Proc. Nati. Acad. Sci. USAVol. 87, pp. 8612-8616, November 1990Physiology/Pharmacology

The metabolism of L-arginine and its significance for thebiosynthesis of endothelium-derived relaxing factor:Cultured endothelial cells recycle L-citrulline to L-arginineMARKUS HECKER*, WILLIAM C. SESSA, HAYLEY J. HARRIS, ERIK E. ANGGARD, AND JOHN R. VANEThe William Harvey Research Institute, Saint Bartholomew's Hospital Medical College, Charterhouse Square, London EC1M 6BQ, United Kingdom

Contributed by John R. Vane, August 7, 1990

ABSTRACT We have investigated the mechanism bywhich cultured endothelial cells generate L-arginine (L-Arg),the substrate for the biosynthesis of endothelium-derived re-laxing factor. When Arg-depleted endothelial cells were incu-bated in Krebs' solution for 60 min, L-Arg levels were signif-icantly (9.7-fold) elevated. The generation of L-Arg coincidedwith a substantial decrease (90%) in intracellular L-glutamine(L-Gln), whereas all other amino acids were virtually unaf-fected. Changes in calcium, pH, or oxygen tension had no effecton L-Arg generation, which was, however, prevented when thecells were incubated in culture medium containing L-Gln.L-Arg generated by endothelial cells labeled with L-['4C]Argwas derived from an unlabeled intracellular source, for thespecific activity of the intracellular L-Arg pool decreasedsubstantially (8.8-fold) over 60 min. Arg-depleted endothelialcells did not form urea or metabolize L-ornithine but convertedL-citrulline (L-Cit) to L-Arg possibly via formation of L-argin-inosuccinic acid. Nondepleted cells stimulated with the calciumionophore A23187 showed only a transient accumulation ofL-Cit, indicating that L-Cit is recycled to L-Arg during thebiosynthesis of endothelium-derived relaxing factor. The gen-eration of L-Arg by Arg-depleted endothelial cells was partially(45%) blocked by protease inhibitors, and various Arg-containing dipeptides were rapidly cleaved to yield L-Arg.Thus, cultured endothelial cells recycle L-Cit to L-Arg andpossibly liberate peptidyl L-Arg. The Arg-Cit cycle appears tobe the equivalent in the endothelial cell to the formation of ureaby the liver. The biosynthesis of endothelium-derived relaxingfactor may, therefore, not only produce a powerful vasodilatorbut also relieve the endothelial cell of excess nitrogen.

Endothelium-derived relaxing factor (EDRF) has been iden-tified as nitric oxide (NO) or a closely related moleculederived from the guanidino group of L-arginine (L-Arg) (1-3).Its biosynthesis involves a cytosolic (4) or microsomal (5)calcium/calmodulin and NADPH-dependent monooxygen-ase, which catalyzes the conversion of L-Arg to L-citrulline(L-Cit) and NO, or an intermediate giving rise to NO forma-tion. Similar enzymes have been identified in cytotoxicmacrophages (6) and various regions of the brain (7, 8).Interestingly, L-Arg relaxes freshly isolated vascular prepa-rations and potentiates the release of EDRF from culturedendothelial cells only when these tissues and cells are de-prived of L-Arg for 24 hr (3, 9-12). These findings indicatethat the endothelial L-Arg pool (0.1-0.8 mM in culturedcompared with 2-4 mM in freshly isolated endothelial cells;refs. 11-13) can be depleted and that its availability thenbecomes rate-limiting for EDRF biosynthesis. Little isknown about the pathway(s) by which the endotheliumsynthesizes or metabolizes L-Arg. We have found (11) thatcultured endothelial cells generate L-Arg from an intracellular

source and that this process is linked to the release ofEDRF.They can also convert L-Cit to L-Arg (13), and we proposedthat this pathway may help endothelial cells to maintainsufficient levels of L-Arg during periods of prolonged EDRFrelease (14). We have now investigated the mechanism bywhich endothelial cells generate L-Arg and whether ureacycle intermediates play a role in L-Arg biosynthesis.

