Soluble Guanylyl Cyclase Inhibitor Research Articles

Mirabegron causes relaxation of human and rat corpus cavernosum: could it be a potential therapy for erectile dysfunction?

To examine the effects of mirabegron, a selective β3 -adrenoceptor agonist that has recently been approved for the treatment of overactive bladder (OAB), on erectile function. Stimulation of β3 -adrenoceptors localised in cavernosal smooth muscle cells may play a physiological role in mediating penile erection, and offer a beneficial pharmacological action for patients who have OAB and erectile dysfunction (ED). Corpus cavernosal (CC) specimens were obtained from patients with ED and Peyronie's disease undergoing penile prosthesis implantation. Erectile responses were also evaluated in vivo after intracavernosal injection (ICI) of mirabegron in anaesthetised rats. Mirabegron-elicited relaxation responses (10(-8) -10(-3) m) on phenylephrine-induced contraction were seen in human CC (HCC) and rat CC strips in isolated organ-bath studies. The effects of inhibitors, namely L-NAME [N(G) -nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase (NOS), 100 μm], ODQ [1H-(1,2,4) oxadiazolo(4,3-α) quinoxalin-1-one, a soluble guanylyl cyclase (sGC) inhibitor, 30μm], methylene blue (a NOS and sGC inhibitor, 20μm), SR59230A (β3 -adrenoceptor blocker, 1 μm), and fasudil [Rho-associated protein kinase (ROCK) inhibitor, 0.1 μm], on mirabegron-induced relaxation responses were evaluated. Responses to mirabegron were compared with responses to isoprenaline and nebivolol. Immunohistochemistry was used to localise β3 -adrenoceptors and ROCK in CC smooth muscle cells. In vivo rat data were expressed as intracavernosal pressure (ICP)/mean arterial pressure, and total ICP. Mirabegron resulted in a relaxation of phenylephrine-evoked CC contractions in a concentration-dependent manner and SR59230A antagonised the mirabegron-induced relaxations in HCC and rat CC. Other inhibitors, L-NAME, ODQ, and methylene blue, did not affect the mirabegron-induced relaxation responses. Mirabegron relaxation responses at concentrations (0.1-10 μm) were enhanced by fasudil (ROCK inhibitor) in rat but not in HCC strips. KCl-induced contractions in HCC and rat CC were partially inhibited by mirabegron. In vivo, ICI of mirabegron (doses of 0.1-1 mg/kg) had a minor effect on ICP when compared with vehicle administration. Immunohistochemistry data showed β3 -adrenoceptors localised in the smooth muscle cells of the HCC and rat CC. Mirabegron markedly relaxed isolated CC strips by activating β3 -adrenoceptors independently of the NO-cGMP pathway. There is also evidence of the existence of a close functional link between β3 -adrenoceptors and the RhoA/ROCK pathway. These results may support further clinical studies using combinations of mirabegron with ROCK and phosphodiesterase type 5 inhibitors (PDE5i) for the treatment of ED, especially in patients who do not respond to PDE5i therapy.

Antidepressant effect of pramipexole in mice forced swimming test: A cross talk between dopamine receptor and NMDA/nitric oxide/cGMP pathway.

Pramipexole is a dopamine D2 receptor agonist indicated for treating Parkinson disorder. This study was aimed to investigate the effect of pramipexole in forced swimming test (FST) in mice and the possible involvement of activation of D2 receptors and inhibition of N-methyl-d-aspartate (NMDA) receptors and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) on this effect. Intraperitoneal administration of pramipexole (1-3mg/kg) reduced the immobility time in the FST similar to fluoxetine (20mg/kg, i.p.). This effect of pramipexole (1mg/kg, i.p.) was ceased when mice were pretreated with haloperidol (0.15mg/kg, i.p,) and sulpiride (5mg/kg, i.p) as D2 receptor antagonists, NMDA (75mg/kg,i.p.), l-arginine (750mg/kg, i.p., a substrate for nitric oxide synthase) or sildenafil (5mg/kg, i.p., a phosphodiesterase 5 inhibitor). The administration of MK-801 (0.05mg/kg, i.p., a NMDA receptor antagonist) l-NG-Nitro arginine methyl ester (l-NAME, 10mg/kg, i.p., a non-specific nitric oxide synthase (NOS) inhibitor), 7-nitroindazole (30mg/kg, i.p., a neuronal NOS inhibitor) and methylene blue (10mg/kg, i.p.), an inhibitor of both NOS and soluble guanylyl cyclase (sGC) in combination with the sub-effective dose of pramipexole (0.3mg/kg, i.p.) reduced the immobility. Altogether, our data suggest that the antidepressant-like effect of pramipexole is dependent on the activation of D2 receptor and inhibition of either NMDA receptors and/or NO-cGMP synthesis. These results contribute to the understanding of the mechanisms underlying the antidepressant-like effect of pramipexole and reinforce the role of D2 receptors, NMDA receptors and l-arginine-NO-GMP pathway in the antidepressant mechanism of this agent.

