cGMP Accumulation Research Articles

Time-Dependent Differences in the Influence of Perivascular Adipose Tissue on Vasomotor Functions in Metabolic Syndrome

Metabolic syndrome (MetS) facilitates the development of cardiovascular disease due to atherosclerosis, which is accelerated by defects of the vascular endothelium. Vascular dysfunction in response to nitric oxide (NO) occurs in the mesenteric arteries of an animal model of MetS, SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats. Vascular responses to vasodilators are affected by perivascular adipose tissue (PVAT) that surrounds the outsides of arteries. In this study, we assessed the role of PVAT in vascular dysfunction observed in SHRSP.ZF. To determine the effects of PVAT on vasodilators in SHRSP.ZF and control Wistar-Kyoto (WKY) rats, we used organ bath bioassay techniques to assay acetylcholine and nitroprusside-induced relaxations of isolated mesenteric arterial ring preparations with PVAT intact or removed. A PVAT-mediated enhancement of relaxations induced by acetylcholine and nitroprusside occurred in SHRSP.ZF at 20 weeks of age, but not at 10 and 30 weeks, and did not occur in WKY. Furthermore, the enhancing effects of PVAT from SHRSP.ZF at 20 weeks could not be substituted by replacement with PVAT from either WKY or 30-week-old SHRSP.ZF, was inhibited by NO synthase inhibitor, and abolished by removal of the arteries' endothelium. Cyclic guanosine monophosphate (cGMP) accumulation elicited by nitroprusside was higher in SHRSP.ZF arteries with PVAT than arteries without PVAT at 20 weeks, but the enhancement of cGMP accumulation did not occur at 30 weeks. PVAT may regulate arterial tone by releasing diffusible vasorelaxing factor(s), which, through endothelium-derived NO production, compensates for impaired vasodilations at early stages of MetS.

Aldosterone does not alter endothelin B receptor signaling in the inner medullary collecting duct.

Recent studies suggest that aldosterone‐mediated sulfenic acid modification of the endothelin B receptor (ETB) promotes renal injury in an ischemia/reperfusion model through reduced ETB‐stimulated nitric oxide production. Similarly, aldosterone inactivation of ETB signaling promotes pulmonary artery hypertension. Consequently, we asked whether aldosterone inhibits collecting duct ETB signaling; this could promote fluid retention since CD ETB exerts natriuretic and diuretic effects. A mouse inner medullary collecting duct cell line (IMCD3) was treated with aldosterone for 48 h followed by sarafotoxin‐6c, an ETB‐selective agonist, and extracellular signal‐related kinase 1/2 (ERK) phosphorylation assessed. S6c increased the phospho/total‐ERK ratio similarly in control and aldosterone‐treated cells (aldosterone alone increased phospho/total‐ERK). Since cultured IMCD cell lines lack ETB inhibited AVP signaling, the effect of S6c on AVP‐stimulated cAMP in acutely isolated IMCD was assessed. Rats (have much higher CD ETB expression than mice) were exposed to 3 days of a normal or low Na+ diet, or low Na+ diet + desoxycorticosterone acetate. S6c inhibited AVP‐stimulated cAMP in rat IMCD by the same degree in the high mineralocorticoid groups compared to controls. Finally, S6c‐stimulated cGMP accumulation in cultured IMCD, or S6c‐stimulated nitric oxide or cGMP in acutely isolated IMCD, was not affected by prior aldosterone exposure. These findings provide evidence that aldosterone does not modify ETB effects on ERK phosphorylation, AVP‐dependent cAMP inhibition, or NO/cGMP accumulation in the IMCD. Thus, while aldosterone can inhibit endothelial cell ETB activity to promote hypertension and injury, this response does not appear to occur in the IMCD.

PDE5 inhibitors promote recovery of peripheral neuropathy in diabetic mice.

PDE5 inhibitors promote recovery of peripheral neuropathy in diabetic mice.

CGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP) has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE) with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2) for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation) mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities) and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

Constitutive cyclic GMP accumulation in Arabidopsis thaliana compromises systemic acquired resistance induced by an avirulent pathogen by modulating local signals.

