Influence of nitric oxide donors and peroxynitrite on the contractile effect and concentration of norepinephrine

Nitric oxide (NO) and peroxynitrite (ONOO) are said to destroy norepinephrine (NE). We studied the role of NE decomposition by NO donors and ONOO as they affect the contractile activity of NE in rat denuded thoracic aorta. First, we determined the relaxing effect of NO donors (SNAP, PROLI/NO, Sodium nitrite, SIN-1) and ONOO after precontraction by NE (1 microM). SNAP and SIN-1 (EC(50) 50-110 nM) were more active than PROLI/NO, Sodium nitrite or ONOO (EC(50) 19-30 microM). The relaxing effect of NO donors and ONOO were decreased by ODQ (10 microM), a guanylate cyclase inhibitor. Second, we compared the contractile activity of NE before and after preincubation with NO donors or ONOO in presence of ODQ. NE (1 microM) was incubated with NO donors or ONOO at the concentrations of 0.1 mM in both Krebs solution or phosphate buffer (pH 7.4; 0.1 M) for 10 minutes at 37 degrees C. NE evoked the aorta contraction in the same concentrations before and after preincubation with NO donors. In contrast, ONOO decreased effect of NE, EC(50) was measured at 4.3+/-0.3 nM and 13.4+/-1.6 nM, before and after preincubation of NE with ONOO respectively. Third, we measured the NE concentration using the HPLC method. We revealed that the concentration of NE after preincubation with NO donors was unaltered. However HPLC measurement revealed that NE concentration after preincubation with ONOO was reduced 2-3-fold. Therefore, under these experimental conditions ONOO, but not NO donors, was capable of destroying NE.     

Nitric oxide inhibits falcipain, the Plasmodium falciparum trophozoite cysteine protease

Nitric oxide (NO) is a pluripotent regulatory molecule possessing, among others, an antiparasitic activity. In the present study, the inhibitory effect of NO on the catalytic activity of falcipain, the papain-like cysteine protease involved in Plasmodium falciparum trophozoite hemoglobin degradation, is reported. In particular, NO donors S-nitrosoglutathione (GSNO), (+/-)-(E)-p6ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenami de (NOR-3), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) inhibit dose-dependently the falcipain activity present in the P. falciparum trophozoite extract, this effect likely attributable to S-nitrosylation of the Cys25 catalytic residue. The results represent a new insight into the modulation mechanism of falcipain activity, thereby being relevant in developing new strategies for inhibition of the P. falciparum life cycle.     

Interaction of heme oxygenase-2 with nitric oxide donors. Is the oxygenase an intracellular 'sink' for NO?

Heme oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281 --> Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm --> 413-419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased ( approximately two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular 'sink' for the heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.     

The survival of skeletal muscle myoblasts in vitro is sensitive to a donor of nitric oxide and superoxide, SIN-1, but not to nitric oxide or peroxynitrite alone.

The survival of skeletal muscle myoblasts in culture after exposure either to a donor of NO, sodium nitroprusside (SNP), or ethanamine, 2,2'-(hydroxynitrosohydrazono)bis-(DETA NONOate), or to a donor of both NO and O(-)(2), 3-morpholinosydnonimine hydrochloride (SIN-1), was investigated. SIN-1 reduced clonogenic survival markedly but donors of NO alone did not. The injurious effect of SIN-1 was prevented by oxyhemoglobin or by uric acid but not by superoxide dismutase. The exposure of myoblasts to authentic peroxynitrite (ONOO(-)) or to DETA NONOate in the presence of an O(-)(2)-generating system did not reduce their survival. The results show that NO or ONOO(-) alone is not detrimental to myoblast survival and suggest that SIN-1 toxicity is, at least in part, mediated by H(2)O(2) in this myoblast culture system.     

