Biomedical subjects

M Feelisch

Publications and source records attributed to M Feelisch.

At least 19 recordsLinked to original sources

PubMed2004

Superoxide dismutase and catalase are required to detect (.-)NO from both coupled and uncoupled neuronal no synthase.

Despite numerous approaches to measuring nitric oxide ((.-)NO) formation from purified NO synthase (NOS), it is still not clear whether (.-)NO is a direct or indirect product of the NO synthase reaction. The direct detection of catalytically formed (.-)NO is complicated by side reactions with reactive oxide species like H(2)O(2) and superoxide. The aim of the present study was therefore to reinvestigate these reactions both electrochemically and by chemiluminescence detection with particular emphasis on the requirement for cofactors and their interference with (.-)NO detection. Flavins were found to generate large amounts of H(2)O(2) and were therefore excluded from subsequent incubations. Under conditions of both coupled and uncoupled catalysis, SOD was absolutely required to detect (.-)NO from NOS. H(2)O(2) formation took place also in the presence of SOD and gave a smaller yet significant interfering signal. Similar data were obtained when the proposed intermediate N(omega)-hydroxy-l-arginine was utilized as substrate. In conclusion, standard Clark-type ()NO electrodes are cross-sensitive to H(2)O(2) and therefore both SOD and catalase are absolutely required to specifically detect (.-)NO from NOS.

Arginine↗

PubMed2001

Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action.

The plasma level of NO(x), i.e., the sum of NO(2)- and NO(3)-, is frequently used to assess NO bioavailability in vivo. However, little is known about the kinetics of NO conversion to these metabolites under physiological conditions. Moreover, plasma nitrite recently has been proposed to represent a delivery source for intravascular NO. We therefore sought to investigate in humans whether changes in NO(x) concentration are a reliable marker for endothelial NO production and whether physiological concentrations of nitrite are vasoactive. NO(2)- and NO(3)- concentrations were measured in blood sampled from the antecubital vein and brachial artery of 24 healthy volunteers. No significant arterial-venous gradient was observed for either NO(2)- or NO(3)-. Endothelial NO synthase (eNOS) stimulation with acetylcholine (1-10 microg/min) dose-dependently augmented venous NO(2)- levels by maximally 71%. This effect was paralleled by an almost 4-fold increase in forearm blood flow (FBF), whereas an equieffective dose of papaverine produced no change in venous NO(2)-. Intraarterial infusion of NO(2)- had no effect on FBF. NOS inhibition (N(G)-monomethyl-l-arginine; 4-12 micromol/min) dose-dependently reduced basal NO(2)- and FBF and blunted acetylcholine-induced vasodilation and NO release by more than 80% and 90%, respectively. In contrast, venous NO(3)- and total NO(x) remained unchanged as did systemic arterial NO(2)- and NO(3)- levels during all these interventions. FBF and NO release showed a positive association (r = 0.85; P < 0.001). These results contradict the current paradigm that plasma NO(3)- and/or total NO(x) are generally useful markers of endogenous NO production and demonstrate that only NO(2)- reflects acute changes in regional eNOS activity. Our results further demonstrate that physiological levels of nitrite are vasodilator-inactive.

Acetylcholine↗

PubMed2001

Nitroxyl anion exerts redox-sensitive positive cardiac inotropy in vivo by calcitonin gene-related peptide signaling.

