of the soluble guanylate cyclase to regulate vascular tone, ..... 2C). When 5774 cells were incubated in H2180 or H2160 the only product observed had an m/z ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 266, No. 35, Issue of December 15, pp. 23790-23795,1991 Printed in U.S. A .
Constitutive and InducibleNitric Oxide Synthases Incorporate Molecular Oxygen into Both Nitric Oxide and Citrulline* (Received for publication, February 6, 1991)
Anna M. Leone, RichardM. J. Palmer, RichardG . Knowles, PeterL. Francis, DavidS. Ashton, and Salvador MoncadaS From the Wellcome Research Laboratories, Langley Court, Beckenham,Kent BR3 3BS, United Kingdom
Nitric oxide (NO) is synthesized by a number ofcells arginine to form ammonia which is then oxidized to NO (3); from a guanidino nitrogen atom of L-arginine by the another suggests the formation of w-hydroxyarginine via an action of either constitutiveor inducible NO synthases, NADPH- and tetrahydrobiopterin-dependentmonooxygenaboth of which form citrulline as a co-product.We have tion (4); and the thirdsuggests that w-hydroxyarginine gives determined the source of the oxygen in bothNO and in rise to hydroxylamine, which is then converted to NO by the citrulline formed by theconstitutiveNO synthase from action of catalase (27). These three mechanisms predict that the vascular endothelium and brain and by the induc- the oxygen in NO would be derived from molecular oxygen, ible NO synthase from the murine macrophage cell line and the ureido oxygen in citrulline would be derived from 5774. All these enzymes incorporate molecular oxygen both into NO and into citrulline. Furthermore, acti- water. Molecular oxygen has been shown recently to be incorvated 5774 cells form NO from w-hydroxyl-L-arginine, porated into citrulline by the inducible NO synthase from confirming the proposal that this compound is an in- activated RAW 264.7cells (28), indicating that these proposed mechanisms are incorrect in terms of the source of the oxygen termediate in the biosynthesis NO. of
in citrulline. In this study we have analyzed NO and citrulline formed by the macrophage cell line 5774 activated with lipopolysaccharide and interferon-y andby vascular endothelial cells and The demonstration of the synthesis of nitric oxide (NO)' brain cytosol in the presence of lSOzor H2lsO.Thus, we have from a guanidino nitrogen atom of L-arginine by vascular attempted to elucidate the mechanism of NO synthesis by endothelial cells (1)and activated macrophages (2) led to the both constitutive andinducible NO synthases. identification of the L-arginine/NO pathway in many other cells and tissues (3-19). Present evidence indicates that there EXPERIMENTALPROCEDURES are at least two distinct NO synthases (provisionally EC Materials--"'02 (>99% pure) and H2180 (>99% pure) were from 1.14.13.39).The vascular endothelium (1,15,16), brain (7-9), MSD Isotopes. "NG-~-Arginine(99% enrichment of both guanidino platelets (ll), and adrenal gland (10) contain a constitutive nitrogen atoms; Cambridge Isotopes), dimethylformamide dimethyl Ca2+/calmodulin-dependentenzyme. The NO formed by this acetal (Aldrich), L-arginine, L-citrulline, morpholine, nitrosomorphoenzyme acts as a transduction mechanism for the stimulation line, 2,6-dimethyl morpholine, NADPH (all Sigma), SCX Bond Elute of the soluble guanylate cyclase to regulate vascular tone, ion