S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide

Nitric oxide (NO) physiologically stimulates the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) to decrease intracellular Ca(2+) concentration and relax cardiac, skeletal and vascular smooth muscle. Here, we show that NO-derived peroxynitrite (ONOO(-)) directly increases SERCA activity by S-glutathiolation and that this modification of SERCA is blocked by irreversible oxidation of the relevant cysteine thiols during atherosclerosis. Purified SERCA was S-glutathiolated by ONOO(-) and the increase in Ca(2+)-uptake activity of SERCA reconstituted in phospholipid vesicles required the presence of glutathione. Mutation of the SERCA-reactive Cys674 to serine abolished these effects. Because superoxide scavengers decreased S-glutathiolation of SERCA and arterial relaxation by NO, ONOO(-) is implicated as the intracellular mediator. NO-dependent relaxation as well as S-glutathiolation and activation of SERCA were decreased by atherosclerosis and Cys674 was found to be oxidized to sulfonic acid. Thus, irreversible oxidation of key thiol(s) in disease impairs NO-induced relaxation by preventing reversible S-glutathiolation and activation of SERCA by NO/ONOO(-).     

Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes

We investigated the potential involvement of peroxynitrite (ONOO(-)) in the modulation of calcium current (I(Ca)) in guinea pig ventricular myocytes with the whole-cell patch clamp technique and with cyclic AMP (cAMP) measurements. Because of the short half-life of ONOO(-) at physiological pH, we induced an increase in its intracellular levels by using donors of the precursors, nitric oxide (NO) and superoxide anion (O(2) (-)). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 microM) or SNAP (300 microM) increased basal I(Ca) (50.3 +/- 4.6%, n = 7 and 46.2 +/- 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 microM) also stimulated basal I(Ca) (44.6 +/- 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 microM), although separately ineffective on I(Ca), enhanced the current if applied together (33.5 +/- 0.7%, n = 7). The simultaneous donor of O(2) (-) and NO, SIN-1 (500 microM), also stimulated basal I(Ca) (22.8 +/- 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 microM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 microM), I(Ca) was still increased by SIN-1 (32.0 +/- 6.1%, n = 4 and 30.0 +/- 5.4%, n = 8). Both Manganese(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP, 100 microM) a ONOO(-) scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN-1 on I(Ca) (respectively -0.6 +/- 4.1%, n = 4 and 3.6 +/- 4.3%, n = 4). Intracellular cAMP level was unaltered by SIN-1, while it was enhanced by blocking the NO-cGMP pathway with the NO synthase inhibitor L-NMMA. These results suggest that peroxynitrite donors increase cardiac calcium current without the involvement of cAMP and cGMP.     

Nitric oxide-related species-induced protein oxidation: reversible, irreversible, and protective effects on enzyme function of papain

Protein oxidation, irreversible modification, and inactivation may play key roles in various neurodegenerative disorders. Therefore, we studied the effects of the potentially in vivo occurring nitric oxide-related species on two different markers of protein oxidation: protein carbonyl generation on bovine serum albumine (BSA) and loss of activity of a cysteine-dependent protease, papain, in vitro by using Angeli's salt, papanonoate, SIN-1, and S-nitrosoglutathione (GSNO) as donors of nitroxyl, nitric oxide, peroxynitrite, and nitrosonium ions, respectively. Angeli's salt, SIN-1, and papanonoate (0-1000 microM) all generated a concentration-dependent increase in carbonyl formation on BSA (107, 60, and 45%, respectively). GSNO did not affect carbonyl formation. Papain was inhibited by Angeli's salt, SIN-1, papanonoate, and GSNO with IC50 values of 0.62, 2.3, 54, and 80 microM, respectively. Angeli's salt (3.16 microM)-induced papain inactivation was only partially reversible, while the effects of GSNO (316 microM) and papanonoate (316 microM) were reversible upon addition of excess DTT. The Angeli's salt-mediated DTT-irreversible inhibition of papain was prevented by GSNO or papanonoate pretreatment, hypothetically through mixed disulfide formation or S-nitrosylation of the catalytically critical thiol group of papain. These results, for the first time, compare the generation of carbonyls in proteins by Angeli's salt, papanonoate, and SIN-1. Furthermore, these results suggest that S-nitrosothiols may have a novel function in protecting critical thiols from irreversible oxidative damage.     

