Cyclic GMP Formation Research Articles

Molecular Mechanism of Blood Pressure Regulation through the Atrial Natriuretic Peptide.

Simple SummaryAtrial natriuretic peptide (ANP) is a cardiac peptide hormone that was identified by Kangawa and Matsuo in 1984. In Japan, ANP has been used as an intravenous drug for the treatment of acute heart failure since 1995. Because ANP has a hypotensive effect, it is important to avoid excessive lowering of blood pressure when ANP is used. Recently, a compound that inhibits neutral endopeptidase, the enzyme that degrades ANP, has been developed (angiotensin receptor-neprilysin inhibitor (ARNI)). ARNI has been approved worldwide for the treatment of chronic heart failure and has been authorized in Japan as an antihypertensive drug. However, it is not understood exactly how ANP exerts its hypotensive effect. In this review, we discuss the molecular mechanism of the blood pressure-regulating effects of ANP, focusing on our recent findings.Natriuretic peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), have cardioprotective effects and regulate blood pressure in mammals. ANP and BNP are hormones secreted from the heart into the bloodstream in response to increased preload and afterload. Both hormones act through natriuretic peptide receptor 1 (NPR1). In contrast, CNP acts through natriuretic peptide receptor 2 (NPR2) and was found to be produced by the vascular endothelium, chondrocytes, and cardiac fibroblasts. Based on its relatively low plasma concentration compared with ANP and BNP, CNP is thought to function as both an autocrine and a paracrine factor in the vasculature, bone, and heart. The cytoplasmic domains of both NPR1 and NPR2 display a guanylate cyclase activity that catalyzes the formation of cyclic GMP. NPR3 lacks this guanylate cyclase activity and is reportedly coupled to Gi-dependent signaling. Recently, we reported that the continuous infusion of the peptide osteocrin, an endogenous ligand of NPR3 secreted by bone and muscle cells, lowered blood pressure in wild-type mice, suggesting that endogenous natriuretic peptides play major roles in the regulation of blood pressure. Neprilysin is a neutral endopeptidase that degrades several vasoactive peptides, including natriuretic peptides. The increased worldwide clinical use of the angiotensin receptor-neprilysin inhibitor for the treatment of chronic heart failure has brought renewed attention to the physiological effects of natriuretic peptides. In this review, we provide an overview of the discovery of ANP and its translational research. We also highlight our recent findings on the blood pressure regulatory effects of ANP, focusing on its molecular mechanisms.

Targeting the paracrine hormone-dependent guanylate cyclase/cGMP/phosphodiesterases signaling pathway for colorectal cancer prevention.

Colorectal cancer (CRC) is one of the leading causes of cancer related-deaths. The risk of development of CRC is complex and multifactorial, and includes disruption of homeostasis of the intestinal epithelial layer mediated though dysregulations of tumor suppressing/promoting signaling pathways. Guanylate cyclase 2C (GUCY2C), a membrane-bound guanylate cyclase receptor, is present in the apical membranes of intestinal epithelial cells and maintains homeostasis. GUCY2C is activated upon binding of paracrine hormones (guanylin and uroguanylin) that lead to formation of cyclic GMP from GTP and activation of downstream signaling pathways that are associated with normal homeostasis. Dysregulation/suppression of the GUCY2C-mediated signaling promotes CRC tumorigenesis. High-calorie diet-induced obesity is associated with deficiency of guanylin expression and silencing of GUCY2C-signaling in colon epithelial cells, leading to tumorigenesis. Thus, GUCY2C agonists, such as linaclotide, exhibit considerable role in preventing CRC tumorigenesis. However, phosphodiesterases (PDEs) are elevated in intestinal epithelial cells during CRC tumorigenesis and block GUCY2C-mediated signaling by degrading cyclic GMP to 5`-GMP. PDE5-specific inhibitors, such as sildenafil, show considerable anti-tumorigenic potential against CRC by amplifying the GUCY2C/cGMP signaling pathway, but cannot achieve complete anti-tumorigenic effects. Hence, dual targeting the elevation of cGMP by providing paracrine hormone stimuli to GUCY2C and by inhibition of PDEs may be a better strategy for CRC prevention than alone. This review delineates the involvement of the GUCY2C/cGMP/PDEs signaling pathway in the homeostasis of intestinal epithelial cells. Further, the events are associated with dysregulation of this pathway during CRC tumorigenesis are also discussed. In addition, current updates on targeting the GUCY2C/cGMP/PDEs pathway with GUCY2C agonists and PDEs inhibitors for CRC prevention and treatment are described in detail.

