Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase

Organic nitrates develop their vasodilating potency by stimulating the enzme guanylate cyclase. There are still several theories concerning the molecular mechanism of enzyme activation, the most likely of which sees nitric oxide (NO·) as the true modulator of the soluble guanylate cyclase. We therefore examined the release of nitric oxide from organic nitrates by means of difference-spectrophotometric method and found that our results correlated well with the extent of enzyme activation. The more NO· was liberated from the compounds in question, the higher was the enzyme activation observed. When the examined nitrates were used in a concentration which caused a half-maximal enzyme stimulation, the result was a NO· liberation of striking uniformity. This correlation also applied to SIN-1 for which it has been assumed up to now that the intact molecule itself is able to stimulate the enzyme and not the nitric oxide released from it. We found the reaction between organic nitrates and cysteine to be highly dependent on temperature, while the extent of the observed enhancement increased with the number of nitrate groups per molecule. We also studied the potential effects of certain compounds on non-enzymatic NO· release and found that, in addition to methylene blue, thionine and brilliantcresyl blue, but not ferricyanide were also effective inhibitors. So it seems likely that both an enzymatic and a non-enzymatic mode of inhibition of enzyme activity does exist. Since oxyhemoglobin is an effective scavenger of nitric oxide, its addition can inhibit enzyme activation by nitrovasodilators. Our results stress the important role of the non-enzymatic liberation of NO· from organic nitrates and related compounds as possible, perhaps even as the principal mode of activation of soluble guanylate cyclase by nitrovasditors.     

Norepinephrine increases cyclic GMP in astrocytes by a mechanism dependent on nitric oxide synthesis

Norepinephrine induces a rapid and concentration-dependent rise (EC₅₀ = 1.21 ± 0.33 μM) in cyclic GMP levels in astrocyte-enriched cultures from rat brain. The response is partially mediated by α₁-adrenoceptors since a marked inhibitory observed in the presence of prarosin while the ß-antagonist propranolol shows a smaller effect and the α₂-antagonist yohimbine is ineffective l-N^G-methylarginine, an inhibitor of nitric oxide synthesis from l-arginine, blocks the norepinephrine-induced cyclic GMP accumulation. This effect is reversed by l-arginine.     

Norepinephrine increases cyclic GMP in astrocytes by a mechanism dependent on nitric oxide synthesis.

Norepinephrine induces a rapid and concentration-dependent rise (EC50 = 1.21 +/- 0.33 microM) in cyclic GMP levels in astrocyte-enriched cultures from rat brain. The response is partially mediated by alpha 1-adrenoceptors since a marked inhibition is observed in the presence of prazosin while the beta-antagonist propranolol shows a smaller effect and the alpha 2-antagonist yohimbine is ineffective. L-NG-methylarginine, an inhibitor of nitric oxide synthesis from L-arginine, blocks the norepinephrine-induced cyclic GMP accumulation. This effect is reversed by L-arginine.     

Effect of cyanide on nitrovasodilator-induced relaxation, cyclic GMP accumulation and guanylate cyclase activation in rat aorta

The effects of sodium cyanide on relaxation, increases in cyclic GMP accumulation and guanylate cyclase activation induced by sodium nitroprusside and other nitrovasodilators were examined in rat thoracic aorta. Cyanide abolished nitroprusside-induced relaxation and the associated increase in cyclic GMP levels. Basal levels of cyclic GMP and cyclic AMP were also depressed. Reversal of nitroprusside-induced relaxation by cyanide was independent of the tissue level of cyclic GMP prior to addition of cyanide. Incubation of nitroprusside with cyanide prior to addition to aortic strips did not alter the relaxant effect of nitroprusside. Sodium azide-, hydroxylamine-, N-methyl-N'-nitro-N-nitrosoguanide-, nitroglycerin- and acetylcholine-induced relaxations and increased levels of cyclic GMP were also inhibited by cyanide. Relaxations induced by nitric oxide were also inhibited by cyanide, although the relaxation with the low concentration of nitric oxide employed was not accompanied by detectable increases in cyclic GMP. Relaxation to 8-bromo-cyclic GMP was essentially unaltered by cyanide; however, isoproterenol-induced relaxation was inhibited. Guanylate cyclase in soluble and particulate fractions of aorta homogenates was activated by nitroprusside and the activation was prevented by cyanide. The present results suggest that cyanide inhibits nitrovasodilator-induced relaxation through inhibition of guanylate cyclase activation; however, cyanide may also have nonspecific effects which inhibit relaxation.     

