627
Biochem. J. (1992) 281, 627-630 (Printed in Great Britain)
Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase Burghard HEINZEL,* Mathias JOHN,* Peter KLATT,t Eycke BOHME* and Bernd MAYER*tt *Institut fur Pharmakologie, Freie Universitat Berlin, Thielallee 67-73, D-1000 Berlin 33, Federal Republic of Germany, and tlnstitut fur Pharmakodynamik und Toxikologie, Karl-Franzens-Universitat-Graz, Universitatsplatz 2, A-8010 Graz, Austria
L-Arginine-derived nitric oxide (NO) acts as an inter- and intra-cellular signal molecule in many mammalian tissues including brain, where it is formed by a flavin-containing Ca2+/calmodulin-requiring NO synthase with NADPH, tetrahydrobiopterin (H4biopterin) and molecular oxygen as cofactors. We found that purified brain NO synthase acted as a Ca2+/calmodulin-dependent NADPH:oxygen oxidoreductase, catalysing the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin, which inhibited the hydrogen peroxide formation with halfmaximal effects at 11 uM and 0.3 /M respectively. Half-maximal rates of L-citrulline formation were observed at closely similar concentrations of these compounds, indicating that the NO synthase-catalysed oxygen activation was coupled to the synthesis of L-citrulline and NO in the presence of L-arginine and H4biopterin. Nw-Nitro-L-arginine, its methyl ester and N'-monomethyl-L-arginine inhibited the synthesis of L-citrulline from L-arginine (100 /tM) with half-maximal effects at 0.74 /M, 2.8 /tM and 15 /M respectively. The N'-nitro compounds also blocked the substrate-independent generation of hydrogen peroxide, whereas N@-monomethyl-L-arginine did not affect this reaction. According to these results, activation of brain NO synthase by Ca2l at subphysiological levels of intracellular L-argimne or H4biopterin may result in the formation of reactive oxygen species instead of NO, and NV-nitro-substituted L-arginine analogues represent useful tools to effectively block NO synthase-catalysed oxygen activation.
INTRODUCTION
EXPERIMENTAL
In a variety of mammalian tissues, L-arginine-derived nitric oxide (NO) acts as signal molecule and represents the endogenous activator of soluble guanylate cyclase [1-3]. NO formation is coupled to the activation of excitatory amino acid receptors in the central nervous system, but apparently NO is also formed post-synaptically and may act as a neurotransmitter [4]. In brain, L-arginine is converted into NO by a Ca2+- and NADPHdependent;cytosolic NO synthase which requires calmodulin and tetrahydrobiopterin (H4biopterin) for enzymic activity [5-7], whereas cytokine-activated macrophages contain a Ca2+-independent NO synthase which apparently exhibits properties similar to those of the brain enzyme, as it also requires NADPH and H4biopterin as cofactors [8,9]. The reaction mechanism of NO formation is not yet fully understood, but it was shown that the macrophage enzyme catalyses the hydroxylation of Larginine, resulting in the formation of N,-hydroxy-L-arginine which apparently reacts with molecular oxygen to yield L-citrulline and NO [10,11]. Recently, we have characterized NO synthase purified from cerebellum as multi-functional oxidoreductase which contains FAD and FMN in equimolar amounts and represents a nonhaem iron protein [12]. Strikingly, the purified enzyme catalysed a Ca2+/calmodulin-dependent oxidation of NADPH in the absence of L-arginine or H4biopterin. Under these conditions, molecular oxygen acted as the electron acceptor instead of Larginine or an intermediary L-arginine derivative. In the present paper we show that NO synthase acts as a Ca2+/calmodulindependent NADPH oxidase which forms hydrogen peroxide in the presence of suboptimal concentrations of L-arginine or H4biopterin.
Materials
L-[2,3-3H]Arglnine (sp. radioactivity 40-70 Ci/mmol) was obtained from DuPont de Nemours, Dreieich, Germany, and was purified by h.p.l.c. using 100 mM-sodium acetate buffer, pH 4.5, as eluant. The ion-exchange resin Dowex-50W, No-nitro-L-arginine (L-NNA), Nw-nitro-L-arginine methyl ester (L-NAME) and catalase from bovine liver were supplied by Sigma, Deisenhofen Germany, and N'-monomethyl-L-arginine (L-NMMA) was from Calbiochem, Frankfurt, Germany. H4Biopterin was obtained from Dr. B. Schircks Laboratories, Jona, Switzerland, and NADPH was from Waldhof, Dusseldorf, Germany. All other reagents, solvents and salts were of analytical grade and were obtained from Sigma or from Merck, Darmstadt, Germany. Purification of NO synthase For the purification of Ca2+-regulated NO synthase from pig cerebellum, a method described previously [7] was scaled-up and slightly modified, resulting in a marked increase in enzyme recovery. About 3 kg of pig cerebella was homogenized with an Ultra-Turrax in 3 vol. of a 50 mM-triethanolamine/HCl buffer, pH 7.5, containing 0.5 mM-EDTA (buffer A). After centrifugation (30 min, 10000 gay), solid ammonium sulphate (176 g/l) was added to the supernatant to precipitate NO synthase. The pellets were washed once with 5 litres of buffer A containing 176 mg of ammonium sulphate/ml and then dissolved in a 20 mM-triethanolamine/HCl buffer, pH 7.5, containing 0.5 mM-EDTA and 10 mM-2-mercaptoethanol so that the conductivity of the suspension was not higher than about 10 mS/cm. After centrifugation (40 min, 10000 g.), the supernatant was
Abbreviations used: H4biopterin, (6R)-5,6,7,8-tetrahydrobiopterin; L-NMMA, N'-monomethyl-L-arginine; L-NNA, Nd-nitro-L-arginine; L-NAME, N'-nitro-L-arginine methyl ester; SOD, superoxide dismutase. t To whom correspondence should be addressed, in Graz.
