Guanylate cyclase activation by organic nitrates is not mediated via nitrite

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.     

Effect of in vitro organic nitrate tolerance on relaxation, cyclic GMP accumulation, and guanylate cyclase activation by glyceryl trinitrate and the enantiomers of isoidide dinitrate

Previously, it was shown that the D enantiomer of isoidide dinitrate was 10-fold more potent than the L enantiomer and 10-fold less potent than glyceryl trinitrate for stimulating cyclic GMP accumulation and relaxation of isolated rat aorta. In the present study, these organic nitrates were tested for their ability to induce tolerance to organic nitrate-induced relaxation, cyclic GMP accumulation, and guanylate cyclase activation in rat aorta in vitro. To compensate for the differences in vasodilator potency, tolerance was induced by incubating isolated rat aorta with concentrations of organic nitrates 1,000-fold greater than the EC50 for relaxation. Under these conditions, the EC50 for relaxation was increased significantly for each organic nitrate and to a similar degree on subsequent reexposure. These data suggest that the potential for inducing in vitro tolerance to relaxation was the same for the three organic nitrates tested. When activation of soluble guanylate cyclase by these compounds was assessed, the enantiomers of isoidide dinitrate were equipotent, but less potent than glyceryl trinitrate, suggesting that the site of enantioselectivity is not guanylate cyclase itself. In blood vessels made tolerant to organic nitrates by pretreatment with glyceryl trinitrate, vasodilator activity, cyclic GMP accumulation, and guanylate cyclase activation were attenuated on reexposure to each organic nitrate. In addition, differences in the potency of the three organic nitrates and the enantioselectivity of isoidide dinitrate for relaxation were abolished in tolerant tissue, whereas the potency difference between glyceryl trinitrate and isoidide dinitrate for activation of guanylate cyclase was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of nitric oxide and atrial natriuretic peptide changes with altered cGMP homeostasis in liver cirrhosis.

BACKGROUND: Cyclic GMP (cGMP) concentration is increased in plasma of patients with liver cirrhosis. Three possible mechanisms may contribute: increased cGMP synthesis by soluble (activated by nitric oxide), or particulate (activated by atrial natriuretic peptide (ANP)) guanylate cyclase or increased release from cells. AIM: The aim of this work was to analyze the possible contributors to increased plasma cGMP and to assess whether changes in the parameters of the system vary with the degree of liver disease (Child Pugh score) or by the presence of ascites. METHODS: We measured cGMP in plasma and lymphocytes, soluble guanylate cyclase activation by nitric oxide in lymphocytes, nitrates and nitrites and ANPs (activator of particulate guanylate cyclase) in plasma. We analyzed the correlation between changes in different parameters to discern which parameters contribute to increased plasma cGMP. RESULTS: The plasma content of nitrates+nitrites, ANP and cGMP are increased. Activation of soluble guanylate cyclase by nitric oxide is increased in patients while basal cGMP in lymphocytes is decreased. CONCLUSIONS: Both increased ANP and increased activation of soluble guanylate cyclase by nitric oxide contribute to increased plasma cGMP in patients. The concentrations of ANP and cGMP in plasma increase with the degree of disease and are higher in patients with ascites.     

Nitric oxide and nitrovasodilators: Similarities,differences, and interactions

The endothelium functions as a semipermeable membrane separating the blood from the body and allowing the transport of macromolecules from the blood to the interstitial space. The endothelium secretes a number of diffusible substances. These include endotheliumderived relaxing factor (EDRF), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin, in addition to vasoconstrictors including endothelin, angiotensin, and endothelium-derived contracting factor. EDRF is now known to be nitric oxide, or a closely related molecule, which affects signaling by stimulation of soluble guanylate cyclase, causing increased intracellular levels of cyclic guanosine monophosphate (cGMP), in turn leading to relaxation of vascular smooth muscle as well as a variety of additional effects that include altered function of platelets and cardiac myocytes. Nitric oxide can be made available to cellular elements in two ways: by endogenous synthesis via one or more of the three nitric oxide syntheses now known to exist in mammalian species; or by exogenous administration of pharmacologic sources of nitric oxide, usually as organic nitrate vasodilators that can be metabolically converted to biologically activated nitric oxide. This process appears to require free sulfhydryl groups. The metabolic machinery necessary to convert organic nitrates to a biologically active form exists mainly in the vasculature and not in the myocardium. Numerous studies have demonstrated that the presence of coronary artery disease is associated with interruption of the endogenous production of nitric oxide. Under these circumstances, exogenous nitrates still produce coronary vasodilation as well as relaxation of vascular smooth muscle in the periphery. Other articles in this supplement will focus on the vascular effects of nitric oxide and nitrovasodilators; this article will conclude with a brief discussion of the role of the nitric oxide pathway in the control of cardiac autonomic responsiveness and the potential role of cytokines and the nitric oxide pathway to impair the ability of the myocardium to respond to catecholamines or other stimuli with a normal increase in contractile function.     

