Presence Of ODQ Research Articles

Novel soluble guanylyl cyclase activators increase glomerular cGMP, induce vasodilation and improve blood flow in the murine kidney.

Background and PurposeGeneration of cGMP via NO‐sensitive soluble guanylyl cyclase (sGC) has been implicated in the regulation of renal functions. Chronic kidney disease (CKD) is associated with decreased NO bioavailability, increased oxidative stress and oxidation of sGC to its haem‐free form, apo‐sGC. Apo‐sGC cannot be activated by NO, resulting in impaired cGMP signalling that is associated with chronic kidney disease progression. We hypothesised that sGC activators, which activate apo‐sGC independently of NO, increase renal cGMP production under conditions of oxidative stress, thereby improving renal blood flow (RBF) and kidney function.Experimental ApproachTwo novel sGC activators, runcaciguat and BAY‐543, were tested on murine kidney. We measured cGMP levels in real time in kidney slices of cGMP sensor mice, vasodilation of pre‐constricted glomerular arterioles and RBF in isolated perfused kidneys. Experiments were performed at baseline conditions, under L‐NAME‐induced NO deficiency, and in the presence of oxidative stress induced by ODQ.Key ResultsMouse glomeruli showed NO‐induced cGMP increases. Under baseline conditions, sGC activator did not alter glomerular cGMP concentration or NO‐induced cGMP generation. In the presence of ODQ, NO‐induced glomerular cGMP signals were markedly reduced, whereas sGC activator induced strong cGMP increases. L‐NAME and ODQ pretreated isolated glomerular arterioles were strongly dilated by sGC activator. sGC activator also increased cGMP and RBF in ODQ‐perfused kidneys.Conclusion and ImplicationsGC activators increase glomerular cGMP, dilate glomerular arterioles and improve RBF under disease‐relevant oxidative stress conditions. Therefore, sGC activators represent a promising class of drugs for chronic kidney disease treatment.LINKED ARTICLESThis article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc

Downregulation of Soluble Guanylate Cyclase and Protein Kinase G With Upregulated ROCK2 in the Pulmonary Artery Leads to Thromboxane A2 Sensitization in Monocrotaline-Induced Pulmonary Hypertensive Rats.

Thromboxane A2 (TXA2) promotes various physiological responses including pulmonary artery (PA) contraction, and pathophysiological implications have been suggested in cardiovascular diseases including pulmonary hypertension. Here, we investigated the role of TXA2 receptor (TP)-mediated signaling in the pathophysiology of pulmonary arterial hypertension (PAH). The sensitivity of PA to the contractile agonist could be set by relaxing signals such as the nitric oxide (NO), soluble guanylate cyclase (sGC), and cGMP-dependent kinase (PKG) pathways. Changes in the TP agonist (U46619)-induced PA contraction and its modulation by NO/cGMP signaling were analyzed in a monocrotaline-induced PAH rat model (PAH-MCT). In the myograph study, PA from PAH-MCT showed higher responsiveness to U46619, that is decreased EC50. Immunoblot analysis revealed a lower expression of eNOS, sGC, and PKG, while there was a higher expression of RhoA-dependent kinase 2 (ROCK2) in the PA from PAH-MCT than in the control. In PAH-MCT, the higher sensitivity to U46619 was reversed by 8-Br-cGMP, a membrane-permeable cGMP analog, but not by the NO donor, sodium nitroprusside (SNP 30 μM). In contrast, in the control PA, inhibition of sGC by its inhibitor (1H− [1,2,4] oxadiazolo [4,3−a] quinoxalin-1-one (ODQ), 10 μM) lowered the threshold of U46619-induced contraction. In the presence of ODQ, SNP treatment had no effect whereas the addition of 8-Br-cGMP lowered the sensitivity to U46619. The inhibition of ROCK by Y-27632 attenuated the sensitivity to U46619 in both control and PAH-MCT. The study suggests that the attenuation of NO/cGMP signaling and the upregulation of ROCK2 increase the sensitivity to TXA2 in the PAH animal, which might have pathophysiological implications in patients with PAH.

