effects and putative guanylyl cyclase stimulation by 2-nitro-1-phenylethanone and 2-nitro-2-phenyl-propane-1,3-diol on rat aorta

Compounds containing a nitro group may reveal vasodilator properties. Several nitro compounds have a NO2 group in a short aliphatic chain connected to an aromatic group. In this study, we evaluated in rat aorta the effects of two nitro compounds, with emphasis on a putative recruitment of the soluble guanylate cyclase (sGC) pathway to induce vasodilation. Isolated aortic rings were obtained from male Wistar rats to compare the effects induced by 2-nitro-1-phenylethanone (NPeth) or 2-nitro-2-phenyl-propane-1,3-diol (NPprop). In aortic preparations contracted with phenylephrine or KCl, NPeth and NPprop induced vasorelaxant effects that did not depend on the integrity of vascular endothelium. NPeth had a lesser vasorelaxant efficacy than NPprop and only the NPprop effects were inhibited by pretreatment with the sGC inhibitors, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or methylene blue. In an ODQ-preventable manner, NPprop inhibited the contractile component of the phenylephrine-induced response mediated by intracellular Ca2+ release or by extracellular Ca2+ recruitment through receptor- or voltage-operated Ca2+ channels. In contrast, NPprop was inert against the transient contraction induced by caffeine in Ca2+-free medium. In an ODQ-dependent manner, NPprop inhibited the contraction induced by the protein kinase C activator phorbol 12,13-dibutyrate or by the tyrosine phosphatase inhibitor sodium orthovanadate. In silico docking analysis of a sGC homologous protein revealed preferential site for NPprop. In conclusion, the nitro compounds NPeth and NPprop induced vasorelaxation in rat aortic rings. Aliphatic chain substituents selectively interfered in the ability of these compounds to induce vasorelaxant effects, and only NPprop relaxed aortic rings via a sGC pathway.

Involvement of the NO/sGC/cGMP/K+ channels pathway in vascular relaxation evoked by two non-quinazoline α1-adrenoceptor antagonists

The aim of this study was to explore the α1-adrenoceptor-independent mechanisms involved in the vasorelaxant properties of two non-quinazoline α1-adrenoceptors antagonists (MH-76 and MH-79). Endothelium intact and endothelium denuded rat aorta was contracted with 1 μM phenylephrine to plateau, and the vasodilatory effect of MH-76 and MH-79 was examined in the absence or presence of inhibitors of the different signal transduction pathways. cGMP concetration was measured in rat aorta (enzyme immunoassay kit). In human aortic endothelial cells (HAEC) NO production was examined using a DAF-FM DA fluorescent indicator, whereas in human aortic smooth muscle cells the influence of the title compounds on K+ efflux was evaluated. The vasorelaxant effect of MH-76 and MH-79 was attenuated by endothelium removal, Nω-Nitro-l-arginine methyl ester (L-NAME) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) pretreatment to the level characteristic for α1-adrenoreceptor blocking activity. In addition, the MH-76 and MH-79 induced relaxation was reduced by K+ channels blockers. In endothelium intact rat aorta, MH-76 and MH-79 caused an increase in cGMP level, whereas in HAEC they increased NO generation. In contrast, the reference, quinazoline based α1-antagonist prazosin, did not influence NO production. Our findings suggest that the mechanisms underlying the vasodilatory properties of non-quinazoline based α1-adrenoceptors antagonists MH-76 and MH-79 involve not only α1-adrenoceptor blocking activity but also the activation of the endothelial NO–cGMP signalling pathway and the subsequent opening of K+ channels. Our studies show that such double mechanism of action is superior to pure α1-adrenoceptor blockade, and may be considered as a promising alternative for the prevention and treatment of cardiovascular diseases.

Stachydrine protects eNOS uncoupling and ameliorates endothelial dysfunction induced by homocysteine

BackgroundHyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases (CVDs). Stachydrine (STA) is an active component in Chinese motherwort Leonurus heterophyllus sweet, which has been widely used for gynecological and cardiovascular disorders. This study is aimed to examine the effects of STA on homocysteine (Hcy)-induced endothelial dysfunction.MethodsThe effects of STA on vascular relaxation in rat thoracic aortas (TA), mesenteric arteries (MA) and renal arteries (RA) were measured by using Multi Myograph System. The levels of nitric oxide (NO), tetrahydrobiopterin (BH4) and guanosine 3′, 5′ cyclic monophosphate (cGMP) were determined. Endothelial nitric oxide synthase (eNOS) dimers and monomers were assayed by using Western blotting. GTP cyclohydrolase 1 (GTPCH1) and dihydrofolate reductase (DHFR) expressions were measured by using quantitative reverse transcriptase-PCR (qRT-PCR) and Western blotting.ResultsSTA effectively blocked Hcy-induced impairment of endothelium-dependent vasorelaxation in rat TA, MA and RA. STA-elicited arterial relaxations were reduced by NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or the NO-sensitive guanylyl cyclase inhibitor 1H- [1, 2, 4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), but not by inducible iNOS inhibitor 1400 W nor the nonselective COX inhibitor indomethacin. Hcy caused eNOS uncoupling and decreases in NO, cGMP and BH4, which were attenuated by STA. Moreover, STA prevented decreases of GTPCH1 and DHFR levels in Hcy-treated BAECs.ConclusionWe demonstrated that STA effectively reversed the Hcy-induced endothelial dysfunction and prevented eNOS uncoupling by increasing the expression of GTPCH1 and DHFR. These results revealed a novel mechanism by which STA exerts its beneficial vascular effects.

Effects of Nitric Oxide on Voltage-Gated K⁺ Currents in Human Cardiac Fibroblasts through the Protein Kinase G and Protein Kinase A Pathways but Not through S-Nitrosylation.

This study investigated the expression of voltage-gated K+ (KV) channels in human cardiac fibroblasts (HCFs), and the effect of nitric oxide (NO) on the KV currents, and the underlying phosphorylation mechanisms. In reverse transcription polymerase chain reaction, two types of KV channels were detected in HCFs: delayed rectifier K+ channel and transient outward K+ channel. In whole-cell patch-clamp technique, delayed rectifier K+ current (IK) exhibited fast activation and slow inactivation, while transient outward K+ current (Ito) showed fast activation and inactivation kinetics. Both currents were blocked by 4-aminopyridine. An NO donor, S-nitroso-N-acetylpenicillamine (SNAP), increased the amplitude of IK in a concentration-dependent manner with an EC50 value of 26.4 µM, but did not affect Ito. The stimulating effect of SNAP on IK was blocked by pretreatment with 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or by KT5823. 8-bromo-cyclic GMP stimulated the IK. The stimulating effect of SNAP on IK was also blocked by pretreatment with KT5720 or by SQ22536. Forskolin and 8-bromo-cyclic AMP each stimulated IK. On the other hand, the stimulating effect of SNAP on IK was not blocked by pretreatment of N-ethylmaleimide or by DL-dithiothreitol. Our data suggest that NO enhances IK, but not Ito, among KV currents of HCFs, and the stimulating effect of NO on IK is through the PKG and PKA pathways, not through S-nitrosylation.

Thiocarbonyl-bound metallonitrosyl complexes with visible-light induced DNA cleavage and promising vasodilation activity

Nitric oxide has been involved in many key biological processes such as vasodilation, platelet aggregation, apoptosis, memory function, and this has drawn attention to the development of exogenous NO donors. Metallonitrosyl complexes are an important class of these compounds. Here, two new ruthenium nitrosyl complexes containing a thiocarbonyl ligand, with the formula cis-[Ru(phen)2(L)(NO)](PF6)3 (phen = phenantroline, L = thiourea or thiobenzamide), were synthesized and characterized by electronic spectroscopy, FTIR, NMR, mass spectrometry and voltammetric techniques. Theoretical calculations using Density Functional Theory (DFT) and Time-dependent Density Functional Theory (TD-DFT) were also used and further supported the characterizations of these complexes. An efficient release of nitric oxide by blue light was validated using a NO/HNO probe: 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, known as cPTIO. Interestingly, the complex containing thiourea cleaved DNA even in the dark, while both complexes showed great DNA photocleavage activity in blue light. This process might work mainly through NO and hydroxyl radical production. Additionally, these complexes showed promising vasodilator activity, whose mechanism of action was investigated using N-Nitro-l-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and compared to sodium nitroprusside. Both compounds were indeed NO-mediated heme-dependent activators of soluble guanylate cyclase. Additionally, they did not show any significant cytotoxicity against cancer cell lines U87 and GBM02. Altogether, these results supported both complexes having potential pharmacological applications that deserve further studies.