MATERIAL AND METHODSMaterials. The amino acids, 3,4,5-trimethoxybenzoic acid

8-(diethylamino)octyl ester (TMB-8), and the protease inhib-itors were obtained from Sigma. L-[14C]Arg, L-[14C]Cit, andL-['4C]glutamine (L-Gln) were purchased from Amersham.Peptides were obtained from Sigma or Bachem, and A23187and NG-monomethyl-L-arginine (MeArg) monoacetate werefrom Nova Biochem (Nottingham, U.K.). All other chemi-cals and solvents were of the highest commercially availablequality from Sigma or BDH.

Determination of Intracellular Amino Acid Levels byHPLC/Fluorescence Detection Analysis. Endothelial cellsfrom bovine aorta (15) were grown on Cytodex 3 microcarrierbeads (Pharmacia) in Dulbecco's modified Eagle's medium(DMEM; Flow Laboratories) containing 0.6 mM L-Arg, 4mM L-Gln, and 10% (vol/vol) fetal calf serum (FCS). Theconfluent cells were either used directly (nondepleted cells)or transferred to Eagle's minimum essential medium (MEM)without L-Arg and FCS but containing 2 mM L-Gln for 24 hrprior to the experiment. They were then washed 5-10 timeswith 9 vol of Krebs' solution (pH 7.4) consisting of 118 mMNaCl, 4.7mM KCI, 1.2mM KH2PO4, 1.17 mM MgSO4-7H2O,2.5 mM CaCl2'6H20, 25 mM NaHCO3, and 5.6 mM glucose.No significant washout of intracellular amino acids wasdetected by using 14C-labeled L-Arg or L-Gln. A portion ofthecells (100 ,l) was directly extracted by adding 5 vol ofmethanol (0-min sample) and the remaining cells (200 ,ul)were incubated at 37°C for periods of 2-60 min in oxygenated(95% 02/5% C02) Krebs' solution (total volume, 1 ml). Insome experiments, a mixture of protease inhibitors wasadded consisting of (final concentration) amastatin (50 pAg/ml), aprotinin (50 ,ug/ml), bestatin (5 ,ug/ml), captopril (50,ug/ml), leupeptin (50 ,ug/ml), phenylmethylsulfonyl fluoride(0.5 mM), phosphoramidon (5 ,ug/ml), and trypsin-chymotrypsin inhibitor (50 ,ug/ml). Incubations were termi-nated by removing 100 ,ul of cells, which was extracted with500 ,ul of ice-cold methanol and centrifuged for 10 min at10,000 x g. A 50-,ul portion of the supernatant was mixedwith 50 ,ul of o-phthalaldehyde reagent for 1 min at ambienttemperature, and 25 ,ul was then applied to a 250 X 4.6(internal diameter) mm Ultratechsphere ODS-5 HPLC col-umn fitted with a 50 x 4.6 (internal diameter) mm guard

Abbreviations: EDRF, endothelium-derived relaxing factor; NO,nitric oxide; FCS, fetal calf serum; Cit. citrulline; Om, ornithine;MeArg, NG-monomethyl-L-arginine; Argsucc, argininosuccinate.*To whom correspondence should be addressed.

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column (HPLC Technology, Macclesfield, U.K.). Materialwas eluted at ambient temperature by using a convex gradient(ACS model 352 ternary gradient system, setting 3) from 0osolvent A (10%o methanol)/lOo solvent B [10 mM KH2-P04/acetonitrile/methanol/tetrahydrofuran, 84.0:7.5:7.5:1(vol/vol)] to 100% solvent A/0% solvent B over 30 minfollowed by 100% solvent A for 5 min. The flow rate was setto 1 ml/min and the fluorescence of the eluate was continu-ously monitored (Waters model 420 fluorescence detector) at425 nm (excitation wavelength, 338 nm).