Vol. 281
628 mixed with 25 ml of 2',5'-ADP-Sepharose which had been preequilibrated with buffer A containing 10 mM-2-mercaptoethanol (buffer B). The slurry was stirred for 30 min, poured into a column, and the Sepharose was washed with 250 ml of buffer B containing 0.5 M-NaCl, followed by 250 ml of buffer B and 50 ml of buffer B containing 0.5 mM-NADPH. NO synthase was eluted with 80 ml of buffer B containing 10 mM-NADPH, and the eluate was immediately concentrated with Centricon-30 microconcentrators (Amicon, Witten, Germany). The concentrate was washed three times with buffer B to decrease the concentration of NADPH, and the enzyme (0.3-0.5 mg/ml) was stored in 20 % (v/v) glycerol at -70 'C. Using this purification procedure, we obtained about 2 mg of NO synthase of a purity of greater than 95 %, as judged from the Coomassie Blue-stained gels. Protein was determined by the method of Bradford [13], with BSA as standard protein.
B. Heinzel and others
superoxide dismutase (SOD)/ml (results not shown). As calculated from the curve shown in Fig. 1, calmodulin exhibited half-maximal effects on NO synthase-catalysed H202 formation at 16 nm, with a Hill coefficient of 4.15 when assayed in the presence of 3 /LM free Ca2+. Thus the concentration-response curve was shifted leftwards when compared with the conversion of L-arginine into L-citrulline, which occurred at half-maximal rates in the presence of about 70 nM-calmodulin [7,14]. In the absence of L-arginine, added H4biopterin (10 ,sM) did not affect the formation of H202. As shown in Fig. 2, L-arginine inhibited this reaction with half-maximal effects at 11.0 /SM, and completely abolished it at concentrations of 0.1 mm. Fig. 2 shows that the enzyme started to act as an NO synthase at L-arginine concentrations of 1 /UM, catalysing the formation of L-citrulline, with half-maximal effects of L-arginine at 8.9 #M. Thus the consumption of NADPH and the reduction of oxygen were
Determination of L-Citrulline Unless otherwise indicated, formation of [3H]citrulline from [3H]arginine was determined as previously described [14] in a final volume of 100 ,ul in triethanolamine/HCI buffer (50 mM, pH 7.0) containing 100 /LM-[3H]arginine (50000-80000 c.p.m.), 1 mm free Mg2+, 10 ,ug of calmodulin/ml, 3/SM free Ca2+, 0.1 mMNADPH, 5 /M-FAD and 10 ,uM-H4biopterin in the presence of 100-300 ng of NO synthase. Blank values were determined in the absence of added enzyme. The concentrations of free Ca2+ were adjusted as described previously [15]. Determination of hydrogen peroxide Hydrogen peroxide was determined as formation of ferric thiocyanate by a method described by Hildebrandt et al. [16], slightly modified. Unless otherwise indicated, purified NO synthase (2 ,ug) was incubated at 37 'C for 10 min in 0.2 ml of a 50 mM-triethanolamine/HCl buffer, pH 7.0, containing 0.1 mmNADPH, 3 /M free Ca2+ and 2 /sg of calmodulin. Reactions were terminated by the addition of 100 1ul of concentrated HCI (about 11.8 M), and aqueous solutions of ferrous ammonium sulphate (20,u1) and potassium thiocyanate (30,1) were added to give final concentrations of 3.2 mm and 180 mm respectively. Samples were incubated for 10 min at ambient temperature, followed by the immediate determination of the absorbance at 492 nm. To achieve optimal specificity of this assay, blank values were determined in the presence of enzyme but with catalase (20 units) additionally present during the incubations. Catalase was without effect on NADPH consumption and on the formation of Lcitrulline or NO. Authentic H202 carried through the incubation and assay procedures was used to record calibration curves, which were linear from 1 to 100 1sM-H202, giving AA values of about 0.01 to 1.0. Data evaluation All values were determined in duplicate or triplicate, and the means+S.E.M. of three separate experiments were calculated. Concentration-response curves were fitted to sigmoidal curves by non-linear regression analysis using the software of GraphPad INPLOT, Version 3.0.
RESULTS Even in the absence of L-arginine and H4biopterin, NO synthase still consumed molecular oxygen at the expense of NADPH [12]. Under these conditions, H202 was formed in a Ca2+/calmodulin-dependent manner at a constant rate for about 15 min, with specific activities of approx. 250 nmol/min per mg, and this reaction was not affected by up to 1000 units of
io-9
lo--,
10-7 104 [Calmodulin] (M) Fig. 1. Effect of calmodulin on NO synthase-catalysed H202 formation in the absence of L-arginine and H4biopterin Purified NO synthase (2 ,ug) was incubated for 10 min at 37 °C in 0.2 ml in the presence of 0.1 mM-NADPH and 3 4tM free Ca2" with increasing amounts of calmodulin. The rate of H202 formation was determined as described in the Experimental section and is given as the mean + S.E.M. of three separate experiments.
500 EE' E
0
0.
0
400 .S
._
E
E200
0
0
300 E
C
.o 200 0E
E 0
s 100
0
0
100 .'
4O
0
zIn [L-Arginine] (M)
Fig. 2. Effects of L-arginime on the NO synthase-catalysed formation of H202 and L-citruife Purified NO synthase (2 ,ug and 100 ng in the H202 and L-citrulline assays respectively) was incubated for 10 min at 37 °C in the presence of 0.1 mM-NADPH, 3/lM free Ca2 , 1O/sg of calmodulin/ml and 10 1sM-H4biopterin with increasing amounts of L-arginine as indicated on the abscissa. Rates of H202 (0) and L-citrulline (0) formation were determined as described in the Experimental section and are given as means + S.E.M. of three separate experiments.