Control of cardiovascular responses to stress by CRF in the bed nucleus of stria terminalis is mediated by local NMDA/nNOS/sGC/PKG signaling

The aims of the present study were to assess an interaction of corticotropin-releasing factor (CRF) neurotransmission within the bed nucleus of the stria terminalis (BNST) with local nitrergic signaling, as well as to investigate an involvement of activation of local NMDA glutamate receptor and nitric oxide (NO) signaling in control of cardiovascular responses to acute restraint stress by BNST CRF neurotransmission in rats. We observed that CRF microinjection into the BNST increased local NO release during restraint stress. Furthermore, bilateral microinjection of CRF into the BNST enhanced both the arterial pressure and heart rate increases evoked by restraint stress, but without affecting the sympathetically-mediated cutaneous vasoconstriction. The facilitation of both pressor and tachycardiac responses to restraint stress evoked by BNST treatment with CRF were completely inhibited by local pretreatment with either the selective NMDA glutamate receptor antagonist LY235959, the selective neuronal nitric oxide synthase (nNOS) inhibitor Nω-Propyl-l-arginine (NPLA), the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or the protein kinase G (PKG) inhibitor KT5823. Taken together, these results provide evidence that BNST CRF neurotransmission facilitates local NMDA-mediated glutamatergic neurotransmission and activates nitrergic signaling, and this pathway is involved in control of cardiovascular responses to stress.

Nitric oxide-cGMP-PKG signaling in the bed nucleus of the stria terminalis modulates the cardiovascular responses to stress in male rats

The bed nucleus of the stria terminalis (BNST) constitutes an important component of neural substrates of physiological and behavioral responses to aversive stimuli, and it has been implicated on cardiovascular responses evoked by stress. Nevertheless, the local neurochemical mechanisms involved in BNST control of cardiovascular responses during aversive threats are still poorly understood. Thus, the aim of the present study was to assess the involvement of activation in the BNST of the neuronal isoform of the enzyme nitric oxide synthase (nNOS), as well as of signaling mechanisms related to nitric oxide effects such as soluble guanylate cyclase (sGC) and protein kinase G (PKG) on cardiovascular responses induced by an acute session of restraint stress in male rats. We observed that bilateral microinjection of either the nonselective NOS inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME), the selective nNOS inhibitor Nω-Propyl-L-arginine (NPLA) or the sGC inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) into the BNST enhanced the tachycardic response and decreased the drop in tail cutaneous temperature evoked by acute restraint stress, but without affecting the increase on blood pressure. Bilateral BNST treatment with the selective PKG inhibitor KT5823 also facilitated the heart rate increase and decreased the drop in cutaneous temperature, in addition to enhancing the blood pressure increase. Taken together, these results provide evidence that NO released from nNOS and activation of sGC and PKG within the BNST play an inhibitory influence on tachycardia to stress, whereas this signaling mechanism mediates the sympathetic-mediated cutaneous vasoconstriction.

Vasoactivity of nitrite in the iliac artery of the toad Rhinella marina.

Nitrite ([Formula: see text]) causes vasodilation in mammals due to the formation of (nitric oxide) NO by endogenous [Formula: see text] reduction in the vascular wall. In this study, we determined if a similar mechanism operates in amphibians. Dual-wire myography of the iliac artery from Rhinella marina showed that applied [Formula: see text] caused a concentration-dependent vasodilation in normoxia (21% O2; EC50: 438 µM). Hypoxia (0.63% O2) significantly increased the maximal dilation to [Formula: see text] by 5% ( P = 0.0398). The addition of oxyhemoglobin significantly increased the EC50 ( P = 0.0144; EC50: 2,236 µM) but did not affect the maximal vasodilation. In contrast, partially deoxygenated hemoglobin (90% desaturation) did not affect the EC50 ( P = 0.1189) but significantly ( P = 0.0012) increased the maximal dilation to [Formula: see text] by 11%. The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) completely abolished the response to [Formula: see text] ( P < 0.0001), and of the nitric oxide synthase inhibitors, only N5-(1-imino-3-butenyl)-l-ornithine (vinyl-l-NIO; P = 0.0028) significantly reduced the [Formula: see text] vasodilation. The xanthine oxidoreductase inhibitor allopurinol ( P = 0.927), the nitric oxide-scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (C-PTIO; P = 0.478), and disruption of the endothelium ( P = 0.094) did not affect the [Formula: see text] vasodilation. Incubation of iliac arteries with 1 mM [Formula: see text] did not a cause a change in the cGMP concentration (P = 0.407). Plasma [Formula: see text] was found to be 0.86 ± 0.20 µmol/l, while nitrate ([Formula: see text]) was 19.55 ± 2.55 µmol/l. Both cygb and ngb mRNAs were expressed in the iliac artery, and it is possible that these globins facilitate [Formula: see text] reduction in hypoxia. In addition, [Formula: see text] intracellular disproportionation processes could be important in the generation of NO from [Formula: see text].