L-Arg, L-Cit, L-Gln, and L-ornithine (L-Orn) were wellseparated from all other amino acids and were eluted at 17.6,11.8, 9.6, and 27.1 min, respectively. The endogenous aminoacids were identified by comparison with authentic standards(detection limit, 0.1 pmol), and their intracellular concentra-tion was calculated by using standard calibration curves andassuming an endothelial cell volume of 0.5 pl. The volumewas calculated from the average size (200-400 Aum2) andheight (1-2 Am) of the cells, as judged by electron micros-copy. The average cell count of the confluent cells was 125cells per bead or 3.5 x 106cells per 100 Al ofbeads. They wereidentified as endothelial cells by their cobblestone-like ap-pearance when seeded onto Petri dishes and by positiveimmunostaining for factor VIII.TLC Analysis of the Conversion of L-[14CJCit to L-[14C]Arg.

L-[carbamoyl-14C]Cit (18.4 AuM; specific activity, 54.5 Ci/mol; 1 Ci = 37 GBq) or L-[ureido-14C]Arg (25 ,tM; specificactivity, 36.6 Ci/mol) was incubated with Arg-depleted en-dothelial cells (200 Al) in 1 ml of Krebs' solution for 60 minat 370C. Incubations were terminated by removing 100 ,ul ofcells, which was extracted with methanol (500 1L/) and cen-trifuged at 10,000 x g for 10 min. A 50-;Ll sample of thesupernatant was spotted on Whatman silica gel 150A TLCplates, which were developed and scanned for radioactivityas described in ref. 16.

Isotope Dilution Analysis of the Generation of L-Arg. Arg-depleted endothelial cells (1.5 ml) were washed 5-10 timeswith Krebs' solution (9 vol) and incubated in Krebs' solutioncontaining 200 ,uM L-Gln (total volume, 7.5 ml) for 5 min at370C. L-[ureido-14C]Arg (6.7 AM; specific activity, 305 Ci/mol) was added for 10 min, excess label was removed bywashing 5 times with Krebs' solution (9 vol), and two 100-.lIportions of cells were extracted with methanol (500 1l). Theremaining cells were incubated for 60 min at 370C in Krebs'solution without L-Gln (total volume, 6.5 ml) followed byextraction of another two portions of cells. Samples wereprocessed for HPLC analysis as described above. The HPLCfraction (1.5 ml) of each sample corresponding to L-Arg wascollected, radioactivity was measured in a Beckman LS 3801liquid scintillation counter, and the specific activity of intra-cellular L-Arg was expressed as dpm/pmol.

Endothelial Cell Homogenates. Homogenates from Arg-depleted endothelial cells were prepared as described in ref.16. A 100-;,l portion was diluted with Krebs' solution (totalvolume, 250 ,l) and either extracted directly with methanol(5 vol) or incubated for 60 min at 37°C. Incubations wereterminated by adding 1.25 ml of methanol and samples wereprocessed for HPLC analysis as described above.

Determination of Ammonia and Urea Formation by Endo-thelial Cells. Ammonia and urea concentrations were deter-mined by using Sigma diagnostics kits no. 170-UV and66-UV, respectively.

Determination of L-Arg Levels in Bovine Aortic SmoothMuscle and J774 Cells. Smooth muscle explants were pre-pared from bovine aortas after removal of the endotheliumand adventitia as described (17). After 4 weeks in DMEMcontaining 4 mM L-Gln and 10% FCS, smooth muscle cellshad grown from the explants. These were removed and thecells were grown to confluence within 1 week, and thencontinuously passaged in a ratio of 1:4. Cells used in this