1992
629
Hydrogen peroxide formation by NO synthase E 'a E 150
0
0 L.
CL
~~~~~-~ ~ .400H0
CD
.E
-
E
o 100 E
~
~
~
~
0250 E
~
Ez
200
0
E 300
0
-
C
0
0 50 E 0
20
x
E 0 .20
o
C
0
o
0
10-7 1io 106o6 [H4Biopterinl (M) 0 Fig. 3. Effects of H4biopterin on the NO synthase-catalysed formation of H202 and L-citrulline Purified NO synthase was incubated as described in the legend to Fig. 2 in the presence of 100 ,UM-L-arginine with increasing amounts of H4biopterin as indicated on the abscissa. Rates of H202 (0) and L-citrulline (0) formation were determined as described in the Experimental section and are given as means + S.E.M. of three separate experiments. i0-9
ID a
0,
E
00
400 a
200
0
o 150 I _ Ec
300
E
S 0 c 0
C
o 100 I _
C
0
200 'm20
100r
0~~~~~~~~~~~~~~~
E
omto 0
10-7
H20adLctuln ioo 10-
10-3 0
10-
10-
10-4
1~~~~~~~~~~~~
[Inhibitor] (m)
Fig. 5. Effects Of L-argifline analogues
on the NO synthase-catalysed formation of H202 and L-Citrulline (a) For the determination of L-Citrulline generation, purified NO synthase (300 ng) was incubated as described in the Experimental section with L-NNA (-), L-NAME (-) and L-NMMA (-) at the concentrations indicated. L-Citrulline-forming enzyme activities were determined as described in the Experimental section and are given as mean values of three separate experiments. At each point, the S.E.M. was < 10 % (not shown). (b) H202 formation was determined by incubation of purified NO synthase (2 ,ug) for 10 min at 37 °C in 0.2 ml in the presence of 0.1 mM-NADPH and 3 1uM free Ca21 with the enzyme inhibitors L-NNA (El), L-NAME (A) and L-NMMA (0) at the concentrations indicated. The rates of H202 formation were determined as described in the Experimental section and are given as mean values of three separate experiments. At each point, the S.E.M. was < 10 % (not shown).
E
E 0 .°- 50 I _ 0
200 2 c
I
6.5
7.0
7.5 pH
8.0
100
,
8.5
Fig. 4. Effects of pH on the NO synthase-catalysed formation of H202 and L-Citruuine
H202 formation (0) was determined by incubation of 2 ,eg of purified NO synthase for 10 min at 37 'C in the presence of 0.1 mmNADPH, 3 /M free Ca2' and 10 /sg of calmodulin/ml at the pH values indicated. For the determination of L-citrulline formation (0), 100 ng of NO synthase was incubated with 100 /tM-L-arginine and 10 /uM-H4biopterin additionally present. Rates of H202 and Lcitrulline formation were determined as described in the Experimental section and are given as means +S.E.M. of three separate experiments.
coupled to the formation of L-citrulline when L-arginine was present during the incubations. NO synthase is considerably stimulated by H4biopterin [7-9], which apparently represents an essential cofactor of the brain enzyme, as NO formation observed in the absence of exogenously added H4biopterin is most likely due to small amounts of this compound present in purified NO synthase preparations [12]. In the absence of added H4biopterin, the formation of H202 was decreased by L-arginine (100 4aM) to about 150 nmol/min per mg. As shown in Fig. 3, addition of H4biopterin resulted in a further inhibition of this reaction (half-maximal at 0.29 ,uM-H4biopterin), whereas the formation of L-citrulline was stimulated from 100 to about 400 nmol/min per mg, with a half-maximally active H4biopterin concentration of 0.27 4uM. Fig. 4 shows that similar effects of pH were observed on the generation of H202 and L-citrulline. Both enzymic activities were Vol. 281
E
10
250
E
C 10-7-150
0 C
maximal at slightly acidic conditions and decreased with increasing pH. As shown in Fig. 5(a), the competitive NO synthase inhibitors L-NNA, L-NAME and L-NMMA inhibited the conversion of 0.1 mM-L-arginine into L-citrulline in a concentration-dependent manner, with half-maximal effects at 0.74 uM, 2.8 /LM and 15 4uM respectively. Fig. 5(b) shows the effects of these inhibitors on the generation of H202 in the absence of L-arginine and H4biopterin. This reaction was inhibited by L-NNA and L-NAME, with halfmaximal effects at 0.2 #M and 0.3 /M respectively. Similarly, the calmodulin-dependent NADPH consumption (determined as decrease in absorbance at 340 nm) was inhibited by these compounds (results not shown). L-NMMA, however, did not affect H202 formation at concentrations up to 1 mm. NO synthase also catalysed a rapid Ca2+/calmodulin-dependent reduction of cytochrome c, but, similar to the observed H202 formation, cytochrome c reduction was insensitive to SOD (results not shown). As NO synthase could be bound to cytochrome c-agarose (results not shown), reduction of cytochrome c apparently results from a direct interaction of these proteins and is not mediated by superoxide anions. DISCUSSION In the presence of Ca2+/calmodulin, NO synthase apparently catalyses an NADPH-dependent reduction of molecular oxygen, as do other flavin-containing enzymes [17]. At saturating concentrations of L-arginine and H4biopterin, this oxygen reduction is fully coupled to the formation of L-citrulline and NO, but it results in the formation of H202 at limiting concentrations of substrate or cofactor. Thus the observed uncoupling of oxygen reduction is only partially prevented by L-arginine in the absence of added H4biopterin. Lack of effect of SOD indicates that
630