The Role of cGMP on Adenosine A 1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus.

Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 μM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 μM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway.

Role of NO/cGMP signaling pathway in dexmedetomidine-induced reduction of neuropathic pain in rats

Objective To investigate the role of nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway in dexmedetomidine-induced reduction of neuropathic pain (NP) in the rats. Methods Forty-two healthy male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 7 groups (n=6 each) using a random number table: normal control group (C group); NP group; dexmedetomidine group (D group); dimethyl sulfoxide (DMSO) group; a non-selective nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) group (L-NAME group); inactive enantiomer D-NAME group (D-NAME group); a soluble guanylyl cyclase inhibitor 1H-[1, 2, 4] oxidazole[4, 3-a] quinoxalin-1-one (ODQ) group (ODQ group). NP was induced by chronic constriction injury to the sciatic nerve in anesthetized rats.On day 7 after chronic constriction injury, dexmedetomidine 1.5 μg/kg was injected intrathecally in group D, and in DMSO, L-NAME, D-NAME and ODQ groups, DMSO 10 μl, L-NAME 100 μg, D-NAME 100 μg and ODQ 10 μg were injected intrathecally, respectively, and 25 min later dexmedetomidine 1.5 μg/kg was injected intrathecally.The thermal paw withdrawal latency was measured immediately after intrathecal administration and at 30, 60, 90, 120, 150, 180 and 210 min after intrathecal administration, and the area under the curve of thermal paw withdrawal latency was calculated to reflect the thermal pain threshold. Results Compared with group C, the thermal pain threshold was significantly decreased in the other six groups (P 0.05). Conclusion The mechanism by which dexmedetomidine reduces NP is related to activation of NO/cGMP signaling pathway in the rats. Key words: Dexmedetomidine; Neuralgia; Nitric oxide; Cyclic GMP; Spinal cord

PARTICIPATION OF ADENYLYL CYCLASE/cAMP PATHWAY IN VASORELAXANT MECHANISM OF THE PYRANOCOUMARIN BRAYLIN IN MESENTERIC ARTERY

IntroductionBraylin is a pyranocoumarin isolated from the species Zanthoxylum tingoassuiba (Rutaceae). Previous studies have shown that Braylin has a possible inhibitory action in phosphodiesterase enzymes.AimsThe aim of this study was to investigate the mechanism involved in the vasorelaxant effect of Braylin.MethodsThe experiments were performed using wistar rats (12–16 weeks/250–300g), euthanized in CO2 camera and the superior mesenteric artery were isolated and free from connective tissue and cut into rings (1–2 mm), suspended by cotton threads for isometric tension recordings in a Tyrode's solution at 37 °C, gassed with a 95% O2 and 5% CO2, under a resting tension of 0.75g. All surgical and experimental procedures were approved by the local Animal use and care ethics committee – CEUA/ICS/UFBA by the number 089/2015.ResultsThe cumulative administration of Braylin (0.1–300 μM) in phenylephrine (Phe 10μM) pre‐contracted mesenteric rings, induced a concentration‐dependent vasorelaxation (Emax = 111.5 ± 7.9%, CE50 = 4.8 (3.0 – 7.7) × 10−5, n = 17), the removal of the endothelium did not change this effect (Emax = 127.6 ± 13.1%, CE50 =1.7 (4.0 – 7.3) × 10−5, n = 16). The presence of ODQ (100 μM), an inhibitor of soluble guanylyl cyclase, also did not change the effect induced by Braylin (Emax = 117.3 ± 2.6%, CE50 = 1.3 (1.9 – 8.9) × 10−5, n = 5). On the other hand, the presence of SQ 22536, an adenylyl cyclase inhibitor, reduced the relaxant effect (Emax = 115.6 ± 2.8, CE50 = 4.3 (3.0 – 6.2) × 10−5, n = 5). The incubation of the superior mesenteric rings with KT‐5720, an inhibitor of PKA, reduced the effect of Braylin (Emax = 110.4 ± 5.3%, CE50 = 2.9 (2.0 – 4.1) × 10−5, n = 5). Pre‐treatment with KCl 20 mM, TEA (1mM), BaCl2 (30μM), or 4‐aminopiridin (1mM) decreased the pharmacological potency of the Braylin significantly.ConclusionThose effects taken together suggest that Braylin induces vasorelaxant effects in isolated superior mesenteric artery rings from normotensive involving AC/cAMP pathway. K+ channels also seem to be important in the relaxant effect, probably BKca channels.Support or Funding InformationFundação de Amparo à Pesquisado Estado da Bahia (fapesb) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tirofiban induces vasorelaxation of the coronary artery via an endothelium-dependent NO-cGMP signaling by activating the PI3K/Akt/eNOS pathway