exchange columns (Analytichem International), L-NMMA (Wellplatelet activation, neurotransmision, and other functions(1, come), RPMI 1640, Dulbecco's modified Eagle's medium, penicillin, (all GIBCO), fetal calf serum (Flow Laboratories), 6-8). In other cells, such as activated macrophages (3,4, 17- streptomycin lipopolysaccharide W from Salmonella typhosa (Difco), 5774 cells 19), Kupffer cells (13), hepatocytes (12, 14), and neutrophils (American Tissue Culture Catalogue TIB-67), A23187 (Calbiochem) (5,6) the NO is synthesized by an enzyme that is induced by and Cytodex 3 (Pharmacia LKB Biotechnology Inc.) were obtained immunological stimuli and is Ca2+-independent. The NO as indicated. 4-Nitroso-2,6-dimethyl morpholine was prepared by formed by this enzyme contributes to the cytotoxic activity nitrosation of 2,6-dimethyl morpholine with sodium nitrite and hyof macrophages against tumor cells and microorganisms (20). drochloric acid (29). The starting material, and therefore the final was a mixture of positional isomers of 4-nitrosodimethyl Recent evidence indicates that both the vascular endothelium product, morpholine. All other chemicals were from BDH. w-Hydroxyl-Land vascular smooth muscle also express this Ca2+-independ- arginine was synthesized by a method based on Sepharose binding of ent enzyme after exposure to endotoxin (lipopolysaccharide) the copper salt of L-ornithine and subsequent displacement by proand interferon-y or interleukin-1(21-26). Both NO synthases tected hydroxylamine (30). J774 Cells-The murine monocytic leukemia cell line 5774was are soluble, require NADPH, form citrulline as a co-product, and are competitively inhibited by NG-monomethyl-L-argi- cultured as described before (6), and NO synthase was induced by culture for 24 h in 4.5-cm3 culture wells (lo6 cells in 3 ml) in the nine (L-NMMA) and otherL-arginine analogues. presence of interferon-y (150 units/ml) and lipopolysaccharide (10 Three mechanisms have beenproposed for the biosynthesis pglml). The source of the ureido oxygen in citrulline was determined ofNOfromL-arginine.Oneinvolves the deimination of in incubates in which HEPES buffer (25 mM, pH 7.2) replaced the sodium bicarbonate. The cells were then incubated a t 37 "C for 24 h under an atmosphere of air or l80,in H2160,or in H2'"O under an the payment of page charges. This article must therefore be hereby atmosphere of air. The final enrichment of the latter incubation was marked "aduertisement" in accordance with 18 U.S.C. Section 1734 90% (v/v), and this was accomplished by preparation of the culture medium from dry powder in H,"0. Incubations to determine the solely to indicate this fact. source of the oxygen in NO were carried out under conditions similar f To whom correspondence should be sent. Fax: 81-658-2278. The abbreviations used are: NO, nitric oxide; L-NMMA, NC- to those for citrulline, except that morpholine (15 mM), neutralized monomethyl-L-arginine; HEPES, 4-(2-hydroxyethyl)-l-piperazine- with 8 N HCI, wasadded to react with NO to form nitrosomorpholine. ethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetra- In some experiments the cells were activated in L-arginine-free culacetic acid; tR, relative retention time; GC, gas chromatography; MS, ture media and were then incubated in the presence of w-hydroxyl+ ) than L-arginine. arginine (100 p ~ rather mass spectroscopy.