Nitric oxide inhibits cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi

Nitric oxide (NO) is a pluripotent regulatory molecule showing, among others, an antiparasitic activity. Moreover, NO inhibits cysteine proteinase action by nitrosylating the Cys catalytic residue. In the present study, the inhibitory effect of the substrate N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methyl coumarin) and of NO on the catalytic activity of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi (the hemoflagellate protozoan parasite which causes the American trypanosomiasis), is reported. In particular, NO-donors S-nitroso-glutathione (GSNO), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), S-nitroso-acetyl-penicillamine (SNAP), and sodium nitroprusside (SNP) dose-dependently inhibited cruzipain, this effect being likely attributable to the S-nitrosylation of the Cys25 catalytic residue. These results were analyzed in parallel with those concerning the inhibitory effect of the substrate and of NO on the catalytic activity of falcipain, the cruzipain-homologous cysteine proteinase from Plasmodium falciparum. The modulation of the cruzipain and falcipain activity by NO may be relevant in developing new strategies against T. cruzi and P. falciparum in human host. As a whole, the NO-mediated S-nitrosylation of pathogenic viral, bacterial, fungal, and parasitic cysteine proteinases may represent a general mechanism of antimicrobial and antiparasitic host defences.     

Mobilisation of Ca2+ stores and flagellar regulation in human sperm by S-nitrosylation: a role for NO synthesised in the female reproductive tract

Generation of NO by nitric oxide synthase (NOS) is implicated in gamete interaction and fertilisation. Exposure of human spermatozoa to NO donors caused mobilisation of stored Ca(2+) by a mechanism that did not require activation of guanylate cyclase but was mimicked by S-nitroso-glutathione (GSNO; an S-nitrosylating agent). Application of dithiothreitol, to reduce protein -SNO groups, rapidly reversed the actions of NO and GSNO on [Ca(2+)](i). The effects of NO, GSNO and dithiothreitol on sperm protein S-nitrosylation, assessed using the biotin switch method, closely paralleled their actions on [Ca(2+)](i). Immunofluorescent staining revealed constitutive and inducible NOS in human oviduct and cumulus (the cellular layer investing the oocyte). 4,5-diaminofluorescein (DAF) staining demonstrated production of NO by these tissues. Incubation of human sperm with oviduct explants induced sperm protein S-nitrosylation resembling that induced by NO donors and GSNO. Progesterone (a product of cumulus cells) also mobilises stored Ca(2+) in human sperm. Pre-treatment of sperm with NO greatly enhanced the effect of progesterone on [Ca(2+)](i), resulting in a prolonged increase in flagellar excursion. We conclude that NO regulates mobilisation of stored Ca(2+) in human sperm by protein S-nitrosylation, that this action is synergistic with that of progesterone and that this synergism is potentially highly significant in gamete interactions leading to fertilisation.     

Cerebrovascular protection by various nitric oxide donors in rats after experimental stroke.

The efficacy of nitric oxide (NO) treatment in ischemic stroke, though well recognized, is yet to be tested in clinic. NO donors used to treat ischemic injury are structurally diverse compounds. We have shown that treatment of S-nitrosoglutathione (GSNO) protects the brain against injury and inflammation in rats after experimental stroke [M. Khan, B. Sekhon, S. Giri, M. Jatana, A. G. Gilg, K. Ayasolla, C. Elango, A. K. Singh, I. Singh, S-Nitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke, J. Cereb. Blood Flow Metab. 25 (2005) 177-192.]. In this study, we tested structurally different NO donors including GSNO, S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (SNP), methylamine hexamethylene methylamine NONOate (MAHMA), propylamine propylamine NONOate (PAPA), 3-morpholinosydnonimine (SIN-1) and compared their neuroprotective efficacy and antioxidant property in rats after ischemia/reperfusion (I/R). GSNO, in addition to neuroprotection, decreased nitrotyrosine formation and lipid peroxidation in blood and increased the ratio of reduced versus oxidized glutathione (GSH/GSSG) in brain as compared to untreated animals. GSNO also prevented the I/R-induced increase in mRNA expression of ICAM-1 and E-Selectin. SNAP and SNP extended limited neuroprotection, reduced nitrotyrosine formation in blood and blocked increase in mRNA expression of ICAM-1 and E-Selectin in brain tissue. PAPA, MAHMA, and SIN-1 neither protected the brain nor reduced oxidative stress. We conclude that neuroprotective action of NO donors in experimental stroke depends on their ability to reduce oxidative stress both in brain and blood.     

