sGC Inhibitor Research Articles

Guanylyl cyclase activation reverses resistive breathing-induced lung injury and inflammation.

Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. The soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is down-regulated in chronic and acute animal models of RB, such as asthma, chronic obstructive pulmonary disease, and in endotoxin-induced acute lung injury. Our objectives were to: (1) characterize the effects of increased concurrent inspiratory and expiratory resistance in mice via tracheal banding; and (2) investigate the contribution of the sGC/cGMP pathway in RB-induced lung injury. Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, marked leukocyte infiltration in the lungs, and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure-volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB down-regulated sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, 1H-[1,2,4]oxodiazolo[4,3-]quinoxalin-l-one, or when RB was performed using sGCα1 knockout mice. Conversely, restoration of sGC signaling by prior administration of the sGC activator BAY 58-2667 (Bayer, Leverkusen, Germany) prevented RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24 hours after RB reversed, within 6 hours, the established lung injury. These findings raise the possibility that pharmacological targeting of the sGC-cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases.

PS-340 Age-related Changes And Effects Of Mild Hypothermia On Carotid Artery Reactivity In Newborn Rats

Introduction Therapeutic hypothermia has become a standard neuroprotective treatment in term newborn infants following perinatal asphyxia. Hypothermia-induced changes in the reactivity of the vessels supplying the brain might play a role in its therapeutic or side effects. We investigated the putative age-related changes and the effect of clinically relevant cooling (33°C) on the reactivity of the newborn rat carotid artery. Methods Carotid artery rings from 2–3 d-old and 9–10 d-old rats were mounted in myographs and studied at 33 and 37°C. Results Hypothermia did not significantly affect the contractions induced by KCl and U46619, nor the relaxations induced by acetylcholine (ACh), the nitric oxide (NO) donor sodium nitroprusside (SNP), the NO-independent stimulator of soluble guanylate cyclase (sGC) BAY 41–2272, the β-adrenoceptor agonist isoproterenol, the adenylate cyclase activator forskolin, and acute hypoxia (PO 2 3 kPa). The relaxations induced by ACh, isoproterenol, the β 2 -adrenoceptor agonist salbutamol, the β 3 -adrenoceptor agonist CL-316243 and hypoxia increased with postnatal age and were impaired by endothelium removal or by inhibition of NO synthase (L-NAME) or sGC (ODQ). By contrast, the relaxations induced by SNP, BAY 41–2272 and forskolin were endothelium-independent and did not change with age. Conclusions Mild hypothermia (33°C) does not affect the reactivity of neonatal rat carotid arteries. Our data suggest a reduced NO bioavailability in the carotid artery during the first days of life. This transient reduction in endothelium-dependent relaxation might play a role in the adaptation of the circulatory system to birth and in the neonatal vascular response to insults such as hypoxia.

Involvement of H1 and H2 Receptors and Soluble Guanylate Cyclase in Histamine‐Induced Relaxation of Rat Mesenteric Collecting Lymphatics

This study investigated the roles of the H1 and H2 histamine receptors, NO synthase, and sGC cyclase in histamine-induced modulation of rat mesenteric collecting lymphatic pumping. Isolated rat mesenteric collecting lymphatics were treated with 1- to 100-μM histamine. Histamine receptors were blocked with either the H1 antagonist mepyramine or the H2 antagonist cimetidine. The role of NO/sGC signaling was tested using the arginine analog L-NAME, the sGC inhibitor ODQ, and SNP as a positive control. Histamine applied at 100 μM decreased tone and CF of isolated rat mesenteric collecting lymphatics. Pharmacologic blockade of either H1 or H2 histamine receptors significantly inhibited the response to histamine. Pretreatment with ODQ, but not L-NAME, completely inhibited the histamine-induced decrease in tone. ODQ pretreatment also significantly inhibited SNP-induced lymphatic relaxation. H1 and H2 histamine receptors are both involved in histamine-induced relaxation of rat mesenteric collecting lymphatics. NO synthesis does not appear to contribute to the histamine-induced response. However, sGC is critical for the histamine-induced decrease in tone and contributes to the drop in CF.