The infection of Arabidopsis thaliana plants with avirulent pathogens causes the accumulation of cGMP with a biphasic profile downstream of nitric oxide signalling. However, plant enzymes that modulate cGMP levels have yet to be identified, so we generated transgenic A. thaliana plants expressing the rat soluble guanylate cyclase (GC) to increase genetically the level of cGMP and to study the function of cGMP in plant defence responses. Once confirmed that cGMP levels were higher in the GC transgenic lines than in wild-type controls, the GC transgenic plants were then challenged with bacterial pathogens and their defence responses were characterized. Although local resistance was similar in the GC transgenic and wild-type lines, differences in the redox state suggested potential cross-talk between cGMP and the glutathione redox system. Furthermore, large-scale transcriptomic and proteomic analysis highlighted the significant modulation of both gene expression and protein abundance at the infection site, inhibiting the establishment of systemic acquired resistance. Our data indicate that cGMP plays a key role in local responses controlling the induction of systemic acquired resistance in plants challenged with avirulent pathogens.

Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC.

Campomanesia adamantium Peel Extract in Antidiarrheal Activity: The Ability of Inhibition of Heat-Stable Enterotoxin by Polyphenols.

Campomanesia adamantium (Myrtaceae) is a medicinal plant distributed in Brazilian Cerrado. Different parts of this plant are used in popular medicine for treatment of several diseases like fever, diarrhea, hypercholesterolemia and rheumatism. The aim of this work was to evaluate the inhibition of heat-stable enterotoxin type A (STa) by gallic acid present in the peel of C. adamantium fruit and assays to assess the antidiarrheal activity, anti-inflammatory and cytotoxic properties of peel extract using the T84 cell line model. The possible inhibition exerted by the gallic acid of the peel extract on the STa peptide was inferred by molecular dynamics simulations. The antidiarrheal effects were investigated measuring cGMP accumulation in cells after stimulation by STa toxin and antibacterial activity was assessed. The anti-inflammatory activity was assessed by inhibition of COX-1 and COX-2. MTT and LDH assays were used to evaluate any possible cytotoxic action while the CyQUANT test was used to investigate the effect on cell proliferation. A representation showing how the possible interactions between STa and the gallic acid of the extract might reduce the action of the enterotoxin is presented. C. adamantium peel extract significantly decreased the levels of cGMP in T84 cells. However, no effect on the species of microorganisms was observed. The extract also inhibited COX-1 (IC50 255.70 ± 0.04 ng/mL) and COX-2 (IC50 569.50 ± 0.11 ng/mL) enzymes. Cytotoxicity assay have shown significant changes in cells treated with the extract, which inhibited the cell proliferation until 72 hours of treatment. Direct interactions of phenolic compounds present in the extract with the STa toxin may limit its activity. Curative effect in the diarrhea treatment and its anti-inflammatory action is based on the pharmacological properties, mechanism of action of the C. adamantium peel extract, and no toxic effects of the peel extract presented on this work.

Age-related changes to vascular protease-activated receptor 2 in metabolic syndrome: a relationship between oxidative stress, receptor expression, and endothelium-dependent vasodilation.

Protease-activated receptor 2 (PAR2) is expressed in vascular endothelium. Nitric oxide (NO) - cyclic GMP-mediated vasodilation in response to 2-furoyl-LIGRLO-amide (2fLIGRLO), a PAR2-activating peptide, is impaired in aortas from aged SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome. Here we investigated mechanisms linking PAR2's vascular effects to phenotypic characteristics of male SHRSP.ZF rats at 10, 20, and 30 weeks of age. We found vasodilation responses to either 2fLIGRLO or enzyme-mediated PAR2 activation by trypsin were sustained until 20 weeks and lessened at 30 weeks. PAR2 protein and mRNA levels were lower in aortas at 30 weeks than at 10 and 20 weeks. PAR2-mediated responses positively correlated with PAR2 protein and mRNA levels. Decreased cGMP accumulation in the presence of 2fLIGRLO paralleled the decreased relaxations elicited by nitroprusside and the cGMP analog 8-pCPT-cGMP, and the less soluble guanylyl cyclase protein at 30 weeks. 2fLIGRLO-induced relaxation was negatively correlated with serum thiobarbituric acid reactive substances, an index of oxidative stress, which increased with age. Forward stepwise data regression supported a model of age-related decreases in PAR2 function resulting from decreased PAR2 mRNA and increased oxidative stress. We conclude that decreased responsiveness of aortic smooth muscle to NO and downregulation of receptor expression impair PAR2 functions at later stages of metabolic syndrome in SHRSP.ZF rats.