Tirofiban, a glycoprotein IIb/IIIa inhibitor, is an antiplatelet drug extensively used in patients with acute coronary syndrome (ACS) and exerts an therapeutic effect on no-reflow phenomenon during percutaneous coronary intervention (PCI). Previous studies elucidated the vasodilation caused by tirofiban in the peripheral artery. However, whether tirofiban exerts a vasodilator effect on the coronary artery is unclear. Our present study found that tirofiban induced endothelium-dependent vasodilation in a concentration- and time-dependent manner in the isolated rat coronary artery pre-constricted by 5-hydroxytryptamine (5-HT). Further study showed that incubation of human umbilical venous endothelial cells (HUVECs) with tirofiban increased NO production, which was ascribed to the increased eNOS phosphorylation. This was confirmed by the loss of the vasorelaxant effect of tirofiban in the presence of l-NAME (eNOS inhibitor) and L-NMMA (NOS inhibitor) but not SMT (iNOS inhibitor) on isolated rat coronary arteries. The vasorelaxation was also blocked by the PI3K inhibitors, wortmannin and LY294002, as well as the Akt inhibitor SH-5, indicating the role of PI3K and Akt in tirofiban-mediated vasodilation. Moreover, further study showed that soluble guanylyl cyclase (sGC) inhibitor ODQ, or blockers of potassium channel (big-conductance calcium-activated potassium channel) blocked tirofiban-induced vasodilation of the coronary artery. These findings suggest that tirofiban induces vasorelaxation via an endothelium-dependent NO-cGMP signaling through the activation of the Akt/eNOS/sGC pathway.

Aged Garlic Extract Inhibits Human Platelet Aggregation by Altering Intracellular Signaling and Platelet Shape Change

Background:Increased platelet aggregation plays a pivotal role in the etiology of cardiovascular disease. Upon platelet aggregation, an increase in free cytoplasmic Ca2+ results in the inhibition of soluble guanylyl cyclase (sGC) and adenylyl cyclase (AC), leading to a decrease in cyclic guaninosine-5′-monophosphate (cGMP) and cAMP, respectively. This leads to the activation of the glycoprotein IIb/IIIa (GPIIb/IIIa) fibrinogen receptor, resulting in platelet shape change. Aged garlic extract (AGE) decreases platelet aggregation; however, the mechanisms involved are not clearly defined.Objective:Our objective was to investigate the effects of AGE on intraplatelet cell signaling and platelet shape change.Methods:Platelets from 14 participants were studied. Platelet aggregation was induced by ADP in the presence of AGE up to a concentration of 6.25% (vol:vol) alone or in combination with 3-morpholinosydnonimine (Sin-1), a nitric oxide donor. The experiments with AGE were repeated in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. In a series of separate experiments, platelet aggregation was induced in the presence of either 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an sGC inhibitor, or 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), an AC inhibitor, or a combination of both in the presence of IBMX and AGE. Intraplatelet cGMP and cAMP were measured. The platelets were also subjected to scanning electron microscopic analysis, and their binding to fibrinogen was determined.Results:AGE decreased platelet aggregation at all concentrations tested; this decrease was more marked in the presence of Sin-1 and ranged between 15% and 67%.The presence of IBMX also led to a decrease (17–35%) in platelet aggregation at all AGE concentrations and a significant decrease in the amounts of cGMP (24–41%) and cAMP (19–70%), respectively, in the presence of ODQ and SQ22536. The presence of AGE significantly inhibited the binding of activated platelets to fibrinogen, preventing changes in platelet shape.Conclusion:These results indicate that AGE inhibits platelet aggregation by increasing cyclic nucleotides and inhibiting fibrinogen binding and platelet shape change.