* The costs of publication of this article were defrayed in part by
23790
Nitric Oxide Synthases Incorporate Molecular Oxygen Endothelial Cells and Cytosol-Vascular endothelial cells from porcine aortae were isolated and cultured on microcarriers as described previously (31). The cells were washed twice with L-arginine-free culture medium containing 20% dialyzed fetal calf serum before incubation of 0.5-ml microcarriers (approximately lo7 cells) in 3 ml of culture medium for 24 h. Incubations to determine the source of oxygen in NO and citrulline were carried out for 24 h as described for 5774 cells except that "NG-~-arginine(300 pM) and A23187 (1 pg/ml) were added. Endothelial cell cytosol was prepared from fresh porcine aortae as described previously (32). Cytosol (1.5 ml) was incubated in a total volume of 2 ml in the presence of NADPH (100 p ~ )"N'-~-arginine , (100 p ~ )and , Ca2+(1 mM) under air or "0, in Hz%, or in H,"0 in air, for 3 h at 37 "C. The final enrichment of H,"O in the incubate was 90%. The latter was accomplished by preparation of the homogenization buffer in H,"O. The incubations were terminated by rapid freezing and citrulline formation determined as described below. Rat Brain Cytosol-Rat brain cytosol was prepared as described previously (7), and 0.5 ml was incubated at 37 "C in a total volume of 2.5 ml in a buffer containing 40 mM potassium phosphate, pH 7.2, MgCl, (1 mM), NADPH (100 pM), and15NG-~-arginine(100 pM) under air or "0, in H2160,or in H2"O inair for 1 h. The final enrichment of H2180in the incubate was 90%. The latterwas accomplished by preparation of the homogenization buffer in H,"0. Incubations were terminated by rapid freezing and citrulline formation determined as described below. Method of Exposure of the Incubates to l8O2--Incubates in 4.5-cm2 culture wellswereplaced in a 125-ml cylindrical glass vessel and connected through a three-way tap system to a 1-liter ''0,bulb. The incubation vessel was evacuated down to 0.1 mbar, and thenlSO2 was introduced to a pressure of 500 mbar. After a 10-min equilibration period, the vessel was sealed prior to incubation as above. Citrulline Analysis-A 1-ml aliquot of the incubation medium was applied to a preconditioned SCX Bond Elute column. The column was washed successively with 6 ml of water, 4 ml of methanol, 2 ml of 2% ammonia in methanol, and 2 ml of 5% ammonia in methanol. Citrulline was eluted from the column by addition of 2 ml of 15% ammonia in methanol. The eluate was taken to dryness under a stream of nitrogen at 40 "Cand stored in uacuo prior to derivatization. Authentic citrulline or dried extracts were derivatized by the addition of 200 pl of a mixture of dimethylformamide dimethyl acetal/ acetonitrile/methano1(3:1:1, v/v/v) and heating for 15 min at 135 "C. The solution was then taken to dryness under a stream of nitrogen at 40 'C and the residue dissolved in 50 p1 of methanol immediately prior to analysis. Citrulline analyses were performed on a Quattro 2000MD mass spectrometer (V.G. Biotech, Altrincham, U. K.) operating in plasma spray mode. Samples were introduced in methanol/ water (1:1 v/v) at a flow rate of 1 ml/min. Mass spectrometer operating conditions were as follows: source temperature, 230 "C; probe temperature, 275 "C; and discharge current, 485 pA. Data were collected over the mass range m/z 299 to m/z 305 at a scan rate of 300 ms at unit mass resolution. NitrosomorpholineAnalysis-A 1-ml aliquot of 5774 cell incubate, to which 4 ng of internal standard(4-nitroso-2,6-dimethyl morpholine containing positional isomers of 4-nitrosodimethyl morpholine) had been added, was mixed with 2 ml of dichloromethane and vortexed for 15 s. The mixture was then centrifuged for 15 min at 500 X g and the aqueous layer discarded. The organic layer was dried by the addition of anhydrous sodium sulfate and filtered through glass wool. The solvent was then dried under a stream of nitrogen at 40 "C to a volume of approximately 20 pl, 2 pl of which was injected into the GCMS. The procedure for endothelial cells was essentially identical to that described above, except that theculture medium from three replicate cultures (total volume of 9 ml) was combined and extracted into 2 equivalent volumes of dichloromethane prior to the final dry down and analysis. Nitrosomorpholine was analyzed without derivatization by GCMS on either a CPSil-43-CB (25-m X 0.32-mm inner diameter (0.12-pm film thickness)) or a CPSil-52-CB (25-m X 0.32-mm inner diameter (0.2-pm film thickness)) column (Chrompack Int., BV Middelburg, Netherlands) mounted in a Mega Series GC (Erba Strumentazione, Milan, Italy). The GC was interfaced directly into the source of an MS5ORFA mass spectrometer (Kratos Analytical, Manchester, UK) through a heated interface. The initial column temperature was set to 80 "C for 2 min and then increased to 200 "C at a rate of 20 "C/ min. The interface temperature was 180 "C, and helium was used as carrier gas at an average velocity of 30 cm/s. The mass spectrometer
