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

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

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.
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PMID:Regulation of neuronal nitric oxide and cyclic GMP formation by Ca2+. 127 21

(6R)-5,6,7,8-Tetrahydro-L-biopterin (R-THBP) is a cofactor not only for aromatic amino acid hydroxylases in mammalian tissues but also for nitric oxide synthase (NOS) induced by endotoxins or cytokines in some kinds of cells. Recently it has been reported that nitric oxide (NO) has biological activity in endothelium and in brain as well. NO activates soluble guanylate cyclase (sGC). Superoxide reacts with NO easily and shortens the half-life of NO actions. We found, in a study using rat cerebellar cytosol fraction, that R-THBP itself did not directly activate sGC, but activated sGC at concentrations ranging from 0.1 to 10 microM only under NO generating conditions of activated NOS and in the presence of sodium nitroprusside. In addition, R-THBP (1 microM) did not alter the NOS activity, which was determined by L-citrulline formation. These results suggest that R-THBP may regulate sGC activity associated with NO formation in the central nervous system.
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PMID:(6R)-5,6,7,8-tetrahydro-L-biopterin modulates nitric oxide-associated soluble guanylate cyclase activity in the rat cerebellum. 135 30

NO synthase (NOS; EC 1.14.23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [32P]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities. 137 27

Nitric oxide (NO) release accounts for the biological activity of endothelium-derived relaxing factor. Given that tumor necrosis factor-alpha (TNF-alpha) has been implicated as an important mediator in septic shock, we explored whether TNF-alpha enhances L-arginine-dependent synthesis of NO and L-citrulline in endothelial cells. The release of NO was detected in a coincubation bioassay where measurement of guanosine 3',5'-cyclic monophosphate (cGMP) production in reporter monolayers, namely glomerular mesangial cells or fetal lung fibroblasts, reflected activation of soluble guanylate cyclase. Reporter monolayer cGMP content was greater in the presence of TNF-alpha-treated bovine aortic and renal artery endothelial cells than in the presence of vehicle-treated endothelial cells. TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were first evident at 8 h and maximal at 16-24 h. In addition, TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were abrogated by hemoglobin and methylene blue, blunted by N omega-nitro-L-arginine and augmented by superoxide dismutase and the calcium agonist bradykinin. These observations suggested that TNF-alpha enhanced release of NO. Furthermore, the formation of L-[14C]citrulline from L-[14C]arginine, as determined by quantitative cation-exchange chromatography and thin-layer chromatography, was enhanced by TNF-alpha in a time- and concentration-dependent manner. Thus it is evident that endothelial cells release NO for a prolonged period in response to TNF-alpha and transiently when stimulated with calcium agonists. The prolonged release of NO from TNF-alpha-stimulated endothelial cells may be implicated in the pathogenesis of septic shock.
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PMID:Nitric oxide synthesis in endothelial cells: evidence for a pathway inducible by TNF-alpha. 165 67

Oxidized low-density lipoprotein (LDLox) is a molecule with strong atherogenic properties. In a concentration dependent fashion, LDLox antagonized the activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor (EDRF), which was produced in vitro by incubation of a partially purified EDRF-forming enzyme in the presence of L-arginine, Ca2+ and NADPH. The inhibitory effect of LDLox was potentiated by preincubation of the soluble guanylate cyclase with LDLox, but not when the EDRF-forming enzyme was pretreated with LDLox. As LDLox did not diminish the calmodulin-dependent conversion of L-arginine into L-citrulline by the EDRF-forming enzyme it would appear that EDRF-biosynthesis was not affected by LDLox. It is suggested that the impaired relaxant response of atherosclerotic blood vessels to endothelium-dependent vasodilators was not due to a reduced formation of EDRF but due to a diminished responsiveness of soluble guanylate cyclase.
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PMID:Oxidized low-density lipoprotein antagonizes the activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor but does not interfere with its biosynthesis. 168 84

The soluble form of guanylyl cyclase-activating-factor (GAF) synthase from rat cerebellum was purified to homogeneity by sequential affinity chromatographic steps on adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin-agarose. Enzyme activity during purification was bioassayed by the L-arginine-, NADPH-, and Ca2+/calmodulin-dependent formation of a plasma membrane-permeable nitric oxide-like factor that stimulated soluble guanylyl cyclase in RFL-6 cells. With calmodulin and NADPH as cofactors, purified soluble GAF synthase induced an increase of 1.05 mumol of cGMP per 10(6) RFL-6 cells per 3 min per mg of protein. The coproduct of this signal-transduction pathway appeared to be L-citrulline. GAF synthase catalyzed the conversion of 107 nmol of L-arginine into L-citrulline per min per mg of protein. Based on these assays, this represents a purification of GAF synthase of approximately 10,076- and 8925-fold with recoveries of 16% and 19%, respectively. Rechromatography of the purified enzyme on Mono P (isoelectric point = 6.1 +/- 0.3), Mono Q, and Superose 12 or 6 resulted in no further purification or increase in specific activity. A Stokes radius of 7.9 +/- 0.3 nm and a sedimentation coefficient s20,w of 7.8 +/- 0.2 S were used to calculate a molecular mass of about 279 +/- 25 kDa for the native enzyme. SDS/PAGE revealed a single protein band with a molecular mass of about 155 +/- 3 kDa. These data suggest that soluble GAF synthase purified from rat cerebellum is a homodimer of 155-kDa subunits and that enzyme activity is dependent upon the presence of calmodulin.
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PMID:Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase. 170 96