Influence of cinaciguat on gastrointestinal motility in apo-sGC mice.

Cinaciguat (BAY 58-2667), an NO- and heme-independent sGC activator, was shown to be more effective when the heme-group of sGC is oxidized in vascular tissue. In apo-sGC mice (sGCβ1 (His105Phe) knockin) both sGC isoforms (sGCα1 β1 and sGCα2 β1 ) are heme-deficient and can no longer be activated by NO; these mice, showing decreased gastrointestinal nitrergic relaxation and decreased gastric emptying, can be considered as a model to study the consequence of heme-oxidation in sGC. Our aim was to compare the influence of cinaciguat, on in vitro muscle tone of gastrointestinal tissues, and on gastric emptying in WT and apo-sGC mice. Gastrointestinal smooth muscle strips were mounted in organ baths for isometric force recording and cGMP levels were determined by enzyme immunoassay. Protein levels of sGC subunits were assessed by immunoblotting. Gastric emptying was determined by phenol red recovery. Although protein levels of the sGC subunits were lower in gastrointestinal tissues of apo-sGC mice, cinaciguat induced concentration-dependent relaxations and increased cGMP levels in apo-sGC fundus and colon to a similar or greater extent than in WT mice. The sGC inhibitor ODQ increased cinaciguat-induced relaxations and cGMP levels in WT fundus and colon. In apo-sGC antrum, pylorus and jejunum, cinaciguat was not able to induce relaxations. Cinaciguat did not improve delayed gastric emptying in apo-sGC mice. Cinaciguat relaxes the fundus and colon efficiently when sGC is in the heme-free condition; the non-effect of cinaciguat in pylorus explains its inability to improve the delayed gastric emptying in apo-sGC mice.

Nitric oxide improves the hemodynamic performance of the hypoxic goldfish (Carassius auratus) heart

Goldfish tolerate prolonged and severe hypoxia, thus representing a well-suited model to study the maintenance of cardiac function when O2 availability represents a limiting factor.Using a working heart preparation, we explored the role of the intracardiac nitric oxide synthase (NOS)-derived nitric oxide (NO) under normoxic and hypoxic conditions. Cardiac performance was examined both under basal (constant preload and afterload) and loading conditions, i.e. preload-induced increases in stroke volume (SV) and hence cardiac output at constant afterload (the Frank–Starling response).Hypoxic hearts showed an increased basal mechanical performance compared to the normoxic counterpart. Under basal conditions, in both normoxic and hypoxic hearts, NOS and soluble guanylyl cyclase (sGC) inhibition increased SV, while exogenous NO supply decreased it.The normoxic heart was very sensitive to filling pressure increases; the maximum SV = 1.08 ± 0.09 mL/kg body mass was obtained at 0.4 kPa. Acute hypoxia increased this sensitivity, SV reaching the maximum value (1.45 ± 0.12 mL/kg body mass) at 0.25 kPa. NOS inhibition by L-NMMA reduced the Frank–Starling response under normoxia, but was ineffective under acute hypoxia, where NO may come from nitrite reduction. In both conditions, sGC inhibition induced a reduction of the cardiac response to preload. Moreover, under acute hypoxia, NO scavenging significantly reduced the Frank–Starling response. The hypoxia-induced hemodynamic patterns were complemented by Western blotting analysis which revealed increased expressions of NOS and hypoxia inducible factor α(HIF-1α). In conclusion, we demonstrated that intracardiac NO/NOS enhances goldfish heart performance, remarkably expanding its hypoxic tolerance.

