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Inhibition of Nitric Oxide Synthesis by Methylene Blue

Inhibition of Nitric Oxide Synthesis by Methylene Blue

Eiochemicol Pharmacology. Vol. 45. No. 2. pp. 367-314. 1993. 0306-295@3 $6.00 + O.OLl Printed in Great Britain. 0 1993. Pergamon Press Ltd

INHIBITION OF SYNTHESIS BY METHYLENE BLUE

BERND MAYER,* FRIEDRICHBRUNNERand KURTSCHMIDT Institut fur Pharmakologie und Toxikologie, Karl-Franzens-Universitat Graz, Universitltsplatz 2, A-8010 Graz, Austria

(Received 29 July 1992; accepted 15 October 1992)

Abstract-Methylene blue appears to inhibit nitric oxide-stimulated and has been widely used for inhibition of cGMP-mediated processes. We report here that - dependent relaxation of isolated blood vessels and NO synthase-dependent cGMP formation in cultured endothelial cells were both markedly more sensitive to inhibition by methylene blue than effects induced by direct activation of soluble guanylyl cyclase. These discrepancies were also observed when superoxide dismutase (SOD) was present to protect NO from inactivation by superoxide anion. Subsequent experiments showed that formation of L-citrulline by purified NO synthase was completely inhibited by 30 PM methylene blue (rcso = 5.3 and 9.2 PM in the absence and presence of SOD, respectively), whereas guanylyl cyclase stimulated by S-nitrosoglutathione was far less sensitive to the drug (50% inhibition at -6OpM, and maximal inhibition of 72% at 1 mM methylene blue). Experimental evidence indicated that oxidation of NADPH, or reduced flavins does not account for the inhibitory effects of methylene blue. Our data suggest that methylene blue acts as a direct inhibitor of NO synthase and is a much less specific and potent inhibitor of guanylyl cyclase than hitherto assumed.

Endothelium-dependent smooth muscle relaxation dependent than -induced relaxation is mediated by L--derived nitric oxide, the [24-261. Moreover, methylene blue was shown to endothelium-derived relaxing factor (EDRF), which inhibit synthesis by endothelial cells and is released from vascular endothelial cells in response isolated arteries independent of its inhibitory to stimulation by hormones and neurotransmitters effects on cGMP accumulation [27,28]. Also, the elevating intracellular Ca*+ concentrations [l]. Ca2+ pronounced inhibitory effects of 5 ,uM methylene blue is required for the activation of - on acetylcholine-induced in cerebral and dependent NO synthase isozymes which synthesize skeletal arterioles were completely prevented by NO together with L-citrulline from L-arginine and simultaneous application of superoxide dismutase are expressed as both soluble and membrane- (SOD?) [29,30]. Under aerobic conditions, reduced associated forms in endothelial cells [2-4]. NO forms of methylene blue are readily reoxidized by apparently exerts its vasodilatory actions via molecular oxygen thereby generating superoxide activation of soluble guanylyl cyclase in smooth anion [9]. Thus, superoxide-mediated inactivation muscle cells adjacent to the endothelial cell layer. of NO [31] may provide an explanation for effects The molecular mechanism of this NO-induced of methylene blue unrelated to guanylyl cyclase stimulation of cGMP formation has been the subject inhibition, although endogenous SOD activity of detailed investigations, and it has been suggested normally protects and stabilizes NO. that NO interacts with the group bound to We speculated about an additional, as yet soluble guanylyl cyclase [5-8]. This hypothesis was unrecognized, mechanism being responsible for the supported by results obtained with methylene blue, powerful effects of methylene blue on NO-mediated which is generally accepted to oxidize protein-bound cellular signalling. We have recently shown that heme and non-heme ferrous iron [%14]. Methylene purified NO synthase, in addition to reduced flavins blue was found to inhibit the stimulation of soluble and tetrahydrohiopterin, also contains tightly bound guanylyl cyclase by NO and in cell- iron [32], but a catalytic role for this iron has not free systems [15,16] as well as to block smooth yet been demonstrated. Provided that iron has an muscle relaxation induced by nitrovasodilators or essential function in NO synthesis, methylene blue acetylcholine [17-191. The compound has been should act as artificial electron acceptor and inhibit extensively used as a “selective” inhibitor of soluble NO synthase. It was the aim of the present work to guanylyl cyclase to demonstrate the involvement of address this question and to investigate the effects cGMP accumulation in vascular relaxation, platelet of methylene blue on the synthesis and the biological aggregation and neurotransmission [20-231. actions of NO. Nevertheless, some studies indicated that methyl- ene blue was more potent in inhibiting endothelium- MATERIALS AND METHODS