Fermented garlic extract decreases blood pressure through nitrite and sGC-cGMP-PKG pathway in spontaneously hypertensive rats

Garlic (Allium sativum L.) has long been used as an anti-hypertensive. The study investigated the effect of fermented garlic extract (Fgarlic) on systolic blood pressure (SBP) and its molecular basis using spontaneously hypertensive rats (SHRs). Fgarlic contained high content of stable nitrite (0.975 mg nitrite/ml). Acute feeding of different amounts (0.3, 0.6, and 0.9 ml) of concentrated Fgarlic (9.75 mg nitrite/ml) reduced SBP dose-dependently. Chronic feeding of Fgarlic (containing 27 mg nitrite/day) for 12 days reduced SBP with increased expressions of eNOS and PKG proteins in aortic tissues, which were attenuated by a soluble guanylyl cyclase (sGC) inhibitor. The relaxation responses of thoracic aorta to acetylcholine and sodium nitroprussside were improved in SHRs fed Fgarlic. These results suggest that nitrite in Fgarlic, which converts to NO in the body, functions as an anti-hypertensive molecule and its effect is mediated through sGC-cGMP-PKG pathway.

Nitric oxide inhibits basolateral 10-pS Cl- channels through the cGMP/PKG signaling pathway in the thick ascending limb of C57BL/6 mice.

We used patch-clamp techniques to examine whether nitric oxide (NO) decreases NaCl reabsorption by suppressing basolateral 10-pS Cl- channels in the thick ascending limb (TAL). Both the NO synthase substrate l-arginine (l-Arg) and the NO donor S-nitroso-N-acetylpenicillamine significantly inhibited 10-pS Cl- channel activity in the TAL. The inhibitory effect of l-Arg on Cl- channels was completely abolished in the presence of the NO synthase inhibitor or NO scavenger. Moreover, inhibition of soluble guanylyl cyclase abrogated the effect of l-Arg on Cl- channels, whereas the cGMP analog 8-bromo-cGMP (8-BrcGMP) mimicked the effect of l-Arg and significantly decreased 10-pS Cl- channel activity, indicating that NO inhibits basolateral Cl- channels by increasing cGMP production. Furthermore, treatment of the TAL with a PKG inhibitor blocked the effect of l-Arg and 8-BrcGMP on Cl- channels, respectively. In contrast, a phosphodiesterase 2 inhibitor had no significant effect on l-Arg or 8-BrcGMP-induced inhibition of Cl- channels. Therefore, we conclude that NO decreases basolateral 10-pS Cl- channel activity through a cGMP-dependent PKG pathway, which may contribute to the natriuretic and diuretic effects of NO in vivo.

Methanolic Extract of Clinacanthus nutans Exerts Antinociceptive Activity via the Opioid/Nitric Oxide-Mediated, but cGMP-Independent, Pathways.

The objectives of the present study were to determine the mechanisms of antinociceptive effect of methanol extract of Clinacanthus nutans (Acanthaceae) leaves (MECN) using various animal nociceptive models. The antinociceptive activity of orally administered 10% DMSO, 100 mg/kg acetylsalicylic acid (ASA), 5 mg/kg morphine, or MECN (100, 250, and 500 mg/kg) was determined using the acetic acid-induced abdominal constriction (ACT), formalin-induced paw licking (FT), and hot plate tests (HPT). The role of opioid and nitric oxide/cyclic guanosine monophosphate (NO/cGMP) systems was also investigated. The results showed that MECN produced a significant (p < 0.05) antinociceptive response in all nociceptive models with the recorded ED50 value of 279.3 mg/kg for the ACT, while, for the early and late phases of the FT, the value was >500 mg/kg or 227.7 mg/kg, respectively. This antinociceptive activity was fully antagonized by naloxone (a nonselective opioid antagonist) but was partially reversed by l-arginine (l-arg; a nitric oxide [NO] precursor), Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME; an NO synthase inhibitor), or their combinations thereof. In contrast, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ; a soluble guanylyl cyclase inhibitor) enhanced the extract's antinociception. UHPLC analysis revealed the presence of several flavonoid-based compounds with antinociceptive action. In conclusion, MECN exerted the peripherally and centrally mediated antinociceptive activity via the modulation of the opioid/NO-mediated, but cGMP-independent, systems.