Mechanisms of action of nitrates

Summary 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 after-load, 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.     

Nitric oxide and nitrovasodilators: Similarities,differences and potential interactions

Many similarities exist between the exogenous nitrates and endothelium-derived relaxing factor, which is nitric oxide or a thiol derivative. Both act by way of guanylate cyclase, which increases intracellular concentrations of cyclic guanosine monophosphate, resulting in smooth muscle cell relaxation and antiplatelet effects. Thiols may be important in the biotransformation of exogenous nitrates and other intracellular processes involving nitric oxide. As such, important interactions might be expected between nitrates and endothelium-dependent processes that involve nitric oxide. This review explores the mechanisms of action, biologic effects and potential interactions between nitrates and endothelium-derived relaxing factor.     

Nitric oxide as a signal in thyroid.

It is now well established that agonist activation of the PIP2/calcium cascade in the thyroid results in the enhancement of cGMP accumulation presumably by activation of the soluble guanylate cyclase. In many tissues the physiological signal controlling soluble guanylate cyclase is nitric oxide (NO) and its synthesis from arginine is controlled by the intracellular Ca2+. In this report we show results that suggest that NO may be the intermediate of the cGMP response to the activation of the PIP2/calcium cascade. In dog thyroid slices, incubation with carbamylcholine or A23187 increases significantly free intracellular Ca2+ levels and the cGMP content of the slices. NG-Monomethyl-L-arginine (NMMA), a competitive inhibitor of arginine for nitric oxide synthase, inhibited these cGMP responses but not the action of sodium nitroprusside which activates soluble guanylate cyclase directly. The inhibition was relieved by arginine. Methylene blue, which blocks the activation of soluble guanylate cyclase by NO, also decreased the three stimulatory effects. NMMA and methylene blue also decreased the basal levels of cGMP. NO may therefore be an important autocrine and paracrine factor in thyroid.     

Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase.

A soluble enzyme obtained from rat forebrain catalyzes the NADPH-dependent formation of nitric oxide (NO) and citrulline from L-arginine. The NO formed stimulates the soluble guanylate cyclase and this stimulation is abolished by low concentrations of hemoglobin. The synthesis of NO and citrulline is dependent on the presence of physiological concentrations of free Ca2+ and is inhibited by NG-monomethyl-L-arginine, but not by its enantiomer NG-monomethyl-D-arginine or by L-canavanine. L-Homoarginine, L-arginyl-L-aspartate, or L-arginine methyl ester can replace L-arginine as substrates for the enzyme. These results indicate that NO is formed from L-arginine in the brain through an enzymic reaction similar to that in vascular endothelial cells, neutrophils, and macrophages, adding support to our hypothesis that the formation of NO from L-arginine is a widespread transduction mechanism for the stimulation of the soluble guanylate cyclase.     

Methylene blue in the treatment of refractory shock from an amlodipine overdose

Amlodipine is a potent vasodilator with a long half-life and delayed onset of action that is particularly concerning after an overdose. Vasodilation occurs through stimulation of nitric oxide release with increased cyclic guanosine monophosphate (cGMP) production. Methylene blue inhibits guanylate cyclase. This enzyme is responsible for the production of cGMP. Methylene blue also has the ability to scavenge nitric oxide, as well as inhibit nitric oxide synthase. We report the use of methylene blue for refractory shock in a patient with amlodipine toxicity.     