Icariin stimulates the osteogenic differentiation of rat bone marrow stromal cells via activating the PI3K–AKT–eNOS–NO–cGMP–PKG

Icariin, a prenylated flavonol glycoside isolated from Epimedii herba, has been found to be a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of icariin is related to the nitric oxide (NO) signal pathway and PI3K/AKT pathway in its upstream. Rat bone marrow stromal cells (rBMSCs) were cultured in osteogenic medium and treated with icariin or together with L-NAME, ODQ, PDE5, and/or LY294002 (the inhibitor of NOS, sGC, cGMP, and PI3K respectively), and effects were examined on the expression of signal messengers (NOS, NO, sGC, cGMP, PKG and PI3K) and the levels of osteogenic markers (alkaline phosphatase or ALP, osteocalcin and calcified nodules). It was found that icariin dose-dependently increased ALP activity, and treatment at the optimal concentration (10−5M) increased NOS activity, iNOS and eNOS expression, NO production, sGC and cGMP contents and PKG expression besides the phosphorylation of AKT. The addition of L-NAME, ODQ and PDE5 significantly inhibited the icariin effects on above markers respectively. The addition of LY294002 decreased the p-AKT level, NOS activity, eNOS expression and NO production significantly, but had no significant effect on iNOS expression. The addition of any of the four inhibitors also abolished the osteogenic effect of icariin on rBMSCs as indicated by ALP activity, osteocalcin synthesis, calcium deposition and the number and areas of calcified nodules. These results suggest that the osteogenic effect of icariin involves the PI3K–AKT–eNOS–NO–cGMP–PKG signal pathway. Furthermore, dosage response studies showed that icariin at 10−6M (a physiologically achievable concentration in vivo) also activated this signal pathway.

CIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries.

cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3',5'-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (Po2: 25-30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5'-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr(853), which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK.

Phenylephrine activates eNOS Ser1177 phosphorylation and nitric oxide signaling in renal hypertensive rat aorta

The endothelial nitric oxide synthase (eNOS) plays an important role in the control of the vascular tone. This work aimed to evaluate the role of an α1-adrenoceptor agonist phenylephrine (PE) on eNOS activity and downstream signaling pathway activation in normotensive (2K) and renal hypertensive (2K-1C) intact-endothelium rat aortas. Concentration-effect curves were performed for PE in intact-endothelium aortas from 2K and 2K-1C rats, in the absence of or in the presence of NOS or soluble guanylyl cyclase (sGC) inhibitor. Intact endothelium aortas were stimulated with PE in organ chambers and eNOS Ser1177/Thr495 phosphorylation expression was evaluated by western blot. Nitric Oxide (NO) production was evaluated in isolated endothelial cells from 2K and 2K-1C rat aortas by flow-cytometry using NO selective fluorescent probe, DAF-2DA. The sGC activity/expression was also evaluated. PE-induced contractile response is lower in 2K-1C than in 2K intact-endothelium rat aorta. This is due to higher eNOS Ser1177 phosphorylation in 2K-1C, which induces the eNOS overactivation. It was abolished by NOS or sGC inhibition. Phenylephrine reduces NO production in 2K as compared to the basal level, but it is not modified in 2K-1C. In PE-stimulated endothelial cells, the NO production is higher in 2K-1C than in 2K. Phenylephrine induces higher cGMP production in 2K-1C than in 2K, despite the lower expression of sGC in 2K-1C. Our results suggest that alpha1-adrenoceptor activation contributes to the increased activity of the enzyme eNOS by Ser1177 phosphorylation in 2K-1C intact-endothelium aorta, which consequently decreases PE-induced contractile response.

Regulation of soluble guanylate cyclase by matricellular thrombospondins: implications for blood flow.

Nitric oxide (NO) maintains cardiovascular health by activating soluble guanylate cyclase (sGC) to increase cellular cGMP levels. Cardiovascular disease is characterized by decreased NO-sGC-cGMP signaling. Pharmacological activators and stimulators of sGC are being actively pursued as therapies for acute heart failure and pulmonary hypertension. Here we review molecular mechanisms that modulate sGC activity while emphasizing a novel biochemical pathway in which binding of the matricellular protein thrombospondin-1 (TSP1) to the cell surface receptor CD47 causes inhibition of sGC. We discuss the therapeutic implications of this pathway for blood flow, tissue perfusion, and cell survival under physiologic and disease conditions.