Soluble guanylate cyclase redox state under oxidative stress conditions in isolated monkey coronary arteries.

Coronary artery disease is associated with oxidative stress due to the excessive generation of free radicals in the vascular wall. This study investigated the impact of tert‐butyl hydroperoxide (t‐BuOOH), a peroxyl radical generator, on the redox state of soluble guanylate cyclase (sGC) in isolated monkey coronary arteries. Helically cut strips of endothelium‐intact monkey coronary arteries treated with the nitric oxide synthase inhibitor NG‐nitro‐L‐arginine (10 μmol/L) were exposed for approximately 60 min to either no drug or t‐BuOOH (100 μmol/L) in the presence and absence of α‐tocopherol (300 μmol/L). Relaxation and cGMP levels in response to the sGC stimulator BAY 41‐2272 and the sGC activator BAY 60‐2770 were assessed by organ chamber technique and enzyme immunoassay, respectively. The relaxant response to BAY 41‐2272 was significantly impaired by the exposure to t‐BuOOH, whereas the response to BAY 60‐2770 was significantly augmented. In addition, vascular cGMP accumulation caused by BAY 41‐2272 was decreased by the exposure to t‐BuOOH, whereas for BAY 60‐2770, it was increased. These effects of t‐BuOOH were abolished by coincubation with α‐tocopherol. Furthermore, correlations were observed between BAY compound‐induced relaxant magnitudes and cGMP levels. Therefore, it is concluded that increased oxidative stress leads to disruption of the sGC redox state in monkey coronary arteries. This finding is of great importance for understanding coronary physiology in primates.

Regulation of soluble guanylyl cyclase redox state by hydrogen sulfide

Soluble guanylate cyclase (sGC) is a receptor for nitric oxide (NO). Binding of NO to ferrous (Fe2+) heme increases its catalytic activity, leading to the production of cGMP from GTP. Hydrogen sulfide (H2S) is a signaling molecule that exerts both direct and indirect anti-oxidant effects. In the present, study we aimed to determine whether H2S could regulate sGC redox state and affect its responsiveness to NO-releasing agents and sGC activators. Using cultured rat aortic smooth muscle cells, we observed that treatment with H2S augmented the response to the NO donor DEA/NO, while attenuating the response to the heme-independent activator BAY58-2667 that targets oxidized sGC. Similarly, overexpression of H2S-synthesizing enzyme cystathionine-γ lyase reduced the ability of BAY58-2667 to promote cGMP accumulation. In experiments with phenylephrine-constricted mouse aortic rings, treatment with rotenone (a compound that increases ROS production), caused a rightward shift of the DEA/NO concentration-response curve, an effect partially restored by H2S. When rings were pre-treated with H2S, the concentration-response curve to BAY 58-2667 shifted to the right. Using purified recombinant human sGC, we observed that treatment with H2S converted ferric to ferrous sGC enhancing NO-donor-stimulated sGC activity and reducing BAY 58-2667-triggered cGMP formation. The present study identified an additional mechanism of cross-talk between the NO and H2S pathways at the level of redox regulation of sGC. Our results provide evidence that H2S reduces sGC heme Fe, thus, facilitating NO-mediated cellular signaling events.