Nitroxyl anion (NO(-)) is the one-electron reduction product of nitric oxide (NO( small middle dot)) and is enzymatically generated by NO synthase in vitro. The physiologic activity and mechanism of action of NO(-) in vivo remains unknown. The NO(-) generator Angeli's salt (AS, Na(2)N(2)O(3)) was administered to conscious chronically instrumented dogs, and pressure-dimension analysis was used to discriminate contractile from peripheral vascular responses. AS rapidly enhanced left ventricular contractility and concomitantly lowered cardiac preload volume and diastolic pressure (venodilation) without a change in arterial resistance. There were no associated changes in arterial or venous plasma cGMP. The inotropic response was similar despite reflex blockade with hexamethonium or volume reexpansion, indicating its independence from baroreflex stimulation. However, reflex activation did play a major role in the selective venodilation observed under basal conditions. These data contrasted with the pure NO donor diethylamine/NO, which induced a negligible inotropic response and a more balanced veno/arterial dilation. AS-induced positive inotropy, but not systemic vasodilatation, was highly redox-sensitive, being virtually inhibited by coinfusion of N-acetyl-l-cysteine. Cardiac inotropic signaling by NO(-) was mediated by calcitonin gene-related peptide (CGRP), as treatment with the selective CGRP-receptor antagonist CGRP(8-37) prevented this effect but not systemic vasodilation. Thus, NO(-) is a redox-sensitive positive inotrope with selective venodilator action, whose cardiac effects are mediated by CGRP-receptor stimulation. This fact is evidence linking NO(-) to redox-sensitive cardiac contractile modulation by nonadrenergic/noncholinergic peptide signaling. Given its cardiac and vascular properties, NO(-) may prove useful for the treatment of cardiovascular diseases characterized by cardiac depression and elevated venous filling pressures.

Animals↗

PubMed2001

Nitroxyl oxidizes NADPH in a superoxide dismutase inhibitable manner.

Nitric oxide synthases (NOS) convert L-arginine and N(omega)-hydroxy-L-arginine to nitric oxide (*NO) and/or nitroxyl (NO(-)) in a NADPH-dependent fashion. Subsequently, *NO/superoxide (O(2-)-derived peroxynitrite (ONOO(-)) consumes one additional mol NADPH. The related stoichiometry of NO(-) and NADPH is unclear. We here describe that NO(-) also oxidizes NADPH in a concentration-dependent manner. In the presence of superoxide dismutase (SOD), which also converts NO(-) to *NO, nitrite accumulation was almost doubled and no oxidation of NADPH was observed. Nitrate yield from NO(-) was low, arguing against intermediate ONOO(-) formation. Thus, biologically formed NO(-) may function as an effective pro-oxidant unless scavenged by SOD and affect the apparent NADPH stoichiometry of the NOS reaction.

Free Radicals↗

PubMed2001

Mechanisms of the antioxidant effects of nitric oxide.

The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury. There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O2- that occurs at physiological levels of NO. In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, appears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress.

Animals↗

PubMed2000

Unique oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion.

The nitroxyl anion (NO-) is a highly reactive molecule that may be involved in pathophysiological actions associated with increased formation of reactive nitrogen oxide species. Angeli's salt (Na2N2O3; AS) is a NO- donor that has been shown to exert marked cytotoxicity. However, its decomposition intermediates have not been well characterized. In this study, the chemical reactivity of AS was examined and compared with that of peroxynitrite (ONOO-) and NO/N2O3. Under aerobic conditions, AS and ONOO- exhibited similar and considerably higher affinities for dihydrorhodamine (DHR) than NO/N2O3. Quenching of DHR oxidation by azide and nitrosation of diaminonaphthalene were exclusively observed with NO/N2O3. Additional comparison of ONOO- and AS chemistry demonstrated that ONOO- was a far more potent one-electron oxidant and nitrating agent of hydroxyphenylacetic acid than was AS. However, AS was more effective at hydroxylating benzoic acid than was ONOO-. Taken together, these data indicate that neither NO/N2O3 nor ONOO- is an intermediate of AS decomposition. Evaluation of the stoichiometry of AS decomposition and O2 consumption revealed a 1:1 molar ratio. Indeed, oxidation of DHR mediated by AS proved to be oxygen-dependent. Analysis of the end products of AS decomposition demonstrated formation of NO2- and NO3- in approximately stoichiometric ratios. Several mechanisms are proposed for O2 adduct formation followed by decomposition to NO3- or by oxidation of an HN2O3- molecule to form NO2-. Given that the cytotoxicity of AS is far greater than that of either NO/N2O3 or NO + O2, this study provides important new insights into the implications of the potential endogenous formation of NO- under inflammatory conditions in vivo.