* Corresponding author. Tel (43) 316 380-5567; FAX Materials. NO synthase was isolated from pig (43) 316 335-41. cerebellum as described previously [33]. Soluble t Abbreviations: GSNO, S-nitrosoglutathione; SNP, guanylyl cyclase purified from bovine lung [34] was ; SOD, superoxide dismutase. a kind gift of Dr E. Bohme, Berlin, Germany. Stock

367 368 B. MAYER,F. BRUNNERand K. SCHMIDT solutions of S-nitrosoglutathione (GSNO) were addition of 1 mL of 0.01 N hydrochloric acid. prepared freshly each day by mixing equimolar Intracellular cGMP was completely released into amounts of glutathione (dissolved in 0.02M the supernatant within 1 hr and measured by hydrochloric acid) and aqueous sodium . SOD radioimmunoassay. from bovine erythrocytes and methylene blue were Determination of guanylyl cyclase activity. Purified from Sigma (Deisenhofen, Germany), all other soluble guanylyl cyclase (0.1-0.2 pg) was incubated reagents were obtained from sources described at 37” for 10min in a total volume of 0.1 mL of previously [32,33,35-371. a triethanolamine-HCI buffer (50 mM, pH 7.4) Relaxation of bovine coronary arteries. Circular containing 0.1 mM [(u-32P]GTP (200,000- strips of bovine coronary arteries were prepared, 300,000 cpm), 1 mM cGMP, 1 mM 3-isobutyl-l- mounted in organ baths, equilibrated and pre- methylxanthine, 3 mM Mg2+, 10 PM NADPH, 3 PM contracted with a combination of the thromboxane tetrahydrobiopterin and 0.1 mM GSNO in the receptor agonist U46619 plus serotonin as described presence of increasing concentrations of methylene previously [36]. Indomethacin (3 PM) was added blue. Reactions were stopped by ZnC03 precipi- prior to precontraction, and methylene blue (30 PM) tation, and [32P]cyclic GMP was isolated by column once a stable muscle length had been reached chromatography [38]. (approximately 30 min after onset of precontraction). Determination of NO syrzthase activity. NO SOD (3OU/mL) and N”-nitro+arginine (0.1 and synthase activity was determined as formation 0.5 mM, respectively) were added 20 min later than of [3H]citrulline from [3H]arginine as described methylene blue, and the A23187 concentration- previously [39]. Unless otherwise indicated, purified response curve was started another 15 min later. NO synthase (0.2-0.4 pg) was incubated at 37” in a Following the highest concentration of A 23187 total volume of 0.1 mL of a triethanolamine-HCI (lOpM), O.lmM GSNO was added, and finally buffer (50 mM, pH 7.0) containing 0.1 mM [3H]- 0.3 mM papaverine. arginine (50,000-70,000 cpm), 0.1 mM NADPH, The following number of tissues (strips) were 10 PM tetrahydrobiopterin, 10 pg/mL calmodulin used (number of hearts in parentheses): A 23187 and 3 ,~LMfree Ca*+ in the presence of increasing concentration-response curves in the absence and concentrations of methylene blue. presence of methylene blue: 16 (6) each; in the Co-incubation of NO synthase and soluble guanylyl presence of SOD: 6 (2); in the combined presence cyclase. Purified NO synthase and soluble guanylyl of SOD and methylene blue: 8 (2). cyclase (0.1 pg each) were co-incubated at 37” for Changes in muscle length were recorded iso- 10 min in a total volume of 0.1 mL of a tonically 1361. Whereas contracted strips to which triethanolamine-HCl buffer (50 mM, pH 7.4) con- no relaxant agents had been added were remarkably taining 0.1 mM [(u-32P]GTP (200,000-300,000 cpm), stable throughout the experiment (2-2.5 hr), methyl- 0.1 mM L-arginine, 1 mM cGMP, 1 mM 3-isobut I- ene blue induced an initial transient increase 1-methylxanthine, 1 mM free Mg2+, 3 PM free Ca J+ (contraction) and