study were taken from passages 3 to 9 and seeded onto 6-welltissue culture plates (Flow Laboratories). They were identi-fied as smooth muscle cells by positive immunostaining fora-actin by using a Sigma SIH903 diagnostics kit. Afterreaching confluence (2.5 x 106 cells per well), the cells werecultured either in DMEM or in Arg-free MEM for 24 hrwithout FCS. The medium was aspirated and the cells werewashed five times with Krebs' solution (2 ml). They werethen harvested with the aid of a rubber policeman eitherdirectly or after a 60-min incubation in 1 ml of Krebs' solutionat 370C in an incubator. The cells were centrifuged for 2 minat 1000 rpm in a MSE Centaur 2 centrifuge, the supernatantwas aspirated, and the cells (resuspended in 100 Al of Krebs'solution) were extracted with 500 Al of methanol. Sampleswere processed for HPLC analysis as described above.The mouse monocyte-macrophage cell line J774.2

(ECACC no. 85011428) was obtained from the EuropeanCollection of Animal Cell Cultures (Salisbury, U.K.), certi-fied to be mycoplasma-free at the time ofpurchase. The cellswere cultured as a semisuspension in DMEM containing 4mM L-Gln and 10% FCS. After reaching 1-2 x 106 cells perml, the cells were either cultured in DMEM without FCS orin Arg-free MEM without FCS for 24 hr. They were thenwashed five times with Krebs' solution (25 ml) and 1 X 106cells in 100 Al were extracted with methanol (500 /l) eitherdirectly or after a 60-min incubation in Krebs' solution (totalvolume, 1 ml) at 370C. The cells were centrifuged for 2 minat 1000 rpm and resuspended in 100 /4 of Krebs' solution towhich methanol (500 /l) was added. Samples were processedfor HPLC or TLC analysis as described above.

Statistical Analysis. Results are shown as mean ± SEM forn experiments. Student's unpaired t test was used to deter-mine the significance of differences between the means, anda P value of

8614 Physiology/Pharmacology: Hecker et al.

absence of L-Gln. As no release of L-[14C]Arg into thesupernatant was detected, these findings demonstrate thatthe intracellular L-Arg pool had been diluted with unlabeledL-Arg derived from an intracellular source.Urea Cycle Intermediates and MeArg as Potential Sources of

L-Arg. To determine whether L-Arg was derived from ureacycle intermediates, Arg-depleted endothelial cells were in-cubated for 60 min with L-Cit, L-Orn, or L-argininosuccinate(L-Argsucc) (Fig. 1). Incubations with L-Orn, in the presenceor absence ofN-acetyl-L-glutamate or with L-Argsucc did notincrease L-Arg levels compared to control cells. However,the generation of L-Arg was significantly potentiated (2.1 ±0.3 times; n = 10) in the presence of L-Cit (50 MM). L-As-partate (L-Asp) inhibited rather than potentiated the netincrease in L-Arg seen with L-Cit. The conversion of L-Cit toL-Arg was confirmed by TLC analysis. Arg-depleted endo-thelial cells converted 8.4 ± 2.1% of L-[14C]Cit (18.4 ,.M) toL-[14C]Arg (n = 3) in 60 min, whereas nondepleted cells wereless active and converted only 3.0 ± 1.0% of L-[14C]Cit toL-[14C]Arg (n = 3). These cells also failed to metabolizeexogenous, unlabeled L-Cit (50 ,tM) to L-Arg (1.1 ± 0.4 timesincrease compared with control; n = 3).The formation by Arg-depleted endothelial cells of L-Arg

from L-Cit but not from L-Argsucc was puzzling and, there-fore, we prepared homogenates from these cells to test thepossibility that, due to its amphiphilic nature, L-Argsucc maynot be transported or diffuse through the plasma membrane.Indeed, homogenates metabolized L-Argsucc (100 ,tM) toL-Arg (7.8 ± 2.8% conversion after 60 min; n = 7) but failedto produce L-Arg from either L-Cit (50 ,uM) or L-Orn (50 ,uM)alone or in the presence of L-Asp (100 ,uM) or N-acetyl-L-glutamate (100 ,uM), respectively (n = 3 for each).