Nitrates↗

PubMed2000

Electron-paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron cannot discriminate between nitric oxide and nitroxyl: implications for the detection of reaction products for nitric oxide synthase.

Purified neuronal nitric oxide synthase (NOS) does not produce nitric oxide (NO) unless high concentrations of superoxide dismutase (SOD) are added, suggesting that nitroxyl (NO(-)) or a related molecule is the principal reaction product of NOS, which is SOD-dependently converted to NO. This hypothesis was questioned by experiments using electron paramagnetic resonance spectroscopy and iron N-methyl-D-glucamine dithiocarbamate (Fe-MGD) as a trap for NO. Although NOS and the NO donor S-nitroso-N-acetyl-penicillamine produced an electron paramagnetic resonance signal, the NO(-) donor, Angeli's salt (AS) did not. AS is a labile compound that rapidly hydrolyzes to nitrite, and important positive control experiments showing that AS was intact were lacking. On reinvestigating this crucial experiment, we find identical MGD(2)-Fe-NO complexes both from S-nitroso-N-acetyl-penicillamine and AS but not from nitrite. Moreover, the yield of MGD(2)-Fe-NO complex from AS was stoichiometric even in the absence of SOD. Thus, MGD(2)-Fe directly detects NO(-), and any conclusions drawn from MGD(2)-Fe-NO complexes with respect to the nature of the primary NOS product (NO, NO(-), or a related N-oxide) are invalid. Thus, NOS may form NO(-) or related N-oxides instead of NO.

Animals↗

PubMed2000

Continuous exposure to high concentrations of nitric oxide leads to persistent inhibition of oxygen consumption by J774 cells as well as extraction of oxygen by the extracellular medium.

Nitric oxide (NO) plays a key role in many physiological and pathophysiological events, including the control of cell respiration. Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of NO by cells. We have exposed the murine macrophage cell line J774 to high concentrations of NO, such as are generated in some pathological conditions, and determined their effect on oxygen consumption. We observed a persistent inhibition of respiration which was due to a redox-dependent, progressive inhibition of complex I activity. No other enzyme of the respiratory chain was inhibited in this way. At the same time, we detected a paradoxical removal of oxygen by the extracellular medium. This removal was due to a chemical interaction between dissolved oxygen and NO-related species released from cells exposed to NO. A similar removal of oxygen by the cell supernatant also occurred following activation of cells with cytokines and bacterial products. Thus, the amounts of NO generated during pathological conditions may contribute to tissue hypoxia both by inhibiting cell respiration and by promoting removal of oxygen from the extracellular medium.

Animals↗

PubMed2000

Dynamic state of S-nitrosothiols in human plasma and whole blood.

In the vasculature, nitrosothiols derived from the nitric oxide (NO)-mediated S-nitrosation of thiols play an important role in the transport, storage, and metabolism of NO. The present study was designed to examine the reactions that promote the decomposition, formation, and distribution of extracellular nitrosothiols in the circulation. The disappearance of these species in plasma and whole blood was examined using a high-performance liquid chromatography method to separate low- and high-molecular weight nitrosothiols. We found that incubation of S-nitrosocysteine (CySNO) or S-nitrosoglutathione (GSNO) with human plasma resulted in a rapid decomposition of these nitrosothiols such that <10% of the initial concentration was recovered after 10-15 min. Neither metal chelators (DTPA, neocuproine), nor zinc chloride (glutathione peroxidase inhibitor), acivicin (gamma-glutamyl transpeptidase inhibitor), or allopurinol (xanthine oxidase inhibitor) inhibited the decomposition of GSNO. With both CySNO and GSNO virtually all NO was recovered as S-nitrosoalbumin (AlbSNO), suggesting the involvement of a direct transnitrosation reaction. Electrophilic attack of the albumin-associated thiols by reactive nitrogen oxides formed from the interaction of NO with O(2) was ruled out because one would have expected 50% yield of AlbSNO. Similar results were obtained in whole blood. The amount of S-nitrosohemoglobin recovered in the presence of 10 microM GSNO or CySNO was less than 100 nM taking into consideration the detection limit of the assay used. Our results suggest that serum albumin may act as a sink for low-molecular-weight nitrosothiols and as a modulator of NO(+) transfer between the vascular wall and intraerythrocytic hemoglobin.