Nitric oxide mediates stretch-induced Ca2+ oscillation in smooth muscle

The stretching of smooth muscle tissue modulates contraction through augmentation of Ca(2+) transients, but the mechanism underlying stretch-induced Ca(2+) transients is still unknown. We found that mechanical stretching and maintenance of mouse urinary bladder smooth muscle strips and single myocytes at 30% and 18% beyond the initial length, respectively, resulted in Ca(2+) oscillations. Experiments indicated that mechanical stretching remarkably increased the production of nitric oxide (NO) as well as the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) oscillations and contractility increases were completely abolished by the NO inhibitor L-NAME or eNOS (also known as NOS3) gene inactivation. Moreover, exposure of eNOS-knockout myocytes to exogenous NO donor induced Ca(2+) oscillations. The stretch-induced Ca(2+) oscillations were greatly inhibited by the selective inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor xestospongin C and partially inhibited by ryanodine. Moreover, the stretch-induced Ca(2+) oscillations were also suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, but not by the soluble guanylyl cyclase (sGC) inhibitor ODQ. These results suggest that stretching myocyte and maintenance at a certain length results in Ca(2+) oscillations that are NO dependent , and sGC and cGMP independent, and results from the activation of PI3K in smooth muscle.

Heterozygous eNOS deficiency is associated with oxidative stress and endothelial dysfunction in diet-induced obesity.

Heterozygous endothelial nitric oxide synthase (eNOS) deficiency is associated with normal endothelium-dependent responses, however, little is known regarding the mechanisms that maintain or impair endothelial function with heterozygous eNOS deficiency. The goals of this study were to (1) determine mechanism(s) which serve to maintain normal endothelial function in the absence of a single eNOS gene; and (2) to determine whether heterozygous eNOS deficiency predisposes blood vessels to endothelial dysfunction in response to a high-fat diet (HFD). Responses of carotid arteries were examined in wild-type (eNOS(+/+)) and heterozygous eNOS-deficient (eNOS(+/-)) treated with either vehicle (saline), N(G)-nitro-L-arginine (L-NNA, 100μmol/L), an inhibitor of nitric oxide synthase, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1μmol/L), an inhibitor of soluble guanylyl cyclase (sGC), and in eNOS(+/+) and eNOS(+/-) mice fed a control (10%) or a 45% HFD (kcal from fat). Responses to acetylcholine (ACh) were similar in vehicle-treated arteries from eNOS(+/+) and eNOS(+/-) mice, and were equally inhibited by L-NNA and ODQ. Phosphorylation of eNOS Ser1176, a site associated with increased eNOS activity, was significantly greater in eNOS(+/-) mice most likely as a compensatory response for the loss of a single eNOS gene. In contrast, responses to ACh were markedly impaired in carotid arteries from eNOS(+/-), but not eNOS(+/+), micefed a HFD. Vascular superoxide levels as well as plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) were selectively increased in HFD-fed eNOS(+/-) mice. In reconstitution experiments, IL-6 produced concentration-dependent impairment of endothelial responses as well as greater increases in NADPH-stimulated superoxide levels in arteries from eNOS(+/-) mice fed a control diet compared to eNOS(+/+) mice. Our findings of increased Ser1176-phosphorylation reveal a mechanism by which NOS- and sGC-dependent endothelial function can be maintained with heterozygous eNOS deficiency. In addition, heterozygous eNOS deficiency predisposes blood vessels to developing endothelial dysfunction in response to a HFD. The impairment produced by a HFD in eNOS(+/-) mice appears to be mediated by IL-6-induced increases in vascular superoxide. These findings serve as an important example of eNOS haploinsufficiency, one that may contribute to the development of carotid artery disease in obese humans.

Cyclic GMP-mediated memory enhancement in the object recognition test by inhibitors of phosphodiesterase-2 in mice.

Cyclic nucleotide phosphodiesterase-2 (PDE2) is a potential therapeutic target for the treatment of cognitive dysfunction. Using the object recognition test (ORT), this study assessed the effects of two PDE2 inhibitors, Bay 60-7550 and ND7001, on learning and memory, and examined underlying mechanisms. To assess the role of PDE2 inhibition on phases of memory, Bay 60-7550 (3 mg/kg) was administered: 30 min prior to training; 0, 1, or 3 h after training; or 30 min prior to recall testing. To assess cyclic nucleotide involvement in PDE2 inhibitor-enhanced memory consolidation, either the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg; intraperitoneal (IP)), soluble guanylyl cyclase inhibitor 1H-[-1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 20 mg/kg; IP), protein kinase G inhibitor KT5823 (2.5 μg; intracerebroventricular (ICV)), or protein kinase A inhibitor H89 (1 μg; ICV) was administered 30 min prior to the PDE2 inhibitor Bay 60-7550 (3 mg/kg) or ND7001 (3 mg/kg). Changes in the phosphorylation of 3'5'-cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) at Ser-133 and vasodilator-stimulated phosphoprotein (VASP) at Ser-239 were determined to confirm activation of cAMP and 3'5'-cyclic guanosine monophosphate (cGMP) signaling. Bay 60-7550 (3 mg/kg) enhanced memory of mice in the ORT when given 30 min prior to training, immediately after training, or 30 min prior to recall. Inhibitors of the cGMP pathway blocked the memory-enhancing effects of both Bay 60-7550 (3 mg/kg) and ND7001 (3 mg/kg) on early consolidation processes. Bay 60-7550 (3 mg/kg) enhanced phosphorylation of CREB and VASP, both targets of cGMP-dependent protein kinase (PKG). These results confirm a potential of PDE2, or components of its signaling pathway, as a therapeutic target for drug discovery focused on restoring memory function.