Nitric oxide dependent signaling via cyclic GMP in dendritic cells regulates migration and T-cell polarization

Allergic airway inflammation is accompanied by excessive generation of nitric oxide (NO). Beside its detrimental activity due to the generation of reactive nitrogen species, NO was found to modulate immune responses by activating the NO-sensitive Guanylyl Cyclases (NO-GCs) thereby mediating the formation of the second messenger cyclic GMP (cGMP). To investigate the contribution of the key-enzyme NO-GC on the development of Th2 immunity in vivo, we sensitized knock-out (KO) mice of the major isoform NO-GC1 to the model allergen ovalbumin (OVA). The loss of NO-GC1 attenuates the Th2 response leading to a reduction of airway inflammation and IgE production. Further, in vitro-generated OVA-presenting DCs of the KO induce only a weak Th2 response in the WT recipient mice upon re-exposure to OVA. In vitro, these NO-GC1 KO BMDCs develop a Th1-polarizing phenotype and display increased cyclic AMP (cAMP) formation, which is known to induce Th1-bias. According to our hypothesis of a NO-GC1/cGMP-dependent regulation of cAMP-levels we further demonstrate activity of the cGMP-activated cAMP-degrading phosphodiesterase 2 in DCs. Herewith, we show that activity of NO-GC1 in DCs is important for the magnitude and bias of the Th response in allergic airway disease most likely by counteracting intracellular cAMP.

Blood Pressure and the Renal Actions of AT2 Receptors

Angiotensin type-2 receptors (AT2Rs) in the renal proximal tubule inhibit sodium (Na+) reabsorption by inducing renal cyclic GMP formation and internalizing and inhibiting major Na+ transporters Na+-H+ exchanger-3 (NHE-3) and Na+/K+ATPase (NKA). Instead of angiotensin II (Ang II), angiotensin III (Ang III) is the predominant endogenous agonist for this response. Exogenous non-peptide AT2R agonist Compound-21 induces natriuresis and lowers blood pressure (BP) in normal and Ang II-infused hypertensive rodents. Spontaneously hypertensive rats (SHR; both pre-hypertensive and hypertensive) have defective natriuretic responses to Ang III, suggesting a defect in AT2R-mediated natriuresis in SHR that leads to hypertension. The mechanisms of deficient AT2R-mediated natriuresis in SHR are unknown but could involve either pre-receptor or receptor/post-receptor defects.

C-type natriuretic peptide prevents activation of perivascular mast cells and inflammation in the postischemic microvasculature

