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)

The cellular mechanism of bioactivation underlying guanylate cyclase activation by organic nitrates was investigated. In cultured rat lung fibroblasts (RFL-6 cells), the inhibitor of cytochrome P-450 proadifen (0.1 mM) decreased cyclic GMP stimulation by glyceryl trinitrate (GTN, 1-100 microM) by up to 81%. Cyclic GMP stimulation by isoidide dinitrate was inhibited to a similar degree under these conditions. However, proadifen did not affect cyclic GMP stimulation by sodium nitroprusside that spontaneously releases nitric oxide. Cyclic GMP stimulation in RFL-6 cells by GTN remained unaltered in the presence of the inhibitor of glutathione S-transferase sulfobromophthalein. In the same cell type, a 24-hr pretreatment with the inducer of cytochrome P-450 3-methylcholanthrene (10 microM) augmented cyclic GMP stimulation by GTN or isoidide dinitrate by up to 102%. Cultured porcine aortic endothelial cells were found to be without a cyclic GMP response to GTN, although sodium nitroprusside produced a marked cyclic GMP elevation in these cells. The endothelial cells remained unresponsive to GTN even in the presence of N-acetylcysteine (5 mM). Moreover, in a cell-free preparation from rat liver, glutathione-dependent biotransformation of GTN was not accompanied by activation of soluble guanylate cyclase. These findings suggest that in intact cells bioactivation of, i.e., nitric oxide formation from organic nitrates is mediated by a cytochrome P-450 enzyme system rather than by glutathione S-transferase or free thiols.
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PMID:Cytochrome P-450 mediates bioactivation of organic nitrates. 135 50

Glyceryl trinitrate, isosorbide dinitrate, and isosorbide-5-mononitrate are organic nitrate esters commonly used in the treatment of angina pectoris, myocardial infarction, and congestive heart failure. Organic nitrate esters have a direct relaxant effect on vascular smooth muscles, and the dilation of coronary vessels improves oxygen supply to the myocardium. The dilation of peripheral veins, and in higher doses peripheral arteries, reduces preload and afterload, and thereby lowers myocardial oxygen consumption. Inhibition of platelet aggregation is another effect that is probably of therapeutic value. Effects on the central nervous system and the myocardium have been shown but not scrutinized for therapeutic importance. Both the relaxing effect on vascular smooth muscle and the effect on platelets are considered to be due to a stimulation of soluble guanylate cyclase by nitric oxide derived from the organic nitrate ester molecule through metabolization catalyzed by enzymes such as glutathione S-transferase, cytochrome P-450, and possibly esterases. The cyclic GMP produced by the guanylate cyclase acts via cGMP-dependent protein kinase. Ultimately, through various processes, the protein kinase lowers intracellular calcium; an increased uptake to and a decreased release from intracellular stores seem to be particularly important.
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PMID:Mechanisms of action of nitrates. 787 67

We investigated the role of glutathione S-transferases (enzymes known to biotransform organic nitrates) in the vascular action of glyceryl trinitrate (GTN). Relaxation of phenylephrine-contracted rat aortic strips was assessed in the presence or absence of the glutathione S-transferase inhibitors Basilen Blue, bromosulfophthalein, Rose Bengal, hematin, chlorotriphenyltin, and (octyloxy)benzoylvinylglutathione. Whereas none of the inhibitors increased the EC50 for GTN relaxation, glutathione S-transferase activity in the 100,000 x g supernatant fraction of rat aorta was inhibited markedly by most of the inhibitors. In addition, GTN-stimulated activation of aortic guanylyl cyclase in broken-cell preparations was attenuated by all of the glutathione S-transferase inhibitors, suggesting a direct inhibitory action on guanylyl cyclase. In other experiments using aortic strips preexposed to phenylephrine, the inhibitors had no effect on GTN-induced cyclic GMP accumulation or on vascular biotransformation of GTN. In contrast, both Basilen Blue and bromosulfophthalein significantly inhibited GTN-induced relaxation of K(+)-contracted aortic strips, and Basilen Blue significantly inhibited GTN biotransformation in aortic strips preexposed to 25 mM K+. This may be due to a more favourable electrochemical gradient for entry of the inhibitors into membrane-depolarized tissues. We conclude that vascular glutathione S-transferases play a role in mediating the vasodilator actions of GTN in intact tissues in vitro, but that this appears to depend upon the nature of the contractile agent used in such studies.
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PMID:Effect of inhibitors of glutathione S-transferase on glyceryl trinitrate activity in isolated rat aorta. 810 Apr 77