Aglycone-rich extracts from citrus by-products induced endothelium-independent relaxation in isolated arteries

Abstract Citrus by-products are rich in flavanone aglycons which have lower bioactivity than aglycone forms. Based on this, the study evaluated the effects of enzyme biotransformation on antioxidant and vasorelaxant capacity of polyphenolic rich-extracts from citrus waste. Enzymatic treatment produced extracts with high hesperetin concentrations and higher antioxidant capacity than non-treated extracts. At the highest concentration tested (3 mg mL−1), citrus juice by-products exhibited only a partial vasorelaxant effect. After the enzymatic treatment, the extract had a maximal effect of 100% relaxation and enhanced their pEC50. The relaxation promoted by citrus pectin by-products was potentiated in the presence of ODQ, suggesting that vasorelaxant effects of these extracts may occur even when sGC is oxidized because of vascular oxidative stress. Thus, the enzyme-assisted extraction appears to enhance antioxidant and vasorelaxant properties of citrus extracts, which could be used to develop new products with antihypertensive properties.

Nitrite may serve as a combination partner and a biomarker for the anti-cancer activity of RRx-001.

RRx-001 is an anti-cancer immunotherapeutic that increases the sensitivity of drug resistant tumors via multiple mechanisms which involve binding to hemoglobin and enhancing nitrite reductase activity of deoxyhemoglobin. In the present study, the effect of clinically used doses of RRx-001 on erythrocyte deformability was examined. A dose dependent effect of RRx-001 (1-1000 micro molar) on erythrocyte deformability was measured by ektacytometer under hypoxia (n =8). Low dose RRx-001 (20 micro molar) in the presence of ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one), L-NAME (L-NG-Nitroarginine methyl ester) or nitrite were examined both in normoxia and hypoxia. Intracellular nitric oxide (NO) levels were measured fluorometrically with DAF-FM-DA. Higher doses of RRx-001 (100, 1000 micro molar) significantly decreased erythrocyte deformability under hypoxia (p <0.01; p <0.05, respectively). RRx-001 (20 micro molar), alone or in combination with ODQ or L-NAME, did not change deformability. However, RRx-001 and nitrite caused an increase in deformability (p <0.01) under hypoxia. RRx-001 induced NO production was more pronounced in the presence of nitrite (p <0.05). Co-administration of RRx-001 and nitrite under hypoxic conditions results in a significant increase in erythrocyte deformability that is related to increased NO production. We suggest that measurement of serum nitrite level in RRx-001 treated cancer patients should be routinely undertaken and supplemented if levels are low for maximal activity.

Kaempferol-induces vasorelaxation via endothelium-independent pathways in rat isolated pulmonary artery.

Kaempferol, a flavonoid, is the essential part of human diet. Flavonoids have different pharmacological activities like cardioprotective, anti-inflammatory and anti-oxidant. The aim of current study was to investigate vasorelaxant potential of kaempferol on rat isolated pulmonary artery and to assess the underling mechanisms. Tension experiments were conducted on both the branches of main pulmonary artery of rats. Experiments were done using isolated organ bath system by recording tension with the help of data acquisition system, Power Lab. Kaempferol (10-8-10-4.5M) caused concentration-dependent relaxation (Emax 124.33±4.37%; pD2 5.03±0.084) of endothelium-intact pulmonary artery. In endothelium-denuded arterial rings, relaxation produced by kaempferol was not different from intact artery. L-NAME, indomethacin, combination of L-NAME and indomethacin did not show any effect on kaempferol-induced relaxation. Kaempferol-induced relaxation was reduced (Emax 55.53±7.72%) in 60mMK+ pre-contracted pulmonary arterial rings. Iberiotoxin significantly decreased (Emax 71.68±11.84%) the relaxation response. However, glibenclamide, BaCl2, 4-AP (1mM) and ICI182780 did not reduce the kaempferol-induced relaxation. TEA (10mM) and 4-AP (5mM) significantly reduced relaxation. Kaempferol-induced relaxation was significantly attenuated (Emax 94.92±19.60%) in presence of ODQ. H89 significantly decreased (Emax, 98.38±8.55%) the kaempferol-induced relaxation in rat pulmonary arterial rings. HC067047 and apamin did not show any effect on kaempferol-induced relaxation. In endothelium-denuded K+ (80mM)-depolarized arterial rings, kaempferol (10μM) markedly reduced CaCl2-induced contractions (Emax 35.14±6.53% vs. control 69.04±15.19%). Kaempferol relaxes rat pulmonary artery in endothelium-independent manner through involvement of BKCa channel, sGC, PKA pathways and inhibition of Ca2+-influx through L-type calcium channels.