Soluble Guanylate Cyclase Modulators in Heart Failure

This review summarizes the role of soluble guanylate cyclase (sGC)-cyclic guanosine 3′, 5′-monophosphate pathways in heart failure and several new drugs that modify guanylate cyclase. The sGC activators and stimulators as modulators of sGC are promising drugs in the therapy for decompensated heart failure and pulmonary hypertension. Cinaciguat is a nitric oxide (NO)–independent direct activator of sGC, which also may be effective under oxidative stress conditions resulting in oxidized or heme-free sGC refractory to organic nitrates. Riociguat is an NO-independent direct stimulator of sGC with beneficial effects in patients with decompensated heart failure and pulmonary hypertension. The sGC modulators play an important role in patients with heart failure and pulmonary hypertension.     

Stimulation of human platelet guanylate cyclase by unsaturated fatty acid peroxides.

Guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] activity of human platelet homogenates was stimulated by the addition of phospholipase A2 or unsaturated fatty acids such as oleic, vaccenic, linoleic, linolenic, eicosenoic, eicosadienoic, and arachidonic acids. The addition of lipoxidase potentiated the fatty acid-induced stimulation of guanylate cyclase purified by DEAE-cellulose column chromatography. The extent of the stimulation was dependent on the concentration of the oxidized form of these fatty acids (peroxides). Saturated fatty acids such as stearic and arachidic acids had no effect on the guanylate cyclase activity in the presence or absence of lipoxidase, indicating that human plateletguanylate cyclase is stimulated by unsaturated fatty acid peroxides rather than by fatty acids.Hemoglobin prevented the enzyme stimulation produced by low concentrations of fatty acid peroxides, but enhanced stimulation of the enzyme activity with high concentrations of fatty acid peroxides. 2-Mercaptoethanol, dithiothreitol, and N-ethylmaleimide inhibited the guanylate cyclase activities both in the presence and absence of unsaturated fatty acidperoxide. The stimulation of guanylate cyclase activity by unsaturated fatty acid peroxidesis attributed to oxidation of sulfhydryl residues of the enzyme protein.     

Nitrates and angina pectoris

This review discusses the mechanisms of action of the organic nitrates, nitrate tolerance, and the effects of nitrates in patients with stable angina pectoris. The nitrates are prodrugs that enter the vascular smooth muscle, where they are denitrated to form the active agent nitric oxide (NO). NO activates guanylate cyclase, which results in cyclic guanosine monophosphate (cGMP) production and vasodilation as a result of reuptake of calcium by the sarcoplasmic reticulum. NO is identical to endothelium-derived relaxing factor (EDRF), which induces vasodilation, inhibits platelet aggregation, reduces endothelium adhesion, and has anticoagulant and fibrinolytic effects. Thus, the nitrates may be more than vasodilators and, in addition to reducing ischemia, may affect the process of atherosclerosis. The vascular effects of nitrates are attenuated during sustained therapy. Although the basis for the phenomenon of nitrate tolerance is not completely understood, sulfhydryl depletion as well as neurohormonal activation and increased plasma volume may be involved. The administration of N-acetylcysteine, angiotensin-converting enzyme (ACE) inhibitors, or diuretics do not consistently prevent nitrate tolerance. At present, intermittent nitrate therapy is the only way to avoid nitrate tolerance. The intermittent administration of nitrates, however, cannot provide continuous therapeutic benefits, and thus monotherapy with nitrates is not suitable for many patients with stable angina pectoris.     

Mutation of His-105 in the beta 1 subunit yields a nitric oxide-insensitive form of soluble guanylyl cyclase.

Soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing); EC 4.6.1.2] is a hemoprotein that exists as a heterodimer; the heme moiety has been proposed to bind nitric oxide, resulting in a dramatic activation of the enzyme. Mutation of six conserved His residues reduced but did not abolish nitric oxide stimulation whereas a change of His-105 to Phe in the beta 1 subunit yielded a heterodimer that retained basal cyclase activity but failed to respond to nitric oxide. Heme was not detected as a component of the mutant heterodimer and protophorphyrin IX failed to stimulate enzyme activity. The activity of the His mutant was almost identical to that of the wild-type enzyme in the presence of KCN, suggesting that disruption of heme binding is the principal effect of the mutation. Thus, the mutation provides a means to inhibit the nitric oxide-sensitive guanylyl cyclase signaling pathway.     