The interplay between heme oxygenase and nitric oxide synthase and downstream PI3K/sGC/ERK signaling ameliorates the estrogen‐dependent depressant effect of chronic nicotine on reflex bradycardia (837.2)

Chronic nicotine impairs reflex bradycardia in estrogen (E2)‐replaced, but not depleted, female rats. We tested the hypothesis that nitric oxide synthase (NOS) and/or heme oxygenase (HO) and their downstream mitogen‐activated protein kinases mediate the nicotine‐baroreflex interaction. The depressant effect of nicotine (2 mg/kg/day i.p., 2 weeks) on reflex bradycardic responsiveness to phenylephrine in E2‐replaced ovariectomized (OVXE2) rats was abolished after treatment with hemin (HO inducer) or L‐arginine (NOS substrate), denoting the importance of reduced availability of their respective gaseous products, CO and NO, in the nicotine effect. The roles of CO and NO appear to be mutually facilitated because the hemin and L‐arginine effect was abolished after NOS (L‐NAME) and HO inhibition (ZnPP), respectively. Estrogen receptor blockade (ICI 182,780) reduced baroreflexes in OVXE2 rats but failed to abolish the improved baroreflex gain caused by hemin or L‐arginine in OVXE2/nicotine rats. Baroreflex facilitation by hemin was eliminated after inhibition of PI3K (wortmannin), sGC (ODQ) or ERK (PD98059) in central neurons, in contrast to no effect for the inhibition of p38 (SB203580) or JNK (SP600125). Together, the upregulation of NOS/HO‐coupled PI3K/sGC/ERK signaling downstream of E2 receptors rectifies the E2‐dependent, nicotine‐evoked impairment of reflex bradycardia in female rats.Grant Funding Source: Supported by Grant STDF ID 502, Egypt

The concomitant coronary vasodilator and positive inotropic actions of the nitroxyl donor Angeli's salt in the intact rat heart: contribution of soluble guanylyl cyclase‐dependent and ‐independent mechanisms

The NO redox sibling nitroxyl (HNO) elicits soluble guanylyl cyclase (sGC)-dependent vasodilatation. HNO has high reactivity with thiols, which is attributed with HNO-enhanced left ventricular (LV) function. Here, we tested the hypothesis that the concomitant vasodilatation and inotropic actions induced by a HNO donor, Angeli's salt (sodium trioxodinitrate), were sGC-dependent and sGC-independent respectively. Haemodynamic responses to Angeli's salt (10 pmol-10 μmol), alone and in the presence of scavengers of HNO (L-cysteine, 4 mM) or of NO [hydroxocobalamin (HXC), 100 μM] or a selective inhibitor of sGC [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10 μM], a CGRP receptor antagonist (CGRP8-37 , 0.1 μM) or a blocker of voltage-dependent potassium channels [4-aminopyridine (4-AP), 1 mM] were determined in isolated hearts from male rats. Angeli's salt elicited concomitant, dose-dependent increases in coronary flow and LV systolic and diastolic function. Both L-cysteine and ODQ shifted (but did not abolish) the dose-response curve of each of these effects to the right, implying contributions from HNO and sGC in both the vasodilator and inotropic actions. In contrast, neither HXC, CGRP8-37 nor 4-AP affected these actions. Both vasodilator and inotropic actions of the HNO donor Angeli's salt were mediated in part by sGC-dependent mechanisms, representing the first evidence that sGC contributes to the inotropic and lusitropic action of HNO in the intact heart. Thus, HNO acutely enhances LV contraction and relaxation, while concomitantly unloading the heart, potentially beneficial actions in failing hearts.