superoxide is not formed (or is at least not released from the enzyme) as an intermediate in H202 formation. This was further confirmed by results showing that NO synthase-catalysed cytochrome c reduction was not inhibited by SOD and that this reaction was due to a direct interaction of these proteins. This latter result is also in good accordance with a recent report describing sequence similarities between rat brain NO synthase and cytochrome P-450 reductase [18]. H202 formation as described here is possibly specific for the brain enzyme, as macrophage NO synthase was reported to lack dihydropteridine reductase activity, as determined by a method based on NADPH uptake in the absence of substrate [11]. The formation of L-citrulline was optimal at slightly acidic conditions, confirming previous results demonstrating a rapid pH-dependent decrease of the L-arginine-dependent activation of purified soluble guanylate cyclase by NO synthase obtained from bovine brain [19]. As shown in Fig. 4, H202 formation exhibited a very similar pH-dependency, indicating that the NADPHdependent reduction of molecular oxygen is decreased at alkaline conditions, resulting in diminished rates of L-citrulline and NO formation. The rank order of potency of L-arginine analogues in inhibiting L-citrulline formation was L-NNA > L-NAME > L-NMMA, confirming previous results obtained with the enzyme from brain, adrenal gland and endothelial cells [5,20-22]. L-NNA and L-NAME also inhibited substrate-independent H202 generation, indicating that binding of the N'-nitro compounds prevented the enzyme from reducing molecular oxygen. As L-NMMA did not affect H202 formation, this compound apparently differs from L-NNA and L-NAME in its inhibitory properties, possibly explaining tissue-specific effects of these compounds [23]. Thus L-NNA and L-NAME, but not the most commonly used LNMMA, ensure a complete inhibition of NO synthase-catalysed oxygen reduction and should represent useful tools to effectively block the formation of both NO and H202 by Ca2+/calmodulinactivated NO synthase. On studying the distribution of NO synthase over various areas of the brain, the enzyme is most abundant in the cerebellum, where it is associated with neurons that make close contact with Purkinje cells [24]. It was reported that all NO synthase neurons also stain for the enzyme NADPH diaphorase [25], a membrane-associated, flavin-containing 73 kDa homodimeric protein which exhibits similarities to cytochrome P-450 reductase [26,27]. Recently it was reported that a 150 kDa monomeric NO synthase purified from rat brain supernatants was recognized and partially precipitated by antibodies directed against NADPH diaphorase [28]. As the purified enzyme catalysed an NADPH-dependent reduction of Nitro Blue Tetrazolium, a reaction on which the diaphorase-staining of brain slices is based, the authors concluded that NO synthase and NADPH diaphorase are identical proteins. Our results apparently explain the observed NO synthase-catalysed NADPH-dependent reduction of Nitro Blue Tetrazolium, especially since we found that the enzyme not only reduces molecular oxygen in a substrate-independent manner, but even more rapidly utilizes artificial electron acceptors (B. Heinzel,
unpublished work). Decreased levels of H4biopterin are found in the cerebrospinal fluid of patients affected with neuronal disorders (for review see
B. Heinzel and others
ref. [29]), and H4biopterin was shown to limit NO formation in mouse fibroblasts [30]. Thus it will be interesting to find out whether Ca2+-activated brain NO synthase also catalyses the described H202 generation in cells and tissues at suboptimal intracellular levels of L-arginine of H4biopterin. We thank Dr. G. Schultz for critical reading of this manuscript. B. M. was a recipient of a fellowship of the Alexander von Humboldt-Stiftung. The financial support of the Deutsche Forschungsgemeinschaft, Osterreichische Nationalbank and Osterreichische Forschungsgemeinschaft is gratefully acknowledged.
REFERENCES 1. Furchgott, R. F. & Vanhoutte, P. M. (1989) FASEB J. 3, 2007-2018 2. Ignarro, L. J. (1991) Biochem. Pharmacol. 41, 485-490 3. Radomski, M. W., Palmer, R. M. J. & Moncada, S. (1991) Trends Pharmacol. Sci. 12, 87-88 4. Garthwaite, J. (1991) Trends Neurol. Sci. 14, 60-67 5. Knowles, R. G., Palacios, M., Palmer, R. M. J. & Moncada, S. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5159-5162 6. Bredt, D. S. & Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,
682-685 7. Mayer, B., John, M. & Bohme, E. (1990} FEBS Lett. 277, 215-219 8. Tayeh, M. A. & Marletta, M. A. (1989) J. Biol. Chem. 264, 19654-19658 9. Kwon, N. S., Nathan, C. F. & Stuehr, D. J. (1989) J. Biol. Chem. 264, 20496-20501 10. Kwon, N. S., Nathan, C. F., Gilker, C., Griffith, 0. W., Matthews, D. E. & Stuehr, D. J. (1990) J. Biol. Chem. 265, 13442-13445 11. Stuehr, D. J., Kwon, N. S., Nathan, C. F., Griffith, 0. W., Feldman, P. L. & Wiseman, J. (1991) J. Biol. Chem. 266, 6259-6263 12. Mayer, B., John, M., Heinzel, B., Werner, E. R., Wachter, H., Schultz, G. & Bohme, E. (1991) FEBS Lett. 288, 187-191 13. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 14. Mayer, B., John, M. & B6hme, E. (1991) J. Cardiovasc. Pharmacol.
17, S46-S51 15. Mayer, B., Schmidt, K., Humbert, P. & B6hme, E. (1989) Biochem. Biophys. Res. Commun. 164, 678-685 16. Hildebrandt, A. G., Roots, I., Tjoe, M. & Heinemeyer, G. (1978) Methods Enzymol. 52, 342-350 17. Ghisla, S. & Massey, V. (1989) Eur. J. Biochem. 181, 1-17 18. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R. & Snyder, S. H. (1991) Nature (London) 351, 714-718 19. Mayer, B., Evers, B., Wilke, P. & Bohme, E. (1990) Arch. Int. Pharmacodyn. Ther. 305, 265 20. Palacios, M., Knowles, R. G., Palmer, R. M. J. & Moncada, S. (1989) Biochem. Biophys. Res. Commun. 165, 802-809 21. Rees, D. D., Palmer, R. M. J., Schulz, R., Hodson, H. F. & Moncada, S. (1990) Br. J. Pharmacol. 101, 746-752 22. Muilsch, A. & Busse, R. (1990) Naunyn-Schmiedeberg's Arch. Pharmacol. 341, 143-147 23. McCall, T. B., Feelisch, M., Palmer, R. M. J. & Moncada, S. (1991) Br. J. Pharmacol. 102, 234-238 24. Bredt, D. S., Hwang, P. M. & Snyder, S. H. (1990) Nature (London) 347, 768-770 25. Snyder, S. H. & Bredt, D. S. (1991) Trends Pharmacol. Sci. 12, 125-128 26. Kuonen, D. R., Kemp, M. C. & Roberts, P. J. (1988) J. Neurochem. 50, 1017-1025 27. Kemp, M. C., Kuonen, D. R., Sutton, A. & Roberts, P. J. (1988) Biochem. Pharmacol. 37, 3063-3070 28. Hope, B. T., Michael, G. J., Knigge, K. M. & Vincent, S. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2811-2814 29. Nichol, C. A., Smith, G. K. & Duch, D. S. (1985) Annu. Rev. Biochem. 54, 729-764 30. Werner-Felmayer, G., Werner, E. R., Hausen, A., Reibnegger, G. & Wachter, H. (1990) J. Exp. Med. 172, 1599-1607