The aqueous garlic, onion and leek extracts release nitric oxide from S-nitrosoglutathione and prolong relaxation of aortic rings

Garlic, onion and leek have beneficial effects in treatment of numerous health disorders. The aim of the present study was to investigate underlying molecular mechanisms. To test the potency of the aqueous garlic, onion and leek extracts to release NO from GSNO we have measured NO oxidation product, NO(2)-, by the Griess reagent method. Further, we studied the ability of garlic extract to relax noradrenaline-precontracted rat aortic rings in the presence of GSNO and effects of garlic extract on electrical properties of rat heart intracellular chloride channels. We have observed that: i) garlic, onion and leek extracts released NO from GSNO in the order: garlic > onion > leek; ii) the ability of garlic extract to release NO was pH-dependent (8.0 > 7.4 > 6.0) and potentiated by thiols (Cys > GSH = N-acetyl-cysteine > oxidized glutathione) at concentration 100 μmol/l; iii) the garlic extract (0.045 mg/ml) prolonged relaxation time of aortic rings induced by GSNO (50 nmol/l) and inhibited intracellular chloride channels. We suggest that NO-releasing properties of the garlic, onion and leek extracts and their interaction with Cys and GSH are involved in NO-signalling pathway which contributes to some of its numerous beneficial biological effects.     

Ascorbate is a potent antioxidant against peroxynitrite-induced oxidation reactions. Evidence that ascorbate acts by re-reducing substrate radicals produced by peroxynitrite

Peroxynitrite (ONOO(((-)))/ONOOH) is expected in vivo to react predominantly with CO(2), thereby yielding NO(2)(.) and CO(3) radicals. We studied the inhibitory effects of ascorbate on both NADH and dihydrorhodamine 123 (DHR) oxidation by peroxynitrite generated in situ from 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). SIN-1 (150 micrometer)-mediated oxidation of NADH (200 micrometer) was half-maximally inhibited by low ascorbate concentrations (61-75 micrometer), both in the absence and presence of CO(2). Control experiments performed with thiols indicated both the very high antioxidative efficiency of ascorbate and that in the presence of CO(2) in situ-generated peroxynitrite exclusively oxidized NADH via the CO(3) radical. This fact is attributed to the formation of peroxynitrate (O(2)NOO(-)/O(2)NOOH) from reaction of NO(2)(.) with O(2), which is formed from reaction of CO(3) with NADH. SIN-1 (25 micrometer)-derived oxidation of DHR was half-maximally inhibited by surprisingly low ascorbate concentrations (6-7 micrometer), irrespective of the presence of CO(2). Control experiments performed with authentic peroxynitrite revealed that ascorbate was in regard to both thiols and selenocompounds much more effective to protect DHR. The present results demonstrate that ascorbate is highly effective to counteract the oxidizing properties of peroxynitrite in the absence and presence of CO(2) by both terminating CO(3)/HO( small middle dot) reactions and by its repair function. Ascorbate is therefore expected to act intracellulary as a major peroxynitrite antagonist. In addition, a novel, ascorbate-independent protection pathway exists: scavenging of NO(2)(.) by O(2) to yield O(2)NOO(-), which further decomposes into NO(2)(-) and O(2).     

