Hydralazine is a powerful inhibitor of peroxynitrite formation as a possible explanation for its beneficial effects on prognosis in patients with congestive heart failure.

The hemodynamic and anti-ischemic effects of nitroglycerin (GTN) are rapidly blunted as a result of the development of nitrate tolerance. Hydralazine has been shown to prevent tolerance in experimental and clinical studies, all of which may be at least in part secondary to antioxidant properties of this compound. The antioxidant effects of hydralazine were tested in cell free systems, cultured smooth muscle cells, isolated mitochondria, and isolated vessels. Inhibitory effects on the formation of superoxide and/or peroxynitrite formation were tested using lucigenin and L-012 enhanced chemiluminescence as well as DHE-fluorescence. The peroxynitrite scavenging properties were also assessed by inhibition of nitration of phenol. Prevention of impairment of NO downstream signaling and GTN bioactivation was determined by measurement of P-VASP (surrogate parameter for the activity of the cGMP-dependent kinase-I, cGK-I) and mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Hydralazine dose-dependently decreased the chemiluminescence signal induced by peroxynitrite from SIN-1 and by superoxide from HX/XO in a cell free system, by superoxide in smooth muscle cells and mitochondria acutely challenged with GTN. Moreover, hydralazine inhibited the peroxynitrite-mediated nitration of phenols as well as proteins in smooth muscle cells in a dose-dependent fashion. Finally, hydralazine normalized impaired cGK-I activity as well as impaired vascular ALDH-2 activity. Our results indicate that hydralazine is a highly potent radical scavenger. Thus, the combination with isosorbide dinitrate (ISDN) will favorably influence the nitroso-redox balance in the cardiovascular system in patients with congestive heart failure and may explain at least in part the improvement of prognosis in patients with chronic congestive heart failure.

The effect of peroxynitrite on the catalytic activity of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is a key enzyme of the *NO/cGMP pathway. Many cardiovascular disorders are associated with reduced *NO-mediated effects, while vascular superoxide (O(2)*(-)) production is increased. Both radicals rapidly react to peroxynitrite. We investigated whether peroxynitrite affects the activity and protein expression of sGC in intact vascular preparations. Catalytic sGC activity and expression of the sGC-beta(1) subunit was measured by conversion of radiolabeled GTP and western blot, respectively, using cytosolic extracts from rat aorta that had been incubated for 4 h with *NO/O(2)*(-) systems (devoid of free *NO) generating either 0.13 microM or 7.5 microM peroxynitrite/min. Incubation of rat aorta with 0.13 microM peroxynitrite/min had no effect. In striking contrast, incubation with 7.5 microM peroxynitrite/min resulted in a shift of the concentration-response curve obtained with a *NO donor (p =.0004) and a reduction of maximal specific activity from 3579 +/- 495 to 2422 +/- 265 pmol cGMP/mg/min (p =.036). The expression of the sGC-beta(1) subunit was unchanged. Exposure of aorta to the O(2)*(-) component had no effect, while exposure to the *NO-component reduced sGC expression to 58.8 +/- 7% (p <.001) and maximal sGC activity from 4041 +/- 992 to 1429 +/- 491 pmol cGMP/mg/min (p =.031). These data suggest that continuous generation of extracellular peroxynitrite might interfere with the *NO/cGMP signaling in vascular cells.

Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta.

BACKGROUND: Chronic in vivo treatment with nitroglycerin (NTG) induces tolerance to nitrates and cross-tolerance to nitrovasodilators and endothelium-derived nitric oxide (NO). We previously identified increased vascular superoxide formation and reduced NO bioavailability as one causal mechanism. It is still controversial whether intracellular downstream signaling to nitrovasodilator-derived NO is affected as well. METHODS AND RESULTS: We therefore studied the effects of 3-day NTG treatment of rats and rabbits on activity and expression of the immediate NO target soluble guanylyl cyclase (sGC) and on the cGMP-activated protein kinase I (cGK-I). Tolerance was induced either by chronic NTG infusion via osmotic minipumps (rats) or by NTG patches (rabbits). Western blot analysis, semiquantitative reverse transcription-polymerase chain reaction, and Northern blot analysis revealed significant and comparable increases in the expression of sGC alpha(1) and beta(1) subunit protein and mRNA. Studies with the oxidative fluorescent dye hydroethidine revealed an increase in superoxide in the endothelium and smooth muscle. Stimulation with NADH increased superoxide signals in both layers. Although cGK-I expression in response to low-dose NTG was not changed, a strong reduction in vasodilator-stimulated phosphoprotein (VASP) serine239 phosphorylation (specific substrate of cGK-I) was observed in tolerant tissue from rats and rabbits. Concomitant in vivo and in vitro treatment with vitamin C improved tolerance, reduced oxidative stress, and improved P-VASP. CONCLUSIONS: We therefore conclude that increased expression of sGC in the setting of tolerance reflects a chronic inhibition rather than an induction of the sGC-cGK-I pathway and may be mediated at least in part by increased vascular superoxide.

MR imaging of nitrosyl-iron complexes: experimental study in rats.