Structure/Activity Relationships of (M)ANT- and TNP-Nucleotides for Inhibition of Rat Soluble Guanylyl Cyclase α1β1

Soluble guanylyl cyclase (sGC) plays an important role in cardiovascular function and catalyzes formation of cGMP. sGC is activated by nitric oxide and allosteric stimulators and activators. However, despite its therapeutic relevance, the regulatory mechanisms of sGC are still incompletely understood. A major reason for this situation is that no crystal structures of active sGC have been resolved so far. An important step toward this goal is the identification of high-affinity ligands that stabilize an sGC conformation resembling the active, "fully closed" state. Therefore, we examined inhibition of rat sGCα1β1 by 38 purine- and pyrimidine-nucleotides with 2,4,6,-trinitrophenyl and (N-methyl)anthraniloyl substitutions at the ribosyl moiety and compared the data with that for the structurally related membranous adenylyl cyclases (mACs) 1, 2, 5 and the purified mAC catalytic subunits VC1:IIC2. TNP-GTP [2',3'-O-(2,4,6-trinitrophenyl)-GTP] was the most potent sGCα1β1 inhibitor (Ki, 10.7 nM), followed by 2'-MANT-3'-dATP [2'-O-(N-methylanthraniloyl)-3'-deoxy-ATP] (Ki, 16.7 nM). Docking studies on an sGCαcat/sGCβcat model derived from the inactive heterodimeric crystal structure of the catalytic domains point to similar interactions of (M)ANT- and TNP-nucleotides with sGCα1β1 and mAC VC1:IIC2. Reasonable binding modes of 2'-MANT-3'-dATP and bis-(M)ANT-nucleotides at sGC α1β1 require a 3'-endo ribosyl conformation (versus 3'-exo in 3'-MANT-2'-dATP). Overall, inhibitory potencies of nucleotides at sGCα1β1 versus mACs 1, 2, 5 correlated poorly. Collectively, we identified highly potent sGCα1β1 inhibitors that may be useful for future crystallographic and fluorescence spectroscopy studies. Moreover, it may become possible to develop mAC inhibitors with selectivity relative to sGC.

Preservation of nitric oxide-induced relaxation of porcine coronary artery: roles of the dimers of soluble guanylyl cyclase, phosphodiesterase type 5, and cGMP-dependent protein kinase

Soluble guanylyl cyclase (sGC), phosphodiesterase type 5 (PDE5), and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) are all dimeric. The present study was to determine the role of their dimeric status in nitric oxide-induced vasodilatation. In isolated porcine coronary arteries, after 20h incubation with serum-free medium, serum-containing medium, or phosphate-buffered saline solution, the protein levels of the dimers of sGC, PDE5, and PKG were diminished while the monomer levels remained unchanged, associated with reduced cGMP elevation in response to DETA NONOate and decreased PDE5 activity; the activity of PKG was not significantly altered. DETA NONOate caused a greater relaxation in arteries incubated for 20 vs. 2h. The relaxant response was largely abolished by 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, an sGC inhibitor. Zaprinast, a PDE5 inhibitor, had no effect on relaxation caused by DETA NONOate of arteries incubated for 20h but augmented the response incubated for 2h. A greater relaxation to 8-bromo-guanosine 3'5'-cyclic monophosphate occurred in arteries incubated for 20 than for 2h. The protein level of the dimers but not monomers of PDE5 was reduced by dithiothreitol and unaffected by hydrogen peroxide, accompanied with decreased PDE5 activity and reduced response to DETA NONOate. These results demonstrate that the dimeric but not monomeric status of sGC and PDE5 of coronary arteries are closely related to their activities. The preserved vasodilator response after 20h incubation may result in part from a synchronous reduction of the dimer levels of sGC and PDE5 as well as an augmented response to cGMP.

The NO/sGC/PKG signaling pathway in the NAc shell is necessary for the acquisition of morphine-induced place preference.