Received 29 May 1991/31 July 1991; accepted 3 September 1991
1992
Biochem. J. (1992) 281, 627-630 (Printed in Great Britain)
Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase Burghard HEINZEL,* Mathias JOHN,* Peter KLATT,t Eycke BOHME* and Bernd MAYER*tt *Institut fur Pharmakologie, Freie Universitat Berlin, Thielallee 67-73, D-1000 Berlin 33, Federal Republic of Germany, and tlnstitut fur Pharmakodynamik und Toxikologie, Karl-Franzens-Universitat-Graz, Universitatsplatz 2, A-8010 Graz, Austria
L-Arginine-derived nitric oxide (NO) acts as an inter- and intra-cellular signal molecule in many mammalian tissues including brain, where it is formed by a flavin-containing Ca2+/calmodulin-requiring NO synthase with NADPH, tetrahydrobiopterin (H4biopterin) and molecular oxygen as cofactors. We found that purified brain NO synthase acted as a Ca2+/calmodulin-dependent NADPH:oxygen oxidoreductase, catalysing the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin, which inhibited the hydrogen peroxide formation with halfmaximal effects at 11 uM and 0.3 /M respectively. Half-maximal rates of L-citrulline formation were observed at closely similar concentrations of these compounds, indicating that the NO synthase-catalysed oxygen activation was coupled to the synthesis of L-citrulline and NO in the presence of L-arginine and H4biopterin. Nw-Nitro-L-arginine, its methyl ester and N'-monomethyl-L-arginine inhibited the synthesis of L-citrulline from L-arginine (100 /tM) with half-maximal effects at 0.74 /M, 2.8 /tM and 15 /M respectively. The N'-nitro compounds also blocked the substrate-independent generation of hydrogen peroxide, whereas N@-monomethyl-L-arginine did not affect this reaction. According to these results, activation of brain NO synthase by Ca2l at subphysiological levels of intracellular L-argimne or H4biopterin may result in the formation of reactive oxygen species instead of NO, and NV-nitro-substituted L-arginine analogues represent useful tools to effectively block NO synthase-catalysed oxygen activation.
INTRODUCTION
EXPERIMENTAL
In a variety of mammalian tissues, L-arginine-derived nitric oxide (NO) acts as signal molecule and represents the endogenous activator of soluble guanylate cyclase [1-3]. NO formation is coupled to the activation of excitatory amino acid receptors in the central nervous system, but apparently NO is also formed post-synaptically and may act as a neurotransmitter [4]. In brain, L-arginine is converted into NO by a Ca2+- and NADPHdependent;cytosolic NO synthase which requires calmodulin and tetrahydrobiopterin (H4biopterin) for enzymic activity [5-7], whereas cytokine-activated macrophages contain a Ca2+-independent NO synthase which apparently exhibits properties similar to those of the brain enzyme, as it also requires NADPH and H4biopterin as cofactors [8,9]. The reaction mechanism of NO formation is not yet fully understood, but it was shown that the macrophage enzyme catalyses the hydroxylation of Larginine, resulting in the formation of N,-hydroxy-L-arginine which apparently reacts with molecular oxygen to yield L-citrulline and NO [10,11]. Recently, we have characterized NO synthase purified from cerebellum as multi-functional oxidoreductase which contains FAD and FMN in equimolar amounts and represents a nonhaem iron protein [12]. Strikingly, the purified enzyme catalysed a Ca2+/calmodulin-dependent oxidation of NADPH in the absence of L-arginine or H4biopterin. Under these conditions, molecular oxygen acted as the electron acceptor instead of Larginine or an intermediary L-arginine derivative. In the present paper we show that NO synthase acts as a Ca2+/calmodulindependent NADPH oxidase which forms hydrogen peroxide in the presence of suboptimal concentrations of L-arginine or H4biopterin.
Materials
L-[2,3-3H]Arglnine (sp. radioactivity 40-70 Ci/mmol) was obtained from DuPont de Nemours, Dreieich, Germany, and was purified by h.p.l.c. using 100 mM-sodium acetate buffer, pH 4.5, as eluant. The ion-exchange resin Dowex-50W, No-nitro-L-arginine (L-NNA), Nw-nitro-L-arginine methyl ester (L-NAME) and catalase from bovine liver were supplied by Sigma, Deisenhofen Germany, and N'-monomethyl-L-arginine (L-NMMA) was from Calbiochem, Frankfurt, Germany. H4Biopterin was obtained from Dr. B. Schircks Laboratories, Jona, Switzerland, and NADPH was from Waldhof, Dusseldorf, Germany. All other reagents, solvents and salts were of analytical grade and were obtained from Sigma or from Merck, Darmstadt, Germany. Purification of NO synthase For the purification of Ca2+-regulated NO synthase from pig cerebellum, a method described previously [7] was scaled-up and slightly modified, resulting in a marked increase in enzyme recovery. About 3 kg of pig cerebella was homogenized with an Ultra-Turrax in 3 vol. of a 50 mM-triethanolamine/HCl buffer, pH 7.5, containing 0.5 mM-EDTA (buffer A). After centrifugation (30 min, 10000 gay), solid ammonium sulphate (176 g/l) was added to the supernatant to precipitate NO synthase. The pellets were washed once with 5 litres of buffer A containing 176 mg of ammonium sulphate/ml and then dissolved in a 20 mM-triethanolamine/HCl buffer, pH 7.5, containing 0.5 mM-EDTA and 10 mM-2-mercaptoethanol so that the conductivity of the suspension was not higher than about 10 mS/cm. After centrifugation (40 min, 10000 g.), the supernatant was
Abbreviations used: H4biopterin, (6R)-5,6,7,8-tetrahydrobiopterin; L-NMMA, N'-monomethyl-L-arginine; L-NNA, Nd-nitro-L-arginine; L-NAME, N'-nitro-L-arginine methyl ester; SOD, superoxide dismutase. t To whom correspondence should be addressed, in Graz.