PURPOSE: To assess the influence of several nitrosyl-iron complexes on proton nuclear spin relaxation rates to establish a magnetic resonance (MR) imaging technique for nitric oxide. MATERIALS AND METHODS: The influence of aqueous phantom solutions of nitrosyl-iron complexes on proton relaxation rates was analyzed for signal enhancement at conventional 1.5-T MR imaging. To induce formation of nitrosyl-iron complexes in a biologic tissue, isolated rat liver was perfused with a saline solution of the NO donor sodium nitroprusside (SNP), and the MR signal intensity was examined thereafter. RESULTS: All investigated nitrosyl-iron complexes shortened the longitudinal, or T1, and transverse, or T2, relaxation times in a concentration-dependent fashion. Relaxivities were highest with a dinitrosyl-iron complex bound to albumin and with a water-soluble mononitrosyl-iron dithiocarbamate complex. The contrast properties of 240 micromol/L of a paramagnetic nitrosyl-iron complex were sufficient to substantially enhance the signal intensity of SNP-perfused rat livers at hydrogen 1 MR imaging. CONCLUSION: Nitrosyl-iron complexes exhibit a contrast effect at MR imaging that can be exploited for NO imaging in living animals and patients with conventional (1)H MR imaging techniques.

Aging and chronic hypertension decrease expression of rat aortic soluble guanylyl cyclase.

We analyzed the influence of aging and genetic hypertension on the function and expression of soluble guanylyl cyclase (sGC) in the aortas of prehypertensive and old spontaneously hypertensive rats (SHR) as well as in age-matched normotensive Wistar-Kyoto rats (WKY). The expression of heterodimeric sGC (alpha(1) and beta(1)) was assessed at the mRNA and protein level, and its function was assessed by the relaxant responses of phenylephrine-contracted endothelium-denuded aortic rings to the nitric oxide (NO) donor sodium nitroprusside. The vasodilator potency of sodium nitroprusside was significantly reduced (P<0.05) with age (3- to 6-fold increase in the EC(50) in old WKY and SHR compared with their young counterparts) as well as with hypertension (3-fold increase in old SHR compared with age-matched WKY), whereas the vasodilator potency of sodium nitroprusside did not differ between young SHR and WKY. A similar influence of aging and hypertension on NO-stimulated GC activity was revealed at the GC expression level: Whereas the beta(1) protein content was similar in young rats of both strains, old WKY exhibited 60% lower and old SHR exhibited 80% lower beta(1) subunit protein compared with young rats (P<0.05). Moreover, the abundance of alpha(1) and beta(1) mRNA (assessed by reverse transcriptase-polymerase chain reaction) was similar in young rats but was 2.5-fold (alpha(1)) and 4.3-fold (beta(1)) lower in old SHR compared with old WKY. In conclusion, our findings show that both aging and hypertension decrease sGC expression and its NO-dependent activation in aortic tissue. Downregulation of sGC may therefore contribute to arterial dysfunction in senescence and chronic hypertension.

Increased nitrovasodilator sensitivity in endothelial nitric oxide synthase knockout mice: role of soluble guanylyl cyclase.

Endogenously produced nitric oxide (NO) modulates nitrovasodilator-induced relaxation. We investigated the underlying mechanism in wild-type (WT) mice and endothelial NO synthase knockout (eNOS(-/-)) mice to determine whether a chronic lack of endothelial NO alters the soluble guanylyl cyclase (sGC) pathway. In aortic segments from eNOS(-/-) mice, the vasodilator sensitivity to sodium nitroprusside (SNP) was significantly greater than that in WT mice. There was no difference in sensitivity to the G-kinase I activator 8-para-chlorophenylthio-cGMP or to cromakalim. N(omega)-Nitro-L-arginine had no effect on the SNP-induced relaxation in eNOS(-/-) but increased the sensitivity in WT mice so it was no longer different than that of eNOS(-/-). Basal cGMP levels in aortic rings were significantly lower in eNOS(-/-) mice than in WT mice. SNP (300 nmol/L) induced a significantly greater cGMP accumulation in eNOS(-/-) mice than in WT mice. The maximal SNP-induced (10 micromol/L) increase in cGMP was similar in both strains. SNP-stimulated sGC activity was significantly greater in eNOS(-/-) mice than in WT mice. Incubation of aortic segments from WT mice with N(omega)-nitro-L-arginine increased sGC activity, an effect prevented by coincubation with SNP (10 micromol/L). The aortic expressions of the sGC alpha1 and beta1 subunits in WT and eNOS(-/-) mice were identical as determined with Western blot analysis. These data suggest that chronic exposure to endothelium-derived NO, as well as acute exposure to nitrovasodilator-derived NO, desensitizes sGC to activation by NO but does not alter sGC expression. Both the acute cessation of endothelial NO formation in WT mice and the chronic deficiency of NO in eNOS(-/-) mice restore the NO sensitivity of sGC and enhance vascular smooth muscle relaxation in response to nitrovasodilator agents.

Detection of nitrosyl-iron complexes by proton-electron-double-resonance imaging.