There is evidence that the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP-dependent protein kinase (PKG) signaling pathway in the basal lateral amygdala and hippocampus plays a key role in memory processing, but it is not known if this NO signaling pathway in the nucleus accumbens (Gomes et al., 2006), a known pivotal region in reward memory, is essential for drug-associated reward memory. We therefore investigated the effect of the NO/sGC/PKG signaling pathway in the nucleus accumbens (NAc) on morphine-induced conditioned place preference (CPP). Results showed that a preconditioning microinjection of the NO synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) into the NAc shell, but not into the core, significantly blocked the acquisition of morphine CPP. The blockage effect of L-NAME on the acquisition of CPP was imitated by the neuronal NOS inhibitor 7-nitroindazole, 3-bromo-, sodium salt (7-NI), the sGC inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), and the PKG inhibitor Rp-8Br-PET-cGMPS. The 7-NI- or ODQ-induced effect was reversed by premicroinjection of the sGC activator YC-1 or the PKG activator 8-Br-cGMP in the NAc shell. However, microinfusion of 7-NI, ODQ, or Rp-8Br-PET-cGMPS into the NAc shell or the core had no effect on the expression of morphine CPP. These findings indicate that the NO/sGC/PKG signaling pathway in the NAc shell is critical for the acquisition of morphine-induced place preference, whereas the same signaling pathway in the NAc shell or core is not involved in the retrieval of morphine-induced place preference.

Activation of soluble guanylyl cyclase prevents foam cell formation and atherosclerosis

Soluble guanylyl cyclase (sGC) is a key modulator in the regulation of vascular tone. However, its role and involving mechanism in cholesterol metabolism of macrophages and atherosclerosis remain unclear. Oil red O staining, Dil-oxidized low-density lipoprotein (oxLDL)-binding assay and cholesterol efflux assay were performed in biology of foam cells. Levels of cytokines or intracellular lipid were evaluated by ELISA or colorimetric kits. Expression of gene or protein was determined by quantitative real-time PCR or Western blotting. Histopathology was examined by haematoxylin and eosin staining. Soluble guanylyl cyclase was expressed in macrophages of mouse atherosclerotic lesions. Treatment with 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, sGC inhibitor) exacerbated oxLDL-induced cholesterol accumulation in macrophages. In contrast, 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1, sGC activator) attenuated the oxLDL-induced cholesterol accumulation because of increased cholesterol efflux. Additionally, YC-1 dose dependently increased the protein expression of ATP-binding cassette transporter A1 (ABCA1) but did not alter that of scavenger receptor class A (SR-A), CD36, SR-BI or ABCG1. Moreover, YC-1-upregulated ABCA1 level depended on liver X receptor α (LXRα). Inhibition of the LXRα-ABCA1 pathway by LXRα small interfering RNA (siRNA), ABCA1 neutralizing antibody or ABCA1 siRNA abolished the effect of YC-1 on cholesterol accumulation and cholesterol efflux. In vivo, YC-1 retarded the development of atherosclerosis, accompanied by reduced serum levels of cholesterol and pro-inflammatory cytokines, in apolipoprotein E-deficient mice. Activation of sGC by YC-1 leads to LXRα-dependent upregulation of ABCA1 in macrophages and may confer protection against atherosclerosis.

Role of endothelial nitric oxide in pulmonary and systemic arteries during hypoxia

Our aim was to investigate the role played by endothelial nitric oxide (NO) during acute vascular response to hypoxia, as a modulator of both vascular tone (through guanylate cyclase (sGC) activation) and mitochondrial O2 consumption (through competitive inhibition of cytochrome-c-oxydase (CcO)). Organ bath experiments were performed and O2 consumption (Clark electrode) was determined in isolated aorta, mesenteric and pulmonary arteries of rats and eNOS-knockout mice. All pre-contracted vessels exhibited a triphasic hypoxic response consisting of an initial transient contraction (not observed in vessels from eNOS-knockout mice) followed by relaxation and subsequent sustained contraction. Removal of the endothelium, inhibition of eNOS (by L-NNA) and inhibition of sGC (by ODQ) abolished the initial contraction without altering the other two phases. The initial hypoxic contraction was observed in the presence of L-NNA+NO-donors. L-NNA and ODQ increases O2 consumption in hypoxic vessels and increases the arterial tone in normoxia but not in hypoxia. When L-NNA+mitochondrial inhibitors (cyanide, rotenone or myxothiazol) were added, the increase in tone was similar in normoxic and hypoxic vessels, which suggests that inhibition of the binding of NO to reduced CcO restored the action of NO on sGC. ConclusionA complex equilibrium is established between NO, sGC and CcO in vessels in function of the concentration of O2: as O2 falls, NO inhibition of mitochondrial O2 consumption increases and activation of sGC decreases, thus promoting a rapid increase in tone in both pulmonary and systemic vessels, which is followed by the triggering of NO-independent vasodilator/vasoconstrictor mechanisms.