Background Atrial- and C-type- natriuretic peptides (ANP, CNP) are both vasodilatatory hormones which are mainly secreted from cardiac atria and vascular endothelium, respectively. Both peptides activate cyclic GMP (cGMP) formation by binding to their specific receptors: the particulate guanylyl cyclases A (GC-A) and B (GC-B). Cyclic GMP via cGMP-dependent protein kinase I (cGKI) was shown to inhibit mast cell (MC) degranulation [1]. During postischemic reperfusion, the activation of MC is detrimental [2]. Due to pleiotropic effects, cytokines released from MC cause tissue damage and vascular leakage. It is unclear whether and how ANP and/or CNP regulate the activity of MC and, thereby, influence ischemia-reperfusion-induced disruption of the microvascular barrier. Methods and results Intravital microscopy was used to visualize the microcirculation within the cremaster muscle of anesthetized mice, and Fluorescein isothiocyanate (FITC)-Dextran was injected intravenously to quantify the microvascular leakage [3,4]. Cremaster blood flow was temporally blocked with an arterial clamp to provoke 30 min of tissue ischemia. During the following 30 min of postischemic reperfusion, the vascular leakage was measured by the ratio of the fluorescent (FITC-Dextran) intensity (RFI) between the adjacent interstitial space and the intravascular lumen. Subsequently resident perivascular degranulated MCs were stained by superfusing Ruthenium Red solution and thereafter counted (number per field). In wildtype mice, ischemia-reperfusion (compared to sham operated cremaster muscles) provoked marked MC degranulation and robust vascular leakage of FITCDextran (as indicated by higher RFI). Local CNP superfusion during reperfusion significantly reduced the amount of degranulated MCs and concomitantly vascular leakage. These effects were mimicked by the membrane-permeable cGKI activator 8-Br-cGMP. The protective effects of 8-Br-cGMP were endothelium-independent, since they were preserved in mice with conditional, endothelial deletion of cGKI. Quantitative real-time RT-PCR revealed local CNP-mRNA expression in the m. cremaster, which was not altered by acute ischemia. In contrast to CNP, ANP superfusion did not significantly alter the amount of degranulated MCs, but still effectively reduced FITC-Dextran leakage resulted from ischemia-reperfusion. Accordingly, in vitro, in cultured human MCs (HMC-1.1, originally developed by Dr. Butterfield and a gift from Dr. Drube) and primary cultured murine bone marrow MC, CNP, but not ANP, induced a marked increase in intracellular cGMP levels and enhanced the phosphorylation of the cytoskeleton-associated vasoactive stimulated phosphoprotein (VASP) at Ser237. Conclusions Our study shows divergent effects of NPs in the postischemic microcirculation. CNP inhibited the ischemic activation of resident, perivascular MCs and consequently the associated microvascular leakage, possibly via MC specific cGMP/cGKI signaling. At difference, ANP had no effect on MCs but still attenuated the postischemic vascular barrier disruption via direct effects on endothelial cells. Our future studies aim to dissect the

Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets.

The platelet-derived soluble CD40L (sCD40L) release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB), has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-β/-γ (PPAR-β/-γ). We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-β/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO) and cyclic GMP formation in a PPAR-β/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2) expression/activity and reactive oxygen species (ROS) formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-β/-γ antagonists. Collectively, these results demonstrate that PPAR-β/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.

The good and the ugly: ANP antagonizes the deleterious effects of aldosterone in hypertensive cardiac remodeling

Background Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and cyclic GMP formation, exerts cardiac antihypertrophic and antifibrotic actions. Conversely, aldosterone promotes pathological cardiac remodeling via the mineralocorticoid receptor (MR). To investigate whether local cardiac ANP/GC-A signaling counteracts the effects of aldosterone during experimental hypertensive cardiac remodeling. Results We studied the impact of the MR antagonist eplerenone (100 mg/kg/day) on cardiac remodeling after transverse aortic constriction (TAC) in mice with conditional, cardiomyocyte-restricted deletion of GC-A (CM GC-A KO) [1] or cGMP-dependent protein kinase I (CM cGK I KO) [2] and respective controls (n=10 in each group). Left ventricular (LV) hypertrophy and interstitial fibrosis were significantly exacerbated in both CM GC-A KO and cGK I KO mice after TAC. These histological changes were accompanied by decreased LV SERCA2a expression, increased CTGF and LV dilatation together with contractile dysfunction. Eplerenone had no effect on systemic blood pressure but fully prevented these pressure-overload induced cardiac morphological, molecular and functional alterations in both CM GC-A KO and CM cGK I KO mice. However, eplerenone did not inhibit TACinduced MAPK ERK1/2 phosphorylation/activation, suggesting that the non-genomic effects of aldosterone are not involved in exacerbated hypertensive cardiac remodeling of the KO mice. Intriguingly, in transfected HEK 293 cells, ANP inhibited the aldosterone-induced nuclear translocation of the MR. Conclusion ANP, via GC-A/cGMP/cGKI signaling in cardiac myocytes, attenuates hypertensive cardiac remodeling and dysfunction. These protective ANP effects seem to be mediated at least in part by counterregulation of the deleterious genomic (MR-mediated) cardiac actions of aldosterone.