Reciprocal regulation of cyclic GMP content by cyclic GMP-phosphodiesterase and guanylate cyclase in SHR with CsA-induced nephrotoxicity

1. The effect of the immunosuppressant drug, cyclosporin A (CsA), on the nitric oxide (NO)-cyclic GMP pathway was examined in spontaneous hypertensive rats (SHR). 2. CsA (50 mg kg(-1)) treatment for 14 days induced typical CsA nephrotoxicity, which was characterized by morphological changes in the glomerulus and proximal tubule as well as an abnormality of creatinine clearance, FENa and BUN. 3. CsA significantly decreased both NOS activity in the kidney and NOx contents in urine, but significantly increased cyclic GMP content in the kidney. 4. A marked change in two kinds of enzyme, which contribute towards the increase in cyclic GMP in tissue, namely, a decrease in cyclic GMP-phosphodiesterase activity and increase in guanylate cyclase activity, was observed in the kidney treated with CsA. 5. In the isolated perfused kidney, a decreased in perfusion pressure induced by SNP in the kidney isolated from CsA group was significantly greater than that of control. 6. There seem to exist a reciprocal mechanism to maintain cyclic GMP content via both a decrease in cyclic GMP degradation and an increase in synthesis of cyclic GMP in the kidney treated with CsA. This mechanism is likely to be playing an important role to regulate the homeostasis in the kidney with CsA nephrotoxicity.     

Inhibition of human platelet aggregation by S-nitrosothiols. Heme-dependent activation of soluble guanylate cyclase and stimulation of cyclic GMP accumulation

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.     

Soluble guanylate cyclase activation by nitric oxide and its reversal. Involvement of sulfhydryl group oxidation and reduction

Pre-incubation of either crude or purified nitric oxide-stimulated soluble lung guanylate cyclase resulted in a temperature-dependent decay of enzyme activity. The decay of nitric oxide-stimulated activity during pre-incubation was associated with a reduced responsiveness of the enzyme to reactivation by a second exposure to nitric oxide. This loss of enzyme responsiveness to reactivation by nitric oxide was greater with purified guanylate cyclase than with the crude enzyme and was highly dependent upon the nitric oxide dose. The addition of dithiothreitol or other thiols to nitric oxide-stimulated enzyme markedly accelerated the decay of activity in a dose-dependent manner. In addition, thiols prevented the loss of responsiveness of guanylate cyclase to reactivation by nitric oxide. Nitric oxide-stimulated enzyme activity was, therefore, reversed by the addition of thiol reducing agents. The addition of the thiol oxidizing agents, diamide or oxidized glutathione, to nitric oxide-stimulated guanylate cyclase caused a rapid and irreversible loss of activity. The effects of diamide or oxidized glutathione on the crude enzyme were prevented by excess dithiothreitol. Dithiothreitol did not prevent the destruction of purified nitric oxide-stimulated guanylate cyclase activity by diamide or oxidized glutathione, however. The results suggest that nitric oxide activation and its reversal are linked to the reversible oxidation and reduction, respectively, of sulfhydryl groups on guanylate cyclase which are involved in enzyme activation. The results further suggest the existence of a second class of sulfhydryl groups involved in the maintenance of enzyme activity.     

Inhibition of low Km cyclic GMP phosphodiesterases and potentiation of guanylate cyclase activators by cicletanine.