23791
was operated in positive chemical ionization mode with ammonia or isobutane as reagent gas. Mass spectrometer operating conditions were: source temperature, 115 "C; ionization energy, 30 eV; and beam current, 500 mA. When ammonia was the reagent gas, the M + H and M + NH, ions were monitored m / z 117 and 134 for l60nitrosomorpholine, 119 and 136 for "0-nitrosomorpholine, and 145 and 162 for the internal standard (narrow scan of3,000 ppm over each ion for 300 ms). When "N'-argininewas used as substrate, isobutane was the reagent gas and the M + H ions were monitored m/z 118, 120,and 145 by selected ion monitoring with a sweep of 500 ppm and a cycle time of 1 s. Nitrosomorpholine produced by cells incubated under an atmosphere of "0, in the presence of w-hydroxyl-L-argininewas analyzed by GCMS using the same column and GC as above. The column was directly interfaced into the source of the Quattro 2000MD mass spectrometer through a heated interface maintained at 220 "C.The initial column temperature was set to 80 "C and increased at 20 "C/ min to 200 "C immediately after injecting the sample. Helium was used as the carrier gas at an average velocity of 33 cm/s. The mass spectrometer was operated in a positive chemical ionization mode with isobutane as the reagent gas. Mass spectrometer operating conditions were: source temperature, 115 'C; ionization energy, 20 eV; and beam current, 500 FA. Data were acquired over the range m/ z 150-110 at 1.0 s/scan, at unit mass resolution. Calculation of "0Incorporation into Citrulline and Nitrosomorpholine-The incorporation of "0 into citrulline was expressed as the ratio between the intensity of the signals at m/z 300 and 302, or 301 and 303 for experiments in which 15N-~-arginine was the substrate and shown as a percentage (mean ? S.E.). The incorporation of "0 into nitrosomorpholine was expressed similarly, using m/z 117 and 119 or 118and 120. Low level signals at other mass ions were assumed to be attributable to thebackground noise and natural abundance of other isotopes, and no correction to the signal intensity was made to allow for these. RESULTS
Inducible NO Synthase in J774 Cells Citrulline-When activated 5774 cells were incubated in air aproduct was formed with an m/z 300, consistent with authentic citrulline (Fig. 1A; 93.4 f 1.7% 0I6, n = 3). This was changed to a product (79.2 f 3.7% "0,n = 3) with an m/z 302 when the cells were incubated in 1 8 0 2 (Fig. 1B). In the presence of L-NMMA no significant peak was observed at either mass ( n = 3 for each). However, when cells were incubated either in H2160 (Fig. L 4 ) or H2"0 (90%; Fig. 1C) the main product observed had an m/z 300. In the presence of H2180 this product was 84.3 f 4.3% 0 I 6 ( n = 4). The formation of this product was inhibited by L-NMMA ( n = 3). Nitrosomorphline-Incubation of activated 5774 cells with morpholine in an atmosphere of air resulted in the formation of a product (92.3 & 3.0% l60, n = 4) with an m/z 117 at the relative retention time ( t R ) of authentic nitrosomorpholine (Fig. M ) .This was changed by 2 atomic mass units to m/z 119 when the cells were incubated in "O2 (81.2 ~fr16.5% lSO, n = 3; Fig. 2B). In the presence of L-NMMA no peak at the t~ of nitrosomorpholine was observed (Fig. 2C). When 5774 cells were incubatedin H2180 or H2160 the only product observed had an m/z 117, and thiswas >92% enriched in I 6 0 in either case. The formation of this product was inhibited by L-NMMA ( n = 3 for each). When 5774 cells were activated for 24 h in L-arginine-free medium and thenincubated with morpholine and w-hydroxyl) 24 h in an atmosphere of "02, they L-arginine (100 p ~ for formed nitrosomorpholine which was enriched by 94 f 3% ( n = 3) with l60(Fig. 3). The formation of nitrosomorpholine was significantly inhibited by L-NMMA ( n = 2). In contrast, ) w-hydroxyl-L-arginine,the when L-arginine (100 p ~ replaced 5774 cells formed nitrosomorpholine, which was enriched by 93 f 2% ( n = 2) with "0.