Soluble Guanylate Cyclase Modulators, BAY 41-2272 and BAY 60-2770, Inhibit Human and Rabbit Prostate Contractility

To evaluate the inhibitory effect of soluble guanylate cyclase (sGC) stimulator, BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluorobenzyl)- 1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine) or activator, BAY 60-2770 (4-({(4- carboxybutyl) [2- (5-fluoro-2-{[40-(trifluoromethyl) biphenyl- 4-yl]methoxy}phenyl)ethyl] amino}methyl)benzoic acid), in human and rabbit prostate smooth muscle contractility. In rabbit or human prostate, contractions induced by electrical field stimulation or phenylephrine (PE) were carried out in the presence of sGC stimulator, BAY 41-2272, or sGC activator, BAY 60-2770. The potency (pEC50) and maximal response (Emax) values were determined. Immunohistochemistry analysis for sGC α1-subunit and quantification of intracellular levels of cyclic guanosine monophosphate (cGMP) were also performed. In rabbit prostate, BAY 60-2770 (30 and 300 nM) inhibited the contractions induced by PE and electrical field stimulation. The coincubation with sGC inhibitor, ODQ, produced greater inhibitions on PE-induced contractions in comparison with BAY 60-2770 alone, mainly due to greater cGMP accumulation (70- and 5.7-fold, respectively). BAY 41-2272 (300 nM) increased and decreased, respectively, cGMP levels and PE-induced contractions, but in the presence of ODQ these effects were reversed. In human prostate, immunohistochemistry analysis revealed the presence of sGC α1-subunit on the transition zone. BAY 60-2770 (300 nM) reduced significantly Emax induced by PE in human prostate. sGC activator seems to be a promising alternative to treat benign prostatic hyperplasia because it increases cGMP levels even when sGC is oxidized.

Extracellular purines' action on glomerular albumin permeability in isolated rat glomeruli: insights into the pathogenesis of albuminuria.

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palb involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes.

Systems Pharmacology and Rational Polypharmacy: Nitric Oxide-Cyclic GMP Signaling Pathway as an Illustrative Example and Derivation of the General Case.

Impaired nitric oxide (NO˙)-cyclic guanosine 3', 5'-monophosphate (cGMP) signaling has been observed in many cardiovascular disorders, including heart failure and pulmonary arterial hypertension. There are several enzymatic determinants of cGMP levels in this pathway, including soluble guanylyl cyclase (sGC) itself, the NO˙-activated form of sGC, and phosphodiesterase(s) (PDE). Therapies for some of these disorders with PDE inhibitors have been successful at increasing cGMP levels in both cardiac and vascular tissues. However, at the systems level, it is not clear whether perturbation of PDE alone, under oxidative stress, is the best approach for increasing cGMP levels as compared with perturbation of other potential pathway targets, either alone or in combination. Here, we develop a model-based approach to perturbing this pathway, focusing on single reactions, pairs of reactions, or trios of reactions as targets, then monitoring the theoretical effects of these interventions on cGMP levels. Single perturbations of all reaction steps within this pathway demonstrated that three reaction steps, including the oxidation of sGC, NO˙ dissociation from sGC, and cGMP degradation by PDE, exerted a dominant influence on cGMP accumulation relative to other reaction steps. Furthermore, among all possible single, paired, and triple perturbations of this pathway, the combined perturbations of these three reaction steps had the greatest impact on cGMP accumulation. These computational findings were confirmed in cell-based experiments. We conclude that a combined perturbation of the oxidatively-impaired NO˙-cGMP signaling pathway is a better approach to the restoration of cGMP levels as compared with corresponding individual perturbations. This approach may also yield improved therapeutic responses in other complex pharmacologically amenable pathways.

Panax notoginseng saponins ameliorate impaired arterial vasodilation in SHRSP.Z-Lepr(fa) /lzmDmcr rats with metabolic syndrome.