Vol. 281
628 mixed with 25 ml of 2',5'-ADP-Sepharose which had been preequilibrated with buffer A containing 10 mM-2-mercaptoethanol (buffer B). The slurry was stirred for 30 min, poured into a column, and the Sepharose was washed with 250 ml of buffer B containing 0.5 M-NaCl, followed by 250 ml of buffer B and 50 ml of buffer B containing 0.5 mM-NADPH. NO synthase was eluted with 80 ml of buffer B containing 10 mM-NADPH, and the eluate was immediately concentrated with Centricon-30 microconcentrators (Amicon, Witten, Germany). The concentrate was washed three times with buffer B to decrease the concentration of NADPH, and the enzyme (0.3-0.5 mg/ml) was stored in 20 % (v/v) glycerol at -70 'C. Using this purification procedure, we obtained about 2 mg of NO synthase of a purity of greater than 95 %, as judged from the Coomassie Blue-stained gels. Protein was determined by the method of Bradford [13], with BSA as standard protein.
B. Heinzel and others
superoxide dismutase (SOD)/ml (results not shown). As calculated from the curve shown in Fig. 1, calmodulin exhibited half-maximal effects on NO synthase-catalysed H202 formation at 16 nm, with a Hill coefficient of 4.15 when assayed in the presence of 3 /LM free Ca2+. Thus the concentration-response curve was shifted leftwards when compared with the conversion of L-arginine into L-citrulline, which occurred at half-maximal rates in the presence of about 70 nM-calmodulin [7,14]. In the absence of L-arginine, added H4biopterin (10 ,sM) did not affect the formation of H202. As shown in Fig. 2, L-arginine inhibited this reaction with half-maximal effects at 11.0 /SM, and completely abolished it at concentrations of 0.1 mm. Fig. 2 shows that the enzyme started to act as an NO synthase at L-arginine concentrations of 1 /UM, catalysing the formation of L-citrulline, with half-maximal effects of L-arginine at 8.9 #M. Thus the consumption of NADPH and the reduction of oxygen were
Determination of L-Citrulline Unless otherwise indicated, formation of [3H]citrulline from [3H]arginine was determined as previously described [14] in a final volume of 100 ,ul in triethanolamine/HCI buffer (50 mM, pH 7.0) containing 100 /LM-[3H]arginine (50000-80000 c.p.m.), 1 mm free Mg2+, 10 ,ug of calmodulin/ml, 3/SM free Ca2+, 0.1 mMNADPH, 5 /M-FAD and 10 ,uM-H4biopterin in the presence of 100-300 ng of NO synthase. Blank values were determined in the absence of added enzyme. The concentrations of free Ca2+ were adjusted as described previously [15]. Determination of hydrogen peroxide Hydrogen peroxide was determined as formation of ferric thiocyanate by a method described by Hildebrandt et al. [16], slightly modified. Unless otherwise indicated, purified NO synthase (2 ,ug) was incubated at 37 'C for 10 min in 0.2 ml of a 50 mM-triethanolamine/HCl buffer, pH 7.0, containing 0.1 mmNADPH, 3 /M free Ca2+ and 2 /sg of calmodulin. Reactions were terminated by the addition of 100 1ul of concentrated HCI (about 11.8 M), and aqueous solutions of ferrous ammonium sulphate (20,u1) and potassium thiocyanate (30,1) were added to give final concentrations of 3.2 mm and 180 mm respectively. Samples were incubated for 10 min at ambient temperature, followed by the immediate determination of the absorbance at 492 nm. To achieve optimal specificity of this assay, blank values were determined in the presence of enzyme but with catalase (20 units) additionally present during the incubations. Catalase was without effect on NADPH consumption and on the formation of Lcitrulline or NO. Authentic H202 carried through the incubation and assay procedures was used to record calibration curves, which were linear from 1 to 100 1sM-H202, giving AA values of about 0.01 to 1.0. Data evaluation All values were determined in duplicate or triplicate, and the means+S.E.M. of three separate experiments were calculated. Concentration-response curves were fitted to sigmoidal curves by non-linear regression analysis using the software of GraphPad INPLOT, Version 3.0.
RESULTS Even in the absence of L-arginine and H4biopterin, NO synthase still consumed molecular oxygen at the expense of NADPH [12]. Under these conditions, H202 was formed in a Ca2+/calmodulin-dependent manner at a constant rate for about 15 min, with specific activities of approx. 250 nmol/min per mg, and this reaction was not affected by up to 1000 units of
io-9
lo--,
10-7 104 [Calmodulin] (M) Fig. 1. Effect of calmodulin on NO synthase-catalysed H202 formation in the absence of L-arginine and H4biopterin Purified NO synthase (2 ,ug) was incubated for 10 min at 37 °C in 0.2 ml in the presence of 0.1 mM-NADPH and 3 4tM free Ca2" with increasing amounts of calmodulin. The rate of H202 formation was determined as described in the Experimental section and is given as the mean + S.E.M. of three separate experiments.
500 EE' E
0
0.
0
400 .S
._
E
E200
0
0
300 E
C
.o 200 0E
E 0
s 100
0
0
100 .'
4O
0
zIn [L-Arginine] (M)
Fig. 2. Effects of L-arginime on the NO synthase-catalysed formation of H202 and L-citruife Purified NO synthase (2 ,ug and 100 ng in the H202 and L-citrulline assays respectively) was incubated for 10 min at 37 °C in the presence of 0.1 mM-NADPH, 3/lM free Ca2 , 1O/sg of calmodulin/ml and 10 1sM-H4biopterin with increasing amounts of L-arginine as indicated on the abscissa. Rates of H202 (0) and L-citrulline (0) formation were determined as described in the Experimental section and are given as means + S.E.M. of three separate experiments.
