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Query: UNIPROT:P06889 (Mol)
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We investigated the roles of cyclic GMP and cyclic AMP in the inhibition of rabbit platelet aggregation and degranulation by two nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1; the active metabolite of molsidomine), with particular reference to the synergistic interaction of these drugs with prostaglandin E1 (PGE1). Changes in platelet cyclic [3H]GMP and cyclic [3H]AMP were measured by rapid and sensitive prelabeling techniques, the validity of which were confirmed by radioimmunoassays. Incubation of the platelets with 0.1 to 10 microM SNP alone for 0.5 min caused progressively greater inhibitions of platelet function associated with large dose-dependent increases in cyclic [3H]GMP and 1.4- to 3.0-fold increases in cyclic [3H]AMP. However, addition of SNP with the adenylate cyclase activator, PGE1, at a concentration of the latter that had little effect alone, caused much larger increases in cyclic [3H]AMP and greatly enhanced the inhibition of platelet aggregation. SIN-1 had effects similar to those of SNP, although it was less active. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) diminished the increases in cyclic [3H]AMP caused by SNP or SIN-1 in both the presence and absence of PGE1 but reduced the inhibition of platelet function caused by the nitrovasodilators only in the presence of PGE1. These results suggest that, although cyclic GMP may mediate the inhibition of rabbit platelet function by high concentrations of nitrovasodilators added alone, the synergistic interaction of lower concentrations with PGE1 depends on an enhanced accumulation of cyclic AMP. Synergistic effects on cyclic [3H]AMP accumulation were also observed on incubation of platelets with SNP and adenosine, another activator of adenylate cyclase. Hemoglobin, which binds nitric oxide, blocked or reversed the increases in both cyclic [3H]GMP and cyclic [3H]AMP in platelets caused by the nitrovasodilators added either alone or with PGE1. Cilostamide, a selective inhibitor of platelet low Km cyclic AMP phosphodiesterase, had effects on platelet cyclic [3H]AMP accumulation identical to those of SNP, suggesting that the action of the latter depends on inhibition of the same enzyme. M&B 22,948, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in both cyclic [3H]GMP and cyclic [3H]AMP caused by SNP. A hyperbolic relationship was found between the increases in cyclic [3H]GMP and cyclic [3H]AMP caused by different concentrations of SNP; this relationship was not affected by addition of M&B 22,948. The results strongly suggest that the increases in platelet cyclic [3H]AMP caused by nitrovasodilators in the presence or absence of activators of adenylate cyclase are mediated by the inhibition by cyclic GMP of cyclic AMP breakdown.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1990 May
PMID:Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP. 216 60

Stimulation of soluble guanylyl cyclase in rat fetal lung fibroblasts (RFL-6 cells) was used as a sensitive assay for endothelium-derived relaxing factor/nitric oxide (EDRF/NO) formation. Intact N1E-115 cells released an EDRF/NO-like material that enhanced cyclic GMP levels in RFL-6 cells. The synthesis of this substance could be stimulated with the receptor agonist neurotensin (10 microM) or by addition of the EDRF/NO substrate L-arginine (100 microM). In Ca2(+)-free Locke's solution, stimulation of EDRF/NO production by both neurotensin and L-arginine was abolished. The EDRF/NO-synthesizing activity was localized in the cytosol of N1E-115 cells. The activity was lost after boiling and it was highly sensitive to Ca2+ with the major increase in activity occurring between 100 and 500 nM Ca2+. L-Arginine and NADPH were required for maximal synthesis of EDRF/NO by the enzyme(s). The synthesis of EDRF/NO was inhibited by the following antagonists of calmodulin-regulated functions (with the approximate IC50 values given in parentheses): calmidazolium (7 microM), trifluoperazine (10 microM), fendiline (80 microM), W-7 (N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide) (120 microM), and compound 48/80 (3 micrograms/ml). The EDRF/NO-synthesizing activity was partially purified from N1E-115 cytosol by DE 52 anion exchange chromatography. The activity was eluted with 0.1 M KCl. The enzyme(s) showed very little activity in the presence of L-arginine (100 microM) and NADPH (100 microM), but the activity could be fully restored by addition of exogenous calmodulin (EC50, approximately 2 units/ml). At 0.3 M KCl, a fraction eluted from the DE 52 column that was also able to fully restore the EDRF/NO-synthesizing activity. Thus, this fraction is likely to contain the endogenous Ca2(+)-binding protein. It is concluded that the activity of the EDRF/NO-synthesizing enzyme(s) in N1E-115 neuroblastoma cells is regulated by Ca2+ and calmodulin.
Mol Pharmacol 1990 Jul
PMID:Hormone-induced biosynthesis of endothelium-derived relaxing factor/nitric oxide-like material in N1E-115 neuroblastoma cells requires calcium and calmodulin. 237 Aug 55