Anomalous Increase in Nitric Oxide Synthase Activity by Certain Nitric Oxide-Generating Compounds in Intact Neuronal Cells

Abstract: It has been shown that nitric oxide (NO) regulates NO synthase (NOS) activity through negative feedback in cytosolic enzyme preparations in various cell types. We compared the effects of the NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) on NOS activity in intact neuroblastoma N1E-115 cells and in the cytosol obtained from the same cells. Enzyme activity was measured by the conversion of l -[³H]arginine into l -[³H]citrulline. At concentrations that elicit almost complete inhibition of NOS activity in cytosolic enzyme preparations of these cells, SIN-1 and SNP did not cause significant attenuation of enzyme activity measured at 45 min in intact cells. It is surprising that SIN-1 and SNP markedly stimulated l -[³H]citrulline formation in a time- and concentration-dependent manner when cells were incubated with the compounds for >1.5 h. Neither inhibitory nor stimulatory effects of SNAP on NOS were observed in intact N1E-115 cells. This is in contrast to the inhibitory effects of SNAP in cytosolic preparations of the enzyme. The increased NOS activity by SIN-1 or SNP in intact cells was dependent on the presence of extracellular Ca²⁺, suggesting that it might be due to increased Ca²⁺ influx. On the other hand, measurements of the activity of lactate dehydrogenase showed that there was no generalized increase in cell permeability in response to SIN-1 or SNP. There was no agreement in the rank order of potencies of these compounds in activating guanylate cyclase and in affecting NOS activity, both in broken-cell preparations and in intact cells. Thus, modulation of NOS activity by NO-releasing compounds is not dependent on cyclic GMP formation and might not be related in a simple fashion to NO generation. Alternatively, activation of guanylate cyclase and stimulation of NOS activity might require different redox species of NO. Our present findings might be of clinical relevance in relation to long-term use of NO-generating compounds as therapeutic agents.     

Interference of S-Nitrosoglutathione with the Binding of Ligands to Ionotropic Glutamate Receptors in Pig Cerebral Cortical Synaptic Membranes

The interactions of S-nitrosoglutathione (GSNO) with the ionotropic glutamate receptors were studied on synaptic membranes isolated from the pig cerebral cortex. GSNO displaced the binding of [³H]glutamate, 3-[(R)-2-carboxypiperazin-4-yl][³H]propyl-1-phosphonate ([³H]CPP), a competitive N-methyl-D-aspartate (NMDA) antagonist, and [³H]kainate, with IC₅₀ values in the low micromolar range. It failed to displace (S)-5-fluoro-[³H]willardiine, a selective agonist of 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Reduced and oxidized glutathione were almost as effective as GSNO in glutamate and CPP binding. Of the three, GSNO was the most potent in kainate binding. They all stimulated [³H]dizocilpine binding in a concentration-dependent manner. This effect was additive to that of glycine and not mimicked by NO donors such as S-nitroso-N-acetylpenicillamine, 5-amino-3-morpholinyl-1,2,3-oxadiazolium chloride (SIN-1) and nitroglycerin. We assume that GSNO may act as an endogenous ligand at the NMDA and non-NMDA classes of glutamate receptors. In this manner it may facilitate NO transfer and target its delivery to specific sites in these receptors.     

Effects of nitric oxide donors on vascular endothelial growth factor gene induction