Inhibition of Platelet Aggregation by Roussin's Black Salt, Sodium Nitroprusside and Other Metal Nitrosyl Complexes

We have examined the action of a range of transition metal nitrosyl compounds in the inhibition of ADP-induced platelet aggregation. Inhibition results from the formation of the activated nitric oxide (NO) complex of guanylate cyclase, hence increasing platelet [cGMP]. Nitrosylation of guanylate cyclase may occur by release of NO from a nitrosyl complex, or, indirectly, by nitrosation of a thiol group followed by decomposition of the S-nitrosyl thiol to give NO. The latter process might be expected to be more efficient for compounds with a greater NO(+)character, and hence nitrosating ability, of the nitrosyl complex, but the results did not show a consistent relationship between NO character and the inhibitory potency on platelets. Inhibition of aggregation by Rousin's black salt, Na[Fe(4)S(3)(NO)(7)], was abolished by haemoglobin, and enhanced in the presence of M&B22948. These findings indicate that activation of guanylate cyclase is mediated by extracellular release of NO. For sodium nitroprusside, inhibition of platelet aggregation became progressively less sensitive to addition of haemoglobin, indicating that another process, such as release of cyanide, became significant as the incubation time was increased.     

Nitric oxide: from endogenous vasodilator to biologic mediator

This paper review the actual knowledges about the physiological role of nitric oxide, sintetized from amino acid L-arginine. The nitric oxide sintetized in the vascular endothelium has a fundamental role in vascular tone, blood flow and arterial pressure control, acting stimulating guanylate cyclase on vascular smooth muscle. Nitric oxide could be considered the endogenous nitrovasodilator. Its action on the cardiovascular system are imitated by nitroglycerine, sodium nitroprusside and related compounds. Probably the disturbance in the synthesis or release of nitric oxide may be involved in the pathophysiology of hypertension, vasospasm and atherosclerosis. Recently has been shown that nitric oxide synthesis from L-arginine also occurs in other different cells like macrophages, central nervous system, liver, neutrophils, adrenal glands, playing different biological effects. Changes in nitric oxide synthesis or action in those systems, could be related to different pathological disorders as inflammation, atherosclerosis and cancer. The found of a substance as simple as nitric oxide, let suppose that we are in the presence of a biological mediator with a very early evolutionary origin, probably widespread in all the animal kingdom, and which represents the universal transduction system for activation of the soluble guanylate cyclase enzyme.     

Hirudin and nitrates inhibit the thrombin-induced release of endothelin from the intact porcine aorta

In intact porcine aorta, endothelium-derived nitric oxide released on thrombin stimulation inhibits the concomitant production of endothelin. Experiments were designed to examine the effect of hirudin (which inactivates thrombin) and the nitrovasodilators nitroglycerin and 3-morpholinosydnonimine on the spontaneous and thrombin-stimulated release of endothelin in intact blood vessels. Endothelin was detected by radioimmunoassay in the incubating medium of intact porcine aortas with endothelium. The spontaneous release of endothelin was not affected by hirudin (0.1 micrograms/ml) but that induced by thrombin (4 units/ml) was prevented. Nitroglycerin (10(-5) M) and the active metabolite of molsidomine, 3-morpholinosydnonimine (10(-5) M), did not modify the basal production of endothelin from the intima of intact porcine aortas. However, the nitrates fully inhibited the release of the peptide induced by thrombin (4 units/ml). The inhibitory effects of both 3-morpholinosydnonimine and nitroglycerin on the thrombin-stimulated release of endothelin were abolished in the presence of an inhibitor of soluble guanylate cyclase, methylene blue (10(-5) M). Thus, the thrombin-stimulated release of endothelin is inhibited by inactivation of thrombin with hirudin or by agents that mimic the effect of endothelium-derived nitric oxide. In contrast, the spontaneous production of endothelin is not modulated by the drugs.     

Increased adhesive properties of neutrophils in sickle cell disease may be reversed by pharmacological nitric oxide donation