Panax notoginseng saponins (PNS) are major components of Panax notoginseng, a herb with established clinical efficacy against vascular diseases. SHRSP.Z-Lepr(fa) /IzmDmcr (SHRSP.ZF) rats, a new animal model for metabolic syndrome, display an impaired vasorelaxation response in aortas and mesenteric arteries that is mediated by nitric oxide (NO). This study investigated whether PNS and its components can ameliorate this vascular dysfunction in SHRSP.ZF rats. In an in vitro study, in the presence or absence of PNS and its components, vasodilation in response to nitroprusside was determined from myographs under isometric tension conditions in aortas and mesenteric arteries from male SHRSP.ZF rats at 18-20 weeks of age. In an in vivo study, PNS (30 mg/kg per day) was orally administered to SHRSP.ZF rats from 8 to 20 weeks of age. In vitro treatment with PNS and Ginsenoside Rb1 increased nitroprusside-induced relaxation of aortas and mesenteric arteries in SHRSP.ZF rats. The PNS-induced increase was not affected by a nitric oxide (NO) synthase inhibitor or endothelium denudation. Relaxation in response to a cell-permeable cGMP analogue was increased by PNS, but cGMP accumulation by nitroprusside was not altered. In vivo treatment with PNS in SHRSP.ZF rats lowered blood pressure and increased relaxation and the expression of soluble guanylyl cyclase protein in arteries, without affecting metabolic abnormalities. These results indicate that PNS causes an increase in vasodilation in response to NO and a decrease in blood pressure, resulting in protection against vascular dysfunction in SHRSP.ZF rats. PNS might be beneficial in alleviating impaired vasodilation in metabolic syndrome.

Enhanced serelaxin signalling in co-cultures of human primary endothelial and smooth muscle cells.

In the phase III clinical trial, RELAX-AHF, serelaxin caused rapid and long-lasting haemodynamic changes. However, the cellular mechanisms involved are unclear in humans. This study examined the effects of serelaxin in co-cultures of human primary endothelial cells (ECs) and smooth muscle cells (SMCs) on cAMP and cGMP signalling. Stimulation of HUVECs or human coronary artery endothelial cells (HCAECs) with serelaxin, concentration-dependently increased cGMP accumulation in co-cultured SMCs to a greater extent than in monocultures of either cell type. This was not observed in human umbilical artery endothelial cells (HUAECs) that do not express the relaxin receptor, RXFP1. Treatment of ECs with l-N(G) -nitro arginine (NOARG; 30μM, 30min) inhibited serelaxin-mediated (30nM) cGMP accumulation in HUVECs, HCAECs and co-cultured SMCs. In HCAECs, but not HUVECs, pre-incubation with indomethacin (30μM, 30min) also inhibited cGMP accumulation in SMCs. Pre-incubation of SMCs with the guanylate cyclase inhibitor ODQ (1μM, 30min) had no effect on serelaxin-mediated (30nM) cGMP accumulation in HUVECs and HCAECs but inhibited cGMP accumulation in SMCs. Serelaxin stimulation of HCAECs, but not HUVECs, increased cAMP accumulation concentration-dependently in SMCs. Pre-incubation of HCAECs with indomethacin, but not l-NOARG, abolished cAMP accumulation in co-cultured SMCs, suggesting involvement of prostanoids. In co-cultures, treatment of ECs with serelaxin caused marked cGMP accumulation in SMCs and with HCAEC also cAMP accumulation. Responses involved EC-derived NO and with HCAEC prostanoid production. Thus, serelaxin differentially modulates vascular tone in different vascular beds.

Enhanced Nitric Oxide Synthase Activation via Protease-Activated Receptor 2 Is Involved in the Preserved Vasodilation in Aortas from Metabolic Syndrome Rats

Endothelium-dependent vasodilation via protease-activated receptor 2 (PAR2) is preserved in mesenteric arteries from SHRSP.Z-Lepr^fa/IzmDmcr rats (SHRSP.ZF) with metabolic syndrome even though nitric oxide (NO)-mediated vasodilation is attenuated. Therefore, we examined the PAR2 mechanisms underlying metabolic syndrome-resistant vasodilation in SHRSP.ZF aortas with ageing. In isolated aortas, the PAR2 agonist 2-furoyl-LIGRLO-amide (2fly) caused vasodilation that was sustained in male SHRSP.ZF until 18 weeks of age, but was attenuated afterwards compared with age-matched Wistar-Kyoto rats (controls) at 23 weeks. In contrast, acetylcholine-induced vasodilation was impaired in SHRSP.ZF already at 18 weeks of age. Treatments of aortas with inhibitors of NO synthase and soluble guanylate cyclase abolished the sustained 2fly- and residual acetylcholine-induced vasodilation in SHRSP.ZF at 18 weeks of age. In the aortas of SHRSP.ZF, 8-bromo-cGMP-induced vasodilation, NO production and cGMP accumulation elicited by 2fly were not different from in the controls. PAR2 agonist increased phospho-Ser¹¹⁷⁷-eNOS protein content only in SHRSP.ZF aortas. These results indicate that vasodilation mediated by PAR2 is sustained even though NO-dependent relaxation is attenuated with ageing/exposure to metabolic disorders in large-caliber arteries from SHRSP.ZF. PAR2 stimulation of NO production via an additional pathway that targets phosphorylation of Ser¹¹⁷⁷-eNOS suggests a regulatory mechanism for sustaining agonist-mediated vasodilation in metabolic syndrome.