Nitric oxide (NO) has been reported to modulate the vascular endothelial growth factor (VEGF) gene by accumulating hypoxia-inducible factor-1alpha (HIF-1alpha) protein, but there is a contradiction among effects of various NO donors. The effects of NO donors including S-nitroso-N-acetyl-penicillamine (SNAP), S-nitroso-glutathione (GSNO), 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), 3-[(+/-)-(E)-ethyl-2(')-[(E)-hydroxyimino]-5-nitro-3-hexenecarbamoyl]-pyridine (NOR4), 3-morpholinosydnonimine (SIN-1), and nitroprusside (SNP) on the VEGF reporter gene were examined. SNAP, GSNO, NOC18, and NOR4 enhanced the VEGF reporter activity under normoxia and modulated the hypoxic induction. In contrast, SNP had only an inhibitory effect. An NO scavenger attenuated the reporter activation by NO donors except NOR4, but did not ameliorate the inhibitory effect of SNP. A reducing compound dithiothreitol suppressed NO-induced activation of the VEGF reporter gene. SNAP, GSNO, and NOC18 induced the accumulation of HIF-1alpha protein, while others did not. These results suggest that SNAP, GSNO, and NOC compounds are suitable for pharmacological studies in HIF-1-mediated VEGF gene activation by NO.     

Nitric oxide-cGMP-mediated vasoconstriction and effects of acetylcholine in the branchial circulation of the eel

Information about the presence and effects of nitric oxide (NO) in fish vasculature is scant and contradictory. We have studied the NO/cGMP system in the branchial circulation of the teleost Anguilla anguilla using a branchial basket preparation under basal conditions and cholinergic stimulation. The effects of endogenous and exogenous NO were tested with L-arginine, the nitric oxide synthase (NOS) substrate, and the NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), respectively. L-arginine (from 10(-11) to 10(-6) M) and the NO donors (starting from 10(-14) M) caused dose-dependent vasoconstriction. Conversely, in the ACh-pre-contracted preparations both donors elicited vasodilation. SIN-1-induced vasoconstriction was due to NO generation: it was increased by superoxide dismutase (SOD) and blocked by NO scavenger hemoglobin. Pre-treatment with sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of SIN-1 and SNP. The stable cGMP analogue 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br cGMP) induced dose-dependent vasoconstriction. Unexpectedly, three NOS inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-monomethyl-L-arginine (L-NMMA), L-N(5)-(1-iminoethyl) ornithine (L-NIO), caused mild vasoconstriction. ACh caused vasoconstriction, but at pico- and nanomolar concentrations it caused mild but significant vasodilation in 40% of the preparations. Both responses, blocked by atropine and pirenzepine, required an intact endothelium. The ACh-induced vasoconstriction was substantially independent of a NO-cGMP mechanism.     

Mechanism of p21Ras S-nitrosylation and kinetics of nitric oxide-mediated guanine nucleotide exchange

Nitric oxide (NO), a highly reactive redox molecule, can react with protein thiols and protein metal centers to regulate a multitude of physiological processes. NO has been shown to promote guanine nucleotide exchange on the critical cellular signaling protein p21Ras (Ras) by S-nitrosylation of a redox-active thiol group (Cys(118)). This increases cellular Ras-GTP levels in vivo, leading to activation of downstream signaling pathways. Yet the process by which this occurs is not clear. Although several feasible mechanisms for protein S-nitrosylation with NO and NO donating have been proposed, results obtained from our studies suggest that Ras can be S-nitrosylated by direct reaction of Cys(118) with nitrogen dioxide (*NO(2)), a reaction product of NO with O(2), via a Ras thiyl-radical intermediate (Ras-S*). Results from our studies also indicate that Ras Cys(118) can be S-nitrosylated by direct reaction of Cys(118) with a glutathionyl radical (GS*), a reaction product derived from homolytic cleavage of S-nitrosoglutathione (GSNO). Moreover, we present evidence that reaction of GS* with Ras generates a Ras-S* intermediate during GSNO-mediated Ras S-nitrosylation. The Ras-S(*) radical intermediate formed from reaction of the Ras thiol with either *NO(2) or GS*, in turn, reacts with NO to complete Ras S-nitrosylation. NO and GSNO modulate Ras activity by promoting guanine nucleotide dissociation from Ras. Our results suggest that formation of the Ras radical intermediate, Ras-S*, may perturb interactions between Ras and its guanine nucleotide substrate, resulting in enhancement of guanine nucleotide dissociation from Ras.     

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.