The nitrogen monoxide radical (NO*) forms paramagnetic mono- and dinitrosyl-iron complexes in biologic tissues. To establish a noninvasive technique for in vivo NO* imaging, we evaluated the suitability of these complexes as magnetic resonance (MR) contrast agents, making use of the ability of the unpaired electrons of the complexes to enter into dynamic nuclear polarization with water protons and hence produce enhancement on images generated by the technique of proton-electron-double-resonance imaging (PEDRI). Phantom solutions of synthetic nitrosyl-iron complexes (NICs) altered the signal intensity of PEDRI images. The dinitrosyl-iron complex (DNIC) with serum albumin induced a significantly larger signal alteration than the mononitrosyl-iron complex (MNIC) with dithiocarbamate. Exposure of rat liver to sodium nitroprusside (SNP) by ex vivo and in situ perfusion induced a composite X-band electron spin resonance (ESR) spectrum of the isolated liver characteristic of a MNIC and DNIC. On storage of the tissue, the MNIC signal disappeared and the DNIC signal intensity increased. Correspondingly, in cross-sectional PEDRI images taken at room temperature, the SNP-exposed livers initially exhibited a weak signal that strongly increased with time. In conclusion, NICs can be detected using PEDRI and could be exploited for in vivo NO* imaging.

Role of superoxide dismutase in in vivo and in vitro nitrate tolerance.

We assessed whether pharmacological inhibition of CuZn-superoxide dismutase (SOD) mimics the molecular mechanism of either in vitro or in vivo nitrovasodilator tolerance. In endothelium-intact aortic rings from in vivo tolerant rabbits the GTN- and acetylcholine (ACh)-induced maximal relaxation was attenuated by 36 and 23%, respectively. In vitro treatment of control rings with GTN (1 h 10 microM) similarly attenuated the vasorelaxant response to GTN, but not to ACh. Formation of superoxide radicals (*O2-) in endothelium-intact rings (lucigenin-chemiluminescence) increased 2.5 fold in in vivo tolerance, but significantly decreased in in vitro tolerance. The membrane associated NADH oxidase activity was increased 2.5 fold in homogenates of in vivo tolerant aortae, but was not changed in in vitro tolerant aorta. Conversely, SOD activity and protein expression was halved in in vivo tolerance, but SOD activity was not altered by in vitro tolerance. The *O2- scavenger tiron (10 mM) effectively restored the vasorelaxant response to GTN in in vivo tolerant aortic rings, but not the reduced response to GTN in in vitro tolerant rings. Pretreatment (1 h) of vessels with diethyldithiocarbamate (DETC; 10 mM) attenuated vasorelaxant responses to GTN and ACh, increased vascular *O2- production, and inhibited SOD activity in vessel homogenates to a similar degree as observed in in vivo tolerance. DETC-treatment of in vivo-tolerant vessels induced an additional increase in *O2- production. Increased *O2- production in in vivo nitrate tolerant aorta is associated with activation of vascular NADH oxidase and inactivation of CuZnSOD. Therefore, in vivo tolerance can be mimicked by in vitro inhibition of CuZnSOD, but not by in vitro exposure to GTN, which does not affect vascular *O2- production, NADH oxidase and CuZnSOD.

Nitric oxide inhibits caspase-3 by S-nitrosation in vivo.

In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. In vitro data have demonstrated that NO inhibits the activity of caspase-3 by S-nitrosation of the enzyme. Here we present evidence for the in vivo occurrence and functional relevance of this novel antiapoptotic mechanism. To demonstrate that the cysteine residue Cys-163 of caspase-3 is S-nitrosated, cells were transfected with the Myc-tagged p17 subunit of caspase-3. After incubation of the transfected cells with different NO donors, Myc-tagged p17 was immunoprecipitated with anti-Myc antibody. S-Nitrosothiol was detected in the immunoprecipitate by electron spin resonance spectroscopy after liberation and spin trapping of NO by N-methyl-D-glucamine-dithiocarbamate-iron complex. Transfection of cells with a p17 mutant, where the essential Cys-163 was mutated into alanine, completely prevented S-nitrosation of the enzyme. As a functional correlate, in human umbilical vein endothelial cells the NO donors sodium nitroprusside or PAPA NONOate (50 microM) significantly reduced the increase in caspase-3-like activity induced by overexpressing caspase-3 by 75 and 70%, respectively. When human umbilical vein endothelial cells were cotransfected with beta-galactosidase, morphological analysis of stained cells revealed that cell death induction by overexpression of caspase-3 was completely suppressed in the presence of sodium nitroprusside, PAPA NONOate, or S-nitroso-L-cysteine (50 microM). Thus, NO supplied by exogenous NO donors serves in vivo as an antiapoptotic regulator of caspase activity via S-nitrosation of the Cys-163 residue of caspase-3.

Validation of lucigenin as a chemiluminescent probe to monitor vascular superoxide as well as basal vascular nitric oxide production.