Relaxant effect of Lippia origanoides essential oil in guinea-pig trachea smooth muscle involves potassium channels and soluble guanylyl cyclase

Ethnopharmacological relevanceLippia origanoides H.B.K. is an aromatic species used in folk medicine to treat respiratory diseases, including asthma. Aim of the studyThe aim of this work was to evaluate the relaxing potential and mechanism of action of the L. origanoides (LOO) essential oil in isolated guinea-pig trachea. Materials and methodsLeaves from L. origanoides were collected at experimental fields under organic cultivation, at the Forest Garden of Universidade Estadual de Feira de Santana. Essential oil was extracted by hydrodistillation, analyzed by GC/FID and GC/MS and the volatile constituents were identified. Spasmolytic activity and relaxant mechanism of LOO were assayed in isolated guinea-pig trachea contracted with histamine, carbachol or hyperpolarizing KCl. ResultsChemical analysis revealed the presence of carvacrol (53.89%) as major constituent. LOO relaxed isolated guinea-pig trachea pre-contracted with KCl 60 mM [EC50 = 30.02 μg/mL], histamine 1 µM [EC50 = 9.28 μg/mL] or carbachol 1 µM [EC50 = 51.80 μg/mL]. The pre-incubation of glibenclamide, CsCl, propranolol, indomethacin, hexamethonium, aminophylline or L-NAME in histamine-induced contractions did not alter significantly the relaxant effect of LOO. However, the presence of 4-aminopyridine, tetraethylammonium or methylene blue reduced LOO effect, while the presence of dexamethasone or atropine potentialized the LOO relaxant effect. LOO pre-incubation inhibited carbachol-evoked contractions, with this effect potentialized in the presence of sodium nitroprusside and blocked in the presence of ODQ. ConclusionsThe relaxant mechanism of LOO on the tracheal smooth muscle possibly involves stimulating of soluble guanylyl cyclase with consequent activation of the voltage-gated and Ca2+-activated K+ channels.

Risk assessment of bis-ethylhexyloxyphenol methoxyphenyl triazine in cosmetic products

Animal toxins are natural resources for pharmacological studies. The venom of Crotalus durissus cascavella (C.d. cascavella) may be a source in the bio-prospecting of new anti-hypertensive agents. The aim of this study was to investigate vascular effects of the venom of C.d. cascavella in normotensive rats. Studies were performed using isolated mesenteric artery segments and aortic endothelial cells. The cumulative administration of the venom of C.d. cascavella (0.001–30 μg/mL) on phenylephrine (Phe; 10 μM) pre-contracted rings induced a concentration-dependent vasorelaxation in the presence of vascular endothelium (Emax = 47.9 ± 5.0% n = 8), and its effect was almost abolished in the absence of endothelium (Emax = 5.8± 2.4% n = 5 (∗∗∗p < 0.001)). Tissue viability was maintained as there was no difference in the contractile capacity of rings before and after the administration of venom. The vasorelaxant effect of the venom was also abolished when arteries were pre-contracted with potassium chloride (KCl; 80 mM) (Emax = 6.4± 0.9% n = 5, ∗∗∗p < 0.001). When assessing the participation of endothelium-derived relaxing factors, it was noted that non-selective COX inhibition with indomethacin (10 μM) caused a significant reduction in the vasorelaxant effect of C.d. cascavella (*p < 0.05). When investigating the participation of NO released by endothelium, there was a significant reduction of the vasorelaxant effect of venom in rings treated with L-NAME (100 μM; Emax = 17.5± 2.2% n = 6; **p < 0.01). Similar results were noted in the presence of ODQ (10 μM), an inhibitor of soluble guanylyl cyclase (Emax = 11.2± 3.5%, n = 6) and PTIO (100 μM), a stable radical scavenger for nitric oxide (Emax = 10.77± 3.6%, n = 6). Moreover, the venom induced the release of NO by isolated aortic endothelial cells through amperometric studies. When assessing the participation of K+ channels on the vasodilatory response of the venom, tyrode solution with 20 mM of KCl caused a significant reduction in the relaxation response (p < 0.001) (Emax = 21.3 ± 8%, n = 7), as did inhibitor of delayed rectifier K+ channels (4-amynopiridine 1 mM; Emax = 9.5 ± 1.3, %, n = 5, ***p < 0.001), and vasorelaxation was almost abolished in the presence of Iberiotoxin (IbTx 100 nM). Therefore, these results suggest that the venom of C.d. cascavella induces vasorelaxation in superior mesenteric artery rings of normotensive rats in an endothelium-dependent manner. Specifically, the venom stimulates the generation of endothelium-derived relaxing factors, especially NO, and activates vascular smooth muscle hyperpolarization through K+ channels. These data illustrate that C.d. cascavella is a source of bioactive molecules and therefore has therapeutic potential in the treatment of cardiovascular diseases such as hypertension.