Allopurinol↗

PubMed2000

A chemiluminescense-based assay for S-nitrosoalbumin and other plasma S-nitrosothiols.

The lack of a simple assay for the quantification of S-nitrosothiols in complex biological matrices has hampered our understanding of their contribution to normal physiology and pathophysiological states. In this paper we describe an assay based upon the release of nitric oxide by reaction with a mixture consisting of Cu(I), iodine and iodide with subsequent quantification by chemiluminescense. With this method we can detect levels of S-nitrosothiols down to 5 nM in plasma. Following alkylation of free thiols with N-ethylmaleimide, and removal of nitrite with acidified sulfanilamide, we were able to measure known amounts of S-nitrosoalbumin added to plasma or whole blood, with an inter-assay variation for plasma S-nitrosothiols of approximately 4%. Further studies showed that the mean concentration of circulating S-nitrosothiols in venous plasma of healthy human volunteers was 28+/-7 nM.

Adult↗

PubMed2000

Mechanisms of cell death governed by the balance between nitrosative and oxidative stress.

Many cellular functions in physiology are regulated by the direct interaction of NO with target biomolecules. In many pathophysiologic and toxicologic mechanisms, NO first reacts with oxygen, superoxide or other nitrogen oxides to subsequently elicit indirect effects. The balance between nitrosative stress and oxidative stress within a specific biological compartment can determine whether the presence of NO will be ultimately deleterious or beneficial. Nitrosative stress can be defined primarily through reactions mediated by N2O3, a reactive nitrogen oxide species generated by high fluxes of NO in an aerobic environment. In contrast, oxidative stress is mediated primarily by superoxide and peroxides. In addition to reactive oxygen species, several reactive nitrogen oxide species such as peroxynitrite, nitroxyl, and nitrogen dioxide can also impose oxidative stress to a cell. We here describe how the mechanisms of cell death are interwoven in the balance between the different chemical intermediates involved in nitrosative and oxidative stress.

Animals↗

PubMed2000

Inhibition of endothelial cell activation by nitric oxide donors.

Because nitric oxide (NO) inhibits the expression of endothelial leukocyte adhesion molecules, NO-generating compounds have major therapeutic potential for use outside their classical indications. We report on the in vitro potential antiatherogenicity of two novel cysteine-containing NO donors, SP/W 3672, a fast spontaneous NO releaser, and its prodrug SP/W 5186, which liberates NO after bioactivation. The ability of these two compounds to inhibit monocyte adhesion and surface expression of endothelial adhesion molecules was evaluated and compared with that of other NO donors. SP/W 5186 and SP/W 3672 inhibited the adhesion of U937 monocytes to cultured human endothelial cells more potently than S-nitrosoglutathione (GSNO) or spermine NONOate, whereas nitroglycerin and isosorbide dinitrate were ineffective at comparable concentrations. A similar rank order of potency was found for the inhibition of expression of the adhesion molecules vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin as well as for major histocompatibility complex class II antigen expression. Estimated IC(50) values for vascular cell adhesion molecule-1 were >400 microM for SP/W 4744 (control for SP/W 3672 lacking the cysteine moiety), 200 microM for GSNO and spermine NONOate, 80 microM for SP/W 3672, and 50 microM for SP/W 5186. Moreover, SP/W 5186 inhibited VCAM-1 mRNA levels more potently than GSNO. This effect was likely to be transcriptional because mRNA degradation was not affected. In conclusion, SP/W 3672 and SP/W 5186 are novel potent inhibitors of endothelial activation, and this effect appears to relate to their ability to liberate NO for prolonged periods of time, either spontaneously or after conversion to active hydrolytic products.