Cardiovascular responses evoked by acetylcholine in prelimbic cortex is mediated by nitric oxide – guanylate cyclase mechanism in rats

The stimulation of PL with acetylcholine (ACh) caused depressor response mediated by muscarinic receptors. Studies in literature demonstrated that stimulation of muscarinic receptors induced cyclic GMP formation mediated by nitric oxide. Hence, the objective of this study was to investigate if cardiovascular response caused by muscarinic receptors stimulation is mediated by nitric oxide and cyclic GMP formation. Male Wistar rats (240–280g) were used and guide cannulae were implanted in the PL for drug injection. The cardiovascular responses evoked by ACh (30nmol/200nL) microinjected into the PL were measured before and 15 min after PL treatment with, J 104129 fumarate ‐ M3 antagonist (6nmol/200nL), pirenzepine ‐ M1 antagonist (6nmol/200nL), N‐Propyl ‐ inhibitor of neuronal nitric oxide synthase (nNOS) (0,04nmol/200nL), carboxy‐PTIO ‐ scavenger NO (1nmol/200nL) or ODQ ‐ guanylate cyclase inhibitor (1nmol/200nL). A polyethylene catheter was implanted in the femoral artery for MAP and HR records. The statistical test utilized was two‐way ANOVA. The pre‐treatment with pirenzepine did not blockade the decrease of the MAP (F1,220=6.526; P=0.0113) and increase of the HR (F1,220=0.2313; P=0.6310), while J 104129 fumarate (MAP: F1,220=200.7; P<0.0001; HR: F1,220=170.1; P<0.0001), N‐Propyl (MAP: F1,264=79.04; P<0.0001; HR: F1,264=257.4; P<0.0001), C‐PTIO (MAP: F1,176=115.4; P<0.0001; HR: F1,176=255.7; P<0.0001) or ODQ (MAP: F1,220=165.7; P<0.0001; HR: F1,220=161.3; P<0.0001) blocked depressor and tachycardic responses caused by ACh microinjection into the PL. This results demonstrated that the cardiovascular responses caused by ACh in PL are mediated by local nitric oxide – guanylate cyclase mechanism. Financial Support: CAPES and FAEPA.

Abstract 511: Nitric Oxide Attenuates Matrix Metalloproteinase-9 Production by Endothelial Cells