NO production and potassium channels activation induced by Crotalus durissus cascavella underlie mesenteric artery relaxation

Animal toxins are natural resources for pharmacological studies. The venom of Crotalus durissus cascavella (C.d. cascavella) may be a source in the bio-prospecting of new anti-hypertensive agents. The aim of this study was to investigate vascular effects of the venom of C.d. cascavella in normotensive rats. Studies were performed using isolated mesenteric artery segments and aortic endothelial cells. The cumulative administration of the venom of C.d. cascavella (0.001–30 μg/mL) on phenylephrine (Phe; 10 μM) pre-contracted rings induced a concentration-dependent vasorelaxation in the presence of vascular endothelium (Emax = 47.9 ± 5.0% n = 8), and its effect was almost abolished in the absence of endothelium (Emax = 5.8± 2.4% n = 5 (∗∗∗p < 0.001)). Tissue viability was maintained as there was no difference in the contractile capacity of rings before and after the administration of venom. The vasorelaxant effect of the venom was also abolished when arteries were pre-contracted with potassium chloride (KCl; 80 mM) (Emax = 6.4± 0.9% n = 5, ∗∗∗p < 0.001). When assessing the participation of endothelium-derived relaxing factors, it was noted that non-selective COX inhibition with indomethacin (10 μM) caused a significant reduction in the vasorelaxant effect of C.d. cascavella (*p < 0.05). When investigating the participation of NO released by endothelium, there was a significant reduction of the vasorelaxant effect of venom in rings treated with L-NAME (100 μM; Emax = 17.5± 2.2% n = 6; **p < 0.01). Similar results were noted in the presence of ODQ (10 μM), an inhibitor of soluble guanylyl cyclase (Emax = 11.2± 3.5%, n = 6) and PTIO (100 μM), a stable radical scavenger for nitric oxide (Emax = 10.77± 3.6%, n = 6). Moreover, the venom induced the release of NO by isolated aortic endothelial cells through amperometric studies. When assessing the participation of K+ channels on the vasodilatory response of the venom, tyrode solution with 20 mM of KCl caused a significant reduction in the relaxation response (p < 0.001) (Emax = 21.3 ± 8%, n = 7), as did inhibitor of delayed rectifier K+ channels (4-amynopiridine 1 mM; Emax = 9.5 ± 1.3, %, n = 5, ***p < 0.001), and vasorelaxation was almost abolished in the presence of Iberiotoxin (IbTx 100 nM). Therefore, these results suggest that the venom of C.d. cascavella induces vasorelaxation in superior mesenteric artery rings of normotensive rats in an endothelium-dependent manner. Specifically, the venom stimulates the generation of endothelium-derived relaxing factors, especially NO, and activates vascular smooth muscle hyperpolarization through K+ channels. These data illustrate that C.d. cascavella is a source of bioactive molecules and therefore has therapeutic potential in the treatment of cardiovascular diseases such as hypertension.

S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism

S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca2+ channels. Western immunoblots showed a larger L-type Ca2+/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca2+/sGC ratio (p = 0.047 and r = −0.906 for correlation between Emax and L-Ca2+/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

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)

Pharmacological characterisation of the relaxation induced by the soluble guanylate cyclase activator, BAY 60-2770 in rabbit corpus cavernosum.