Arg-depleted or nondepleted endothelial cells did not con-vert L-[14C]Arg to L-[14C]Orn (n = 3). Moreover, L-Orn levelswere the same in Arg-depleted (188 ± 29 ,uM; n = 6) andnondepleted cells (194 ± 31 ,uM; n = 3) and did not change[1.0 ± 0.2 times (n = 6) and 1.3 ± 0.3 times (n = 3) increase]when the cells were incubated in Krebs' solution for 60 min.Thus, there is no evidence that L-Orn is a precursor for L-Arg.In addition, Arg-depleted or nondepleted cells did not containor produce urea when incubated in Krebs' solution, providingfurther evidence for a lack of a complete urea cycle. How-ever, they contained relatively high amounts of ammonia [6.8± 2.0 nmol per 106 cells (n = 3) for Arg-depleted and 4.4 ±

1.8 nmol per 106 cells (n = 3) for nondepleted cells] andreleased ammonia when incubated in Krebs' solution (3.2 ±0.6 nmol per 106 cells per hr and 2.3 ± 0.6 nmol per 106 cellsper hr; n = 3).

Incubations of MeArg (50 ,uM) with Arg-depleted endo-thelial cells for 60 min resulted in a concomitant increase inL-Arg and L-Cit (5.2 ± 1.6 times and 17.9 ± 4.5 times; n = 8;Fig. 1). Whereas the metabolism ofMeArg produced a similarnet increase in L-Arg (9.8 ± 2.7% conversion) and L-Cit (6.6± 1.9% conversion), homogenates of Arg-depleted cells aswell as nondepleted whole cells prcduced only L-Cit fromMeArg [11.7 ± 4.2% (n = 4) and 11.9 ± 6.4% (n = 3)conversion].

Recycling of L-Cit to L-Arg During EDRF Biosynthesis.When transferred to Krebs' solution, nondepleted endothe-lial cells also showed an increase in L-Arg (4.5 ± 1.1 timesfrom 160 ± 21 ,uM to 641 ± 125 ,uM after 60 min; n = 6),whereas L-Cit levels were not significantly affected. In adifferent set of experiments, these cells were incubated inKrebs' solution for 2-60 min, in the absence (control incu-bation) or presence of the calcium ionophore A23187 at 1 ,uM(to stimulate EDRF biosynthesis). There was a time-dependent increase in L-Arg (2.5 ± 0.8 times after 60 min; n= 3) but not L-Cit (Fig. 2) in control cells. The calciumionophore elicited not only a more pronounced increase inL-Arg (4.5 ± 0.4 times after 60 min; n = 3) but also, after ashort lag phase, a transient increase in L-Cit. Coincubationsof A23187 with L-Arg (10 ,uM) led to an immediate increasein L-Cit, which was similar in magnitude and duration to thatwith the ionophore alone, whereas L-Arg levels were onlymarginally elevated (1.4 ± 0.2 times after 60 min; n = 3). Inthe presence of A23187, L-Arg, and L-Gln (200 ,gM), changesin L-Arg were similar to control (2.7 ± 0.9 times increase after60 min; n = 3), but L-Cit was further elevated and remainedat this level throughout the experiment. As L-Gln inhibits theconversion of L-Cit to L-Arg (16), these findings imply thatduring EDRF biosynthesis L-Cit accumulates and is recycledto L-Arg.Metabolism of L-Arg Via Deiminase Reactions. L-Arg was

not converted to L-Cit in nonstimulated Arg-depleted (1.2 ±0.1 times increase in L-Cit; n = 3) or nondepleted (n = 3)endothelial cells or by homogenates (n = 3). Moreover,neither Arg-depleted nor nondepleted endothelial cells me-tabolized L-['4C]Arg to L-[14C]Cit in the absence of A23187(n = 3). Furthermore, Na-benzoyl-L-arginine (100 ,uM), the

750 r

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400 [

300 F

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Orn +NAG Argsucc MeArg

I

0 1 0 20 30 40 50 60Time (min)

