Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:4.6.1.2 (guanylate cyclase)
8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide, a messenger molecule with multiple clinical implications. Understanding the activation of sGC is an important step for establishing new therapeutic principles. We have now overexpressed sGC in a baculovirus/Sf9 system optimized for high protein yields to facilitate spectral and kinetic studies of the activation mechanisms of this enzyme. It was expressed in a batch fermenter using a defined mixture of viruses encoding the alpha and beta1 subunits of the rat lung enzyme. The expressed enzyme was purified from the cytosolic fraction by anion exchange chromatography, hydroxyapatite chromatography, and size exclusion chromatography. By use of this new method 2.5 l culture yielded about 1 mg of apparently homogeneous sGC with a content of about one heme per heterodimer without the need of a heme reconstitution step. The enzyme did not contain stoichiometric amounts of copper. The basal activities of the purified enzyme were 153 and 1259 nmol min(-1) mg(-1) in the presence of Mg2+ and Mn2+, respectively. The nitric oxide releasing agent 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) stimulated the enzyme 160-fold with Mg2+, whereas the NO-independent activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) induced an increase in the activity of 101-fold at a concentration of 300 microM. The combination of DEA/NO (10 microM) and YC-1 (100 microM) elicited a dose-dependent synergistic stimulation with a maximum of a 792-fold increase over the basal activity in the presence of Mg2+, resulting in a specific activity of 121 micromol min(-1) mg(-1). The synergistic stimulation of DEA/NO and YC-1 was attenuated by the sGC inhibitor 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ) (10 microM) by 94%. In a different experimental setup a saturated carbon monoxide solution in the absence of ambient oxygen or NO stimulated the enzyme 15-fold in the absence and 1260-fold in the presence of YC-1 compared to an argon control. The heme spectra of the enzyme showed a shift of the Soret peak from 432 to 399 and 424 nm in the presence of DEA/NO or carbon monoxide, respectively. The heme spectra were not affected by YC-1 in the absence or in the presence of DEA/NO or of carbon monoxide, which reflects the fact that YC-1 does not interact directly with the heme group of the enzyme. In summary, this study shows that our expression/purification procedure is suitable for producing large amounts of highly pure sGC which contains one heme per heterodimer without a reconstitution step. The activator experiments show that in a synergistic stimulation with YC-1 sGC can be activated maximally both by nitric oxide and by carbon monoxide and that YC-1 does not directly act via heme. The described method should help to facilitate the investigation of the new therapeutic principle of NO-independent guanylyl cyclase activators.
...
PMID:Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide. 993 Sep 22

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.
...
PMID:Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line. 1035 96

To investigate the effect of nitric oxide (NO) on the release of serotonin and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA), the posterior hypothalamus of the conscious rat was superfused through a push-pull cannula with drugs which either liberate NO, or inhibit NO synthase (NOS). The NO donors, linsidomine, diethylamine/nitric oxide (DEA/NO), S-nitroso-N-acetylpenicillamine (SNAP), S-nitroso-glutathione (SNOG) and sodium nitroprusside influenced the release of serotonin in a biphasic way. Low concentrations of drugs diminished, while higher concentrations of these compounds enhanced the outflow of serotonin. The NOS inhibitors N(G)-methyl-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NINA) enhanced the serotonin release. A high concentration of L-NAME slightly diminished the outflow of serotonin. Inhibition of the guanylyl cyclase by oxodiazolo[4, 3]quinoxaline-one (ODQ) abolished the changes in serotonin outflow induced by both low and high concentrations of linsidomine. The extracellular concentration of the 5-HIAA was not influenced by the compounds used. These data suggest that endogenous NO modulates the release of serotonin in a biphasic and cGMP-dependent way.
...
PMID:Nitric oxide modulates the release of serotonin in the rat hypothalamus. 1041 93

The effects of the different types of soluble guanylate cyclase (sGC) stimulators on the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) in both human and rat platelets were studied under in vitro and in vivo conditions. sGC-dependent VASP phosphorylation (at Ser(239) and Ser(157)) both by the new direct sGC stimulator YC-1 and by NO donors was examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS/PAGE) with different antibodies. One antibody, which recognizes VASP independent of its phosphorylation state, was used to detect the mobility shift of VASP caused by Ser(157) phosphorylation. The other antibody was specifically directed against VASP phosphorylated at Ser(239), the cGMP-dependent protein kinase (PKG) preferred phosphorylation site of VASP. In vitro YC-1 increased both VASP phosphorylation and cyclic guanosine monophosphate (cGMP) levels as did the NO donors 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) and sodium nitroprusside (SNP). The combination of both types induced a synergistic effect in both VASP phosphorylation and cGMP increase. In rat platelets, similar effects could be shown in vitro. In vivo we observed a significant increase in cGMP and a distinct effect on VASP phosphorylation in rat platelets 1 h after oral administration of YC-1. These biochemical alterations are supported by a significant prolongation in rat-tail bleeding time. Direct stimulators of sGC like YC-1 are on the one hand direct potent stimulators of the cGMP/PKG/VASP pathway in platelets and on the other hand synergize with NO, the physiologic stimulator of sGC. Therefore YC-1-like substances are interesting tools for the development of new cardiovascular drugs with vasodilatory and antithrombotic properties.
...
PMID:The vasodilator-stimulated phosphoprotein (VASP): target of YC-1 and nitric oxide effects in human and rat platelets. 1071 Jan 23