To characterise the relaxation induced by the soluble guanylate cyclase (sGC) activator, BAY 60-2770 (4-({(4-carboxybutyl) [2- (5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl]methoxy}phenyl)ethyl] amino}methyl)benzoic acid) in rabbit corpus cavernosum (CC). The penis from male New Zealand rabbits was removed and fours strips of CC were obtained. Concentration-response curves to BAY 60-2770 were constructed in the absence and presence of inhibitors of nitric oxide synthase, N (G)-nitro-L- arginine methyl ester (L-NAME, 100 μm), sGC, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μm) and phosphodiesterase type 5 (PDE-5), tadalafil (0.1 μm). The potency (pEC50 ) and maximal response (Emax ) values were determined. Then, electrical-field stimulation (EFS)-induced contraction or relaxation was tested in the absence and presence of BAY 60-2770 (0.1 or 1 μm) alone or combined with ODQ (10 μm). For EFS-induced relaxation two protocols were used: (i) ODQ (10 μm) was first incubated for 20 min and then BAY 60-2770 (1 μm) was added for another 20 min (ODQ + BAY 60-2770); (ii) in different CC strips, BAY 60-2770 was incubated for 20 min followed by another 20 min with ODQ (BAY 60-2770 + ODQ). The intracellular levels of cyclic guanosine monophosphate (cGMP) were also determined. BAY 60-2770 potently relaxed rabbit CC with mean (sem) pEC50 and Emax values of 7.58 (0.19) and 81 (4)%, respectively. The inhibitors ODQ (n = 7) or tadalafil (n = 7) produced 4.2- and 6.3-leftward shifts, respectively in BAY 60-2770-induced relaxation without interfering with the Emax values. The intracellular levels of cGMP were augmented after stimulation with BAY 60-2770 (1 μm) alone, whereas its co-incubation with ODQ produced even higher levels of cGMP. The EFS-induced contraction was reduced in the presence of BAY 60-2770 (1 μm) and this inhibition was even greater when BAY 60-2770 was co-incubated with ODQ. The nitrergic stimulation induced CC relaxation, which was abolished in the presence of ODQ. BAY 60-2770 alone increased the amplitude of relaxation. Co-incubation of ODQ and BAY 60-2770 did not alter the relaxation in comparison with ODQ alone. Interestingly, when BAY 60-2770 was incubated before ODQ, EFS-induced relaxation was partly restored in comparison with ODQ alone or ODQ + BAY 60-2770. The relaxation induced by the sGC activator, BAY 60-2770 was increased after sGC oxidation and unaltered in the absence of nitric oxide. Thus, this class of substances may have advantages over sGC stimulators or PDE-5 inhibitors for treating patients with erectile dysfunction and extensive endothelial damage.

Lack of effect of ODQ does not exclude cGMP signalling via NO‐sensitive guanylyl cyclase

Nitric oxide (NO) is known to activate NO-sensitive guanylyl cyclase (NO-GC) and to elicit cGMP production. However, NO has also been proposed to induce cGMP-independent effects. It is accepted practice to use specific NO-GC inhibitors, such as ODQ or NS2028, to assess cGMP-dependent NO effects. Consequently, NO-induced reactions seen in the presence of these inhibitors commonly serve as an affirmation of cGMP independence. We evaluated the use of ODQ to discriminate between cGMP-dependent and cGMP-independent NO effects. NO-GC-expressing HEK cells, platelets and tissues from wild type (WT) and NO-GC-deficient mice (GCKO) were used. NO donors led to accumulation of cGMP in platelets and GC-expressing HEK cells and induced phosphorylation of the vasodilator-stimulated phosphoprotein in platelets; both effects were reduced by ODQ. High concentrations of NO donors, however, overrode the inhibitory effect of ODQ. Correspondingly, ODQ inhibited but did not fully eliminate NO-induced relaxation of aorta and fundus from WT mice. Relaxation induced by endogenously released NO was fully or partially inhibited by ODQ in fundus and aorta, respectively. In aorta and fundus of GCKO mice NO-induced relaxation was absent and served as standard for complete NO-GC inhibition. High NO concentrations can overcome the inhibitory effect of ODQ on NO-GC. Smooth muscle relaxation induced by NO donors/endogenously released NO in the presence of ODQ in WT was absent in GCKO animals indicating involvement of NO-GC. Accordingly, NO-induced effects in the presence of ODQ do not necessarily prove cGMP independence.