Relaxant effect of a metal-based drug in human corpora cavernosa and its mechanism of action.

We studied the mechanisms involved in the human corpora cavernosa (HCC) relaxation induced by a new metal-based nitric oxide (NO) donor, the ruthenium complex cis-[Ru(bpy)2Imn(NO)](+3) (FOR0811). FOR0811 produced relaxation in phenylephrine (PE)-precontracted HCC with a maximal response that achieved 112.9 ± 10.6%. There was no difference between the maximal relaxation induced by FOR0811 when compared with sodium nitroprusside (SNP) (106.8 ± 7.3%), BAY41-2272 (107.6 ± 4.1%) or vardenafil (103.4 ± 3.8%), however, FOR0811 was less potent than SNP and vardenafil. L-N(G)-nitroarginine methyl ester (L-NAME), a NO synthase inhibitor, had no effect in the concentration-response curve elicited by FOR0811. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a heme-site inhibitor of soluble guanylyl cyclase (sGC) was able to either block or reverse the relaxation induced by FOR0811. On the other hand, the relaxation induced by FOR0811 was not affected by glibenclamide, a blocker of ATP-sensitive potassium channels. FOR0811 (10 μM) was able to increase cyclic guanosine monophosphate (cGMP) levels in corpora cavernosa strips. FOR0811 completely relaxes HCC by a sGC-cGMP-dependent mechanism and can be a lead compound in the development of new stable NO donors.

Carbon Monoxide (CO) Released from Tricarbonyldichlororuthenium (II) Dimer (CORM-2) in Gastroprotection against Experimental Ethanol-Induced Gastric Damage.

The physiological gaseous molecule, carbon monoxide (CO) becomes a subject of extensive investigation due to its vasoactive activity throughout the body but its role in gastroprotection has been little investigated. We determined the mechanism of CO released from its donor tricarbonyldichlororuthenium (II) dimer (CORM-2) in protection of gastric mucosa against 75% ethanol-induced injury. Rats were pretreated with CORM-2 30 min prior to 75% ethanol with or without 1) non-selective (indomethacin) or selective cyclooxygenase (COX)-1 (SC-560) and COX-2 (celecoxib) inhibitors, 2) nitric oxide (NO) synthase inhibitor L-NNA, 3) ODQ, a soluble guanylyl cyclase (sGC) inhibitor, hemin, a heme oxygenase (HO)-1 inductor or zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1 activity. The CO content in gastric mucosa and carboxyhemoglobin (COHb) level in blood was analyzed by gas chromatography. The gastric mucosal mRNA expression for HO-1, COX-1, COX-2, iNOS, IL-4, IL-1β was analyzed by real-time PCR while HO-1, HO-2 and Nrf2 protein expression was determined by Western Blot. Pretreatment with CORM-2 (0.5–10 mg/kg) dose-dependently attenuated ethanol-induced lesions and raised gastric blood flow (GBF) but large dose of 100 mg/kg was ineffective. CORM-2 (5 mg/kg and 50 mg/kg i.g.) significantly increased gastric mucosal CO content and whole blood COHb level. CORM-2-induced protection was reversed by indomethacin, SC-560 and significantly attenuated by celecoxib, ODQ and L-NNA. Hemin significantly reduced ethanol damage and raised GBF while ZnPPIX which exacerbated ethanol-induced injury inhibited CORM-2- and hemin-induced gastroprotection and the accompanying rise in GBF. CORM-2 significantly increased gastric mucosal HO-1 mRNA expression and decreased mRNA expression for iNOS, IL-1β, COX-1 and COX-2 but failed to affect HO-1 and Nrf2 protein expression decreased by ethanol. We conclude that CORM-2 released CO exerts gastroprotection against ethanol-induced gastric lesions involving an increase in gastric microcirculation mediated by sGC/cGMP, prostaglandins derived from COX-1, NO-NOS system and its anti-inflammatory properties.

Protective effect of exogenous nitrite in postoperative ileus.