Cicletanine is an antihypertensive/vasorelaxant/natriuretic agent of unknown mechanism. We wished (a) to determine if cicletanine interacts with guanylate cyclase activators that modulate vasomotor tone and sodium balance [i.e., atriopeptin II (AP II), endothelium-derived relaxing factor (EDRF), and sodium nitroprusside (SNP)], and (b) to define the subcellular basis for this interaction by quantitating the effects of cicletanine on low Km cyclic GMP phosphodiesterase (PDE) activity. In phenylephrine-contracted rat aortic smooth muscle, the vasorelaxant potency of cicletanine was increased twofold in the presence of a threshold-relaxant concentration of AP II, and functional cyclic GMP PDE inhibition was also evident from the three- to sixfold potentiation by cicletanine of AP II- or SNP-induced vasorelaxation. Vasorelaxation produced by cicletanine was not endothelium dependent, however. In further studies, intravenous (i.v.) administration of cicletanine or the low Km cyclic GMP PDE inhibitor, zaprinast, decreased blood pressure (BP) greater than or equal to 20% in conscious spontaneously hypertensive rats (SHR). These results are consistent with the additional finding that cicletanine inhibited Ca2(+)-calmodulin (CaM) cyclic GMP PDE and zaprinast-sensitive cyclic GMP specific PDE over a concentration range (10-600 microM) similar to that for vasorelaxation. Thus, inhibition of low Km cyclic GMP PDEs by cicletanine may be partly responsible for the vasorelaxant effect of cicletanine as well as the potentiation by cicletanine of the vasorelaxant actions of guanylate cyclase activators. The extent to which this mechanism contributes to the antihypertensive efficacy of cicletanine has not yet been fully determined.     

Enhanced nitric oxide and cyclic GMP formation plays a role in the anti-platelet activity of simvastatin

BACKGROUND AND PURPOSE: It has been found that 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert various vascular protective effects, beyond their cholesterol-lowering property, including inhibition of platelet-dependent thrombus formation. The objective of the present study was to determine whether the nitric oxide (NO)/cyclic GMP-mediated processes in platelets contribute to the anti-aggregatory activity of simvastatin. EXPERIMENTAL APPROACH: After rabbit platelets were incubated with simvastatin for 5 min, aggregation was induced and the platelet aggregation, nitric oxide synthase activity, guanylyl cyclase activity, NO and cyclic GMP formation were measured appropriately. KEY RESULTS: Treatment with simvastatin concentration-dependently inhibited platelet aggregation induced by collagen or arachidonic acid with an IC(50) range of 52-158 microM. We also demonstrated that simvastatin (20-80 microM) concentration-dependently further enhanced collagen-induced NO and cyclic GMP formation through increasing NOS activity (from 2.64+/-0.12 to 3.52+/-0.21-5.10+/-0.14 micromol min(-1) mg protein(-1)) and guanylyl cyclase activity (from 142.9+/-7.2 to 163.5+/-17.5-283.8+/-19.5 pmol min(-1) mg protein(-1)) in the platelets. On the contrary, inhibition of platelet aggregation by simvastatin was markedly attenuated (by about 50%) by addition of a nitric oxide synthase inhibitor, a NO scavenger or a NO-sensitive guanylyl cyclase inhibitor. The anti-aggregatory effects of simvastatin were significantly increased by addition of a selective inhibitor of cyclic GMP phosphodiesterase. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that enhancement of a NO/cyclic GMP-mediated process plays an important role in the anti-aggregatory activity of simvastatin.     

Nitric oxide and cyclic GMP signaling pathway as a focus for drug development

Recent progress in understanding of the nitric oxide and cGMP signaling pathway provided evidence for mechanism of action of known drugs and identified novel targets for drug development. These discoveries resulted in numerous efforts in drug and formulation discovery. Some of the most promising approaches were applied for efficient therapies of various diseases.     

Modulation of neuroeffector transmission by endogenous nitric oxide: a role for acetylcholine receptor-activated nitric oxide formation, as indicated by measurements of nitric oxide/ nitrite release

Nitric oxide (NO) synthase inhibitors enhanced nerve-mediated contractile responses in guinea pig ileum longitudinal muscle, likely via a prejunctional effect on substance P-like neuroeffector transmission. Supporting a modulatory role for NO, application of NO through administration of acid sodium nitrite evoked marked inhibitory effects on responses to transmural nerve stimulation. Substance P-like responses to nerve stimulation were abolished by substance P receptor antagonists and were enhanced by atropine, indicating a cholinergic influence on substance P-like neuroeffector transmission. Since acetylcholine can evoke release of NO from endothelium, the possible role of acetylcholine in NO release in ileum was examined. The release of NO/nitrite, determined by chemiluminescence, was inhibited by N^G-monomethyl-L-arginine (L-NMMA), by calcium removal, by tetrodotoxin or by atropine, indicating a nerve-mediated control of NO production. A basis for the NO release is likely to be spontaneous neuronal activity, where release of acetylcholine, with subsequent muscarinic receptor activation, contributes to stimulation of NO formation.     