Imidazole-induced contractility of vascular smooth muscle cells in the presence of U-73122, ODQ, indomethacin and 7-nitroindazole

The aim of the study was to assess the impact of modulating factors on vascular smooth muscle cells reactivity. Vascular resistance was induced by the administration of increasing concentrations of imidazole. The experiments were performed on isolated and perfused tail artery of Wistar rats (weight 250 g - 350 g). Rats were been narcotized by urethane (intraperitoneal injection) at a dose of 120 mg/kg, stunned and then sacrificed by cervical dislocation. In the following investigation classical pharmacometric methods were used. Relationships between concentration-response curves (CRCs) for imidazole observed in the presence of ODQ [(1H-(1,2,4)oxadiazolo-[4,3-a]quinoxalin-1-one)], 7-nitroindazole and indomethacin were analyzed. Imidazole-induced contractility of vascular smooth muscle cells was independent from alpha-adrenergic receptors and PLC activity. Reactivity of VSMCsinduced by imidazole, was significantly changed in the presence of ODQ and 7-nitroindazole.

Investigation of the role of the NO–cGMP pathway on YC-1 and DEA/NO effects on thoracic aorta smooth muscle responses in a rat preeclampsia model

The present study was designed to investigate the effects of YC-1, a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO, a NO donor, on smooth muscle responses in the preeclampsia model with suramin-treated rats and on the levels of cyclic guanosine monophosphate (cGMP) of thoracic aorta rings isolated from term-pregnant rats. Rats of 2 groups, control group and suramin group, were given intraperitoneal injection of saline or suramin, respectively. Suramin injection caused increased blood pressure, protein in urine, and fetal growth retardation. Thoracic aorta rings were exposed to contractile and relaxant agents. KCl contraction and papaverine relaxation responses were similar. Relaxation responses of YC-1 and DEA/NO decreased in suramin group. In both groups in the presence of ODQ, a sGC inhibitor, the relaxation responses of YC-1 and DEA/NO decreased. The cGMP content was determined by radioimmunoassay technique. The content of cGMP in the suramin group decreased. In the presence of YC-1 and DEA/NO in both groups, cGMP content increased, but in ODQ-added groups, there was a significant decrease. We conclude that in preeclampsia, the decrease of relaxation responses and the decrease of cGMP content could be due to the reduction in stimulation of sGC and the decrease in cGMP levels.

The role of cGMP‐dependent nitric oxide signalling on cardiac repolarisation

Recently, roles for nitric oxide (NO) signalling pathways have been linked with regulation of cardiac repolarisation and protection from arrhythmias. In this study, cGMP‐dependent regulation of repolarisation was investigated using BAY 60–2770, a novel NO and haem‐independent soluble guanylyl cyclase (sGC) activator. Action potentials (AP) were recorded from isolated guinea‐pig ventricular myocytes using the perforated‐patch clamp technique. Cellular cGMP was quantified by radioimmunoassay. 1 μM BAY 60–2770 increased cellular cGMP by 2.1 ± 0.2 (n=5) fold on its own, by 7.9 ± 0.7 (n=4) fold in the presence of 100 μM IBMX a non‐selective phosphodiesterase (PDE) inhibitor, and by 38.7 ± 0.2 (n=2) fold in the presence of ODQ (which strongly potentiates the action of BAY 60–2770 on sGC). Despite substantial increases in cellular cGMP, changes in AP duration were modest. BAY 60–2770 shortened AP duration from 223.3 ± 8.1 ms in basal conditions to 213.0 ± 7.4 ms (n=6, P&lt;0.001) ms and ODQ + BAY 60–2770 caused no further shortening. Importantly, when cGMP hydrolysis was blocked by IBMX, BAY 60–2770 prolonged APs by 19.5 ± 5.6 ms (p &lt; 0.01, n = 8). Overall, these findings demonstrate the complex interplay between cGMP and cAMP mediated effects on cardiac repolarisation. Our results further demonstrate that PDEs limit cGMP accumulation close to ion channels in the sarcolemma, thus compartmentalising cGMP signalling.KEB is supported by a British Heart Foundation Fellowship and REC by a University of Leicester PhD studentship

Vascular relaxation induced by NO donor in mesenteric resistance artery from 2K‐1C rats involves reactive oxygen species and potassium channels activation