Cell Adhesion↗

PubMed1999

Opposite effects of nitric oxide and nitroxyl on postischemic myocardial injury.

Recent experimental evidence suggests that reactive nitrogen oxide species can contribute significantly to postischemic myocardial injury. The aim of the present study was to evaluate the role of two reactive nitrogen oxide species, nitroxyl (NO(-)) and nitric oxide (NO(.)), in myocardial ischemia and reperfusion injury. Rabbits were subjected to 45 min of regional myocardial ischemia followed by 180 min of reperfusion. Vehicle (0.9% NaCl), 1 micromol/kg S-nitrosoglutathione (GSNO) (an NO(.) donor), or 3 micromol/kg Angeli's salt (AS) (a source of NO(-)) were given i.v. 5 min before reperfusion. Treatment with GSNO markedly attenuated reperfusion injury, as evidenced by improved cardiac function, decreased plasma creatine kinase activity, reduced necrotic size, and decreased myocardial myeloperoxidase activity. In contrast, the administration of AS at a hemodynamically equieffective dose not only failed to attenuate but, rather, aggravated reperfusion injury, indicated by an increased left ventricular end diastolic pressure, myocardial creatine kinase release and necrotic size. Decomposed AS was without effect. Co-administration of AS with ferricyanide, a one-electron oxidant that converts NO(-) to NO(.), completely blocked the injurious effects of AS and exerted significant cardioprotective effects similar to those of GSNO. These results demonstrate that, although NO(.) is protective, NO(-) increases the tissue damage that occurs during ischemia/reperfusion and suggest that formation of nitroxyl may contribute to postischemic myocardial injury.

Acetylcholine↗

PubMed1999

Biochemical characterization of S-nitrosohemoglobin. Mechanisms underlying synthesis, no release, and biological activity.

S-Nitrosohemoglobin (SNO-Hb) has been suggested to act as an endogenous NO donor and physiological regulator of blood pressure. However, the mechanisms responsible for the formation of SNO-Hb and those underlying the release of NO and subsequent biological activity have yet to be elucidated. In the present study, a number of nitrosated oxyhemoglobin (HbO(2)) derivatives have been synthesized and characterized. HbO(2) can be nitrosated at up to three distinct residues, one in the alpha-globin chain and two in the beta-chain. A beta-chain mononitrosated species (designated "SNO-Hb"), generated by the reaction of HbO(2) and S-nitrosoglutathione, released NO via a thiol-dependent mechanism involving nucleophilic attack at the nitrosated thiol functionality of SNO-Hb; in the case of glutathione, this process was associated with the formation of a mixed disulfide. In contrast, multinitrosated hemoglobin species released NO and relaxed vascular smooth muscle by a thiol-independent mechanism. HbO(2) scavenged potently NO released from SNO-Hb and inhibited its vasorelaxant properties. These data show that the predominant vasoactive species released from SNO-Hb is NO, with HNO a putative intermediate; the presence of a low molecular weight thiol is a prerequisite for this process. Such observations have important implications for the generation, metabolic fate, and biological activity of S-nitrosothiols.

2,3-Diphosphoglycerate↗

PubMed1999

Autoinhibition of neuronal nitric oxide synthase: distinct effects of reactive nitrogen and oxygen species on enzyme activity.