Relaxation of guinea-pig trachea by sodium nitroprusside: cyclic GMP and nitric oxide not involved.

1. Sodium nitroprusside (SNP) completely relaxed the guinea-pig isolated, perfused trachea in a concentration-dependent manner. Although SNP was less potent by about 2 orders of magnitude, its maximal effect was 25% higher compared to isoprenaline. 2. SNP (3.2 microM) increased cyclic GMP levels by 300% and relaxed guinea-pig isolated, perfused trachea by 54%. The SNP-induced relaxations of the preparations were not affected by the guanylate cyclase inhibitor, methylene blue. Moreover, zaprinast, a cyclic GMP-specific phosphodiesterase inhibitor which was supposed to enhance SNP-induced relaxations, decreased the maximal relaxation by 22% (P < 0.001). 3. In contrast, 8Br-cyclic GMP (10 microM) increased the cyclic GMP levels by 1100% without inducing a marked relaxation. 4. SNP (10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; a direct donor of nitric oxide; 10 microM), relaxed the tissues by 75% and 25%, respectively, without any nitric oxide (NO) release by SNP (< 1 pmol 100 microliters-1), but a substantial NO release by SNAP (560 pmol 100 microliters-1). 5. It is concluded that the SNP-induced tracheal relaxations are probably not mediated by cyclic GMP and NO.     

Human alpha-calcitonin gene-related peptide stimulates adenylate cyclase and guanylate cyclase and relaxes rat thoracic aorta by releasing nitric oxide.

1. The signal transduction pathway for vasorelaxation induced by human alpha-calcitonin gene-related peptide (human alpha-CGRP) was studied in rat thoracic aortic rings preconstricted with noradrenaline (10(-7) M). 2. Vasorelaxation by human alpha-CGRP was inhibited by haemoglobin (10(-6) M) and methylene blue (10(-5) M) but was unaffected by ibuprofen (10(-5) M). 3. Acetylcholine caused a 16 fold increase in levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) with levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) being unaltered. Human alpha-CGRP caused a 12 fold increase in levels of cyclic GMP but, in contrast to acetylcholine, evoked a 2.5 fold rise in levels of cyclic AMP. The rises in cyclic nucleotides evoked by human alpha-CGRP and acetylcholine were dependent on the presence of an intact endothelium. 4. NG-nitro-L-arginine (L-NOARG: 10(-5) M), which inhibits nitric oxide synthetase, inhibited the relaxant response to human alpha-CGRP and cyclic GMP accumulation without affecting the cyclic AMP accumulation. 5. The data presented in this paper suggests that human alpha-CGRP relaxes the rat thoracic aorta by releasing nitric oxide and stimulating guanylate cyclase. The stimulation of adenylate cyclase by human alpha-CGRP probably precedes the activation of nitric oxide synthase but could be unrelated to the relaxant response.     

Endothelin and a Ca2+ ionophore raise cyclic GMP levels in a neuronal cell line via formation of nitric oxide.

1. The vasoconstrictor peptide endothelin-1 caused a fast, transient rise in guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in a neuronal cell line (mouse neuroblastoma x rat glioma hybrid cells 108CC15). The mechanism of activation of guanylate cyclase by endothelin-1 was investigated. The endothelin-1-induced rise depended on the release of internal Ca2+. 2. The stimulation of cyclic GMP synthesis induced by endothelin-1 was suppressed after preincubating the cells in medium containing haemoglobin (IC50 3 microM). Similarly, pretreatment of the cells with the L-arginine analogues, L-canavanine (IC50 60 microM) or NG-monomethyl-L-arginine (IC50 2.5 microM), inhibited the cyclic GMP response to endothelin-1. Therefore, endothelin-1 activates guanylate cyclase most probably via formation of nitric oxide, which is released from L-arginine. 3. The Ca2+ ionophore ionomycin induced a transient rise in cyclic GMP levels, which was also suppressed by preincubation in the presence of either haemoglobin or the L-arginine analogues L-canavanine or NG-monomethyl-L-arginine. Therefore, we conclude that ionomycin can activate guanylate cyclase by a mechanism involving nitric oxide formation, similar to that induced by endothelin-1. 4. The alkaloid veratridine, which activates Na+ channels and also causes influx of Ca2+ induced a transient rise of cyclic GMP levels in the neuronal cell line. This stimulation was blocked by pretreating the cells with L-canavanine, NG-monomethyl-L-arginine or haemoglobin. 5. Loading the cells with the Ca2+ chelator BAPTA suppresed the cyclic GMP response to application of endothelin-1, ionomycin, or veratridine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of sulfhydryl groups in the oxidative modulation of particulate lung guanylate cyclase by nitric oxide and N-methyl-N'nitro-N-nitrosoguanidine