later slow decrease (relaxation) of 10 pg/mL calmodulin, 30 PM NADPH and 0.3 PM muscle length. N”-Nitro-L-arginine rapidly increased tetrahydrobiopterin in the presence of increasing muscle length to a stable level. SOD had no influence concentrations of methylene blue. Guanylyl cyclase on muscle length. The results are expressed as per activities were determined in triplicates as described cent of control relaxation obtained with papaverine. above. The base line was the stable length resulting from Cytochrome c reduction. Reduced forms of precontraction (controls in absence and presence of methylene blue directly reduce cytochrome c [9]. SOD), or (in the presence of methylene blue or N*- We used this assay to determine to what extent the nitro+arginine) the length at the beginning of the dye reacts with the NO synthase cofactors NADPH A 23187 concentration-response curve. The gradual (0.1 mM), FAD (10 PM) and tetrahydrobiopterin loss of muscle length induced by methylene blue was (10 PM). Reduction of cytochrome c (0.1 mM) was subtracted for each A23187 concentration. determined in the presence of lOU/mL SOD and Cell culture techniques and cGMP measurements. 10 PM methylene blue by continuously monitoring Intracellular cGMP levels were measured in the increase in absorbance at 550nm against cultured porcine aortic endothelial cells as described appropriate blank samples as described previously previously [37]. Briefly, endothelial cells were [351. isolated by enzymic treatment (0.1% collagenase) Presentation of data. Data points were averaged and cultured up to three passages in Opti- and concentration-response curves fitted by non- MEM (GIBCO/BRLGmbH, Eggenstein, Germany) linear least squares regression using the Hill equation. containing 3% foetal calf serum and antibiotics. In relaxation studies, the ECss represents the Prior to the experiments, endothelial cells were concentration of A 23187 producing half maximal subcultured in 6-well lastic plates. After reaching effects irrespective of the absence or presence of confluence ( -lo6 cells Pdish) the culture medium was additional compounds. In all other experiments, ICY removed and the cells were preincubated at 37” with refers to that concentration of methylene blue which isotonic HEPES buffer (10 mM, pH 7.4) containing reduced the formation of cGMP or L-citrulline by 2.5 mM CaQ, 1 mM MgC12, 1 mM 3-isobutyl-l- 50%. methylxanthine and 1 PM indomethacin. Methylene blue and SOD were present as indicated. After RESULTS 15 min, A 23187, GSNO or sodium nitroprusside (SNP) was added and the reaction was stopped 4 min Methylene blue (30 PM) antagonized the relaxation later by removal of the incubation medium and of bovine coronary arteries induced by A 23187. In Novel actions of methylene blue 369

+ without SOD --t +sov -ct-withoutSOD+MB + +SOD+MB

t loa lo-7 10” 10” - SOD + SOD A23187 # Fig. 2. Effects of methylene blue (MB) and SOD on the relaxation of bovine coronary artery induced by 0.3 PM A Fig. 1. Inhibition of A 23187-induced relaxation of bovine 23187 or 0.1 mM GSNO. The A 23187 data are the same coronary artery by methylene blue (30pM) and effect of as in Fig. 1. The columns show mean values f SEM (of SOD (30 U/mL). Endothelium-preserved tissues were the number of experiments given in Materials and precontracted, A 23187 added cumulatively (30 nM-10 PM) Methods). and the relaxant effect determined as per cent of maximum relaxation (=lOO%) induced by papaverine (see Materials and Methods). The symbols show mean values f SEM for the number of tissues given in Materials and Methods.