Lucigenin has been widely used as a chemiluminescent substrate to monitor vascular superoxide (O*-2) formation. The validity of lucigenin for detection of O*-2 has been questioned because O*-2 is generated by lucigenin itself. It has been shown that the concentration of lucigenin is a critical parameter affecting the validity of this assay. In the present studies we evaluated a reduced concentration of lucigenin (5 microM) as a tool to quantify O*-2 production in vascular tissue. Lucigenin-induced effects on endothelial function were assessed by isometric tension recording of isolated aortic rings suspended in organ baths. The effects of lucigenin on O*-2 production were studied using spin trapping and electron spin resonance spectroscopy. Lucigenin at 250 microM but not at 5 microM caused a significant attenuation of endothelium-dependent relaxations to acetylcholine, which was prevented by pretreatment with superoxide dismutase. Spin-trapping studies revealed that lucigenin at 250 microM increased vascular O*-2 production several fold while 5 microM lucigenin did not stimulate O*-2 production. Inhibition of NO synthase by NG-momomethyl-l-arginine as well as the removal of the endothelium almost doubled lucigenin-derived chemiluminescence (LDCL), indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. Thus, lucigenin at a concentration of 5 microM seems to be a sensitive and valid probe for assessing O*-2 in vascular tissue. It can also be used as an indirect probe to estimate basal vascular NO release.

Normoxic cardiopulmonary bypass reduces oxidative myocardial damage and nitric oxide during cardiac operations in the adult.

OBJECTIVE: Hyperoxic cardiopulmonary bypass is widely used during cardiac operations in the adult. This management may cause oxygenation injury induced by oxygen-derived free radicals and nitric oxide. Oxidative damage may be significantly limited by maintaining a more physiologic oxygen tension strategy (normoxic cardiopulmonary bypass). METHODS: During elective coronary artery bypass grafting, 40 consecutive patients underwent either hyperoxic (oxygen tension = 400 mm Hg) or normoxic (oxygen tension = 140 mm Hg) cardiopulmonary bypass. At the beginning and the end of bypass this study assessed polymorphonuclear leukocyte elastase, nitrate, creatine kinase, and lactic dehydrogenase, antioxidant levels, and malondialdehyde in coronary sinus blood. Cardiac index was measured before and after cardiopulmonary bypass. RESULTS: There was no difference between groups with regard to age, sex, severity of disease, ejection fraction, number of grafts, duration of cardiopulmonary bypass, or ischemic time. Hyperoxic bypass resulted in higher levels of polymorphonuclear leukocyte elastase (377 +/- 34 vs 171 +/- 32 ng/ml, p = 0.0001), creatine kinase 672 +/- 130 vs 293 +/- 21 U/L, p = 0.002), lactic dehydrogenase (553 +/- 48 vs 301 +/- 12 U/L, p = 0.003), antioxidants (1.97 +/- 0.10 vs 1.41 +/- 0.11 mmol/L, p = 0.01), malondialdehyde (1.36 +/- 0.1 micromol/L,p = 0.005), and nitrate (19.3 +/- 2.9 vs 10.1 +/- 2.1 micromol/L, p = 0.002), as well as reduction in lung vital capacity (66% +/- 2% vs 81% +/- 1%,p = 0.01) and forced 1-second expiratory volume (63% +/- 10% vs 93% +/- 4%, p = 0.005) compared with normoxic management. Cardiac index after cardiopulmonary bypass at low filling pressure was similar between groups (3.1 +/- 0.2 vs 3.3 +/- 0.3 L/min per square meter). [Data are mean +/- standard error (analysis of variance), with p values compared with an oxygen tension of 400 mm Hg.] CONCLUSIONS: Hyperoxic cardiopulmonary bypass during cardiac operations in adults results in oxidative myocardial damage related to oxygen-derived free radicals and nitric oxide. These adverse effects can be markedly limited by reduced oxygen tension management. The concept of normoxic cardiopulmonary bypass may be applied to surgical advantage during cardiac operations.

Vasodilator dysfunction in aged spontaneously hypertensive rats: changes in NO synthase III and soluble guanylyl cyclase expression, and in superoxide anion production.

OBJECTIVE/METHODS: Genetic hypertension is associated with an apparent endothelial dysfunction and impaired endothelium-dependent vasodilatation in response to increased flow and receptor-dependent agonists. However, the link between impaired vasodilatation and nitric oxide (NO) synthase expression is still unclear. In the present study, dilator responses were determined in the aorta and coronary circulation of 16 month old spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY). Changes in vascular reactivity were compared with alterations in superoxide anion production as well as endothelial NO synthase (NOS III) and soluble guanylyl cyclase expression. RESULTS: In the isolated perfused heart both the bradykinin- and sodium nitroprusside-induced vasodilator responses were attenuated in SHR compared to WKY. Western blot analysis revealed a parallel reduction in NOS III expression in coronary microvascular endothelial cells from SHR. Superoxide anion production in aortae from SHR was markedly elevated over that of aortae from WKY, and was almost completely abolished by pretreatment with superoxide dismutase. Superoxide dismutase induced similar relaxations in phenylephrine-preconstricted aortic rings from both SHR and WKY, but failed to restore the attenuated acetylcholine- and sodium nitroprusside-induced relaxations in SHR. No difference in NOS III expression was detected in the aortae from either strain whereas soluble guanylyl cyclase expression was markedly decreased in SHR. CONCLUSIONS: These results demonstrate that NOS III expression in different tissues is differentially affected by hypertension. Moreover, although an elevated superoxide anion production is apparent in the aorta, a reduced soluble guanylyl cyclase expression appears to account for the observed vasodilator dysfunction in SHR.