As the pathogenesis of postoperative ileus (POI) involves inflammation and oxidative stress, comparable to ischaemia/reperfusion injury which can be ameliorated with nitrite, we investigated whether nitrite can protect against POI and explored the mechanisms involved. We used intestinal manipulation (IM) of the small intestine to induce POI in C57BL/6J mice. Sodium nitrite (48 nmol) was administered intravenously just before IM. Intestinal transit was assessed using fluorescent imaging. Bethanechol-stimulated jejunal circular muscle contractions were measured in organ baths. Inflammatory parameters, neutrophil infiltration, inducible NOS (iNOS) activity, reactive oxygen species (ROS) levels, mitochondrial complex I activity and cGMP were measured in the intestinal muscularis. Pre-treatment with nitrite markedly improved the delay in intestinal transit and restored the reduced intestinal contractility observed 24 h following IM. This was accompanied by reduced protein levels of TNF-α, IL-6 and the chemokine CCL2, along with reduced iNOS activity and ROS levels. The associated neutrophil influx at 24 h was not influenced by nitrite. IM reduced mitochondrial complex I activity and cGMP levels; treatment with nitrite increased cGMP levels. Pre-treatment with the NO scavenger carboxy-PTIO or the soluble guanylyl cyclase inhibitor ODQ abolished nitrite-induced protective effects. Exogenous nitrite deserves further investigation as a possible treatment for POI. Nitrite-induced protection of POI in mice was dependent on NO and this effect was not related to inhibition of mitochondrial complex I, but did involve activation of soluble guanylyl cyclase.

Evidence that NO/cGMP/PKG signalling cascade mediates endothelium dependent inhibition of IP3R mediated Ca2+ oscillations in myocytes and pericytes of ureteric microvascular network in situ

In ureteric microvessels the antagonistic relationship between Ca2+ signalling in endothelium and Ca2+ oscillations in myocytes and pericytes of arterioles and venules involves nitric oxide (NO), but the underlying mechanisms are not well understood. In the present study we investigated the effects of carbachol and NO donor SNAP on Ca2+ signalling and vasomotor responses of arterioles and venules in intact urteric microvascular network in situ using confocal microscopy. Vasomotor responses of arterioles and venules induced by AVP correlated with the occurrence of Ca2+ oscillations in the myocytes and pericytes and were not abolished by the removal of Ca2+ from extracellular fluid. Carbachol-induced rise of intracellular Ca2+ in endothelium was accompanied by the termination of the Ca2+ oscillations in myocytes and pericytes. This carbachol-induced inhibitory effect on Ca2+ oscillations in myocytes and pericytes was reversed by ODQ, an inhibitor of soluble guanylyl cyclase (sGC) and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Ca2+ oscillations in myocytes and pericytes were also effectively blocked by NO donor SNAP. An Inhibitory effect of SNAP was markedly enhanced by zaprinast, a selective inhibitor of cGMP-specific phosphodiesterase-5, and reversed by sGC inhibitor, ODQ and PKG inhibitor, Rp-8-pCPT-cGMPS. The cGMP analogue and selective PKG activator 8pCPT-cGMP also induced inhibition of the AVP-induced Ca2+ oscillations in myocytes and pericytes. SNAP had no effects on Ca2+ oscillations induced by caffeine in distributing arcade arterioles. Consequently, we conclude that NO- mediated inhibition of Ca2+ oscillations in myocytes and pericytes predominantly recruits the cGMP/PKG dependent pathway. The inhibitory effect of NO/cGMP/PKG cascade is associated with suppressed Ca2+ release from the SR of myocytes and pericytes selectively via the inositol triphosphate receptor (IP3R) channels.

A Novel Arginase Inhibitor Derived from Scutellavia indica Restored Endothelial Function in ApoE-Null Mice Fed a High-Cholesterol Diet.