3A). The drug inhibited the actions of direct guanylyl cyclase activators less effectively: in the presence of SOD, cGMP levels raised by SNP (Fig. 3B) and GSNO (Fig. 3C) were reduced to 50% of controls the absence of the dye, the mean ECsOfor the calcium at 1 mM and 13 PM methylene blue, respectively. ionophore was 45 nM, and the maximum relaxation As further shown in Fig. 3B and C, both was 59% of that induced by papaverine (open circles concentration-response curves were markedly in Fig. 1). In the presence of methylene blue (open shifted leftward when SOD had been omitted (open squares), the Eqo was 30-fold higher (1.7 PM) and symbols), and, in contrast to A 23187-induced E reduced. In the presence of SOD control responses, the effects of the direct NO donors were rengation was increased (79%, solid circles in Fig. not fully inhibited by methylene blue. 1) and the relaxation curve in the presence of We used NO synthase purified from porcine methylene blue (solid squares) was shifted leftward. cerebellum (sp. act. -400 nmol L-citrulline/mg/min) The potency of A 23187 to relax bovine coronary to investigate the effects of methylene blue on NO artery strips was compared with that of the nitric synthesis. activity was assayed both as oxide donor GSNO (Fig. 2). At 0.3pM, A 23187- conversion of radiolabelled L-arginine into L- induced relaxation was 54 + 6% in the absence and citrulline and NO formation by co-incubation of NO 9 + 2% in the presence of methylene blue. In the synthase with purified guanylyl cyclase. Figure 4 presence of 30 U/mL SOD, both control and (open symbols) shows that methylene bluecompletely methylene blue-inhibited relaxation was increased inhibited L-citrulline formation with an lcsovalue of to a similar extent, indicating that the effect of 5.3 PM and maximal effects at about 30 PM. In the methylene blue was not attenuated by SOD. GSNO presence of 10 U/mL SOD, the inhibition curve was (0.1 mM) relaxed the tissues almost as potently as only slightly shifted rightward (IQ, = 9.2pM; Fig. papaverine both in the absence (81 C 1%) and 2, solid symbols). presence (80 + 2%) of 30pM methylene blue, Under the conditions used for the co-incubation and SOD only slightly increased GSNO-induced with NO synthase, the basal activity of purified relaxation (Fig. 2). soluble guanylyl cyclase was -0.005 pmol cGMP/ The concentration-dependent effects of methylene mg/min, and addition of 0.1 pg of purified NO blue on the NO/cGMP system were studied by synthase increased this activity to 1.82.2pmol determination of intracellular cGMP levels in cGMP/mg/min. Methylene blue completely blocked cultured porcine aortic endothelial cells which had the NO synthase-induced stimulation of cGMP been stimulated with NO synthase-dependent (A formation (1~s~value -70 nM; Fig. 5A, open circles). 23187) and -independent (SNP and GSNO) activators In the presence of SOD (solid circles), the methylene of soluble guanylyl cyclase. The ionophore A 23187 blue concentration-response curve was markedly (1 PM) raised endothelial cGMP levels from 2.1 2 0.2 shifted rightward (50% inhibition at 2.1 PM). to 30.0 + 1.4pmol/106 cells, and methylene blue To ascertain whether guanylyl cyclase inhibition completely blocked this response with half-maximally was also involved in the inhibitory actions of effective concentrations of 0.4 and 5.8pM in the methylene blue, we determined the effects of the absence and presence of SOD, respectively (Fig. dye on purified guanylyl cyclase which had been B. MAYER, F. BRUNNER and K. SCHMIDT