Characterization of NS 2028 as a specific inhibitor of soluble guanylyl cyclase.

1 The haeme-containing soluble guanylyl cyclase (alpha1beta1-heterodimer) is a major intracellular receptor and effector for nitric oxide (NO) and carbon monoxide (CO) and mediates many of their biological actions by increasing cyclic GMP. We have synthesized new oxadiazolo-benz-oxazins and have assessed their inhibitory actions on guanylyl cyclase activity in vitro, on the formation of cyclic GMP in cultured cells and on the NO-dependent relaxation of vascular and non-vascular smooth muscle. 2 Soluble guanylyl cyclase, purified to homogeneity from bovine lung, was inhibited by 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS 2028) in a concentration-dependent and irreversible manner (IC50 30 nM for basal and 200 nM for NO-stimulated enzyme activity). Evaluation of the inhibition kinetics according to Kitz & Wilson yielded a value of 8 nM for Ki, the equilibrium constant describing the initial reversible reaction between inhibitor and enzyme, and 0.2 min(-1) for the rate constant k3 of the subsequent irreversible inhibition. Inhibition was accompanied by a shift in the soret absorption maximum of the enzyme's haem cofactor from 430 to 390 nm. 3 S-nitroso-glutathione-enhanced soluble guanylyl cyclase activity in homogenates of mouse cerebellum was inhibited by NS 2028 (IC50 17 nM) and by 17 structural analogues in a similar manner, albeit with different potency, depending on the type of substitution at positions 1, 7 and 8 of the benzoxazin structure. Small electronegative ligands such as Br and Cl at position 7 or 8 increased and substitution of the oxygen at position 1 by -S-,- NH- or -CH2- decreased the inhibition. 4 In tissue slices prepared from mouse cerebellum, neuronal NO synthase-dependent activation of soluble guanylyl cyclase by the glutamate receptor agonist N-methyl-D-aspartate was inhibited by NS 2028 (IC50 20 nM) and by two of its analogues. Similarly, 3-morpholino-sydnonimine (SIN-1)-elicited formation of cyclic GMP in human cultured umbilical vein endothelial cells was inhibited by NS 2028 (IC50 30 nM). 5 In prostaglandin F2alpha-constricted, endothelium-intact porcine coronary arteries NS 2028 elicited a concentration-dependent increase (65%) in contractile tone (EC50 170 nM), which was abolished by removal of the endothelium. NS 2028 (1 microM) suppressed the relaxant response to nitroglycerin from 88.3+/-2.1 to 26.8+/-6.4% and induced a 9 fold rightward shift (EC50 15 microM) of the concentration-relaxation response curve to nitroglycerin. It abolished the relaxation to sodium nitroprusside (1 microM), but did not affect the vasorelaxation to the KATP channel opener cromakalim. Approximately 50% of the relaxant response to sodium nitroprusside was recovered after 2 h washout of NS 2028. 6 In phenylephrine-preconstricted, endothelium-denuded aorta of the rabbit NS 2028 (1 microM) did not affect relaxant responses to atrial natriuretic factor, an activator of particulate guanylyl cyclase, or forskolin, an activator of adenylyl cyclase. 7 NO-dependent relaxant responses in non-vascular smooth muscle were also inhibited by NS 2028. The nitroglycerin-induced relaxation of guinea-pig trachea preconstricted by histamine was fully inhibited by NS 2028 (1 microM), whereas the relaxations to terbutaline, theophylline and vasoactive intestinal polypeptide (VIP) were not affected. The relaxant responses to electrical field stimulation of non-adrenergic, non-cholinergic nerves in the same tissue were attenuated by 50% in the presence of NS 2028 (1 microM). 8 NS 2028 and its analogues, one of which is the previously characterized 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), appear to be potent and specific inhibitors of soluble guanylyl cyclase present in various cell types. Oxidation and/or a change in the coordination of the haeme-iron of guanylyl cyclase is a likely inhibitory mechanism.

Dinitrosyl-dithiol-iron complexes, nitric oxide (NO) carriers in vivo, as potent inhibitors of human glutathione reductase and glutathione-S-transferase.