Nitric Oxide Synthases Incorporate Molecular Oxygen
23792
IS
A.
A' :::
"
I
C.
-
299
301 mh
300
302
303
FIG. 1. Partial mass spectrum of derivatized citrulline formed by the inducible NO synthase in 5774 cells. A, cells incubated in an atmosphere of air formed a product with an m/z 300 consistent with authentic citrulline. B, cells incubated in an atmosformed a product with an m/z 302. C, cells incubated in phere of '*02 H21R0in an atmosphere of air synthesized a product with an m/z 300. Truces are representative of three similar experiments.
Constitutive NO Synthase in Vascular Endothelium
-
117
IS
850 R-tim
119
-
h
8:23
119
9:W
117
e43
Tim bin)
FIG. 2. Selected ion mass chromatograms from the analysis of nitrosomorpholine on a CPSil-43 column at m/z 117, 119, and 145. A, 5774 cells incubated in anatmosphere of air formed "0 nitrosomorpholine (m/z 117). B, cells incubated in '"02 formed "0-
nitrosomorpholine (mlt 119). C, the formation of this product was inhibited by L-NMMA (1mM). IS denotes the m/z 145 for the internal standard, 4-nitroso-2,6-dimethyl morpholine, and a positional isomer of 4-nitroso-dimethyl morpholine. 5 indicates the t R of nitrosomorpholine. Truces are representative of three similar experiments.
A B Citrulline-The formation of citrulline was studied in endothelial cytosol to avoid previously observed problems associated with resynthesis of L-arginine from citrulline (1).Furthermore, since the background at m/z 300 was higher than in 5774 cells, "N'-~-arginine was used as substrate in order to obtain clearer signals. As a result all m/z values were increased by 1 atomic mass unit compared with those from 5774 cells. Incubation of endothelial cytosol in air gave a major peak (84.2 f 0.1% l60, n = 3) at m/z 301 consistent with authentic 0 "N-citrulline (Fig. 4A). In the presence of "02, the major 120 119 118 117 120 119 118 117 product (81.6 f 15.0% "0, n = 4) observed was at m/z 303 mh mh (Fig. 4B). The formation of both of these products was inhibFIG. 3. Partial mass spectrum of nitrosomorpholine formed ited by L-NMMA ( n = 3 for each). However, when endothelial by the inducible NO synthase in 5774 cells. A, cells incubated cytosol was incubated either in H2180or in H2160,only prod- in an atmosphere of "0,in the presence of w-hydroxyarginine (100 ucts with an m/z 301 (>80.9 1.6% l60, n = 3 in both cases) PM). B, cells incubated in an atmosphere of '"02in the presence of Lwere observed, and production of these was inhibited by L- arginine (100 PM). Truces are representative of three similar experiments. NMMA (n = 2 and n = 3, respectively). Nitrosomorpholine-Incubation of endothelial cells with morpholine, "NG-~-arginine, andA23187 in an atmosphere was inhibited by L-NMMA (n = 2 and n = 3, respectively). of air resulted in the formation of a product (97.3 f 0.7%l6O, The formation of nitrosomorpholine was not detected in n = 3) at the tR of nitrosomorpholine with an m/z 118 (Fig. incubations of endothelial cytosol ( n = 3). 5A). This was increased to m/z 120 when the cells were Constitutive NO Synthase of Brain incubated in 1802 (Fig. 5B; 73.7 f 18.1%"0, n = 4), and its production was inhibited by L-NMMA (Fig. 5C). Citrulline-Incubation of brain cytosol in an atmosphere of However, when endothelial cells were incubated either in air resulted in the formation of a product (87.9 f 6.4% l60, n HZ1'0 or in H2160only a product at m/z 118 (>95.6 f 0.6% = 3) at m/z 301 (Fig. 6A). This was abolished by L-NMMA l60, n = 3 in both cases) was observed, and this production or by EGTA ( n = 3 and n = 2, respectively). Incubation of
*
23793
Nitric Oxide Synthases Incorporate Molecular Oxygen A.