BAY 41-2272, a Soluble Guanylate Cyclase Stimulator, Relaxes Isolated Human Ureter in a Standardized In Vitro Model

To characterize the relaxation induced by BAY 41-2272 in human ureteral segments. Ureter specimens (n= 17) from multiple organ human deceased donors (mean age 40 ± 3.2 years, male/female ratio 2:1) were used to characterize the relaxing response of BAY 41-2272. Immunohistochemical analysis for endothelial and neuronal nitric oxide synthase, guanylate cyclase stimulator (sGC) and type 5 phosphodiesterase was also performed. The potency values were determined as the negative log of the molar to produce 50% of the maximal relaxation in potassium chloride-precontracted specimens. The unpaired Student t test was used for the comparisons. Immunohistochemistry revealed the presence of endothelial nitric oxide synthase in vessel endothelia and neuronal nitric oxide synthase in urothelium and nerve structures. sGC was expressed in the smooth muscle and urothelium layer, and type 5 phosphodiesterase was present in the smooth muscle only. BAY 41-2272 (0.001-100 μM) relaxed the isolated ureter in a concentration dependent manner, with a potency and maximal relaxation value of 5.82 ± 0.14 and 84% ± 5%, respectively. The addition of nitric oxide synthase and sGC inhibitors reduced the maximal relaxation values by 21% and 45%, respectively. However, the presence of sildenafil (100 nM) significantly potentiated (6.47 ± 0.10, P<.05) this response. Neither glibenclamide or tetraethylammonium nor ureteral urothelium removal influenced the relaxation response by BAY 41-2272. BAY 41-2272 relaxes the human isolated ureter in a concentration-dependent manner, mainly by activating the sGC enzyme in smooth muscle cells rather than in the urothelium, although a cyclic guanosine monophosphate-independent mechanism might have a role. The potassium channels do not seem to be involved.

Critical role of nitric oxide in the modulation of prepulse inhibition in Swiss mice

Nitric oxide (NO) modulates the dopamine uptake and release processes and appears to be implicated in dopamine-related pathologies, such as schizophrenia. However, it is unclear whether there is excess or deficient NO synthesis in schizophrenia pathophysiology. Analyses of the intracellular pathways downstream of NO system activation have identified the cyclic nucleotide cyclic guanosine monophosphate (cGMP) as a possible target for drug development. Defects in the sensorimotor gating of the neural mechanism underlying the integration and processing of sensory information have been detected across species through prepulse inhibition (PPI). The aim of this study was to investigate the effects of NO/cGMP increase on sensorimotor gating modulation during dopamine hyperfunction. Mice were treated with NO donors and subjected to the PPI test. Treatment with the NO donor sodium nitroprusside was preceded by pretreatment with a soluble guanylate cyclase (sGC) inhibitor. Additionally, the mice were treated with NO donors and phosphodiesterases inhibitors prior to amphetamine treatment. Pretreatment with the NO donors enhanced the PPI response and attenuated the amphetamine-disruptive effects on the PPI. The sGC inhibitor did not modify the sodium nitroprusside effects. Additionally, the cGMP increase induced by a specific phosphodiesterase inhibitor did not modify the amphetamine-disruptive effect. This study provides the first demonstration that an increase in NO can improve the PPI response and block the amphetamine-disruptive effects on the PPI response. Our data are consistent with recent clinical results. However, these effects do not appear to be related to an increase in cGMP levels, and further investigation is thus required.