PPARα induced NOS1 phosphorylation via PI3K/Akt in guinea pig antral mucous cells: NO-enhancement in Ca(2+)-regulated exocytosis.

A PPARα (peroxisome proliferation activation receptor α) agonist (GW7647) activates nitric oxide synthase 1 (NOS1) to produce NO leading to cGMP accumulation in antral mucous cells. In this study, we examined how PPARα activates NOS1. The NO production stimulated by GW7647 was suppressed by inhibitors of PI3K (wortmannin) and Akt (AKT 1/2 Kinase Inhibitor, AKT-inh), although it was also suppressed by the inhibitors of PPARα (GW6471) and NOS1 (N-PLA). GW7647 enhanced the ACh (acetylcholine)-stimulated exocytosis (Ca(2+)-regulated exocytosis) mediated via NO, which was abolished by GW6471, N-PLA, wortmannin, and AKT-inh. The Western blotting revealed that GW7647 phosphorylates NOS1 via phosphorylation of PI3K/Akt in antral mucous cells. The immunofluorescence examinations demonstrated that PPARα existing with NOS1 co-localizes with PI3K and Akt in the cytoplasm of antral mucous cells. ACh alone and AACOCF3, an analogue of arachidonic acid (AA), induced the NOS1 phosphorylation via PI3K/Akt to produce NO, which was inhibited by GW6471. Since AA is a natural ligand for PPARα, ACh stimulates PPARα probably via AA. In conclusion, PPARα activates NOS1 via PI3K/Akt phosphorylation to produce NO in antral mucous cells during ACh stimulation.

Nitrite is a cGMP generator in isolated platelets

cGMP is generated in blood platelets following activation of soluble guanylate cyclase (sGC) by nitric oxide (NO). Once synthesised, cGMP activates cGMP-dependent protein kinase (PKG) and causes an increase in intracellular cAMP concentration by inhibiting phosphodiesterase 3 (PDE3), responsible for its degradation. PKG and the cAMP-dependent protein kinase (PKA) phosphorylate a number of substrates with inhibitory effects on platelet function [1]. While NO is mainly generated by endothelial NO synthase (eNOS) in the endothelium, reduction of nitrite constitutes an alternative vascular source of NO during hypoxic and acidic conditions. Nitrite originates from reduction of dietary nitrate, found in foods such as beetroot and green leafy vegetables. Nitrite in the circulation can be converted to NO by several nitrite reductases in blood and tissues [2], and triggers vasodilatation [3] thus lowering blood pressure [4]. It has also been demonstrated that when eNOS activity is impaired, NO generated through the nitrate/nitrite pathway exerts negative effects on platelet function [5]. Whilst deoxyhaemoglobin is a candidate nitrite-reductase in blood [6], we aimed to characterise the effects of nitrite in isolated platelets. As such, we used a preparation of washed platelets and supraphysiological concentrations of nitrite in vitro and characterised nitrite-dependent cGMP generation and inhibition of platelet function. Our data show that nitrite at high concentrations (1mM) is a powerful generator of cGMP in platelets in the absence of extracellular reductases. While cGMP accumulation is inhibited by sGC inhibitor ODQ, NO scavengers have a partial effect. This seems to indicate that nitrite acts through both NO-dependent and independent mechanisms. Accordingly, phosphorylation of the the cAMP and cGMP-dependent substrate VASP is strongly increased and, similarly to cGMP generation, is completely dependent on sGC activity but not NO. The increase of cGMP induced by high concentrations of nitrite inhibits platelet function as measured by platelet aggregation and secretion. However, whilst being dependent on sGC activity, the effect of nitrite on aggregation is not dependent on NO.generation. Lower concentrations of nitrite synergise with inhibition of PDEs, in particular PDE5 responsible for cGMP degradation, to trigger detectable VASP phosphorylation and inhibition of aggregation. Nitrite triggers generation of cGMP in platelets and exerts inhibitory effects independently of extracellular reductases and other blood cells. Nitrite acts partly through reduction to NO and partly through an uncharacterised direct effect on sGC.