Nitric oxide (NO) acts as a neurotransmitter and neuromodulator in the nervous system of many vertebrates and invertebrates. The effects of extracellularly applied sodium nitroprusside (SNP) and diethylamine NO (C(2)H(5))(2)N[N(O)NO]-Na(+) (DEA/NO), NO donors, on a glutamate (Glu)-induced K(+) current in identified Onchidium neurons were investigated using voltage clamp and pressure ejection techniques. Bath-applied SNP (10 microM) and DEA/NO (5-10 microM) reduced the Glu-induced K(+) current without affecting the resting membrane conductance and holding current. The Glu-induced K(+) current also was inhibited by the focal application of SNP to the neuron somata. The suppressing effects of NO donors were concentration-dependent and completely reversible. Pretreatment with hemoglobin (50 microM), a nitric oxide scavenger, and 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), a specific inhibitor of NO-stimulated guanylate cyclase, decreased the SNP-induced inhibition of the Glu-induced current. Bath-applied 50 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, or intracellular injection of 1 mM guanosine 3',5'-cyclic monophosphate (cGMP) inhibited the Glu-induced current, mimicking the effect of NO donors. These results demonstrate that SNP and DEA/NO inhibit the Glu-induced K(+) current and that the mechanism of NO inhibition of the Glu-induced current involves cGMP-dependent protein kinase.
...
PMID:Inhibition of the glutamate-induced K(+) current in identified Onchidium neurons by nitric oxide donors. 1082 Apr 35

Extracellular recording techniques were used to study the effects of the nitric oxide releasing agents diethylamine-NO (DEA-NO) and S-nitroso-N-acetyl-penicillamine (SNAP) on synaptic transmission in the intermediate and medial part of the hyperstriatum ventrale (IMHV), a part of the domestic chick forebrain that is essential for some forms of early learning. The field response evoked by local electrical stimulation was recorded in the IMHV in an in vitro slice preparation. DEA-NO (100-200 mgr) significantly depressed the field response in a concentration dependent and reversible manner. However, the depression produced by perfusion with 400 mgr DEA-NO, was not reversed following washout of the drug. With 400 mgr DEA-NO, NO reaches a maximum concentration of 10 mgr at 2 min of perfusion, and then declines slowly. SNAP (400 mgr) produced an effect similar to 400 mgr DEA-NO. Neither the immediate nor the longer-term depressive effect of NO is mediated by activation of guanylyl cyclase because in the presence of both low and high doses of ODQ, a potent and selective inhibitor of NO-stimulated guanylyl cyclase, NO produced the same depression of the field response. There is evidence however that the IMHV possesses c-GMP responsive elements since direct perfusion of 8-Br-cGMP (1 mM) produced a long-term but not an immediate depression. The long-term depression produced by 400 mgr DEA-NO was eliminated in the presence of either a selective adenosine A(1) receptor antagonist or an ADP-ribosyltransferase inhibitor. It was also possible to prevent the long-term effect in the presence of tetraethyl ammonium a K(+)-channel blocker. These results suggest that the NO may be acting presynaptically in a synergistic fashion with the adenosine A(1) receptor to depress transmitter release.
...
PMID:Effects of nitric oxide release in an area of the chick forebrain which is essential for early learning. 1083 95

Dopamine-beta-hydroxylase (DbetaH) is a copper-containing enzyme that uses molecular oxygen and ascorbate to catalyze the addition of a hydroxyl group on the beta-carbon of dopamine to form norepinephrine. While norepinephrine causes vasoconstriction following reflex sympathetic stimulation, nitric oxide (NO) formation results in vasodilatation via a guanylyl cyclase-dependent mechanism. In this report, we investigated the relationship between NO and DbetaH enzymatic activity. In the initial in vitro experiments, the activity of purified DbetaH was inhibited by the NO donor, diethylamine/NO (DEA/NO), with an IC(50) of 1 mm. The inclusion of either azide or GSH partially restored DbetaH activity, suggesting the involvement of the reactive nitrogen oxide species, N(2)O(3). Treatment of human neuroblastoma cells (SK-N-MC) with diethylamine/NO decreased cellular DbetaH activity without affecting their growth rate and was augmented by the depletion of intracellular GSH. Co-culture of the SK-N-MC cells with interferon-gamma and lipopolysaccharide-activated macrophages, which release NO, also reduced the DbetaH activity in the neuroblastoma cells. Our results are consistent with the hypothesis that nitrosative stress, mediated by N(2)O(3), can result in the inhibition of norepinephrine biosynthesis and may contribute to the regulation of neurotransmission and vasodilatation.
...
PMID:Inhibitory effects of nitric oxide and nitrosative stress on dopamine-beta-hydroxylase. 1088 4

