Mechanism of Superoxide Generation by Neuronal Nitric-oxide Synthase

Neuronal nitric-oxide synthase (NOS I) in the absence of l-arginine has previously been shown to generate superoxide (O·̄2) (Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173–24176). In the presence ofl-arginine, NOS I produces nitric oxide (NO⋅). Yet the competition between O2 and l-arginine for electrons, and by implication formation of O·̄2, has until recently remained undefined. Herein, we investigated this relationship, observing O·̄2 generation even at saturating levels ofl-arginine. Of interest was the finding that the frequently used NOS inhibitor N G-monomethyll-arginine enhanced O·̄2 production in the presence of l-arginine because this antagonist attenuated NO⋅formation. Whereas diphenyliodonium chloride inhibited O·̄2, blockers of heme such as NaCN, 1-phenylimidazole, and imidazole likewise prevented the formation of O·̄2 at concentrations that inhibited NO⋅ formation from l-arginine. Taken together these data demonstrate that NOS I generates O·̄2 and the formation of this free radical occurs at the heme domain. Neuronal nitric-oxide synthase (NOS I) in the absence of l-arginine has previously been shown to generate superoxide (O·̄2) (Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173–24176). In the presence ofl-arginine, NOS I produces nitric oxide (NO⋅). Yet the competition between O2 and l-arginine for electrons, and by implication formation of O·̄2, has until recently remained undefined. Herein, we investigated this relationship, observing O·̄2 generation even at saturating levels ofl-arginine. Of interest was the finding that the frequently used NOS inhibitor N G-monomethyll-arginine enhanced O·̄2 production in the presence of l-arginine because this antagonist attenuated NO⋅formation. Whereas diphenyliodonium chloride inhibited O·̄2, blockers of heme such as NaCN, 1-phenylimidazole, and imidazole likewise prevented the formation of O·̄2 at concentrations that inhibited NO⋅ formation from l-arginine. Taken together these data demonstrate that NOS I generates O·̄2 and the formation of this free radical occurs at the heme domain. nitric oxide superoxide hydrogen peroxide neuronal nitric-oxide synthase inducible nitric-oxide synthase endothelial nitric-oxide synthase 5,5-dimethyl-1-pyrroline-N-oxide electron paramagnetic resonance N G-nitro-l-arginine methyl ester N G-monomethyll-arginine 2,2,-dimethyl-5-hydroperoxy-1-pyrrolidinyloxyl diphenyliodonium chloride peroxynitrite superoxide dismutase tetrahydrobiopterin At a time before the physiologic properties of endothelium-derived relaxation factor were associated with nitric oxide (NO⋅)1 (1Ignarro L.J. Buga G.M. Wood K.S. Byrns R.E. Chaudhuri G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9265-9269Crossref PubMed Scopus (4273) Google Scholar, 2Palmer R.M.J. Ferrige A.G. Moncada S. Nature. 1987; 327: 524-526Crossref PubMed Scopus (9225) Google Scholar, 3Furchgott R.F. Vanhoutte P.M. Vasodilatation: Vascular Smooth Muscle, Peptides, Autonomic Nerves and Endothelium. Raven Press, New York1988: 401-414Google Scholar), it was found that this free radical activated soluble guanylate cyclase from crude homogenates of brain tissue (4Miki N. Kawabe Y. Kuriyama K. Biochem. Biophys. Res. Commun. 1977; 75: 851-856Crossref PubMed Scopus (159) Google Scholar). The significance of this observation would, surprisingly, remain dormant for nearly a decade, even though it was known that l-arginine was the endogenous activator of this enzyme (5Deguchi T. Yoshioka M. J. Biol. Chem. 1982; 257: 10147-10151Abstract Full Text PDF PubMed Google Scholar). With the purification and characterization of a unique monooxygenase, NOS I, capable of oxidizingl-arginine to l-citrulline, and NO⋅ (6Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3111) Google Scholar,7Bredt D.S. Hwang P.M. Snyder S.H. Nature. 