TRPV4 channel activation leads to endothelium-dependent relaxation mediated by nitric oxide and endothelium-derived hyperpolarizing factor in rat pulmonary artery

The purpose of the present study was to characterize TRPV4 channels in the rat pulmonary artery and examine their role in endothelium-dependent relaxation. Tension, Real-Time polymerase chain reaction (Real-Time PCR) and Western blot experiments were conducted on left and right branches of the main pulmonary artery from male Wistar rats. TRPV4 channel agonist GSK1016790A (GSK) caused concentration-related robust relaxation (Emax 88.6±5.5%; pD2 8.7±0.2) of the endothelium-intact pulmonary artery. Endothelium-denudation nearly abolished the relaxation (Emax 5.6±1.3%) to GSK. TRPV4 channel selective antagonist HC067047 significantly attenuated GSK-induced relaxation (Emax 56.2±6.6% vs. control Emax 87.9±3.3%) in endothelium-intact vessels, but had no effect on either ACh-induced endothelium-dependent or SNP-induced endothelium-independent relaxations. GSK-induced relaxations were markedly inhibited either in the presence of NO synthase inhibitor L-NAME (Emax 8.5±2.7%) or sGC inhibitor ODQ (Emax 28.1±5.9%). A significant portion (Emax 30.2±4.4%) of endothelium-dependent relaxation still persisted in the combined presence of L-NAME and cyclooxygenase inhibitor indomethacin. This EDHF-mediated relaxation was sensitive to inhibition by 60mM K+ depolarizing solution or K+ channel blockers apamin (SKCa; KCa2.3) and TRAM-34 (IKCa; KCa3.1). GSK (10−10−10−7M) caused either modest decrease or increase in the basal tone of endothelium-intact or denuded rings, respectively. We found a greater abundance (>1.5 fold) of TRPV4 mRNA and protein expressions in endothelium-intact vs. denuded vessels, suggesting the presence of this channel in pulmonary endothelial and smooth muscle cells as well. The present study demonstrated that NO and EDHF significantly contributed to TRPV4 channel-mediated endothelium-dependent relaxation of the rat pulmonary artery.

Notch activation augments nitric oxide/soluble guanylyl cyclase signaling in immortalized ovarian surface epithelial cells and ovarian cancer cells

Nitric oxide (NO) is generated by tumor, stromal and endothelial cells and plays a multifaceted role in tumor biology. Many physiological functions of NO are mediated by soluble guanylyl cyclase (sGC) and NO/sGC signaling has been shown to promote proliferation and survival of ovarian cancer cells. However, how NO/sGC signaling is modulated in ovarian cancer cells has not been studied. The evolutionarily conserved Notch signaling pathway plays an oncogenic role in ovarian cancer. Here, we report that all three ovarian cancer cell lines we examined express a higher level of GUCY1B3 (the β subunit of sGC) compared to non-cancerous immortalized ovarian surface epithelial (IOSE) cell lines. Interestingly, the highest expression of GUCY1B3 in ovarian cancer OVCAR3 cells is concurrent with the expression of Notch3. In IOSE cells, forced activation of Notch3 increases the expression of GUCY1B3, NO-induced cGMP production, and the expression of cGMP-dependent protein kinase (PKG), thereby enhancing NO- and cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP, a direct PKG substrate protein). In contrast, inhibition of Notch by DAPT reduces GUCY1B3 expression and NO-induced cGMP production and VASP phosphorylation in OVCAR3 cells. Finally, we confirmed that inhibition of sGC by ODQ decreases growth of ovarian cancer cells. Together, our work demonstrates that Notch is a positive regulator of NO/sGC signaling in IOSE and ovarian cancer cells, providing the first evidence that Notch and NO signaling pathways interact in IOSE and ovarian cancer cells.