Human glutathione reductase (GR) and rat liver glutathione-S-transferases (GSTs) had been shown to be inhibited by the nitric oxide (NO) carrier S-nitroso-glutathione (GSNO). We have now extended these studies by measuring the effects of dinitrosyl-iron complexed thiols (DNIC-[RSH]2) on human GR, GST and glutathione peroxidase. DNIC-[RSH]2 represent important transport forms of NO but also of iron ions and glutathione in vivo. Human GR was found to be inhibited by dinitrosyl-iron-di-glutathione (DNIC-[GSH]2) and dinitrosyl-iron-di-L-cysteine (DNIC-Cys2) in two ways: both compounds were competitive with glutathione disulfide (GSSG), the inhibition constant (Ki) for reversible competition of DNIC-[GSH]2 with GSSG being approximately 5 microM; preincubating GR for 10 min with 4 microM DNIC-[GSH]2 and 40 microM DNIC-Cys2, respectively, led to 50% irreversible enzyme inactivation. More than 95% GR inactivation was achieved by incubation with 36 microM DNIC-[GSH]2 for 30 min. This inhibition depended on the presence of NADPH. Absorption spectra of inhibited GR showed that the charge-transfer interaction between the isoalloxazine moiety of the prosthetic group flavin adenine dinucleotide (FAD) and the active site thiol Cys63 is disturbed by the modification. Cys2 and FAD could be ruled out as sites of the modification. Isolated human placenta glutathione-S-transferase and GST activity measured in hemolysates were also inhibited by DNIC-[GSH]2. This inhibition, however, was reversible and competitive with reduced glutathione, the Ki being 20 nM. The inhibition of GST induced by GSNO was competitive with reduced glutathione (GSH) (Ki = 180 microM) and with the second substrate of the reaction, 1-chloro-2,4,-dinitrobenzene (Ki = 170 microM). An inhibition of human glutathione peroxidase by GSNO or DNIC-[RSH]2 was not detectable. Inactivation of GR by DNIC-[GSH]2 is by two orders of magnitude more effective than modification by GSNO; this result and the very efficient inhibition of GST point to a role of DNIC-[RSH]2 in glutathione metabolism.

Inhibition of glutathione reductase by dinitrosyl-iron-dithiolate complex.

The biological signal molecule nitric oxide (NO) exists in a free and carrier-bound form. Since the structure of the carrier is likely to influence the interaction of NO with macromolecular targets, we assessed the interaction of a dinitrosyl-iron-dithiolate complex carrying different thiol ligands with glutathione reductase. The enzyme was irreversibly inhibited by dinitrosyl-iron-di-L-cysteine and dinitrosyl-iron-di-glutathione in a concentration- and time-dependent manner (IC50 30 and 3 microM, respectively). Evaluation of the inhibition kinetics according to Kitz-Wilson yielded a Ki of 14 microM, and a k3 of 1.3 x 10(-3) s-1. A participation of catalytic site thiols in the inhibitory mechanism was indicated by the findings that only the NADPH-reduced enzyme was inhibited by dinitrosyl-iron complex and that blockade of these thiols by Hg2+ afforded protection against irreversible inhibition. This inhibition was not accompanied by formation of a protein-bound dinitrosyl-iron complex and/or S-nitrosation of active site thiols (Cys-58 and Cys-63). However, one NO moiety exhibiting an acid lability similar to a secondary N-nitrosamine was present per mol of inhibited monomeric enzyme. These findings suggest specifically N-nitrosation of glutathione reductase as a likely mechanism of inhibition elicited by dinitrosyl-iron complex and demonstrate in general that structural resemblance of an NO carrier with a natural ligand enhances NO+ transfer to the ligand-binding protein.

N-alpha-tosyl-L-lysine chloromethylketone prevents expression of iNOS in vascular smooth muscle by blocking activation of NF-kappa B.

Certain cytokines and lipopolysaccharide stimulate expression of inducible nitric oxide synthase (iNOS) in vascular smooth muscle, an event that is regulated at the transcriptional level and appears to involve several transcription factors, including nuclear factor kappa B (NF-kappa B). Since proteases play an essential role in NF-kappa B activation, experiments were designed to clarify, in both cultured rat aortic smooth muscle cells (SMCs) and isolated rat aortas, whether protease inhibitors affect the interleukin-1 beta (IL-1 beta)-elicited expression of iNOS. The formation of NO was assessed by nitrite release in cultured SMCs and the attenuation of phenylephrine-induced contraction in aortic rings, the expression of iNOS by Western blot analysis and reverse transcription-polymerase chain reaction, and NF-kappa B activity in nuclear extracts by gel electrophoretic mobility shift assya. Exposure of cultured SMCs to IL-1 beta increased NF-kappa B binding activity within 30 minutes and was associated with nitrite accumulation and the appearance of iNOS protein 24 hours later. These responses were abolished in cells that had been exposed to the cytokine in the presence of the protease inhibitor N-alpha-tosyl-L-lysine chloromethylketone. Aprotinin and p-toluenesulfonyl-L-arginine methyl ester, two other protease inhibitors, also reduced the cytokine-stimulated release of nitrite and the level of iNOS protein. Exposure of rat aortic segments without endothelium to IL-1 beta activated NF-kappa B within 30 minutes and was associated with the appearance of iNOS mRNA and an attenuation of phenylephrine-induced contraction 6 hours later. These responses were blunted when the segments were incubated with the cytokine and N-alpha-tosyl-L-lysine chloromethyl ketone. The present observations indicate that protease inhibitors prevent iNOS expression in both cultured and native vascular SMCs by blocking the activation of NF-kappa B.

Regional measurements of NO formed in vivo during brain ischemia.