1990; 347: 768-770Crossref PubMed Scopus (2671) Google Scholar), a new class of small molecules (8Dawson T.M. Snyder S.H. J. Neurosci. 1994; 14: 5147-5159Crossref PubMed Google Scholar) acting as transient second messengers in the brain was discovered (9Jaffrey S.R. Snyder S.H. Annu. Cell. Dev. Biol. 1995; 11: 417-440Crossref PubMed Scopus (304) Google Scholar). The versatility of this free radical in controlling a myriad of brain functions will undoubtedly result in new and provocative findings. Of special interest will be research that can distinguish NO⋅ from NOS I-secreting neurons versus NO⋅ from NOS III-containing endothelial cells and NO⋅ from NOS II-stimulated microglial cells. Until the recent development of specific antagonists for each of the NOS isozymes (10Moore P.K. Wallace P. Garren Z. Hart S.L. Babbedge R.C. Br. J. Pharmacol. 1993; 110: 219-224Crossref PubMed Scopus (462) Google Scholar, 11Garvey E.P. Oplinger J.A. Furfine E.S. Kiff R.J. Laszlo F. Whittle B.J.R. Knowles R.G. J. Biol. Chem. 1997; 272: 4959-4963Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar, 12Marletta M.A. J. Med. Chem. 1994; 37: 1899-1907Crossref PubMed Scopus (176) Google Scholar), it has been difficult to address this question, which is of particular significance when one considers, as described in this article, that NOS I, NOS II, and NOS III may produce NO⋅ and O·̄2 under differing cell conditions. Nitric-oxide synthase is known to catalyze the production of NO⋅from l-arginine (13Marletta M.A. J. Biol. Chem. 1993; 268: 12231-12234Abstract Full Text PDF PubMed Google Scholar, 14Masters B.S.S. McMillan K. Sheta E.A. Nishimura J.S. Roman L.J. Martasek P. FASEB J. 1996; 10: 552-558Crossref PubMed Scopus (191) Google Scholar). In the absence of substrate, we have previously demonstrated that purified NOS I can use O2as the terminal electron acceptor, generating O·̄2 (15Pou S. Pou W.S. Bredt D.S. Snyder S.H. Rosen G.M. J. Biol. Chem. 1992; 267: 24173-24176Abstract Full Text PDF PubMed Google Scholar). These findings have been confirmed because O·̄2 has been spin-trapped in l-arginine-depleted NOS I-transfected human kidney cells (16Xia Y. Dawson V.L. Dawson T.M. Snyder S.H. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6770-6774Crossref PubMed Scopus (647) Google Scholar). During the course of our earlier studies (15Pou S. Pou W.S. Bredt D.S. Snyder S.H. Rosen G.M. J. Biol. Chem. 1992; 267: 24173-24176Abstract Full Text PDF PubMed Google Scholar), we noted to our surprise that purified NOS I appeared to produce O·̄2 even in the presence of l-arginine. The current study, therefore, explores this phenomenon in depth. Herein, we demonstrate that NOS I, like NOS II (17Xia Y. Roman L.J. Masters B.S.S. Zweier J.L. J. Biol. Chem. 1998; 273: 22635-22639Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar) and NOS III (18Vásquez-Vivar J. Kalyanaraman B. Martásek P. Hogg N. Masters B.S.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1213) Google Scholar, 19Xia Y. Tsai A.-L. Berka V. Zweier J.L. J. Biol. Chem. 1998; 273: 25804-25808Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar, 20Wever R.M.F. van Dam T. van Rijn H.J.M. de Groot F. Rabelink T.J. Biochem. Biophys. Res. Commun. 1959; 237: 340-344Crossref Scopus (245) Google Scholar), can generate O·̄2 and NO⋅ despite saturating levels of substrate. However, unlike NOS II, the heme of NOS I is the locus for the production of both free radicals. Finally, we discuss the implications of our findings; particularly relevant is the ability of l-NMMA to enhance NOS I-derived O·̄2 even in the presence of saturating concentrations of l-arginine. NADPH, calmodulin, l-arginine, phenylmethylsulfonyl fluoride, diethylenetriaminepentaacetic acid, ferricytochrome c, xanthine,N G-nitro-l-arginine methyl ester (l-NAME), N G-monomethyll-arginine (l-NMMA), EGTA, HEPES, and penicillin G-streptomycin solution were purchased from Sigma. Imidazole, 