Increased leukocyte adhesion to vascular endothelium contributes to vaso-occlusion in sickle cell disease. Since nitric oxide bioavailability is decreased in sickle cell disease and nitric oxide may inhibit leukocyte adhesion, we investigated whether stimulation of NO-signaling pathways can reduce the adhesive properties of neutrophils from sickle cell disease individuals (sickle cell diseaseneu). sickle cell diseaseneu presented greater adhesion in vitro to both fibronectin and ICAM-1 than control neutrophils. Co-incubation of sickle cell diseaseneu with the nitric oxide-donor agents, sodium nitroprusside and dietheylamine NONOate (DEANO), and the guanylate cyclase stimulator, BAY41-2272, all significantly reduced the increased adhesion to fibronectin/ICAM-1. Oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, reversed sodium nitroprusside/DEANO-diminished adhesion to fibronectin, implicating cGMP-dependent signaling in this mechanism. Interestingly, intracellular cGMP was significantly higher in neutrophils from sickle cell disease individuals on hydroxyurea (sickle cell diseaseHUneu). Accordingly, sickle cell diseaseHUneu adhesion to fibronectin/ICAM-1 was significantly lower than that of sickle cell diseaseneu. Agents that stimulate the nitric oxide/cGMP-dependent pathway may have beneficial effects on leukocyte function if used in these subjects.     

Antiplatelet and antithrombotic effects of organic nitrates.

Organic nitrates are believed to provide relief from angina principally by dilating the coronary vasculature. Substantial evidence exists, however, to support a potent antiplatelet effect for these agents as well. Each of these compounds ultimately is metabolized to nitric oxide (or an S-nitrosothiol congener thereof), and this metabolite, in turn, is a potent activator of platelet guanylate cyclase. Activation of guanylate cyclase increases platelet cyclic guanosine monophosphate (cGMP), and is accompanied by inhibition of agonist-mediated calcium flux, and, in turn, reduction of fibrinogen binding to the glycoprotein IIb/IIIa receptor. Since fibrinogen binding is essential for platelet aggregation regardless of the agonist involved, its inhibition appears to be the critical mechanism by which platelet function is impaired by these agents. The recently recognized role that platelet-dependent thrombotic processes play in acute coronary syndromes suggests that the inhibition of platelets by nitrates may offer an additional mechanism by which these compounds improve perfusion to ischemic myocardium.     

Organic nitrates: New formulations and their clinical advantages

The organic nitrates have been used for more than a century in the management of patients with myocardial ischemia. The most commonly used agents at this time include nitroglycerin, isosorbide dinitrate, and isosorbide-5-mononitrate. These agents all exert their therapeutic effects through biodegradation to nitric oxide, which stimulates guanylate cyclase in vascular smooth muscle cells with the production of cyclic guanosine monophosphate. The latter induces vasodilation by reducing the availability of ionized calcium to the contractile proteins. Tolerance to the organic nitrates occurs when the agents are administered in an attempt to provide therapeutic effects throughout 24 hours each day. There are probably several mechanisms responsible for nitrate tolerance, but there is no evidence at this time that concurrent medications will modify the development of tolerance. The only available method at this time is to give these agents intermittently to provide a period of washout. In so doing, it is possible to provide therapeutic nitrate effects for approximately 12 hours throughout each 24-hour period.     

Nitric oxide. A novel signal transduction mechanism for transcellular communication

Nitric oxide first captured the interest of biologists when this inorganic molecule was found to activate cytosolic guanylate cyclase and stimulate cyclic guanosine monophosphate (GMP) formation in mammalian cells. Further studies led to the finding that nitric oxide causes vascular smooth muscle relaxation and inhibition of platelet aggregation by mechanisms involving cyclic GMP and that several clinically used nitrovasodilators owe their biological actions to nitric oxide. Nitric oxide possesses physicochemical and pharmacological properties that make it an ideal candidate for a short-term regulator or modulator of vascular smooth muscle tone and platelet function. Nitric oxide is synthesized by various mammalian tissues including vascular endothelium, macrophages, neutrophils, hepatic Kupffer cells, adrenal tissue, cerebellum, and other tissues. Nitric oxide is synthesized from endogenous L-arginine by a nitric oxide synthase system that possesses different cofactor requirements in different cell types. The nitric oxide formed diffuses out of its cells of origin and into nearby target cells, where it binds to the heme group of cytosolic guanylate cyclase and thereby causes enzyme activation. This interaction represents a novel and widespread signal transduction mechanism that links extracellular stimuli to the biosynthesis of cyclic GMP in nearby target cells. The small molecular size and lipophilic nature of nitric oxide enable communication with nearby cells containing cytosolic guanylate cyclase. The extent of transcellular communication is limited by the short half-life of nitric oxide, thereby ensuring a localized response. Labile nitric oxide-generating molecules such as S-nitrosothiols may be involved as precursors or effectors. Further research will provide a deeper understanding of the biology of nitric oxide and the nature of associated pathophysiological states.