Impaired nitric oxide (NO) bioavailability and imbalanced matrix metalloproteinase (MMP) activity are key pathogenetic mechanisms involved in cardiovascular diseases. However, there is little evidence supporting a direct link between these mechanisms, although NO is known to interfere with nuclear factor kappa B (NFB) activity, an important modulator of MMP-9 expression. Moreover, it is not known whether the possible effects of NO on MMPs is dependent on cyclic GMP formation. Objective: We examined the effect of NO donors on MMP-9 production by endothelial cells, and if this effect is dependent on cGMP formation or NFB activation. Methods: Human umbilical vein endothelial cells were cultured in appropriate medium and treated for 24 hours with 10 nM phorbol myristate acetate (PMA; a MMP-9 inducer) and other drugs: NO donors (S-nitroso-N-acetylpenicillamine; SNAP, or DetaNONOate), 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a soluble guanylyl cyclase inhibitor), BAY 11-7082 (a NFB inhibitor), or their vehicles. Conditioned media were analyzed by gelatin zimography. Results: PMA increased MMP-9 activity from 0.10±0.08 arbitrary units (AU) to 0.94±0.18 AU ( P< 0.05). Treatment with SNAP (200 or 320 μM) attenuated the increases in MMP-9 activity induced by PMA (0.52±0.16 AU or 0.25±0.10 AU, respectively; both P< 0.05 vs. PMA). DetaNONOate (320 or 400 μM) exerted similar inhibitory effects on MMP-9 activity (0.57±0.13 AU and 0.45±0.06 AU, respectively; both P< 0.05 vs. PMA). Conversely, treatment with ODQ (10 or 32 μM) had no effects on 200 μM SNAP-induced inhibition of PMA-stimulated MMP-9 activity (0.25±020 AU and 0.14±0.11 AU, respectively; both P< 0.05 vs. PMA). Nevertheless, 3.2 and 5 μM BAY 11-7082 decreased PMA-stimulated MMP-9 activity (0.54±0.07 AU and 0.27±0.09 AU, respectively; both P< 0.05 vs. PMA). Conclusion: Our results suggest that NO attenuates MMP-9 production by endothelial cells in a concentration-dependent manner. While this effect is independent of soluble guanylate cyclase activation, it apparently involves inhibition of NFB activity.

Regulation by mitochondrial superoxide and nadph oxidase of cellular formation of nitrated cyclic GMP: Potential implications for NO and ROS signaling

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of oxidative stress response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of reactive oxygen species (ROS). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of hydrogen peroxide were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and hydrogen peroxide. Using the mitochondria-targeted chemical probe MitoSOX Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that NADPH oxidase 2 (Nox2)-generated hydrogen peroxide regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent hydrogen peroxide production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger that may be implicated in regulating ROS signaling in the presence of NO.

Mechanism of Binding of NO to Soluble Guanylyl Cyclase: Implication for the Second NO Binding to the Heme Proximal Site

Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP, is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of binding of NO to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used (15)NO and (14)NO and anaerobic sequential mixing-freeze-quench electron paramagnetic resonance to unambiguously confirm that the heme Fe is the target of the second NO. The linear dependence on NO concentration up to 600 s(-1) for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e., the proximal site of the heme, but also supports a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of five-coordinate NO complexes with the ligand on either the distal or proximal site and supports the selective release of NO from the distal side of the transient bis-NO-sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC complex containing a proximal NO is formed after the binding of the second NO.

Discovery of nitric oxide and cyclic GMP in cell signaling and their role in drug development