1-phenylimazole, sodium cyanide, and diphenyliodonium chloride (DPI) were obtained from Aldrich. Tetrahydrobiopterin was purchased from Alexis Biochemicals (San Diego, CA). Cation exchange resin Dowex 50W-X8 hydrogen form resin was obtained from Bio-Rad. 2′,5′-ADP-Sepharose was obtained from Pharmacia (Uppsala, Sweden).l-14CArginine was purchase from ICN Radiochemicals (Costa Mesa, CA). Dulbecco's modified Eagle's medium:nutrient mixture F-12 (1:1) and phosphate-buffered saline were obtained from Life Technologies, Inc. Bovine calf serum was purchased from Hyclone (Logan, UT). Superoxide dismutase and xanthine oxidase were obtained from Boehringer Mannheim. The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was synthesized according to the procedure of Bonnett et al. (21Bonnett R. Brown R.F.C. Clark V.M. Sutherland I.O. Todd A. J. Chem. Soc. 1959; : 2094-2102Crossref Google Scholar). NOS I-transfected kidney 293 cells were cultured in Dulbecco's modified Eagle's medium:nutrient mixture F-12 containing 10% fetal calf serum, penicillin G (100 units/ml), and streptomycin (100 μg/ml). NOS I was purified from these cells by the method of Bredt and Snyder (6Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3111) Google Scholar). Briefly, cells were removed from the culture flasks and washed three times with phosphate-buffered saline via centrifugation. The pellet was resuspended in buffer containing phenylmethylsulfonyl fluoride (4 mg/ml) and homogenized with a Polytron (Brinkmann Instruments, model PCU-2 at setting 2 for 10 s). The remaining mixture was centrifuged at 15,000 rpm for 20 min to separate unbroken cells, and the supernatant was applied to a 2′,5′-ADP-Sepharose affinity column. After washing the column three times with 0.45 m NaCl and standard buffer, NOS was with standard buffer containing 10 was removed by washing and the with until the of was as at was by the method Biochem. PubMed Scopus Google Scholar) serum as a NOS I was by the formation of from as described previously D.S. Hwang P.M. Snyder S.H. Nature. PubMed Scopus Google Scholar). Briefly, purified NOS I was a mixture containing (100 free as described in A. F. J. 75: Google Scholar), and standard buffer at After at for 10 the was by 2 containing was by column of and was a with purified NOS I were by the described in the to a of The was by purified NOS were to a cell and the of the model and were at min of the in the The of the the ability of oxidase to produce O·̄2 was as described previously S. Rosen G.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Briefly, xanthine oxidase was to a solution containing xanthine and ferricytochrome such that the of O·̄2 as the of ferricytochrome at H. V. Biochem. J. 1982; PubMed Scopus Google Scholar) was The of concentrations of the of was The of by NOS I was at the ability of NOS I to generate O·̄2, our of studies were to m and the for NOS I, because these will these the m and were found to be and the m of of by FASEB J. 1992; PubMed Scopus Google Scholar). In the absence of l-arginine, NOS I has previously been shown to generate O·̄2 (15Pou S. Pou W.S. Bredt D.S. Snyder S.H. Rosen G.M. J. Biol. Chem. 1992; 267: 24173-24176Abstract Full Text PDF PubMed Google Scholar, Y. Dawson V.L. Dawson T.M. Snyder S.H. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6770-6774Crossref PubMed Scopus (647) Google Scholar), at concentrations of l-arginine of O·̄2 production been noted (15Pou S. Pou W.S. Bredt D.S. Snyder S.H. Rosen G.M. J. Biol. Chem. 1992; 267: 24173-24176Abstract Full Text PDF PubMed Google Scholar). the that concentrations of l-arginine have the ability of NOS I to generate shown in l-arginine, in a O·̄2 from NOS I, of NOS O·̄2 at was nearly these the of as l-arginine from in the absence of to at the of remained result that for NOS I the of from to O2 in the absence of is the of to catalyze the formation of NO⋅ and from l-arginine. In to NOS I, O·̄2 generation by purified NOS II was by In it was to this free radical even in the presence of (17Xia Y. Roman L.J. Masters B.S.S. Zweier J.L. J. Biol. Chem. 1998; 273: 22635-22639Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). findings have been for NOS III (18Vásquez-Vivar J. Kalyanaraman B. Martásek P. Hogg N. Masters B.S.