This study aimed to evaluate the effect of the inhibition of soluble guanylyl‐cyclase (sGC) by ODQ, K+ channels by TEA and ROS production by apocynin on the relaxation to the NO donor RuBPY in mesenteric resistance artery from normotensive (2K) and 2K‐1C rats. Concentration‐effect curves to RuBPY were constructed in isolated denuded mesenteric arteries from 2K and 2K‐1C rats contracted with phenylephrine, in the absence or in the presence of ODQ, TEA, or Apocynin. The maximum relaxation induced by RuBPY was similar in 2K (77.6±8.9%, n=7) and 2K‐1C (76.9±4.5%,n=13), that was inhibited by ODQ in 2K (36.5±2.9%,n=5) and 2K‐1C arteries (24.6±5.5%, n=5). The relaxation induced by RuBPY was reduced by TEA (50.5±10.6%, n =8) and apocynin (45.7±3.9%,n=4) only in 2K‐1C arteries. These results indicate that although RuBPY induces relaxation with similar efficacy in mesenteric arteries from normotensive and hypertensive rats and that the NO released from RuBPY activates sGC, the NO signaling can differ. It involves ROS production and K+ channels activation only in 2K‐1C arteries. Supported by FAPESP and CNPq.

Cytosolic Ca2+–Induced Apoptosis in Rat Cardiomyocytes via Mitochondrial NO-cGMP-Protein Kinase G Pathway

Previously, we showed that in adult rat cardiomyocytes, nitric oxide (NO) donors stimulate mitochondrial cGMP production, followed by cytochrome c release, independently of the mitochondrial permeable transition pore. We investigated whether mitochondrial cGMP-induced cytochrome c release from cardiac mitochondria is Ca(2+)-sensitive. Mitochondria and primary cultured cardiomyocytes were prepared from left ventricles of male Wistar rats. The cytosolic Ca(2+) concentration was adjusted with Ca(2+)-EGTA buffers. Cytochrome c released from mitochondria was measured by Western blotting. Cardiomyocyte apoptosis was assessed by Annexin V staining. Cytochrome c release from cardiac mitochondria was evoked by buffered Ca(2+) (1 μM); this was inhibited by NO-cGMP pathway inhibitors such as N(G)-monomethyl-l-arginine monoacetate (inhibitor of NO synthase), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NO scavenger), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, NO-sensitive guanylyl cyclase inhibitor) and voltage-dependent anion channel (VDAC) inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, but not by cyclosporin A (mitochondrial permeable transition pore inhibitor). Furthermore, this release was significantly and dose dependently inhibited by 0.3-3 μM KT5823 (protein kinase G inhibitor). At the cellular level, intracellular perfusion of cardiomyocytes with buffered Ca(2+) (1 μM) also induced apoptosis, which was inhibited in the presence of ODQ. A membrane-permeable cGMP analog, 8-Br-cGMP, but not cGMP itself, mimicked buffered Ca(2+) actions in both cardiac mitochondria and cardiomyocytes. We further confirmed an increase in protein kinase G activity by adding cGMP in mitochondrial protein fraction. Our results suggest that mitochondrial NO-cGMP pathway-induced cytochrome c release from cardiac mitochondria, triggered by increased cytosolic Ca(2+), occurs through VDAC via the stimulation of an undiscovered mitochondrial protein kinase G.

Melatonin inhibits NO‐dependent activation of large conductance, calcium‐activated K channels (BKCa) in porcine coronary artery smooth muscle cells

We recently demonstrated that melatonin inhibits NO‐induced relaxation of porcine coronary arteries by activating MT2 melatonin‐receptors (JPET 336: 127, 2011). We tested the hypothesis that melatonin inhibits NO/cGMP‐dependent activation of BKCa channels in coronary artery smooth muscle cells. Melatonin (10−7M) inhibited sodium nitroprusside (SNP)‐induced relaxation of isolated coronary arteries but had no effect on relaxation induced by 8‐Br‐cGMP, a stable, cell permeable cGMP analog. ODQ (10−5 M) completely inhibited SNP‐induced relaxation, as well as the increase in intracellular cGMP levels in response to SNP. In patch clamp studies, SNP (10−5 M)‐induced increases in BKCa currents were abolished in the presence of ODQ (10−5 M). Melatonin (10−7M) inhibited both the voltage‐dependent and SNP‐induced increase in BKCa currents, but had no effect on BKCa currents elicited by 8‐Br‐cGMP (10−3M). The inhibitory effect of melatonin on the voltage‐dependent and SNP‐induced increase in BKCa currents was abolished by either the PDE inhibitor, zaprinast (10−5M), or the MT2‐receptor antagonist, 4P‐PDOT (10−7 M). Coronary artery relaxation in response to NO involves the cGMP‐dependent activation of BKCa channels. Melatonin impairs NO‐induced relaxation of porcine coronary arteries by inhibiting the cGMP‐dependent activation of BKCa channels in coronary smooth muscle cells. (Supported by NIH HL77204)