Nitric oxide (NO) synthases (NOSs), which catalyse the oxidation of L-arginine to L-citrulline and an oxide of nitrogen, possibly NO or nitroxyl (NO-), are subject to autoinhibition by a mechanism that has yet to be fully elucidated. In the present study we investigated the actions of NO and other NOS-derived products as possible autoregulators of enzyme activity. With the use of purified NOS-I, L-arginine turnover was found to operate initially at Vmax (0-15 min, phase I) although, despite the presence of excess substrate and cofactors, prolonged catalysis (15-90 min, phase II) was associated with a rapid decline in L-arginine turnover. Taken together, these observations suggested that one or more NOS products inactivate NOS. Indeed, exogenously applied reactive nitrogen oxide species (RNSs) decreased Vmax during phase I, although with different potencies (NO->NO> ONOO-) and efficacies (NO>NO-=ONOO-). The NO scavengers oxyhaemoglobin (HbO2; 100 microM) and 1H-imidazol-1 - yloxy - 2 - (4-carboxyphenyl) - 4,5 - dihydro - 4,4,5,5 - tetramethyl - 3 -oxide (CPTIO; 10 microM) and the ONOO- scavenger GSH (7 mM) had no effect on NOS activity during phase I, except for an endogenous autoinhibitory influence of NO and ONOO-. However, superoxide dismutase (SOD; 300 units/ml), which is thought either to increase the half-life of NO or to convert NO- to NO, lowered Vmax in an NO-dependent manner because this effect was selectively antagonized by HbO2 (100 microM). This latter observation demonstrated the requirement of SOD to reveal endogenous NO-mediated autoinhibition. Importantly, during phase II of catalysis, NOS became uncoupled and began to form H2O2 because catalase, which metabolizes H2O2, increased enzyme activity. Consistent with this, exogenous H2O2 also inhibited NOS activity during phase I. Thus during catalysis NOS is subject to complex autoinhibition by both enzyme-derived RNS and H2O2, differentially affecting enzyme activity.

Animals↗

PubMed1999

Regulation of the L-type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition.

In adult mammalian cardiomyocytes, stimulation of muscarinic receptors counterbalances the beta-adrenoceptor-mediated increase in myocardial contractility and heart rate by decreasing the L-type Ca2+ current (ICa) (1, 2). This effect is mediated via inhibition of adenylyl cyclase and subsequent reduction of cAMP-dependent phosphorylation of voltage-dependent L-type Ca2+ channels (3). Little is known, however, about the nature and origin of this pivotal inhibitory pathway. Using embryonic stem cells as an in vitro model of cardiomyogenesis, we found that muscarinic agonists depress ICa by 58 +/-3% (n=34) in early stage cardiomyocytes lacking functional beta-adrenoceptors. The cholinergic inhibition is mediated by the nitric oxide (NO)/cGMP system since it was abolished by application of NOS inhibitors (L-NMA, L-NAME), an inhibitor of the soluble guanylyl cyclase (ODQ), and a selective phosphodiesterase type II antagonist (EHNA). The NO/cGMP-mediated ICa depression was dependent on a reduction of cAMP/protein kinase A (PKA) levels since application of the catalytic subunit of PKA or of the PKA inhibitor PK) prevented the carbachol effect. In late development stage cells, as reported for ventricular cardiomyocytes (2, 4), muscarinic agonists had no effect on basal ICa but antagonized beta-adrenoceptor-stimulated ICa by 43 +/-4% (n=16). This switch in signaling pathways during development is associated with distinct changes in expression of the two NO-producing isoenzymes, eNOS and iNOS, respectively. These findings indicate a fundamental role for NO as a signaling molecule during early embryonic development and demonstrate a switch in the signaling cascades governing ICa regulation.

Adenine↗

PubMed1999

The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a] quinoxalin-1-one is a nonselective heme protein inhibitor of nitric oxide synthase and other cytochrome P-450 enzymes involved in nitric oxide donor bioactivation.