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1213) Google Scholar, 19Xia Y. Tsai A.-L. Berka V. Zweier J.L. J. Biol. Chem. 1998; 273: 25804-25808Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). These the that the m for NOS II between to for NOS m has been to be FASEB J. 1992; PubMed Scopus Google Scholar). These data to a between the three isozymes of NOS with to O·̄2 NOS II, it was found that O·̄2 was by electron from the (17Xia Y. Roman L.J. Masters B.S.S. Zweier J.L. J. Biol. Chem. 1998; 273: 22635-22639Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), the finding that as by spin (17Xia Y. Roman L.J. Masters B.S.S. Zweier J.L. J. Biol. Chem. 1998; 273: 22635-22639Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). In Y. Tsai A.-L. Berka V. Zweier J.L. J. Biol. Chem. 1998; 273: 25804-25808Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar) (18Vásquez-Vivar J. Kalyanaraman B. Martásek P. Hogg N. Masters B.S.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1213) Google Scholar) O·̄2 from NOS These data that the heme of NOS III is the of O·̄2 production (18Vásquez-Vivar J. Kalyanaraman B. Martásek P. Hogg N. Masters B.S.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1213) Google Y. Tsai A.-L. Berka V. Zweier J.L. J. Biol. Chem. 1998; 273: 25804-25808Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). The in the generation of NO⋅ by NOS is the of from to the in of a from the between the to The electron from to the and R. Clark P. 1996; PubMed Scopus Google Scholar). of the the R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In the absence ofl-arginine, O2 electron from generating O·̄2 (15Pou S. Pou W.S. Bredt D.S. Snyder S.H. Rosen G.M. J. Biol. Chem. 1992; 267: 24173-24176Abstract Full Text PDF PubMed Google Scholar, Y. Roman L.J. Masters B.S.S. Zweier J.L. J. Biol. Chem. 1998; 273: 22635-22639Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, J. Kalyanaraman B. Martásek P. Hogg N. Masters B.S.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1213) Google Scholar, 19Xia Y. Tsai A.-L. Berka V. Zweier J.L. J. Biol. Chem. 1998; 273: 25804-25808Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). l-arginine is is a of the in the heme Martasek P. Masters B.S.S. J. Chem. Soc. 1998; Google Scholar), which the to a m of for the NOS I of l-arginine and data in NO⋅ and O·̄2 both The of each free radical be by spin that NOS I at l-arginine m is capable of O·̄2 at of the of that in the absence of l-arginine. NOS II, electron from to to be because from the in the formation of O·̄2, even in the presence of (17Xia Y. Roman L.J. Masters B.S.S. Zweier J.L. J. Biol. Chem. 1998; 273: 22635-22639Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). NOS I, the of O·̄2 generation has been that the of l-arginine to and competition with O2 in O·̄2 At a of the of NO⋅ and O·̄2 is therefore, the ofl-arginine. the of O·̄2 generation by NOS I, it is to the we a of the of known of O·̄2 these we the of these NOS to formation by the generation of The formation by NOS I in the presence and l-NMMA shown in l-NMMA was found to be 10 NOS has been to be a inhibitor of this l-NMMA E.A. R. R.G. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). With these data in we the ability of to O·̄2 production by NOS the of concentrations of the ability of NOS I to generate to the spin of this free radical by in a with In l-NMMA the formation of O·̄2 by NOS I even at concentrations as as 10 with these the of by NOS I was inhibited by as the of the of remained at of with to 10 These inhibited the formation of O·̄2 by the electron to this the of the oxidase production of O·̄2, as by the of was this was These data that O·̄2 that NOS I, generation of this free is a antagonist of we l-NMMA the ability to NOS I production of These findings in l-arginine (100 inhibited the NOS I formation of O·̄2 which confirmed earlier studies (15Pou S. Pou W.S. Bredt D.S. Snyder S.H. Rosen G.M. J. Biol. Chem. 1992; 267: 24173-24176Abstract Full Text PDF PubMed Google Scholar, B. M. P. B. Biochem. J. 1992; PubMed Scopus Google Scholar). in a the properties of l-arginine in the absence ofl-arginine, at findings the that the of to l-arginine l-NMMA the by for the the enzyme Clark P. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). NO⋅ production is inhibited by the presence of NOS I has the to from to O2 and of l-NMMA O·̄2 generation by purified NOS I as by spin as of levels in the absence of (100 free (100 and NOS for data a the purified of NOS of l-NMMA the of by purified NOS I, as described under NOS was a containing free (100 and The of by NOS was NOS was a containing free (100 units/ml), and The of by NOS was data and l-NMMA a the purified of NOS of l-NMMA by purified NOS I in the presence of l-arginine as by spin to under a of The mixture in of (100 free (100 units/ml), and NOS μg/ml). were under to for the of l-arginine (100 l-arginine (100 l-arginine (100 l-NMMA (100 (100 l-NMMA was under to for the was 20 was with of time was time was and was 10 were to the NOS I O·̄2 production and NOS I of the of B.S.S. McMillan K. Sheta E.A. Nishimura J.S. Roman L.J. Martasek P. FASEB J. 1996; 10: 552-558Crossref PubMed Scopus Google Scholar), we to to as to for NOS I-derived and NOS I O·̄2 may be the of the and the of the the heme is the of O·̄2 formation R.E. and Scholar). NOS I in a we to demonstrate that our NOS I was capable of from the to the heme domain. this we investigated the of a known to the electron at the locus J.A. R. FASEB J. PubMed Scopus Google Scholar), NO⋅ and O·̄2 formation by NOS shown in is a inhibitor generation by NOS I with 10 at 10 inhibited the spin of NOS In the of with O·̄2 from though these data that the electron is the these O·̄2 is at this at the heme domain. address this we investigated the of and to and NOS S. R. J. Chem. Soc. 1994; Scopus Google Scholar, T.M. S. J.A. 1996; PubMed Scopus Google Scholar) by the heme the generation of this free we to demonstrate that NaCN, and inhibited the to by NOS the formation by NOS I in the presence of NaCN, and was the of these with 10 imidazole and were with for 20 and we were that properties O·̄2 generation were the heme was the of this free radical At NaCN, was a in the of at 10 of O·̄2 was nearly In of at 10 was found to the spin of O·̄2 from the model O·̄2 generating of is known to with (21Bonnett R. Brown R.F.C. Clark V.M. Sutherland I.O. Todd A. J. Chem. Soc. 1959; : 2094-2102Crossref Google Scholar), the of to with O·̄2, the findings in that the heme in NOS I was the of O·̄2 antagonists for NOS were have been to the heme of NOS T.M. S. 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Finally, we investigated the that in production of O·̄2 by NOS I in the absence ofl-arginine. at 10 and inhibited O·̄2 by NOS I by shown in and the of and to a of The of to be At 10 we were to even though the At 10 10 was the spin-trapped These data that at concentrations the of is to NOS I production of However, at in to the of NOS O·̄2, generates O·̄2 The K. A. H. A. M. J. Chem. 1992; Google P.M. W. Biochem. Biophys. 1992; PubMed Scopus Google Scholar). from our we were to spin trap O·̄2 even in the presence of saturating levels of l-arginine. is that O·̄2 and NO⋅ at R.E. S. Res. Commun. 1993; PubMed Scopus Google Scholar, S. G. Biol. Med. 1995; PubMed Scopus Google Scholar), O·̄2 with at Rosen G.M. J. Chem. Soc. Scopus Google Scholar). 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