We have proposed that glutathione S-transferases (GSTs), especially the mu isozyme, play a critical role in the metabolism of nitroglycerin (glyceryl trinitrate, GTN), leading to pharmacologic effects. Here we study this enzyme(s) during tolerance development in male New Zealand white rabbits. Each aorta was divided into two segments designated as GTN pretreated and buffer control. Tolerance was induced in rabbit aortic strips so assigned by incubation with GTN (0.22 mM). The activity of the mu isozyme and of total GSTs was determined in portions f each segment. In each rabbit aorta, the response to GTN (0.5 microM) was determined in GTN-pretreated and buffer-pretreated strips by measuring cyclic GMP levels (N = 7 pairs) and percent relaxation (N = 4 pairs). In GTN-pretreated strips, a significant decrease was observed in the activity of the mu isozyme of GST, while the total GST activity was unchanged as compared with control strips. The decrease in isozyme activity correlated very well with the decrease in response to GTN. Two rabbit aortae did not become tolerant, and the activity of the mu isozyme was also not affected. The levels of thiols were not affected by GTN pretreatment and aortae tolerant to GTN did not develop tolerance to S-nitroso acetylpenicillamine (SNAP), indicating that thiol depletion and guanylate cyclase desensitization probably play a minor role in tolerance development to GTN in our model. These studies suggest that tolerance to GTN in rabbit aorta in vitro is associated with a decrease in GST mu activity, which correlates well with the decrease in GTN response.
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PMID:Tolerance to nitroglycerin in rabbit aorta. Investigating the involvement of the mu isozyme of glutathione S-transferases. 878 52

The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions. Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography. Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor. Purified antibodies recognized a single protein of Mr 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells. These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E. coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation. The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction.
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PMID:Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies. 881 50

It is generally accepted that the biotransformation of organic nitrates to an activator of soluble guanylyl cyclase (presumably NO) is a prerequisite for their vasodilator actions. The glutathione S-transferases (GSTs) mediate glyceryl trinitrate (GTN) biotransformation, but whether this results in guanylyl cyclase activation and relaxation of vascular smooth muscle is equivocal. We used electroporation of adherent cultured cells to deliver the membrane-impermeable GST inhibitor basilen blue (BB) into porcine kidney epithelial cells. This resulted in significant inhibition of GTN biotransformation because of a reduction in the formation of glyceryl-1,2-dinitrate, but not glyceryl-1, 3-dinitrate. In the 105,000 x g supernatant fraction of porcine kidney epithelial cells, BB significantly inhibited the formation of both GTN metabolites. Electroporation of porcine kidney epithelial cells with BB also inhibited GTN-induced cyclic GMP accumulation. This was caused in part by inhibition of soluble guanylyl cyclase by BB. To differentiate BB-mediated inhibition of the bioactivation of GTN from its inhibitory effect on guanylyl cyclase, inhibition of cyclic GMP accumulation induced by GTN and that induced by the spontaneous NO-releasing compound, t-butyl-S-nitrosothiol were compared. Maximum inhibition of cyclic GMP accumulation by BB was 80% and 40% with GTN and t-butyl-S-nitrosothiol as the stimulating compounds, respectively. These data suggest that GSTs mediate the biotransformation of GTN to an activator of guanylyl cyclase and support the contention that vascular GSTs participate in mediating the relaxant effects of organic nitrates.
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PMID:Inhibition of the pharmacological actions of glyceryl trinitrate after the electroporetic delivery of a glutathione S-transferase inhibitor. 896 80