Phospholipase A2 (PLA2) produced slow dose dependent relaxation in intact and endothelium-deprived precontracted rabbit aortic strips. In endothelium-deprived preparations, relaxation induced by PLA2 is inhibited by hemoglobin, methylene blue and parabromophenacylbromide (PBPB), and is potentiated by superoxide dismutase (SOD). Indomethacin has no effect. Relaxation is accompanied by a rise in c-GMP. Phospholipase C causes a significant increase in tension, while Phospholipase D has no effects. In intact aortic strips PLA2 causes a biphasic response with no elevation in c-GMP. The results indicate several common features of the PLA2 released factor with endothelium-derived relaxing factor (EDRF). However, PLA2 induced relaxation is not dependent on endothelial cells. Apparently in addition to nitric oxide which may be the endothelium-derived relaxing factor, a second smooth muscle relaxing factor exists which is initiated by PLA2 and is independent of endothelium. The production of the PLA2 produced relaxation is dependent on its specific hydrolytic activity. We call this relaxing factor the phospholipid-derived relaxing factor (PDRF).
Mol Cell Biochem 1987 Nov
PMID:A relaxing factor released by phospholipase A2 in the absence of endothelium. 284 57

According to our present understanding organic nitrates like glycerine trinitrate mediate their pharmacological effect by an intracellular stimulation of the enzyme guanylate cyclase (E.C. 4.6.1.2.) [1, 10]. The exact molecular mechanism underlying the process of enzyme activation is still a matter of controversial discussion. But there is general agreement in literature about the fact that organic nitrate compounds are able to activate the enzyme guanylate cyclase only in the presence or by the interaction of the amino acid cysteine [3, 5]. The stimulatory activity of nitric oxide-containing compounds may be due, at least in part, to the formation of active, unstable intermediate S-nitrosothiols, i.e. S-nitrosocysteine in case of the organic nitrates [7]. According to Craven and DeRubertis [2], the active intermediates of guanylate cyclase stimulation are represented by nitric oxide-heme complexes. There is, however, substantial evidence that the organic nitrates have to be cleaved before they become biologically active. During the transformation which takes place in the presence of cysteine or by means of enzymatic catalysis, nitric oxide radicals are reductively split off the molecule from which (via the intermediate formation of salpetric acid) the nitric oxide is liberated as the essential stimulatory agent. In this study we examined the transformation of glycerine trinitrate and other organic nitrates under the influence of different thiols and a purified soluble rat liver guanylate cyclase preparation. At the same time the stimulation of guanylate cyclase in the presence of the thiols mentioned was quantitatively estimated. Only in case of cysteine did we find a strict correlation between the liberation of nitric oxide from different organic nitrates and the degree of enzyme activation. Several other thiols were also able to liberate nitric oxide, but surprisingly enough, there was no equivalent stimulation of guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Cell Cardiol 1985 Sep
PMID:Evidence for a correlation between nitric oxide formation by cleavage of organic nitrates and activation of guanylate cyclase. 286 57

Nitrovasodilators relax vascular smooth muscle by stimulating guanylate cyclase. Ignarro et al. (1981) proposed a mechanistic scheme according to which organic nitrates release nitrite in the presence of thiols. The corresponding nitrous acid would decay leading to nitric oxide, which then would react with another thiol to nitrosothiol. Dose-response relations with regard to guanylate cyclase stimulation of organic nitrates and sodium nitrite were compared in the presence of cysteine and its closely related methylester. Nitrite formation from ED95 concentrations of organic nitrates was also measured and compared with that present under an equi-effective concentration of sodium nitrite. In addition, the proposed formation of nitrosothiol from nitric oxide was re-examined. In the presence of cysteine, organic nitrates as well as sodium nitrite stimulated guanylate cyclase, but nitrite formation under ED95 concentrations of organic nitrates was 1000-fold smaller than that present under an equi-effective concentration of sodium nitrite. In the presence of cysteinemethylester, liberation of nitrite from organic nitrates was similar but no stimulation of guanylate cyclase was obtained. Sodium nitrite, however, showed a stimulating activity similar to that in the presence of cysteine. These results clearly demonstrate that guanylate cyclase stimulation by organic nitrates is not mediated by nitrite and subsequent formation of nitrosothiol. Since nitrous acid did not decay to nitric oxide in the pH range studied, the formation of nitrosothiol is apparently due to a direct reaction of nitrous acid with thiol.
J Mol Cell Cardiol 1988 May
PMID:Guanylate cyclase activation by organic nitrates is not mediated via nitrite. 290 90