I
A.
i\
loo 50
E.
301
302
303
304
306
d z
FIG. 4. Partial mass spectrum of derivatizedcitrulline formed by the constitutive NO synthase from vascular endothelial cells. A, endothelial cytosol incubated with 15NG-~-arginine in anatmosphere of air formed a product with an m/z 301 consistent with "N-citrulline. B, incubation of endothelial cell cytosol in " 0 2 resulted in the formation of a product with an m/z 303. Traces are representative of three similar experiments.
the cytosol in 1802 resulted in the formation of a product (68.9 f 6.6%"0, n = 3) with an m/z 303 (Fig. 6B) which was also inhibited in the presence of L-NMMA or EGTA ( n = 3 and n = 2, respectively). Incubation in either H2180or H2160 led only to thegeneration of a product with an m/z 301 (X37.6 & 0.7% l60, n = 3 in both cases), and formation of this product was inhibited by L-NMMA or EGTA ( n = 3 and n = 2, respectively). Nitrosomorpholine-Nitrosomorpholine was not detected in incubations of brain cytosol (n = 3).
I
5:09
5:19
5:29
5:39 5:49 Rnantion Time h i n l
5:59
FIG. 5. Selected ion mass chromatograms from the analysis of "N-nitrosomorpholine on a CPSil-62 column at m/z 118 and 120. A , endothelial cells incubated in an atmosphere of air B, formed 160-nitrosomorpholine ( m / z 118) from 15NG-~-arginine. when cells were incubated in 1802 the major product formed was "0nitrosomorpholine (m/z 120). C, this was inhibited by L-NMMA (1 mM). 1denotes the t R of authentic nitrosomorpholine, and peak II is a background peak in the sample. Truces are representative of three similar experiments.
DISCUSSION
Inthepresentstudy we have used " 0 2 and H2180to investigate whether O2 or the oxygen in H 2 0 is incorporated into NO and into the ureido position of citrulline formed by both the constitutive and inducible NO synthases. The use of plasma spray mass spectrometry allowed the determination of the pseudomolecular ion of citrulline with high sensitivity and specificity. Nitric oxide formation by cells was analyzed as its reaction product with morpholine, nitrosomorpholine, by GCMS since this method allowed the accurate identification of nitrosomorpholine at a specific retention time relative to the internal standard. Furthermore, the use of chemical ionization mass spectrometry also allowed the specific and sensitive detection of the pseudomolecular ion of nitrosomorpholine. The murine monocytic leukemia cell line 5774, in which the Ca2+-independent NO synthase had been induced by lipopolysaccharide and interferon-y, synthesized NO and citrulline from L-arginine. When these cells were incubated in an atmosphere of 1802, rather than air, the pseudomolecular ions of both citrulline and NO increased by 2 atomic mass units,andthissynthesis was inhibited by L-NMMA, an inhibitor of NO synthase (33,34). There was no mass shift in the pseudomolecular ion of either nitrosomorpholine or citrulline when the cells were incubated in Hz"O.These data
indicate that the oxygen atom in both NO and in the ureido position of citrulline formed by the inducible NO synthase is derived from molecular oxygen. Similar findings on the source of the oxygen in citrulline formed by the inducible NO synthase from activated RAW 264.7 cells have been reported recently (28). We have also shown that the oxygen incorporated into citrulline is confined to theureido position.' The possibility that NO, or nitrosomorpholine, maybe scrambling with the 1802 in theincubation atmosphere to give artifactual resultsis excluded by the finding that thesynthesis by 5774 cells of nitrosomorpholine from w-hydroxyl-L-arginine incubated in an atmosphere of 1802 gave rise to only l60nitrosomorpholine. The N-OH in w-hydroxy-L-arginine has been proposed as thesource of NO (4), and the N in NO has recently been shown to come from the N-OH and the 0 in citrulline from 0' (35). The present data show that the 0 in NO, formed from w-hydroxyl-L-arginine,is not derived from molecular oxygen, and this provides conclusive evidence that the formation of nitrosomorpholine is a direct reflection of NO synthesis. These findings further support w-hydroxyl+ arginine as an intermediate in the formation of NO (4, 35). Endothelial cells stimulated with A23187 synthesized NO A. M. Leone, P. L. Francis, R. M. J. Palmer, D. S. Ashton, and S. Moncada, submitted for publication.