The role of nitric oxide in cellular signaling in the past three decades has become one of the most rapidly growing areas in biology. Nitric oxide is a gas and a free radical with an unshared electron that can regulate an ever-growing list of biological processes. Nitric oxide is formed from L-arginine by a family of enzymes called nitric oxide synthases. These enzymes have a complex requirement for a number of cofactors and regulators including NADPH, tetrahydrobioterin, flavins, calmodulin and heme. The enzymes are present in most cells and tissues. In many instances, nitric oxide mediates its biological effects by activating the soluble isoform of guanylyl cyclase and increasing cyclic GMP synthesis from GTP. Cyclic GMP, in turn, can activate cyclic GMP-dependent protein kinase (PKG) and can cause smooth muscles and blood vessels to relax, decrease platelet aggregation, alter neuron function, etc. These effects can decrease blood pressure, increase blood flow to tissues, alter memory and behavior, decrease blood clotting, etc. The list of effects of nitric oxide that are independent of cyclic GMP formation is also growing at a rapid rate. For example, nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids, and other molecules. Some of these reactions result in the oxidation of nitric oxide to nitrite and nitrate to terminate the effect, while other reactions can lead to altered protein structure function and/or catalytic capacity. These effects probably regulate bacterial infections, inflammation of tissues, tumor growth, and other disorders. These diverse effects of nitric oxide that are cyclic GMP dependent or independent can alter and regulate numerous important physiological events in cell regulation and function. Nitric oxide can function as an intracellular messenger, an antacoid, a paracrine substance, a neurotransmitter, or as a hormone that can be carried to distant sites for effects. Thus, it is a unique molecule with an array of signaling functions. However, with any messenger molecule, there can be too little or too much of the substance, resulting in pathological events. Some of the methods to regulate either nitric oxide formation metabolism, or function have been in clinical use for more than a century, as with the use of organic nitrates and nitroglycerin in angina pectoris that was initiated in the 1870s. Inhalation of low concentrations of nitric oxide can be beneficial in premature infants with pulmonary hypertension and increase survival rates. Ongoing clinical trials with nitric oxide synthase inhibitors and nitric oxide scavengers are examining the effects of these agents in septic shock, hypotension with dialysis, inflammatory disorders, cancer therapy, etc. Recognition of additional molecular targets in the areas of nitric oxide and cyclic GMP research will continue to promote drug discovery and development programs in the field. Current and future research will undoubtedly expand the clinician’s therapeutic armamentarium to manage a number of important diseases by perturbing nitric oxide formation and metabolism. Such promise and expectations have obviously fueled the interests in nitric oxide research for a growing list of potential therapeutic applications. There have been and will continue to be many opportunities from nitric oxide and cyclic GMP march to develop novel and important therapeutic agents. There are presently more than 80,000 publications in the area of nitric oxide research. The lecture will discuss our discovery of the first biological effects of nitric oxide and how the field has evolved since our original reports in 1977. The possible utility of this signaling pathway to facilitate novel drug development and the creation of numerous projects in the Pharmaceutical and biotechnology industrials will also be discussed.

Regulation by mitochondrial superoxide and NADPH oxidase of cellular formation of nitrated cyclic GMP: potential implications for ROS signalling

8-Nitro-cGMP (8-nitroguanosine 3',5'-cyclic monophosphate) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of an antioxidant adaptive response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of ROS (reactive oxygen species). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of H2O2 were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and H2O2. Using the mitochondria-targeted chemical probe MitoSOX Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that Nox2 (NADPH oxidase 2)-generated H2O2 regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent H2O2 production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger that may be implicated in regulating ROS signalling in the presence of NO.

Knockdown of natriuretic peptide receptor-A enhances receptor C expression and signalling in vascular smooth muscle cells

Natriuretic peptide receptor-A (NPR-A) knockout mice exhibited an increased blood pressure that may also be attributed to the up-regulation of NPR-C and associated signalling; however, the interaction between the two receptors has not been investigated. In the present study, we investigated the effect of knockdown of NPR-A using NPR-A antisense (AS) on the expression of NPR-C and adenylyl cyclase (AC) signalling in A10 vascular smooth muscle cells (VSMC). The receptor and G protein expression was determined by western blotting, and AC activity was determined by measuring [(32)P]cAMP formation from [α-(32)P]ATP. Treatment of A10 VSMC with NPR-A AS decreased NPR-A and enhanced NPR-C expression without altering the levels of angiotensin II AT1 and muscarinic M2 receptors. In addition, siRNA-NPR-A also resulted in the up-regulation of NPR-C. The re-expression of NPR-A in AS-treated cells reversed the enhanced expression of NPR-C to control levels. In addition, NPR-C-, AT1, and M2 receptor-mediated inhibition of AC and Giα protein expression was enhanced in AS-treated cells, whereas NPR-A-mediated cyclic GMP (cGMP) formation and Gsα-mediated stimulation of AC were significantly reduced. Pertussis toxin treatment attenuated the AS-induced enhanced inhibition of AC to control levels. Furthermore, the enhanced levels of NPR-C and Giα proteins were reversed to control levels by 8-bromo-cGMP (8Br-cGMP) and PD98059, an MEK inhibitor. In addition, 8Br-cGMP also attenuated AS-induced enhanced ERK1/2 phosphorylation to control levels. These results demonstrate that knockdown of NPR-A up-regulates the expression of NPR-C, Giα proteins, and NPR-C-linked AC signalling and suggests a cross-talk between NPR-A and NPR-C.