FIG. 1. Effect of urea cycle intermediates and MeArg on thegeneration of L-Arg by Arg-depleted endothelial cells. The cells wereincubated in Krebs' solution for 60 min at 37°C in the absence (barControl; n = 30) or presence of L-Cit (bar Cit; 50 ,uM; n = 10), L-Orn(bar Orn; 50 AM; n = 4), L-Argsucc (bar Argsucc; 50 ,tM; n = 4), orMeArg (bar MeArg; 50 ,uM; n = 8) followed by HPLC analysis. Theopen bars represent incubations of L-Cit and L-Orn in the presenceof L-Asp (bar +Asp; 100 uM; n = 4) or N-acetyl-L-glutamate (bar+NAG; 100 ,uM; n = 3). The changes in intracellular L-Arg werecalculated as percent of 60 min control and expressed as mean ±SEM for the number of experiments indicated above. Values signif-icantly different (P < 0.05) from control (*) are indicated.

FIG. 2. Accumulation of L-Cit in nondepleted endothelial cellsduring EDRF biosynthesis. The figure shows the changes in intra-cellular L-Cit in nondepleted endothelial cells incubated in Krebs'solution for 2-60 min at 37°C in the absence (open circles) orpresence of A23187 (1 ,uM; open squares), A23187 plus L-Arg (10,uM; open triangles), or A23187, L-Arg, plus L-Gln (200 ,uM; solidsquares). Values are calculated as percent of 0 min control andexpressed as mean + SEM (n = 3). Significant differences (bytwo-way analysis of variance) between incubations in the presenceof A23187, L-Arg, plus L-Gln and control incubations at P < 0.01 (**)or incubations in the presence of A23187 and L-Arg at P < 0.05 (+)are indicated.

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synthetic substrate for peptidylarginine deimwell as NI-benzoyl-L-citrulline (100 uM) werelized to L-Cit or L-Arg [1.1 ± 0.1 times (n = 3)times (n = 3) increase].

Peptidyl L-Arg or L-Cit as a Precursor ofgeneration of L-Arg was partially inhibited (45 :by a combination of protease inhibitors, suggesof the newly generated L-Arg may be derived fior protein. Therefore, we incubated four dipepL-Phe, L-Ala-L-Arg, L-Arg-L-Arg, and L-Cit-L-Pdepleted endothelial cells. All Arg-containing prapidly cleaved to yield L-Arg [e.g., L-Ala-L-iverted to L-Argby 17.1 ± 7.6%, 31.1 ± 4.0o, ancafter 2, 10, and 60 min, respectively (n = 3)]. TIa significant increase in both L-Cit and L-Arg inof L-Cit-L-Phe (Fig. 3), and coincubation of tiwith the protease inhibitors completely preventeage. Moreover, Arg-containing dipeptides werepotentiate basal and stimulated EDRF release fiendothelial cells (data not shown).

Generation of L-Arg by J774 Monocyte-MacrCultured Smooth Muscle Cells. The intracellulation of L-Arg and L-Cit in nonstimulated J774 cel145 AM (n = 4) and 128 ± 40,uM (n = 3), respecttransferred to Arg-free MEM for 24 hr, L-Arcreased by 36 ± 9% to 366 ± 104 uM (n = 4).showed a 2.5 ± 0.6 times (n = 3) increase in L-Asolution over 60 min, which was further potentiattimes; n = 4) in the presence of L-Cit (50 IJMeArg (50 AM) was not metabolized to L-Arg or0.2 times increase; n = 4). The conversion of L-by J774 cells was confirmed by TLC analysis (Iconversion of L-[14C]Cit to L-[14C]Arg over 60 rwhereas no significant formation of L-[14C]Cit o0was detected in the presence of L-[14C]Arg (n =Bovine aortic smooth muscle cells contained 2