23794
Nitric Oxide Synthases Incorporate Molecular Oxygen A.
formation, only occurs to a significant extent when intact cells are present. The reason for this is not known. We may conclude, therefore, that molecular oxygen is the source of the oxygen in both NO and citrulline, formed either by the inducible or by the constitutive NO synthases. As a result, the three previously proposed mechanisms (3, 4, 29) that require that HZ1'0 and not "OZ should give rise to "0 in the ureido position of citrulline are incorrect. Furthermore, these results indicate that theN-OH oxygen in w-hydroxy+ arginine gives rise to NO, confirming that this compound is an intermediate in the synthesis of NO (4,35). Acknowledgments-We are indebted to Neale Foxwell and Tara Andrews for assistance with the cell culture.
REFERENCES 1. Palmer, R. M. J., Ferrige, A. G., and Moncada, S. (1988) Nature
301
302
303 306 mlr
304
FIG. 6. Partial mass spectrum of derivatizedcitrulline formed by the constitutive NO synthase in brain cytosol. A, brain cytosol incubated in an atmosphere of air formed "N-citrulline (m/z 301) from "N'-~-arginine. B, when incubated in the major product had an m / z 303. Traces are representative of three similar experiments.
from L-arginine, as indicated by the formation of "N-nitrosomorpholine from "NG-L-arginine. When these cells were stimulated in an atmosphere of "OZ, the pseudomolecular ion shifted by 2 atomic mass units, indicating that theoxygen in nitrosomorpholine is derived from molecular oxygen. Nosuch shift was observed when the cells were incubated in medium enriched in HZ1'0. That the nitrosomorpholine was formed by the NO synthase was indicated by the finding that the formation of this product was inhibited by L-NMMA. The formation of citrulline by the constitutive NO synthase from vascular endothelial cells was studied in endothelial cytosol to avoid the resynthesis of L-arginine from citrulline thought to occur in intact cells (1).Incubation of endothelial cytosol in the presence of "NG-~-arginine, NADPH, and Ca" resulted in the formation of 15N-citrullinewhich wasinhibited by L-NMMA. When incubated in an atmosphere of l8OZ,but not when incubated in HZ1'O,a 2-atomic mass unit shift in the pseudomolecular ionwas observed, indicating that the oxygen in NO, synthesized by the constitutive endothelial NO synthase, was also derived from molecular oxygen. The formation of 15N-citrullinefrom I5NG-~-arginine was also observed in brain cytosol incubated in the presence of NADPH and ea2+. This was inhibited by both L-NMMA and EGTA, confirming that the Ca2+-dependentconstitutive NO synthase was responsible for this synthesis. As with endothelial cells, incubation of brain cytosol in an atmosphere of "O2 resulted in a 2-atomic mass unit shift of the pseudomolecular ion, indicating that the ureido oxygen in citrulline is derived from molecular oxygen. The formation of nitrosomorpholine by brain cytosol was not detected. Similar findings with cytosol preparations from the vascular endothelium3 and from activated RAW 264.7 cells (28) have also been observed. This suggests that the reaction between NO and morpholine, used to detect NO A. M. Leone, R. M. J. Palmer, R. G. Knowles, P. L. Francis, D. S. Ashton, and S. Moncada, unpublished results.
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