Nitric oxide (NO) has concentration-dependent biphasic myocardial contractile effects. We tested the hypothesis, in isolated rat hearts, that NO cardiostimulation is primarily non-cGMP dependent. Infusion of 3-morpholinosydnonimine (SIN-1, 10(-5) M), which may participate in S-nitrosylation (S-NO) via peroxynitrite formation, increased the rate of left ventricular pressure rise (+dP/dt; 19 +/- 4%, P < 0.001, n = 11) without increasing effluent cGMP or cAMP. Superoxide dismutase (SOD; 150 U/ml) blocked SIN-1 cardiostimulation and led to cGMP elaboration. Sodium nitroprusside (10(-10)-10(-7) M), an iron nitrosyl compound, did not augment +dP/dt but increased cGMP approximately eightfold (P < 0.001), whereas diethylamine/NO (DEA/NO; 10(-7) M), a spontaneous NO. donor, increased +dP/dt (5 +/- 2%, P < 0.05, n = 6) without augmenting cGMP. SIN-1 and DEA/NO +dP/dt increase persisted despite guanylyl cyclase inhibition with 1H-(1,2,4)oxadiazolo-(4,3,-a)quinoxalin-1-one (10(-5) M, P < 0.05 for both donors), suggesting a cGMP-independent mechanism. Glutathione (5 x 10(-4) M, n = 15) prevented SIN-1 cardiostimulation, suggesting S-NO formation. SIN-1 also produced SOD-inhibitable cardiostimulation in vivo in mice. Thus peroxynitrite and NO donors can stimulate myocardial contractility independently of guanylyl cyclase activation, suggesting a role for S-NO reactions in NO/peroxynitrite-positive inotropic effects in intact hearts.
...
PMID:cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation. 1100 88

Nitric oxide (NO) donors increase heart rate (HR) through a guanylyl cyclase-dependent stimulation of the pacemaker current I(f), without affecting basal I(Ca-L). The activity of I(f)is known to be enhanced by cyclic nucleotides and by an increase in cytosolic Ca(2+). We examined the role of cGMP-dependent signaling pathways and intracellular Ca(2+)stores in mediating the positive chronotropic effect of NO donors. In isolated guinea pig atria, the increase in HR in response to 1-100 micromol/l 3-morpholino-sydnonimine (SIN-1; with superoxide dismutase, n=6) or diethylamine-NO (DEA-NO, n=8) was significantly attenuated by blockers of the cGMP-inhibited phosphodiesterase (PDE3; trequinsin, milrinone or Ro-13-6438, n=22). In addition, the rate response to DEA-NO or sodium nitroprusside (SNP) was significantly reduced following inhibition of PKA (KT5720 or H-89, n=15) but not PKG (KT5728 or Rp-8-pCPT-cGMPs, n=16). Suppression of sarcoplasmic (SR) Ca(2+)release by pretreatment of isolated atria with ryanodine or cyclopiazonic acid (2 micromol/l and 60 micromol/l, n=16) significantly reduced the chronotropic response to 1-100 micromol/l SIN-1 or DEA-NO. Moreover, in isolated guinea pig sinoatrial node cells 5 micromol/l SNP significantly increased diastolic and peak Ca(2+)fluorescence (+13+/-1% and +28+/-1%, n=6, P<0.05). Our findings are consistent with a functionally significant role of cAMP/PKA signaling (via cGMP inhibition of PDE3) and SR Ca(2+)in mediating the positive chronotropic effect of NO donors.
...
PMID:Role of cGMP-inhibited phosphodiesterase and sarcoplasmic calcium in mediating the increase in basal heart rate with nitric oxide donors. 1101 27

Inhibition of platelet activation by nitric oxide (NO) is not exclusively cGMP-dependent. Here, we tested whether inhibition of platelet aggregation by structurally distinct NO donors is mediated by different mechanisms, partly determined by the site of NO release. Glyceryl trinitrate (GTN), sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO), diethylamine diazeniumdiolate (DEA/NO), and a novel S-nitrosothiol, RIG200, were examined in ADP (8 microM)- and collagen (2.5 microgram/ml)-activated human platelet rich plasma. GTN was a poor inhibitor of aggregation whilst the other NO donors inhibited aggregation, irrespective of agonist. These effects were abolished by the NO scavenger, hemoglobin (Hb; 10 microM, P < 0.05, n = 6), except with high concentrations of DEA/NO, when NO concentrations exceeded the capacity of Hb. However, experiments with the soluble guanylate cyclase inhibitor, ODQ (100 microM), indicated that only SNP-mediated inhibition was exclusively cGMP-dependent. Furthermore, the cGMP-independent effects of S-nitrosothiols were distinct from those of DEA/NO, suggesting that different NO-related mediators (e.g., nitrosonium and peroxynitrite, respectively) are responsible for their actions.
...
PMID:Inhibition of human platelet aggregation by nitric oxide donor drugs: relative contribution of cGMP-independent mechanisms. 1111 1


<< Previous 1 2 3 4 5 6 7 Next >>

---