Particulate guanylate cyclase from rat lung was activated by nitric oxide or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a dose-dependent manner that was enhanced by dithiothreitol. Nitric oxide-stimulated guanylate cyclase activity decayed during a 60-min preincubation at 37 degrees, but did not decay at 24 degrees or 4 degrees. Dithiothreitol enhanced the decay of nitric oxide-stimulated enzyme at all temperatures by potentiating the reversal of nitric oxide activation. Following the reversal of nitric oxide activation at 24 degrees by dithiothreitol, the particulate enzyme could be reactivated by a second exposure to nitric oxide. Preincubation of basal particulate guanylate cyclase activity at 37 degrees resulted in the loss of enzyme responsiveness to activation by nitric oxide or MNNG that was potentiated by diamide or oxidized glutathione. The inhibitory effects of the thiol oxidants on enzyme responsiveness to activation by MNNG were prevented by dithiothreitol. The results suggest that activation of particulate guanylate cyclase by nitric oxide or MNNG involves the oxidation of key enzyme sulfhydryl groups.     

Involvement of nitric oxide/cyclic GMP signaling pathway in the regulation of fatty acid metabolism in rat hepatocytes

The role of nitric oxide (NO)/guanosine 3',5'-cyclic monophosphate (cGMP) signaling pathway in the regulation of fatty acid metabolism was investigated in rat hepatocytes. Treatment with NO donors, which are known to activate soluble guanylyl cyclase, inhibited in parallel fatty acid synthesis de novo and acetyl-CoA carboxylase activity. This effect was mimicked by 8-Br-cGMP and abolished by KT5823, a selective inhibitor of cGMP-dependent protein kinase (PKG). Furthermore, specific and hydrolysis-resistant activators of PKG, and inhibitors of Ca2+ release from endoplasmic reticulum, were also effective in inhibiting both fatty acid-synthesizing activities. These results suggest that this biological action of NO is regulated by a signaling cascade involving soluble guanylyl cyclase, cGMP, and PKG, and may be mediated, at least in part, by inhibition of Ca2+ release from endoplasmic reticulum. In addition, 8-Br-cGMP was able to stimulate fatty acid oxidation by two different mechanisms: the relieving of malonyl-CoA-dependent inhibition by lowering levels of this product of acetyl-CoA carboxylase, and a malonyl-CoA-independent stimulation of carnitine palmitoyltransferase I. Taken together, results of this study suggest that NO/cGMP signaling pathway is endowed with regulatory properties in fatty acid metabolism, and may have a physiological role in the control of this metabolism in liver.     

Effects of cysteine and nitroglycerin on bovine heart guanylate cyclase and on tissue cyclic GMP and lactate of rat atria

The effects of thiols on guanylate cyclase activation by nitroglycerin were studied in bovine heart and the effects of cysteinee and nitroglycerin on the tissue levels of cyclic GMP and lactate were studied in beating rat atria. Cysteine (2.5 X 10(-3) M) together with nitroglycerin (1 X 10(-3) M), increased 15-fold the activity of guanylate cyclase. In hypoxia, nitroglycerin (1 X 10(-3) M) together with cystein (5 X 10(-3) M) increased cyclic GMP and decreased the tissue lactate level. It is concluded that cysteine potentiates the effect of nitroglycerin on cyclic GMP formation even in integrated cell systems with an intact physiological function.