C #*lb --o-A 23187 -o-.sNP -o- GSNO --e A 23187 + SOD --tSNP+SOD ---t GSNO+ SOD -II> 1 1 0 0 u I-IIt 8 1 1 1 8 --II7 1 1 1 9 1 0 10s 10” lo4 0 10% 10” 10” 0 10s 10” lo4 Methylene blue 0 Methylene blue (M) Methylene blue (M)

Fig. 3. Effects of methylene blue on endothelial cGMP levels raised by A 23187 (A), SNP (B) and GSNO (C). Endothelial cells were preincubated as described in Materials and Methods with various concentrations of methylene blue in the absence (0) or presence (0) of 10 U/mL SOD. After 15 min, maximally active concentrations of A 23187 (1 m), SNP (1 mM) or GSNO (100 PM) were added and the reaction was terminated 4 min later. Results are expressed as per cent of control activities obtained in the absence of methylene blue. Mean values k SEM of four separate experiments are shown.

the inhibitory effects of the dye were not significantly affected by SOD. As in the experiments with intact 100 cells, methylene blue failed to block cGMP formation 1 completely. Even in the presence of 1 mM, the highest concentration of the dye used in the present ‘=8 80 study, GSNO still produced about 80-fold increases in cGMP generation, so that approximately 30% of maximal enzyme activity was not affected by methylene blue. As it seemed possible that the apparent inhibition of NO synthase was due to a chemical reaction of methylene blue with one of the redox-active compounds required for full enzyme activity, redox- 0 cycling of 1OpM methylene blue was assayed as -o- 1OU/mlSOD reduction of cytochrome c [9] in the presence of 1 I L-,/l 7 NADPH, FAD and tetrahydrobiopterin. In the 0 KS’ absence of these compounds, we observed no Methyle~~kte f$ lo’ reduction of cytochrome c, and FAD (10 PM) failed to induce such a reaction. In the presence of NADPH Fig. 4. Inhibition of purified NO synthase by methylene (0.1 mM) and tetrahydrobiopterin (10 PM) we blue in the absence (0) and presence (0) of lOU/mL observed moderate methylene blue-dependent reac- SOD. Assay conditions were as described in Methods. tion rates of 1.0 and O&PM cytochrome c Results are expressed as per cent of control activities obtained in the absence of methylene blue. Mean values reduced/min, respectively. Considering that the 2 SEM of three separate experiments are shown. concentrations of the NO synthase cofactors used saturate the enzyme at least lo-fold [33], these reaction rates could hardly account for the observed effects of methylene blue. Moreover, we failed to overcome methylene blue-induced enzyme inhibition in the presence of lo-fold increased concentrations stimulated 200-300-fold by GSNO. NADPH (30 PM) of tetrahydrobiopterin, FAD or NADPH (not and tetrahydrobiopterin (0.3 MM), which were shown). present in some experiments to ensure conditions rcsovalues and maximal effects of methylene blue, comparable with the reconstituted system described as calculated from Figs 3-5, are summarized in Table above, had no significant effect (not shown). AS 1. Only NO synthase-mediated processes seemed to shown in Fig. SB, methylene blue inhibited GSNO- be fully inhibited by methylene blue, whereas soluble stimulated guanylyl cyclase in a concentration- guanylyl cyclase remained considerably sensitive to dependent manner (50% inhibition at -60 PM), and stimulating compounds even at methylene blue Novel actions of methylene blue 371

+ GSNO --e-- NOS+SOD --c GSNO + SOD I- 1-J - ’ ’ * 8 8 --II, ’ ’ 0 IO8 1f.P lo4 0 10-s IO4 lo4 M~ylene blue (MI Methylene blue (MI

Fig. 5. Effects of methyiene blue on purified soluble guanylyl cyelase activities.(A) Purified NO synthase (NOS) and soluble guanylyl cyclase were co-incubated as described in Materials and Methods in the absence (0) and presence (0) of 10 U/mL SOD. (B) Purified soluble guanylyl cyclase was incubated in the presence of 0.1 mM GSNO with (0) and without (0) 10 U/mL SOD. Results are expressed as per cent of control activities obtained in the absence of methyfene blue. Mean values + SEM of three separate experiments are shown.