Nitric oxide formed in vivo in the rat brain regions of hippocampus, striatum, neocortex and cerebellum was spin trapped and measured ex vivo by cryogenic electron paramagnetic resonance spectroscopy. In non-ischemic control animals the rate of nitric oxide (NO) formation in the individual brain regions ranged from 15 to 42 pmol.g-1.min-1. During exposure to global ischemia for 7 min the generation of NO increased in all parts of the brain. In the hippocampus the rate of NO formation during ischemia increased by 6-fold from a control rate of 19 pmol.g-1.min-1. This increase was attenuated 47% by pretreatment with the NO synthase antagonist 7-nitroindazole, whereas pretreatment with the non-NMDA receptor anatogonist NBQX and the Ca2+ channel blocker NS638 did not influence the NO formation. The data show that short-duration ischemia elicits a significant, NO-synthase-dependent formation of NO in all brain regions.

Effect of YC-1, an NO-independent, superoxide-sensitive stimulator of soluble guanylyl cyclase, on smooth muscle responsiveness to nitrovasodilators.

1. We studied the effects of 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) on the activity of purified soluble guanylyl cyclase (sGC), the formation of guanosine-3':5' cyclic monophosphate (cyclic GMP) in vascular smooth muscle cells (VSMC), and on the tone of rabbit isolated aortic rings preconstricted by phenylephrine (PE). In addition, we assessed the combined effect of YC-1, and either NO donors, or superoxide anions on these parameters. 2. YC-1 elicited a direct concentration-dependent activation of sGC (EC50 18.6 +/- 2.0 microM), which was rapid in onset and quickly reversible upon dilution. YC-1 altered the enzyme kinetics with respect to GTP by decreasing KM and increasing Vmax. Activation of sGC by a combination of sodium nitroprusside (SNP) and YC-1 was superadditive at low and less than additive at high concentrations, indicating a synergistic activation of the enzyme by both agents. A specific inhibitor of sGC, 1H-(1,2,4)-oxdiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (NS 2028), abolished activation of the enzyme by either compound. 3. YC-1 induced a concentration-dependent increase in intracellular cyclic GMP levels in rat cultured aortic VSMC, which was completely inhibited by NS 2028. YC-1 applied at the same concentration as SNP elicited 2.5 fold higher cyclic GMP formation. Cyclic GMP-increases in response to SNP and YC-1 were additive. 4. YC-1 relaxed preconstricted endothelium-denuded rabbit aortic rings in a concentration-dependent manner (50% at 20 microM) and markedly increased cyclic GMP levels. Relaxations were inhibited by NS 2028. A concentration of YC-1 (3 microM), which elicited only minor effects on relaxation and cyclic GMP, increased the vasodilator potency of SNP and nitroglycerin (NTG) by 10 fold and markedly enhanced SNP- and NTG-induced cyclic GMP formation. 5. Basal and YC-1-stimulated sGC activity was sensitive to inhibition by superoxide (O-2) generated by xanthine/xanthine oxidase, and was protected from this inhibition by superoxide dismutase (SOD). YC-1-stimulated sGC was also sensitive to inhibition by endogenously generated (O-2 in rat preconstricted endothelium-denuded aortic rings. Relaxation to YC-1 was significantly attenuated in aortae from spontaneously hypertensive rats (SHR), which generated O-2 at a higher rate than aortae from normotensive Wistar Kyoto rats (WKY). SOD restored the vasodilator responsiveness of SHR rings to YC-1. 6. In conclusion, these results indicate that YC-1 is an NO-independent, O-2-sensitive, direct activator of sGC in VSMC and exerts vasorelaxation by increasing intracellular cyclic GMP levels. The additive or even synergistic responses to NO-donors and YC-1 in cultured VSMC and isolated aortic rings apparently reflect the direct synergistic action of YC-1 and NO on the sGC. The synergism revealed in this in vitro study suggests that low doses of YC-1 may be of therapeutic value by permitting the reduction of nitrovasodilator dosage.

In vivo nitrate tolerance is not associated with reduced bioconversion of nitroglycerin to nitric oxide.

BACKGROUND: In vitro data suggest that reduced bioconversion of nitroglycerin (NTG) to nitric oxide (NO) contributes to the development of vascular and hemodynamic tolerance to NTG. We examined the in vivo validity of this hypothesis by measuring NTG-derived NO formation by in vivo spin-trapping of NO in vascular tissues from nitrate-tolerant and -nontolerant rats. METHODS AND RESULTS: Five groups (n = 6 to 8 each) of conscious chronically catheterized rats received NTG (0.2 or 1 mg/h IV) for 72 hours (nitrate-tolerant groups). Four other groups received either NTG vehicle (placebo, for 72 hours) or were left untreated (control). Nitrate tolerance was substantiated by a reduced (55% to 85%) hypotensive response to NTG in vivo and a reduced relaxation to NTG in isolated aortic rings. NTG-derived NO formation in aorta, vena cava, heart, and liver was measured as NOFe(DETC)2 and NO-heme complexes formed in vivo during 35 minutes combined with ex vivo cryogenic electron spin resonance spectroscopy. NO formation was significantly (P < .05) increased in all tissues in nitrate-tolerant rats in an NTG dose-dependent manner. Furthermore, the amount of NO formed from a bolus dose of NTG (6.5 mg/kg over 20 minutes) was similar in nitrate-tolerant and -nontolerant rats. CONCLUSIONS: The results suggest that vascular and hemodynamic NTG tolerance occurs despite high and similar rates of NO formation by NTG in tolerant and nontolerant target tissues. This finding is compatible with the assumption that reduced biological activity of NO, rather than reduced bioconversion of NTG to NO, contributes to in vivo development of nitrate tolerance.