Decrease of guanylyl cyclase β1 subunit and nitric oxide (NO)-induced relaxation in mouse rectum with colitis and its reproduction on long-term NO treatment

Nitric oxide (NO) influences motility in the colon in patients with ulcerative colitis, but the exact mechanism involved remains unknown. Colitis was induced in mice by the oral administration of 2.5% dextran sodium sulfate (DSS), and the motility in longitudinal preparations from rectum and distal colon and expression of β1 subunit of soluble guanylyl cyclase (sGCβ1) were analyzed. Electrical stimulation (ES) caused a transient relaxation via the NO pathway in both rectum and colon from control mice. Stimulation with sodium nitroprusside (SNP) caused relaxation in the two regions, and the half-time (T (1/2)) of the maximal relaxation induced by 100μM SNP was 8.1 ± 1.0s in rectum. DSS treatment (1) abolished the ES-induced relaxation, but not dibutyryl cyclic GMP-induced response, in both regions, (2) decreased the maximal response to SNP accompanied by a loss of immunoreactive sGCβ1 protein in rectum, but did not affect the amplitude of the relaxant response or the protein in distal colon, and (3) caused an increase in the T (1/2) value in response to SNP in both regions. Pretreatment of both preparations from control mice with 600μM SNP for 30min decreased both ES- and SNP-induced relaxation, SNP-induced cyclic GMP formation, and immunoreactive sGCβ1 levels. NO-mediated relaxation was impaired by a dysfunctional sGC with and without a loss of immunoreactivity to sGCβ1 in rectum and colon from DSS-treated mice, respectively. Long-term exposure of the tissues with an excess amount of NO changes the sGC-mediated relaxation.

Atrial natriuretic peptide increases the albumin permeability of mouse skeletal muscle microcirculation by the caveolae-mediated transcellular pathway

Background Cardiac atrial natriuretic peptide (ANP) is an essential physiological regulator of arterial blood pressure and intravascular volume. Unlike other “natriuretics”, which reduce interstitial fluid volume with little change in plasma volume, ANP has important extrarenal, endothelial actions that enable it to reduce plasma volume preferentially. Thus ANP, via its guanylyl cyclase-A (GC-A) receptor and cyclic GMP formation, enhances microvascular endothelial permeability to albumin, ultimately moving plasma fluid into interstitial pools. However, the cellular pathways mediating this effect are unknown.v Here we investigated the role of caveolae-mediated endothelial albumin transcytosis in the mechanism of increased permeability induced by ANP.

Dietary Spinach Saponin-Enriched Lipophilic Fraction Inhibits Platelet Aggregation and Blood Coagulation

In this study, we investigated the effect of spinach saponin-enriched lipophilic fraction (SSEF) on collagen (10 μg/mL)-stimulated platelet aggregation in vivo. Dietary SSEF dose-dependently inhibited collagen-induced platelet aggregation by decreasing thromboxane A₂ production and intracellular Ca²⁺ agonist activity as an aggregation-inducing autacoidal molecule. In addition, SSEF significantly increased the formation of cyclic AMP and cyclic GMP, intracellular Ca²⁺ antagonists that are aggregation-inhibiting molecules in collagen-stimulated platelets. These results suggest that SSEF is a potent inhibitor of collagen-stimulated platelet aggregation in vivo. Prothrombin time and activated partial thromboplastin time, indicators of blood coagulation, were potently prolonged by dietary SSEF in vivo. These findings suggest that SSEF prolongs the interval time between the conversion of fibrinogen to fibrin. Dietary SSEF also inhibited 0.4 M sucrose-induced hemolysis. Accordingly, our data demonstrate that SSEF might be a useful tool for inhibiting platelet activation and blood coagulation in thrombotic diseases.