Soluble guanylyl cyclase (sGC) is an important effector for nitric oxide (NO). It acts by increasing intracellular cyclic GMP (cGMP) levels to mediate numerous biological functions. Recently, 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) was identified as a novel and selective inhibitor of this enzyme. Therefore, ODQ may represent an important pharmacological tool for differentiating cGMP-mediated from cGMP-independent effects of NO. In the present study, we examined the inhibitory action of ODQ both functionally and biochemically. In phenylephrine-preconstricted, endothelium-intact, isolated aortic rings from the rat, ODQ, in a concentration-dependent manner, increased contractile tone and inhibited relaxations to authentic NO with maximal effects at 3 microM. Pretreatment of vascular rings with ODQ induced a parallel, 2-log-order shift to the right of the concentration-response curves (CRCs) to histamine, ATP, NO, the NO-donors S-nitrosoglutathione, S-nitroso-N-acetyl-D,L-penicillamine, and spermine NONOate [N-[4-[1-(3-amino propyl)-2-hydroxy-2-nitroso hydrazino]butyl]-1, 3-propane diamine], and the direct sGC-stimulant [3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole] YC-1 but did not affect relaxations induced by papaverine and atriopeptin II. Moreover, the rightward shift of the CRCs to Angeli's salt, peroxynitrite, and linsidomine was similar to that of NO. These results suggested that ODQ is specific for sGC. Furthermore, they indicate that NO can cause vasorelaxation independent of cGMP. Three interesting exceptions were observed to the otherwise rather uniform inhibitory effect of ODQ: the responses to acetylcholine, glycerol trinitrate, and sodium nitroprusside. The latter two agents are known to require metabolic activation, possibly by cytochrome P-450-type proteins. The 3- to 5-log-order rightward shift of their CRCs suggests that, in addition to sGC, ODQ may interfere with heme proteins involved in the bioactivation of these NO donors and the mechanism of vasorelaxation mediated by acetylcholine. In support of this notion, ODQ inhibited hepatic microsomal NO production from both glycerol trinitrate and sodium nitroprusside as well as NO synthase activity in aortic homogenates. The latter effect seemed to require biotransformation of ODQ. Collectively, these data reveal that ODQ interferes with various heme protein-dependent processes in vascular and hepatic tissue and lacks specificity for sGC.

Animals↗

PubMed1998

Redox generation of nitric oxide to radiosensitize hypoxic cells.

PURPOSE: Previous studies have shown that nitric oxide (NO) delivered from NO donor agents sensitizes hypoxic cells to ionizing radiation. In the present study, nitroxyl (NO-), a potential precursor to endogenous NO production, was evaluated for hypoxic cell radiosensitization, either alone or in combination with electron acceptor agents. METHODS AND MATERIALS: Radiation survival curves of Chinese hamster V79 lung fibroblasts under aerobic and hypoxic conditions were assessed by clonogenic assay. Hypoxia induction was achieved by metabolism-mediated oxygen depletion in dense cell suspensions. Cells were treated with NO- produced from the nitroxyl donor Angeli's salt (AS, Na2N2O3, sodium trioxodinitrate), in the absence or presence of electron acceptor agents, ferricyanide, or tempol. NO concentrations resulting from the combination of AS and ferricyanide or tempol were measured under hypoxic conditions using an NO-sensitive electrode. RESULTS: Treatment of V79 cells under hypoxic conditions with AS alone did not result in radiosensitization; however, the combination of AS with ferricyanide or tempol resulted in significant hypoxic radiosensitization with SERs of 2.5 and 2.1, respectively. Neither AS alone nor AS in combination with ferricyanide or tempol influenced aerobic radiosensitivity. The presence of NO generated under hypoxic conditions from the combination of AS with ferricyanide or tempol was confirmed using an NO-sensitive electrode. CONCLUSION: Combining NO- generated from AS with electron acceptors results in NO generation and substantial hypoxic cell radiosensitization. NO- derived from donor agents or endogenously produced in tumors, combined with electron acceptors, may provide an important strategy for radiosensitizing hypoxic cells and warrants in vivo evaluation.

Animals↗