1992
629
Hydrogen peroxide formation by NO synthase E 'a E 150
0
0 L.
CL
~~~~~-~ ~ .400H0
CD
.E
-
E
o 100 E
~
~
~
~
0250 E
~
Ez
200
0
E 300
0
-
C
0
0 50 E 0
20
x
E 0 .20
o
C
0
o
0
10-7 1io 106o6 [H4Biopterinl (M) 0 Fig. 3. Effects of H4biopterin on the NO synthase-catalysed formation of H202 and L-citrulline Purified NO synthase was incubated as described in the legend to Fig. 2 in the presence of 100 ,UM-L-arginine with increasing amounts of H4biopterin as indicated on the abscissa. Rates of H202 (0) and L-citrulline (0) formation were determined as described in the Experimental section and are given as means + S.E.M. of three separate experiments. i0-9
ID a
0,
E
00
400 a
200
0
o 150 I _ Ec
300
E
S 0 c 0
C
o 100 I _
C
0
200 'm20
100r
0~~~~~~~~~~~~~~~
E
omto 0
10-7
H20adLctuln ioo 10-
10-3 0
10-
10-
10-4
1~~~~~~~~~~~~
[Inhibitor] (m)
Fig. 5. Effects Of L-argifline analogues
on the NO synthase-catalysed formation of H202 and L-Citrulline (a) For the determination of L-Citrulline generation, purified NO synthase (300 ng) was incubated as described in the Experimental section with L-NNA (-), L-NAME (-) and L-NMMA (-) at the concentrations indicated. L-Citrulline-forming enzyme activities were determined as described in the Experimental section and are given as mean values of three separate experiments. At each point, the S.E.M. was < 10 % (not shown). (b) H202 formation was determined by incubation of purified NO synthase (2 ,ug) for 10 min at 37 °C in 0.2 ml in the presence of 0.1 mM-NADPH and 3 1uM free Ca21 with the enzyme inhibitors L-NNA (El), L-NAME (A) and L-NMMA (0) at the concentrations indicated. The rates of H202 formation were determined as described in the Experimental section and are given as mean values of three separate experiments. At each point, the S.E.M. was < 10 % (not shown).
E
E 0 .°- 50 I _ 0
200 2 c
I
6.5
7.0
7.5 pH
8.0
100
,
8.5
Fig. 4. Effects of pH on the NO synthase-catalysed formation of H202 and L-Citruuine
H202 formation (0) was determined by incubation of 2 ,eg of purified NO synthase for 10 min at 37 'C in the presence of 0.1 mmNADPH, 3 /M free Ca2' and 10 /sg of calmodulin/ml at the pH values indicated. For the determination of L-citrulline formation (0), 100 ng of NO synthase was incubated with 100 /tM-L-arginine and 10 /uM-H4biopterin additionally present. Rates of H202 and Lcitrulline formation were determined as described in the Experimental section and are given as means +S.E.M. of three separate experiments.
coupled to the formation of L-citrulline when L-arginine was present during the incubations. NO synthase is considerably stimulated by H4biopterin [7-9], which apparently represents an essential cofactor of the brain enzyme, as NO formation observed in the absence of exogenously added H4biopterin is most likely due to small amounts of this compound present in purified NO synthase preparations [12]. In the absence of added H4biopterin, the formation of H202 was decreased by L-arginine (100 4aM) to about 150 nmol/min per mg. As shown in Fig. 3, addition of H4biopterin resulted in a further inhibition of this reaction (half-maximal at 0.29 ,uM-H4biopterin), whereas the formation of L-citrulline was stimulated from 100 to about 400 nmol/min per mg, with a half-maximally active H4biopterin concentration of 0.27 4uM. Fig. 4 shows that similar effects of pH were observed on the generation of H202 and L-citrulline. Both enzymic activities were Vol. 281
E
10
250
E
C 10-7-150
0 C
maximal at slightly acidic conditions and decreased with increasing pH. As shown in Fig. 5(a), the competitive NO synthase inhibitors L-NNA, L-NAME and L-NMMA inhibited the conversion of 0.1 mM-L-arginine into L-citrulline in a concentration-dependent manner, with half-maximal effects at 0.74 uM, 2.8 /LM and 15 4uM respectively. Fig. 5(b) shows the effects of these inhibitors on the generation of H202 in the absence of L-arginine and H4biopterin. This reaction was inhibited by L-NNA and L-NAME, with halfmaximal effects at 0.2 #M and 0.3 /M respectively. Similarly, the calmodulin-dependent NADPH consumption (determined as decrease in absorbance at 340 nm) was inhibited by these compounds (results not shown). L-NMMA, however, did not affect H202 formation at concentrations up to 1 mm. NO synthase also catalysed a rapid Ca2+/calmodulin-dependent reduction of cytochrome c, but, similar to the observed H202 formation, cytochrome c reduction was insensitive to SOD (results not shown). As NO synthase could be bound to cytochrome c-agarose (results not shown), reduction of cytochrome c apparently results from a direct interaction of these proteins and is not mediated by superoxide anions. DISCUSSION In the presence of Ca2+/calmodulin, NO synthase apparently catalyses an NADPH-dependent reduction of molecular oxygen, as do other flavin-containing enzymes [17]. At saturating concentrations of L-arginine and H4biopterin, this oxygen reduction is fully coupled to the formation of L-citrulline and NO, but it results in the formation of H202 at limiting concentrations of substrate or cofactor. Thus the observed uncoupling of oxygen reduction is only partially prevented by L-arginine in the absence of added H4biopterin. Lack of effect of SOD indicates that
630