NADPH oxidase and eNOS control cardiomyogenesis in mouse embryonic stem cells on ascorbic acid treatment

Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1+) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4–7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-ß and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis.

Guanylate Cyclase Activators, Cell Volume Changes and IOP Reduction

Glaucoma afflicts millions of people worldwide and is a major cause of blindness. The risk to develop glaucoma is enhanced by increases in IOP, which result from deranged flow of aqueous humor. Aqueous humor is a fluid located in the front of the eye that gives the eye its buoyancy and supplies nutrients to other eye tissues. Aqueous humor is secreted by a tissue called ciliary processes and exits the eye via two tissues; the trabecular meshwork (TM) and Schlemm’s canal. Because the spaces through which the fluid flows get smaller as the TM joins the area of the Schlemm’s canal, there is resistance to aqueous humor outflow and this resistance creates IOP. There is a correlation between changes in TM and Schlemm’s canal cell volume and rates of aqueous humor outflow; agents that decrease TM and Schlemm’s canal cell volume, increase the rate of aqueous humor outflow, thus decreasing IOP. IOP is regulated by guanylate cyclase activators as shown in humans, rabbits and monkeys. There are two distinct groups of guanylate cyclases, membrane guanylate cyclase and soluble guanylate cyclase (sGC); activation of both have been shown to decrease IOP. Members of the membrane guanylate cyclase family of receptors bind to peptide ligands, while the sGC responds to gases (such as NO and CO₂) and compounds (such as YC1, [3-(5’-hydroxymethyl-2’furyl)-1-benzyl indazole), a benzyl indazole derivative, and BAY-58-2667); activation of either results in formation of cyclic GMP (cGMP) and activation of protein kinase G (PKG) and subsequent phosphorylation of target proteins, including the high conductance calcium activated potassium channel (BKca channel). While activators of both membrane guanylate cyclase and sGC have the ability to lower IOP, the IOP lowering effects of sGC are noteworthy because sGC activators can be topically applied to the eye to achieve an effect. We have demonstrated that activators of sGC increase the rate at which aqueous humor exits the eye in a time course that correlates with the time course for sGC-induced decreases in TM and Schlemm’s canal cell volume. Additionally, sGC-induced decrease in cell volume is accompanied by both K⁺ and Cl^- efflux induced by activation of K⁺ and Cl^- channels, including the BKca channel and/or K⁺Cl^- symport. This suggests that parallel K⁺Cl^- efflux, and resultant H₂O efflux result in decreases in cell volume. These observations suggest a functional role for sGC activators, and suggest that the sGC/cGMP/PKG systems are potential therapeutic targets in the treatment of glaucoma.

A splice variant of the guanylyl cyclase-A receptor interferes with atrial natriuretic peptide (ANP) signaling

Background Activation of the homodimeric transmembrane guanylyl cyclase-A (GC-A) receptor upon binding of its extracellular ligands, atrial (ANP) and B-type (BNP) natriuretic peptides, leads to cyclic GMP formation in many types of cells. This NP/GC-A pathway has a critical role in the endocrine regulation of arterial blood pressure and volume and in the local counter-regulation of cardiac hypertrophy and fibrosis. Alterations of this system result in arterial hypertension, hypervolemia and cardiac hypertrophy. Many studies have shown that exposure of GC-A to high concentrations of ANP/BNP or to growth hormones such as angiotensin II (Ang II) or endothelin provokes homologous versus heterologous desensitization of the receptor [1]. However, the mechanisms accounting for this loss of function of GC-A in vivo are largely unknown. In the present study we identified and characterized a novel isoform of GC-A (GC-AΔLys-Gln 330) with unique structural properties. Our data reveal that this splice variant functions as dominant negative isoform and suggest that increased alternative splicing of GC-A may contribute to homologous or heterologous desensitization of the NP/GC-A system.