Amphiphiles are known to modulate the activity of ATPase, phospholipase A2, adenylate and guanylate cyclase amongst others and relax vascular smooth muscle. The effect of two amphiphiles, lysophosphatidylcholine (LPC) and digitonin on the activity of nitric oxide synthase (NOS), as measured by conversion of radiolabeled L-arginine to L-citrulline, has been studied. Neither digitonin (0.01 mmol/l) nor LPC (0.01 mmol/l) influenced NOS activity in endothelial cell homogenates. Digitonin but not LPC stimulated NOS in intact endothelial cells. NOS activity was markedly inhibited by L- but not by D-omega-nitroarginine (D-NNA, 0.1 mmol/l). L-NNA or D-NNA data demonstrate no effect of amphiphiles on isolated NOS. NOS activation may occur as a result of detergent action on the membrane.
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PMID:Effect of amphiphiles on nitric oxide synthase in endothelial cells. 751 49

Nitric oxide is known to function as a neurotransmitter in the central nervous system. It is also known to be involved in the central nervous system excitatory amino acid neurotransmission cascade. Activation of excitatory amino acid receptors causes an influx of calcium, which activates nitric oxide synthase. The resulting increase in intracellular nitric oxide activates soluble guanylate cyclase, leading to a rise in cyclic guanosine monophosphate. The excitatory amino acids glutamate and aspartate are found in the vestibular system and have been postulated to function as vestibular system neurotransmitters. Although nitric oxide has been investigated as a neurotransmitter in other tissues, no published studies have examined the role of nitric oxide in the vestibular system. Neuronal NADPH-diaphorase has been characterized as a nitric oxide synthase. This enzyme catalyzes the conversion of L-arginine to L-citrulline, producing nitric oxide during the reaction. We used a histochemical stain characterized by Hope et al. (Proc Natl Acad Sci 1991;88:2811) as specific for neuronal nitric oxide synthase to localize the enzyme in the rat vestibular system. An immunocytochemical stain was used to examine rat inner ear tissue for the presence of the enzyme's end product, L-citrulline, thereby demonstrating nitric oxide synthase activity. Staining of vestibular ganglion sections showed nitric oxide synthase presence and activity in ganglion cells and nerve fibers. These results indicate the presence of active nitric oxide synthase in these tissues and suggest modulation of vestibular neurotransmission by nitric oxide.
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PMID:Nitric oxide in the rat vestibular system. 752 6

Methylene blue appears to inhibit nitric oxide-stimulated soluble guanylyl cyclase and has been widely used for inhibition of cGMP-mediated processes. We report here that endothelium-dependent relaxation of isolated blood vessels and NO synthase-dependent cGMP formation in cultured endothelial cells were both markedly more sensitive to inhibition by methylene blue than effects induced by direct activation of soluble guanylyl cyclase. These discrepancies were also observed when superoxide dismutase (SOD) was present to protect NO from inactivation by superoxide anion. Subsequent experiments showed that formation of L-citrulline by purified NO synthase was completely inhibited by 30 microM methylene blue (IC50 = 5.3 and 9.2 microM in the absence and presence of SOD, respectively), whereas guanylyl cyclase stimulated by S-nitrosoglutathione was far less sensitive to the drug (50% inhibition at approximately 60 microM, and maximal inhibition of 72% at 1 mM methylene blue). Experimental evidence indicated that oxidation of NADPH, tetrahydrobiopterin or reduced flavins does not account for the inhibitory effects of methylene blue. Our data suggest that methylene blue acts as a direct inhibitor of NO synthase and is a much less specific and potent inhibitor of guanylyl cyclase than hitherto assumed.
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PMID:Inhibition of nitric oxide synthesis by methylene blue. 767 77

The mechanism for myometrial quiescence during pregnancy is unknown. cGMP plays an integral role in the relaxation of smooth muscle, and nitric oxide (NO) is the most important endogenous activator of soluble guanylate cyclase. The purpose of this study was to determine the effect of gestational age on myometrial cGMP and NO synthase (NOS) activity in the guinea pig. Myometrial cGMP content (measured by RIA) rose slowly until 0.49 (fraction of pregnancy completed) gestation before abruptly increasing to 200 times the non-pregnant control value. It then declined precipitously after 0.87 gestation. Of the known isoenzymes of NOS, the messenger RNAs coding for both endothelial and neuronal NOS could be amplified from the myometrium of pregnant and nonpregnant animals using reverse transcriptase-polymerase chain reaction, but inducible NOS messenger RNA was not found. Myometrial calcium-dependent NOS activity (measured by the conversion of L-[U-14C]arginine to [U-14C]citrulline) declined slowly with advancing gestation (r2 = 0.096; slope = -0.34; P = 0.01), but never differed significantly from the activity in nonpregnant animals [31.1 +/- 11 (term pregnancy) vs. 56.9 +/- 16 (nonpregnant) pmol/min.g; P = NS]. Calcium-independent activity declined shortly after conception, and then rose toward the nonpregnant level (r2 = 0.19; slope = 0.45; P = 0.0009). However, at no time was it significantly different from that in the nonpregnant animal. Pregnancy had no effect on myometrial L-arginine and L-citrulline content. The administration of L-nitro-arginine methyl ester (200 mg/kg) to inhibit NOS dramatically increased blood pressure and reduced fetal renal NOS activity, but had no effect on the myometrial cGMP content. Estradiol (500 micrograms/kg for 5 days) modestly increased cGMP, but in contrast to many tissues in which estradiol increases NOS, it had no effect on myometrial NOS activity. We conclude that pregnancy dramatically increases cGMP by a mechanism independent of NOS. The stimulus remains to be identified. The temporal change in cGMP concentration is consistent with the hypothesis that cGMP mediates myometrial quiescence during pregnancy.
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PMID:Pregnancy increases guanosine 3',5'-monophosphate in the myometrium independent of nitric oxide synthesis. 798 34


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