Receptor guanylyl cyclase-G is a sensory protein activated by cool temperatures and predator odor 2,4,5-trimethylthiazoline

We show that GC-G is a dual thermosensory and pheromone receptor that can be maximally stimulated by cool temperatures of about 15°C and TMT in both in vivo cellular cGMP accumulation assays and in vitro GC assays. Mechanistically, while coolness enhances dimerization of GC-G, TMT directly binds the extracellular domain of GC-G to stimulate its enzymatic activity. Consistent with these findings, we observed substantially reduced coolness-induced responses of GG neurons and coolness-evoked ultrasonic vocalization in GC-G-KO pups. Likewise, TMT-induced calcium transients in the GG as well as TMT-evoked innate fear behaviors and an increase of serum corticosterone (a stress hormone) were markedly attenuated in the GC-G-KO mice compared to wild-type littermates. Conclusions Our data suggest that GC-G is a double functional receptor in the GG for sensing cool temperatures and predator scents in an age-specific manner. Whereas GC-G activation in the GG by coolness is critical for the generation of ultrasound calls by isolated pups to elicit maternal care [8], GC-G can detect the predator odor TMT to evoke innate defensive responses in adult mice to maximize species survival.

Abstract P195: Transforming Growth Factor beta 1-Mediated Repression of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Gene Expression and Function Involving TGF-beta 1 and Delta EF1 in Vascular Smooth Muscle Cells

The binding of atrial and brain natriuretic peptides (ANP and BNP) to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPR-A) produces second messenger cGMP, which lowers blood pressure and prevents cardiovascular events. The objective of the present study was to examine the repressive effect of transforming growth factor (TGF-β1) in the regulation of Npr1 (coding for GC-A/NPRA) gene expression and function. The rat thoracic aortic vascular smooth muscle cells (RTASMC) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and treated with TGF-β1. The luciferase assay results showed that TGF-β1 significantly repressed Npr1 promoter activity in a dose- and time-dependent manner by 82% and 85% (2.5 ng/ml, 25.51 ± 2.2 and 24 h, 7.0 ± 0.6 vs. untreated control 70.06 ± 4.6, p < 0.001, respectively). Treatment with TGF-β1 decreased NPRA mRNA and protein levels by 62% (treated, 0.42 ± 0.05 vs. control, 0.9 ± 0.02, p < 0.01) and 55% (treated, 9603 ± 860 vs. untreated, 22211 ± 1449, p < 0.01), respectively. TGF-β1 attenuated ANP-dependent intracellular accumulation of cGMP by 59% (TGF-β1 + ANP-treated, 8.66 ± 0.9 pmol/1x 106 cells vs. ANP-treated cells 23.51 ± 2.2; p < 0.001). Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that TGF-β1 enhanced the recruitment of transcription factor delta EF1 (δEF1) to form a transcriptional repressor complex with their binding sites in Npr1 promoter. Western blot analysis showed significant increase in δEF1 protein expression by 2.4-fold (treated, 907.9 ± 36.5. vs. untreated, 378.5 ± 10.3; p < 0.001) and phosphorylation of mothers against decapentaplegic homolog 2/3 (SMAD2/3) proteins by 2.3-fold (treated, 620.9 ± 10.6 vs. untreated, 269 ± 9.7; p < 0.01) in TGF-β1-treated cells. Collectively, the present results demonstrate that TGF-β1 represses Npr1 gene transcription and expression by interactive actions of δEF1 and phosphorylated SMADs. Together, the present results suggest that TGF-β1 and δEF1 inhibit Npr1 gene expression and function, which may be critical in regulating the blood pressure and cardiovascular homeostasis.