superoxide is not formed (or is at least not released from the enzyme) as an intermediate in H202 formation. This was further confirmed by results showing that NO synthase-catalysed cytochrome c reduction was not inhibited by SOD and that this reaction was due to a direct interaction of these proteins. This latter result is also in good accordance with a recent report describing sequence similarities between rat brain NO synthase and cytochrome P-450 reductase [18]. H202 formation as described here is possibly specific for the brain enzyme, as macrophage NO synthase was reported to lack dihydropteridine reductase activity, as determined by a method based on NADPH uptake in the absence of substrate [11]. The formation of L-citrulline was optimal at slightly acidic conditions, confirming previous results demonstrating a rapid pH-dependent decrease of the L-arginine-dependent activation of purified soluble guanylate cyclase by NO synthase obtained from bovine brain [19]. As shown in Fig. 4, H202 formation exhibited a very similar pH-dependency, indicating that the NADPHdependent reduction of molecular oxygen is decreased at alkaline conditions, resulting in diminished rates of L-citrulline and NO formation. The rank order of potency of L-arginine analogues in inhibiting L-citrulline formation was L-NNA > L-NAME > L-NMMA, confirming previous results obtained with the enzyme from brain, adrenal gland and endothelial cells [5,20-22]. L-NNA and L-NAME also inhibited substrate-independent H202 generation, indicating that binding of the N'-nitro compounds prevented the enzyme from reducing molecular oxygen. As L-NMMA did not affect H202 formation, this compound apparently differs from L-NNA and L-NAME in its inhibitory properties, possibly explaining tissue-specific effects of these compounds [23]. Thus L-NNA and L-NAME, but not the most commonly used LNMMA, ensure a complete inhibition of NO synthase-catalysed oxygen reduction and should represent useful tools to effectively block the formation of both NO and H202 by Ca2+/calmodulinactivated NO synthase. On studying the distribution of NO synthase over various areas of the brain, the enzyme is most abundant in the cerebellum, where it is associated with neurons that make close contact with Purkinje cells [24]. It was reported that all NO synthase neurons also stain for the enzyme NADPH diaphorase [25], a membrane-associated, flavin-containing 73 kDa homodimeric protein which exhibits similarities to cytochrome P-450 reductase [26,27]. Recently it was reported that a 150 kDa monomeric NO synthase purified from rat brain supernatants was recognized and partially precipitated by antibodies directed against NADPH diaphorase [28]. As the purified enzyme catalysed an NADPH-dependent reduction of Nitro Blue Tetrazolium, a reaction on which the diaphorase-staining of brain slices is based, the authors concluded that NO synthase and NADPH diaphorase are identical proteins. Our results apparently explain the observed NO synthase-catalysed NADPH-dependent reduction of Nitro Blue Tetrazolium, especially since we found that the enzyme not only reduces molecular oxygen in a substrate-independent manner, but even more rapidly utilizes artificial electron acceptors (B. Heinzel,
unpublished work). Decreased levels of H4biopterin are found in the cerebrospinal fluid of patients affected with neuronal disorders (for review see
B. Heinzel and others
ref. [29]), and H4biopterin was shown to limit NO formation in mouse fibroblasts [30]. Thus it will be interesting to find out whether Ca2+-activated brain NO synthase also catalyses the described H202 generation in cells and tissues at suboptimal intracellular levels of L-arginine of H4biopterin. We thank Dr. G. Schultz for critical reading of this manuscript. B. M. was a recipient of a fellowship of the Alexander von Humboldt-Stiftung. The financial support of the Deutsche Forschungsgemeinschaft, Osterreichische Nationalbank and Osterreichische Forschungsgemeinschaft is gratefully acknowledged.
REFERENCES 1. Furchgott, R. F. & Vanhoutte, P. M. (1989) FASEB J. 3, 2007-2018 2. Ignarro, L. J. (1991) Biochem. Pharmacol. 41, 485-490 3. Radomski, M. W., Palmer, R. M. J. & Moncada, S. (1991) Trends Pharmacol. Sci. 12, 87-88 4. Garthwaite, J. (1991) Trends Neurol. Sci. 14, 60-67 5. Knowles, R. G., Palacios, M., Palmer, R. M. J. & Moncada, S. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5159-5162 6. Bredt, D. S. & Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,
682-685 7. Mayer, B., John, M. & Bohme, E. (1990} FEBS Lett. 277, 215-219 8. Tayeh, M. A. & Marletta, M. A. (1989) J. Biol. Chem. 264, 19654-19658 9. Kwon, N. S., Nathan, C. F. & Stuehr, D. J. (1989) J. Biol. Chem. 264, 20496-20501 10. Kwon, N. S., Nathan, C. F., Gilker, C., Griffith, 0. W., Matthews, D. E. & Stuehr, D. J. (1990) J. Biol. Chem. 265, 13442-13445 11. Stuehr, D. J., Kwon, N. S., Nathan, C. F., Griffith, 0. W., Feldman, P. L. & Wiseman, J. (1991) J. Biol. Chem. 266, 6259-6263 12. Mayer, B., John, M., Heinzel, B., Werner, E. R., Wachter, H., Schultz, G. & Bohme, E. (1991) FEBS Lett. 288, 187-191 13. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 14. Mayer, B., John, M. & B6hme, E. (1991) J. Cardiovasc. Pharmacol.
17, S46-S51 15. Mayer, B., Schmidt, K., Humbert, P. & B6hme, E. (1989) Biochem. Biophys. Res. Commun. 164, 678-685 16. Hildebrandt, A. G., Roots, I., Tjoe, M. & Heinemeyer, G. (1978) Methods Enzymol. 52, 342-350 17. Ghisla, S. & Massey, V. (1989) Eur. J. Biochem. 181, 1-17 18. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R. & Snyder, S. H. (1991) Nature (London) 351, 714-718 19. Mayer, B., Evers, B., Wilke, P. & Bohme, E. (1990) Arch. Int. Pharmacodyn. Ther. 305, 265 20. Palacios, M., Knowles, R. G., Palmer, R. M. J. & Moncada, S. (1989) Biochem. Biophys. Res. Commun. 165, 802-809 21. Rees, D. D., Palmer, R. M. J., Schulz, R., Hodson, H. F. & Moncada, S. (1990) Br. J. Pharmacol. 101, 746-752 22. Muilsch, A. & Busse, R. (1990) Naunyn-Schmiedeberg's Arch. Pharmacol. 341, 143-147 23. McCall, T. B., Feelisch, M., Palmer, R. M. J. & Moncada, S. (1991) Br. J. Pharmacol. 102, 234-238 24. Bredt, D. S., Hwang, P. M. & Snyder, S. H. (1990) Nature (London) 347, 768-770 25. Snyder, S. H. & Bredt, D. S. (1991) Trends Pharmacol. Sci. 12, 125-128 26. Kuonen, D. R., Kemp, M. C. & Roberts, P. J. (1988) J. Neurochem. 50, 1017-1025 27. Kemp, M. C., Kuonen, D. R., Sutton, A. & Roberts, P. J. (1988) Biochem. Pharmacol. 37, 3063-3070 28. Hope, B. T., Michael, G. J., Knigge, K. M. & Vincent, S. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2811-2814 29. Nichol, C. A., Smith, G. K. & Duch, D. S. (1985) Annu. Rev. Biochem. 54, 729-764 30. Werner-Felmayer, G., Werner, E. R., Hausen, A., Reibnegger, G. & Wachter, H. (1990) J. Exp. Med. 172, 1599-1607
Received 29 May 1991/31 July 1991; accepted 3 September 1991
1992