Table 1. Effects of methylene blue (MB) and SOD on cGMP accumulation in endothelial cells (EC), NO and L-citrulline (L-Cit) formation by purified brain NO synthase (NOS), and on the generation of cGMP by GSNO-stimulated purified soluble guanyfyl cyclase (sGC)

Inhibition at 1 mM MB (&ZB) (% of control)

System Activator or product -SOD +SOD -SOD +SOD

cGMP in EC A 23187 0.4 5.8 100 100 SNP 56 >looo 69 47 GSNO 0.5 12.8 83 NOS activity L-Cit 9.2 100 1; NO Z7 2.1 loo 100 sGC activity GSNO 60 60 72 72

concentrations as high as 1 mM. Interestingly, relaxations of bovine coronary arteries. In contrast endothelial responses to SNP differed markedly from to endothelium-dependent relaxations, the relaxant those to GSNO with respect to the inhibitory potency responses of the vessels to GSNO were not inhibited of methylene blue, which blocked the actions of by 3OpM methylene blue, indicating that smooth GSNO at -lOO-fold lower ~ncentrations than those muscle guanyly1 cyclase was not affected by the drug. of SNP. Moreover, it seemed unlikely that methylene blue acted via superoxide anion-mediated inactivation of DISCUSSION NO, since SOD failed to prevent the inhibitory effects of the dye on endothelium-dependent Methylene blue (3O@f) markedly inhibited A relaxation. 231874nduced endothelium-dependent relaxation of Similarly, we observed striking differences in the bovine coronary arteries, but small relaxant effects inhibitor action profiles of methylene blue on of the ionophore were still observed in the presence endothelial cGMP levels raised by either direct or of the dye. Since these methylene blue-insensitive indirect activators of soluble guanylyl cyclase (see relaxations were also largely insensitive to inhibition Fig. 3). It is remarkable that the effects of both SNP by W-nitro+arginine (not shown), a mechanism and GSNO were not completely inhibited by unrelated to the t-arginine/NO pathway may methylene blue. As we obtained similar results using additionally be involved in A 231874nduced purified guanylyl cyclase (see Fig. SB), it appears