Effect of cyclic GMP-dependent vasodilators on the expression of inducible nitric oxide synthase in vascular smooth muscle cells: role of cyclic AMP.

1. In the present study we examined whether interleukin-1 beta (IL-1 beta) increases the activity of adenylyl cyclase in vascular smooth muscle cells and determined its role in the cytokine-induced expression of the inducible nitric oxide synthase (iNOS) and activation of nuclear transcription factor-kappa B (NF-kappa B). In addition the interaction between cyclic AMP- and cyclic GMP-elevating agonists on the IL-1 beta-stimulated expression of iNOS was examined. 2. Exposure of vascular smooth muscle cells to IL-1 beta stimulated the formation of cyclic AMP but not of cyclic GMP. The intracellular level of cyclic AMP reached a maximum within 1 h and then gradually declined over the next 5 h. This IL-1 beta (60 u ml-1)-stimulated formation of cyclic AMP was modest (about 3 fold at 60 u ml-1 for 1 h) compared to that evoked by isoprenaline (about 9 fold at 3 x 10(-6) M for 2 min). 3. The IL-1 beta (60 u ml-1 for 24 h)-stimulated accumulation of nitrite, which was taken as an index of NO production, was concentration-dependently increased by preferential inhibitors of cyclic AMP-dependent phosphodiesterases (rolipram and trequinsin). This effect was reproduced by a specific activator of the cyclic AMP-dependent protein kinase(s) A, Sp-8-CPT-cAMPS (10(-4) M) but was prevented by a specific inhibitor of cyclic AMP-dependent protein kinase(s) A, Rp-8-CPT-cAMPS (10(-4) M). These compounds alone [rolipram (10(-6) M), trequinsin (3 x 10(-6) M) and Sp-8-CPT-cAMPS (10(-4) M)] slightly but significantly increased the release of nitric oxide while Rp-8-CPT-cAMPS elicited no such effect. 4. Inducible NOS protein was expressed in IL-1 beta (30 u ml-1, 24 h)-stimulated smooth muscle cells as assessed by Western blot analysis. The level of iNOS protein was markedly increased in smooth muscle cells which had been exposed to IL-1 beta in combination with either rolipram (3 x 10(-6) M) or Sp-8-CPT-cAMPS (10(-4) M) but was reduced in those exposed to IL-1 beta and Rp-8-CPT-cAMPS (10(-4) M). A weak expression of iNOS protein was found in smooth muscle cells which had been exposed to either Sp-8-CPT-cAMPS or rolipram alone for 24 h while Rp-8-CPT-cAMPS elicited no such effect. 5. Exposure of smooth muscle cells to IL-1 beta (30 u ml-1) for 30 min increased the level of NF-kappa B-DNA complexes in nuclear extracts as detected by electrophoretic mobility shift assay. Similar levels of NF-kappa B-DNA complexes were found in cells which had been exposed to IL-1 beta in combination with either Sp-8-CPT-cAMPS (10(-4) M), trequinsin (10(-6) M) or rolipram (10(-6) M). None of the modulators alone affected the basal level of NF-kappa B binding activity. 6. NO-donors [sodium nitroprusside (SNP) 10(-4) M; dinitrosyl-iron-di-L-cysteine-complex (DNIC), 10(-4) M; 3-morpholino-sydnonimine (SIN-1), 10(-4) M] and atrial natriuretic factor (10(-6) M) significantly increased the IL-1 beta (30 or 60 u ml-1, 24 h)-stimulated expression of iNOS protein and activity as assessed indirectly by the conversion of oxyhaemoglobin to methaemoglobin. In the absence of IL-1 beta, SNP (10(-4) M, 24 h) but not the other cyclic GMP-dependent vasodilators caused a modest expression of iNOS protein. No such effect was found in smooth muscle cells exposed to SNP in combination with Rp-8-CPT-cAMPS (10(-4) M) while an increased level of iNOS protein was found in those exposed to SNP in combination with either Sp-8-CPT-cAMPS (10(-4) M) or rolipram (3 x 10(-6) M). 7. Exposure of vascular smooth muscle cells to either S-nitroso-L-cysteine (Cys-SNO, 10(-4) M), SNP (10(-4) M) or SIN-1 (10(-4) M) for 35 min affected minimally the basal activation of NF-kappa B but abolished that evoked by IL-1 beta (30 u ml-1 added during the last 30 min). However, addition of Cys-SNO following the stimulation with IL-1 beta (during the last 5 min of the 30 min exposure period) reduced the level of NF-kappa B-DNA complexes only slightly. 8. These data indicate that the cyclic AMP-dependent pathway plays a decisi