Reactive oxygen species modulate the cell growth of a wide variety of mammalian cells. To determine whether oxidative metabolism is altered during the differentiation process, we studied the expression of pro- and antioxidant proteins in proliferating and differentiated CaCo-2 cells, a human colon adenocarcinoma cell line. Nitric oxide synthase type 2 (iNOS) produces nitric oxide (NO). Depending on its rate of synthesis, NO may either promote cellular and DNA damage or reduce the ability of other free radicals to induce cell injury. Using Western and Northern blot analysis and arginine conversion assay, we demonstrate that the expression of iNOS decreases when cells undergo differentiation. This biological event entails a diminished production of NO metabolites and correlates with the loss of activation of soluble guanylate cyclase activity. In differentiated cells, a 2-fold down-regulation of the nuclear factor kappa B activity was observed, suggesting that nuclear factor kappa B could be one of the iNOS gene regulatory factors in the CaCo-2 model. In parallel, we studied the expression of other antioxidant proteins including glutathione S-transferase alpha (GST alpha), bcl-2, and the metallothioneins (MTs). We show that the protein levels of GST alpha and MT increase during the differentiation of CaCo-2 cells, whereas bcl-2 levels decrease. Our investigation indicates that the expression of iNOS, GST alpha, bcl-2, and MT is associated with the enterocytic differentiation. The shift in the expression of specific antioxidant genes during CaCo-2 cell differentiation may occur to avoid alterations in the cell redox potential.
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PMID:Decreased activity of inducible nitric oxide synthase type 2 and modulation of the expression of glutathione S-transferase alpha, bcl-2, and metallothioneins during the differentiation of CaCo-2 cells. 904 Sep 48

Soluble guanylyl cyclase (sGC) is an alpha/beta-heterodimeric hemoprotein that, upon interaction with the intercellular messenger molecule NO, generates cGMP. Although the related family of particulate guanylyl cyclases (pGCs) forms active homodimeric complexes, it is not known whether homodimerization of sGC subunits occurs. We report here the expression in Sf9 cells of glutathione S-transferase-tagged recombinant human sGCalpha1 and beta1 subunits, applying a novel and rapid purification method based on GSH-Sepharose affinity chromatography. Surprisingly, in intact Sf9 cells, both homodimeric GSTalpha/alpha and GSTbeta/beta complexes were formed that were catalytically inactive. Upon coexpression of the respective complementary subunits, GSTalpha/beta or GSTbeta/alpha heterodimers were preferentially formed, whereas homodimers were still detectable. When subunits were mixed after expression, e.g. GSTbeta and beta or GSTalpha and beta, no dimerization was observed. In conclusion, our data suggest the previously unrecognized possibility of a physiological equilibrium between homo- and heterodimeric sGC complexes.
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PMID:Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione s-transferase affinity tag. 1037 11

We examined the effect of nipradilol on contraction of the posterior ciliary artery induced by high potassium or norepinephrine and on cyclic GMP (cGMP) levels in the posterior ciliary artery of dogs. Nipradilol caused dose-dependent relaxation of KCl-and norepinephrine-induced contractions of posterior ciliary artery. The relaxant effect of nipradilol on norepinephrine-contracted ciliary artery was significantly greater than that on KCl-contracted ciliary artery. In KCl-contracted ciliary artery, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 10(-4) M) did not alter the relaxant effect of nipradilol, whereas 1H-1,2,4-oxadiazolo-4,3-a-quinoxalin-1-one (ODQ, 10(-6) M) significantly inhibited this effect. Ethacrynic acid at 10(-5) M, sulfasalazine at 10(-4) M and S-decylglutathione at 10(-4) M (glutathione S-transferase inhibitors) did not inhibit the relaxant effect of nipradilol. In addition, nipradilol produced dose-dependent increases in cGMP levels in the canine posterior ciliary artery. These findings indicate that nipradilol-induced vasorelaxation in the canine posterior ciliary artery occurs via stimulation of the guanylyl cyclase-cGMP pathway.
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PMID:Nipradilol induces vasodilation of canine isolated posterior ciliary artery via stimulation of the guanylyl cyclase-cGMP pathway. 1209 33

Soluble guanylate cyclase (sGC) is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the alpha- or beta-subunit of sGC show that the sGC beta-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.
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PMID:Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase. 1267 72


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