BP 4!ir2-II 372 8. MAYER,F. BRUNNERand K. SCHMIDT that NO and/or NO-containing compounds activate reported that the presence of 25 PM methylene blue the enzyme by two different mechanisms. Accord- reduced citrulline formation in homogenates of ingly, binding of NO to the heme moiety of guanylyl porcine aortic endothelial cells by 60% 1421. cyclase may be inhibited by methylene blue, and S- Differences in the enzyme assay conditions, such as of regulatory sulphydryl groups [40,41] a different substrate concentration, may account for may account for the methylene blue-insensitive the less pronounced effects of the dye described component of NO-induced enzyme stimulation. therein. In assays with endothelial cells, methylene blue Methylene blue is known to affect iron-containing was considerably less potent when SOD was present, [g-14], Stoichiometrical amounts of iron i.e. 1~~0values were increased, whereas the maximal are present in both soluble guanylyl cyclase and NO inhibitory effect of the drug was decreased synthase [6,32]. The heme group of soluble guanylyl by exogenously added SOD. Given that SOD cyclase has been suggested as a target site of predominantly acts by increasing the steady state methylene blue, resulting in a reduced responsiveness concentrations of extracellular NO, these data clearly of the enzyme to activation by NO [17,18]. suggest that SNP and GSNO, at least partially, Consistently, heme-independent basal and arachi- elevate endothelial cGMP levels via release of free donic acid-stimulated cGMP formation were found NO. to be insensitive to inhibition by methylene blue Surprisingly, the actions of SNP and GSNO on (431. Xanthine oxidase, another iron-containing endothelial cGMP levels were rather differently enzyme, is inhibited by methylene blue most likely affected by methylene blue (Table l), whereas no due to a competition of the dye with moiecular oxygen such discrepancies were observed in experiments for xanthine-delved electrons [14]. Methylene blue with purified guanylyl cyciase (not shown). Thus, in apparently interacts with the iron-sulphur centre of the presence of SOD the effect of methyiene blue xanthine oxidase, since its reduction was also on SNP-elevated endothelial cGMP levels was similar catalysed by flavin- and molybdenum-free forms of to the effects of the drug on purified guanylyl cyclase. the enzyme [lo, 111. Accordingly, methylene blue Accumulation of endothelial cGMP induced by seems to act as a powerful oxidant of ferrous iron GSNO, however, was much more susceptible to bound to various enzymes either in heme or non- inhibition by methylene blue. Presently, we have no heme forms. The present results show for the first conclusive explanation for these data, but it seems time that this compound is a potent inhibitor of NO possible that an as yet unknown iron-containing synthase and indicate a catalytic function of enzyme- enzyme may catalyse the liberation of NO from S- bound iron in NO synthesis. nitrosothiols in endothelial cells. This explanation is Experimental evidence suggests that enzymic compatible with our finding that SOD enhanced the liberation of NO accounts for the potent vasodilatory inhibitory effect of methylene blue on GSNO- properties of organic [44-46]. We are induced cGMP accumulation in endothelial cells, presently investigating the effects of methylene blue while leaving GSNO-induced relaxation and guanylyl on metabolism in smooth muscle tissues to cyclase stimulation unaffected (see Figs 3C vs 2 and determine if hemeproteins cataiyse NO release from 5B). nitrovasodilators in vascular tissues, as has been The experiments using purified NO synthase suggested for the metabolism of glycerol trinitrate further confirmed the results obtained with isolated in liver microsomes [47,48]. blood vessels and cultured endothelial cells. When In conclusion, our data show that methylene the enzyme was incubated together with purified blue, besides generation of superoxide anion, soluble guanylyl cyclase in the presence of all predominantly acts via a direct inhibition of NO required substrates, cosubstrates and enzyme synthase and other iron-containing enzymes and activators, this reconstituted system produced cGMP does not represent either a selective or a potent with reaction rates even exceeding those observed inhibitor of NO-stimulated soluble guanylyl cyclase by directly stimulating guanylyl cyclase with NO- as previously assumed. containing compounds. Methylene blue inhibited this cGMP formation with half-maximal effects Acknowledgemenrs-We wish to thank Dr E. Biihme for observed at 0.07 and 2,uM in the absence and providing purified guanylyl cyclase. The excellent technical presence of SOD, respectively (see Fig. 5A assistance of Eva Leowld, Mar&t Rehn and Heike Stessel and Table 1). We have previously used NO is gratefully acknowledged. Th& work was supported by chemiiuminescence under non-reducing conditions grants from the.Fonds zur Fiirderung der wissenschaftlichen Forschung in Osterreich (P 8836 and P 8581). to specifically demonstrate the generation of free NO by isolated NO synthase [33], making it highly likely that the pronounced effects of SOD in the REFERENCES reconstituted system were due to a stabilization of enzymatically formed free NO. The methylene blue 1. Moncada S, Palmer RMJ and Higgs EA, Nitric oxide- concentrations required for a complete inhibition of physiology, pathophysiology, and pha~aco~ogy. P&r- cGMP generation by this system were about 30 FM macol Reu 43: 109-142, 1991. in the presence of SOD, i.e. equal to those necessary 2. Mayer 3, Schmidt K, Humbert P and Biihme E, Biosynthesis of endothehum-derived relaxing factor: a for full inhibition of citrulline formation (see Fig, cytosolic enzyme in porcine aortic endothelial cells 4). 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