Recent studies have suggested that cyclic GMP accumulation in platelets mediates the antiaggregatory effects of certain nitrogen oxide-containing agents such as sodium nitroprusside, nitric oxide, nitrosoguanidines, and related agents. The vasodilator effect of these agents may involve the formation of S-nitrosothiol intermediates which relax vascular smooth muscle, elevate tissue levels of cyclic GMP, and activate guanylate cyclase. The purpose of this study was to investigate the effects of various synthetic S-nitrosothiols on human platelet aggregation. The S-nitroso derivatives of N-acetylpenicillamine, cysteine, and beta-D-thioglucose inhibited human platelet aggregation in a concentration-dependent fashion when ADP, collagen, U46619, or sodium arachidonate was employed as the aggregating agent. The antiaggregatory effects of the S-nitrosothiols were associated with a rapid and marked increase in intracellular platelet cyclic GMP levels, whereas cyclic AMP levels remained unchanged. Additionally, S-nitrosothiols disaggregated platelets which had been aggregated while concomitantly elevating platelet cyclic GMP levels. Moreover, guanylate cyclase, partially purified from the soluble fraction of human platelets, was markedly activated by S-nitrosothiols in a heme-dependent manner. Methemoglobin, a hemoprotein with a high affinity for nitric oxide, partially reversed the antiaggregatory effects, attenuated the accumulation of cyclic GMP, and inhibited the activation of guanylate cyclase by S-nitrosothiols. These data are consistent with the hypothesis that S-nitrosothiols could serve as active intermediates in the inhibitory action of sodium nitroprusside, nitric oxide, and related nitrogen oxides on platelet aggregation.
Mol Pharmacol 1983 May
PMID:Inhibition of human platelet aggregation by S-nitrosothiols. Heme-dependent activation of soluble guanylate cyclase and stimulation of cyclic GMP accumulation. 613 48

The effects of L-arginine on recovery of myocardial contractile function and oxidative metabolism were investigated in a model of reversible global normothermic, ischemic injury using an isolated, buffer-perfused rabbit heart preparation. One mM L-arginine was infused into hearts for 2 min at the onset (group 1) of a 35 min period of ischemia or at the onset of reperfusion (group 2). In non-ischemic hearts, L-arginine caused a slight increase in developed pressure but had no effects on diastolic pressure, oxygen consumption (MVO2), coronary flow, or lactate production. When administered either before or after ischemia-reperfusion. L-arginine caused a significant increase in the diastolic pressure-volume relationship (PVR) and decline in systolic function when compared to untreated control hearts receiving the same ischemic injury. Recovery of MVO2 and high energy phosphates (phosphocreatine and ATP), measured by 31P-NMR spectroscopy, were significantly impaired in L-arginine-treated hearts compared to reperfused control hearts. Lactate release on reperfusion was also higher in both arginine-treated groups. Nitric oxide release into the coronary circulation (measured in separate experiments by the conversion of [15N]L-arginine to [15N]nitrate/nitrite using gas chromatography/mass spectroscopy) was not increased by L-arginine administration. Thus, we conclude that L-arginine acts synergistically with ischemia reperfusion to augment myocardial injury, which includes inhibition of oxidative metabolism and mitochondrial function.
J Mol Cell Cardiol 1995 Jul
PMID:Direct detrimental effects of L-arginine upon ischemia--reperfusion injury to myocardium. 747 86