Involvement of Guanylate Cyclase and K+ Channels in Relaxation Evoked by Ferulate Nitrate in Rat Aorta Artery

Vasorelaxant properties of N-2-(ferulamidoethyl)-nitrate (ferulate nitrate, FLNT), a newly synthesized nitrate, were compared with those of isosorbide dinitrate, nicorandil, nitroglycerin, and 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) in rat aorta pre-contracted by phenylephrine. FLNT produced vasorelaxation in a concentration-dependent manner (0.1 – 100 μM). The degree of relaxation induced by FLNT was similar to that induced by isosorbide dinitrate. In addition, removal of endothelium did not affect the relaxant effect of FLNT. FLNT caused a rightward shift of the cumulative concentration–response curves of phenylephrine and reduced the maximal efficacy of contraction. 1H-[1,2,4]Oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 μM) and K+-channel blockers charybdotoxin (CHT, 0.1 μM) and BaCl2 (1 μM) reduced the relaxant effect of FLNT in the endothelium-denuded arteries, whereas glibenclamide (1 μM) and 4-aminopyridine (1 mM) failed to influence FLNT-induced vasorelaxation. Furthermore, in the presence of ODQ, both CHT (0.1 μM) and BaCl2 (1 μM) still significantly reduced the relaxation evoked by FLNT. Pretreatment of vessels with hydroxocobalamin, a nitric oxide scavenger, abolished the FLNT effect. These findings demonstrate that FLNT induces relaxation of the rat aorta rings endothelium-independently. Furthermore, we demonstrated that FLNT-induced vasorelaxation is related to its stimulation of soluble guanylate cyclase and activation of K+ channels.

Nitric oxide activates hypoglossal motoneurons by cGMP-dependent inhibition of TASK channels and cGMP-independent activation of HCN channels

Nitric oxide (NO) is an important signaling molecule that regulates numerous physiological processes, including activity of respiratory motoneurons. However, molecular mechanism(s) underlying NO modulation of motoneurons remain obscure. Here, we used a combination of in vivo and in vitro recording techniques to examine NO modulation of motoneurons in the hypoglossal motor nucleus (HMN). Microperfusion of diethylamine (DEA; an NO donor) into the HMN of anesthetized adult rats increased genioglossus muscle activity. In the brain slice, whole cell current-clamp recordings from hypoglossal motoneurons showed that exposure to DEA depolarized membrane potential and increased responsiveness to depolarizing current injections. Under voltage-clamp conditions, we found that NO inhibited a Ba(2+)-sensitive background K(+) conductance and activated a Cs(+)-sensitive hyperpolarization-activated inward current (I(h)). When I(h) was blocked with Cs(+) or ZD-7288, the NO-sensitive K(+) conductance exhibited properties similar to TWIK-related acid-sensitive K(+) (TASK) channels, i.e., voltage independent, resistant to tetraethylammonium and 4-aminopyridine but inhibited by methanandamide. The soluble guanylyl cyclase blocker 1H-(1,2,4)oxadiazole(4,3-a)quinoxaline-1-one (ODQ) and the PKG blocker KT-5823 both decreased NO modulation of this TASK-like conductance. To characterize modulation of I(h) in relative isolation, we tested effects of NO in the presence of Ba(2+) to block TASK channels. Under these conditions, NO activated both the instantaneous (I(inst)) and time-dependent (I(ss)) components of I(h). Interestingly, at more hyperpolarized potentials NO preferentially increased I(inst). The effects of NO on I(h) were retained in the presence of ODQ and blocked by the cysteine-specific oxidant N-ethylmaleimide. These results suggest that NO activates hypoglossal motoneurons by cGMP-dependent inhibition of a TASK-like current and S-nitrosylation-dependent activation of I(h).