Elevated endothelial arginase activity decreases nitric oxide (NO) production by competing with the substrate l-arginine, previously reported, and reciprocally regulating endothelial nitric oxide synthase (eNOS) activity. Thus, arginase inhibitors may help treat vascular diseases associated with endothelial dysfunction. A screening of metabolites from medicinal plants revealed that (2S)-5,2',5'-trihydroxy-7,8-dimethoxy flavanone (TDF) was a noncompetitive inhibitor of arginase. We investigated whether TDF reciprocally regulated endothelial NO production and its possible mechanism. TDF noncompetitively inhibited arginase I and II activity in a dose-dependent manner. TDF incubation decreased arginase activity and increased NO production in human umbilical vein endothelial cells and isolated mouse aortic vessels and reduced reactive oxygen species (ROS) generation in the endothelium of the latter. These TDF-mediated effects were associated with increased eNOS phosphorylation and dimerization but not with changes in protein content. Endothelium-dependent vasorelaxant responses to acetylcholine (Ach) were significantly increased in TDF-incubated aortic rings and attenuated by incubation with soluble guanylyl cyclase inhibitor. Phenylephrine-induced vasoconstrictor responses were markedly attenuated in TDF-treated vessels from wild-type mice. In atherogenic-prone ApoE(-/-) mice, TDF attenuated the high-cholesterol diet (HCD)-induced increase in arginase activity, which was accompanied by restoration of NO production and reduction of ROS generation. TDF incubation induced eNOS dimerization and phosphorylation at Ser1177. In addition, TDF improved Ach-dependent vasorelaxation responses and attenuated U46619-dependent contractile responses but did not change sodium nitroprusside-induced vasorelaxation or N-NAME-induced vasoconstriction. The findings suggest that TDF may help treat cardiovascular diseases by reducing pathophysiology derived from HCD-mediated endothelial dysfunction.

Physiological recycling of endogenous nitrate by oral bacteria regulates gastric mucus thickness

BackgroundInorganic nitrate from exogenous and endogenous sources is accumulated in saliva, reduced to nitrite by oral bacteria and further converted to nitric oxide (NO) and other bioactive nitrogen oxides in the acidic gastric lumen. To further explore the role of oral microbiota in this process we examined the gastric mucus layer in germ free (GF) and conventional mice given different doses of nitrate and nitrite. MethodsMice were given either nitrate (100mg/kg/d) or nitrite (0.55–11mg/kg/d) in the drinking water for 7 days, with the lowest nitrite dose resembling the levels provided by swallowing of fasting saliva. The gastric mucus layer was measured in vivo. ResultsGF animals were almost devoid of the firmly adherent mucus layer compared to conventional mice. Dietary nitrate increased the mucus thickness in conventional animals but had no effect in GF mice. In contrast, nitrite at all doses, restored the mucus thickness in GF mice to the same levels as in conventional animals. The nitrite-mediated increase in gastric mucus thickness was not inhibited by the soluble guanylyl cyclase inhibitor ODQ. Mice treated with antibiotics had significantly thinner mucus than controls. Additional studies on mucin gene expression demonstrated down regulation of Muc5ac and Muc6 in germ free mice after nitrite treatment. ConclusionOral bacteria remotely modulate gastric mucus generation via bioactivation of salivary nitrate. In the absence of a dietary nitrate intake, salivary nitrate originates mainly from NO synthase. Thus, oxidized NO from the endothelium and elsewhere is recycled to regulate gastric mucus homeostasis.

Endothelium-Dependent Relaxation Effect of Apocynum venetum Leaf Extract via Src/PI3K/Akt Signalling Pathway.

Botanical herbs are consumed globally not only as an essential diet but also as medicines or as functional/recreational food supplements. The extract of the Apocynum venetum leaves (AVLE), also known as Luobuma, exerts its antihypertensive effect via dilating the blood vessels in an endothelium- and concentration-dependent manner with optimal effect seen at as low as 10 µg/mL. A commercial Luoboma “antihypertensive tea” is available commercially in the western province of China. The present study seeks to investigate the underlying cellular mechanisms of the nitric oxide (NO)-releasing property of AVLE in rat aortas and human umbilical vein endothelial cells (HUVECs). Endothelium-dependent relaxation induced by AVLE was assessed in organ chambers in the presence or absence of polyethyleneglycol catalase (PP2, 20 µM; inhibitor of Src kinase), wortmannin (30 nM) and LY294002 (20 µM; PI3 (phosphatidylinositol3)-Kinase inhibitor), NG-nitro-l-arginine (L-NAME, 100 µM; endothelial NO synthase inhibitor (eNOS)) and ODQ (1 µM; soluble guanylyl cyclase inhibitor). Total nitrite and nitrate (NOx) level and protein expression of p-Akt and p-eNOS were measured. AVLE-induced endothelium-dependent relaxation was reduced by PP2, wortmannin and LY294002 and abolished by L-NAME and ODQ. AVLE significantly increased total NOx level in rat aortas and in HUVECs compared to control. It also instigated phosphorylation of Akt and eNOS in cultured HUVECs in a concentration-dependent manner and this was markedly suppressed by PP2, wortmannin and LY294002. AVLE also inhibited superoxide generated from both NADPH oxidase and xanthine/xanthine oxidase system. Taken together, AVLE causes endothelium-dependent NO mediated relaxations of rat aortas through Src/PI3K/Akt dependent NO signalling pathway and possesses superoxide scavenging activity.