Injection of small volumes of N-methyl-D-aspartate (NMDA) or Sin-1 molsidomine (a nitric oxide releasing agent) onto the dendrites of granule cells in the hippocampal dentate gyrus leads to changes in the level of expression of a number of genes. There is a fall in prodynorphin mRNA levels with a corresponding increase in proenkephalin mRNA levels. Similar changes in opioid gene expression occur following the induction of long-term potentiation (LTP). We report here that at short time periods (1-6 h) after injections of NMDA or sin-1 molsidomine, there is an increase in the levels of the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKII alpha), consistent with a report of elevated CaMKII alpha mRNA in postsynaptic neurons in the CA1 region of the hippocampus following LTP induction [54]. However, we also report that 24 h after injection of NMDA or sin-1, there is a dramatic decrease in CaMKII alpha mRNA levels in the vicinity of the injection. This effect is specific for CaMKII alpha mRNA, in that many other mRNA species are not affected, and occurs in the dendritic population of CaMKII alpha mRNA as well as in the pool of mRNA in the granule cell bodies. The effect is blocked by an inhibitor of cGMP-dependent protein kinase. The biphasic regulation of CaMKII alpha mRNA may be of considerable functional importance for the long-term response of granule cells to local stimulation of NMDA receptors or NO release.
Brain Res Mol Brain Res 1995 Jul
PMID:N-methyl-D-aspartate and nitric oxide regulate the expression of calcium/calmodulin-dependent kinase II in the hippocampal dentate gyrus. 747 22

Functional roles of peroxynitrite in N-methyl-D-aspartate (NMDA)- and sodium nitroprusside (SNP)-evoked releases of acetylcholine (ACh) from cerebral cortical neurons in primary culture have been investigated. NMDA increased the release of ACh in a dose-dependent manner, which was significantly suppressed by (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cycloheptan-5,10-imine (MK-801), a non-competitive antagonist specific for the NMDA receptor complex, and NO synthase inhibitors. SNP also showed a concentration-dependent increase in ACh release. Hemoglobin significantly abolished the stimulatory effects of both NMDA and SNP on ACh release. In addition, superoxide anion scavengers such as superoxide dismutase and ceruloplasmin significantly reduced the increased ACh release evoked by NMDA and SNP. Synthesized peroxynitrite dose-dependently elevated the release of ACh. These results indicate that the increased release of ACh by NMDA and SNP is mediated through peroxynitrite formed in the reaction of superoxide anion with nitric oxide produced by NMDA receptor activation and liberated from SNP rather than nitric oxide itself.
Brain Res Mol Brain Res 1995 Jul
PMID:Involvement of peroxynitrite in N-methyl-D-aspartate- and sodium nitroprusside-induced release of acetylcholine from mouse cerebral cortical neurons. 747 28

Previous studies in our laboratory demonstrated that murine cerebral microvessel smooth muscle cells (SMC) activate syngeneic CD4+ T-cells in vitro. These T-cells, or their culture supernatants, in turn, strongly inhibit proliferation of the SMC. The present study focuses on IFN-gamma as a mediator of inhibition of SMC proliferation, and addresses the molecular mechanism of this inhibition. IFN-gamma profoundly reduced the proliferation of murine brain microvessel smooth muscle cells in vitro. Three lines of evidence indicate that nitric oxide contributed to this effect: (1) IFN-gamma-mediated inhibition of proliferation correlated with the quantity of nitrite, a stable breakdown product of nitric oxide, in culture supernatants; (2) the addition of N(g)- monomethyl-l-arginine, and inhibitor of nitric oxide synthesis, restored proliferation to control or near control levels; and (3) the addition of hemoglobin, which has a high affinity for, and thus sequesters nitric oxide, also resulted in significant restoration of the proliferative response. However, the nitric oxide donating chemical sodium nitro-prusside, at concentrations up to 100 microM, had no direct cytostatic effect. These results suggest that nitric oxide is a necessary but insufficient component in IFN-gamma-mediated inhibition of microvessel smooth muscle cell proliferation. TNF-alpha also stimulated nitric oxide production by the smooth muscle cells, but was not as potent as IFN-gamma at inhibiting proliferation. Knowledge of the physiological effects of lymphokines on cells of the brain microvasculature will contribute towards a better understanding of inflammatory processes in diseases such as multiple sclerosis and infectious encephalitis.
Mol Immunol 1995 Sep
PMID:Involvement of nitric oxide in IFN-gamma-mediated reduction of microvessel smooth muscle cell proliferation. 747 2


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