L-Arg (n = 4), and L-Arg levels decreased by 3533 ILM (n = 4) when they were cultured for 24 hiMEM. Unlike endothelial cells or J774 cells, 1decrease (rather than an increase) in L-Arg over 6smooth muscle cells were transferred to Krel[from 220 ± 21 ttM to 121 ± 9 ,uM (n = 4) for nondfrom 144 ± 33 ,AM to 76 ± 17 AuM (n = 4) for dep

0

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FIG. 3. Protease-mediated cleavage of L-Arg-L-PhegM; n = 6) and L-Cit-L-Phe (bars CF; 50 gM; n = 6) tL-Cit by Arg-depleted endothelial cells compared withL-Arg and L-Cit in control cells (bars C; n = 6). Controlare represented by the solid bars, whereas the openincubations (n = 3 for L-Arg-L-Phe and L-Cit-L-Phe; n = 4performed in the presence of a mixture of protease inhitand L-Cit levels were calculated as percent of 60 minexpressed as mean + SEM. Values significantly differenfrom control (*) or between incubations with and withcinhibitors (+) are indicated.

inase (18) asa nnt mi-tqhn-

DISCUSSIONand 13 ±0V2V- This study demonstrates that cultured endothelial cells gen-

erate L-Arg through an Arg-Cit cycle. The concept of a der L-Arg. The novo synthesis of L-Arg from an intracellular source wasL-Arg.The

8616 Physiology/Pharmacology: Hecker et al.

[Arg]oX-Arg

4-Arg NO

Argsucc " (DATP CM

ci MeArg

NH3 Lt-,llJo

IL-Arg to NO and L-Cit. Argininosuccinate synthetase (step 2)forms L-Argsucc in the presence of L-Asp, L-Cit, and ATP.L-Argsucc in turn is cleaved by argininosuccinate lyase (step3) to yield fumarate and L-Arg. Fumarate can be recycled toL-Asp by the mitochondrial citric acid cycle involving malate,oxaloacetate, and ammonia. The present findings do not ruleout the possibility that L-Cit is directly or indirectly con-verted to L-Arg by an unknown transamination reaction.Under quiescent conditions, ammonia may be incorporatedinto L-Arg but will predominantly be released. The increaseddemand for L-Arg during EDRF biosynthesis, however, mayultimately lead to the excretion of ammonia as NO. If thishypothesis is correct, then the biosynthesis of EDRF couldserve two functions, the formation of a powerful vasodilator(physiologic) and the removal of excess nitrogen (metabolic).Our experiments in which protease inhibitors reduced the

generation of L-Arg by =50%6 suggest that the Arg-Cit cycleis not the only source of intracellular L-Arg. Some may enterthe cell from exogenous sources ([Arg]0) but some may alsobe derived from peptidyl L-Arg (X-Arg) or L-Cit (X-Cit).The presence of a dimethylarginine deiminase (step 4; ref.

27) in endothelial cells (13) is particularly interesting, as wecould not detect any other deiminase activity [e.g., for L-Arg(28) or protein-bound L-Arg (18)]. NG-Dimethyl-L-arginineand possibly MeArg are found in a variety of post-translationally methylated proteins (29). If MeArg also oc-curs in endothelial cells, it could act as an endogenousinhibitor of EDRF biosynthesis and, when metabolized,provide L-Cit as a precursor for the formation of L-Arg.The generation of L-Arg is not restricted to endothelial

cells. Nonstimulated J774 cells have an amino acid compo-sition similar to endothelial cells but with a higher level ofL-Arg. Like endothelial cells, they generate L-Arg whenincubated in Krebs' solution and metabolize L-Cit to L-Arg.In contrast to the generation of L-Arg, however, the conver-sion of L-Cit to L-AMg was not inhibited by L-Gln (16) and,unlike endothelial cells, J774 cells did not metabolize MeArgto L-Cit.The amino acid pattern of cultured smooth muscle cells

was similar to that of endothelial cells, but L-Arg levelsdecreased upon incubation in Krebs' solution. We concludefrom these differences that the metabolism of L-Arg may beof special significance for the biosynthesis of EDRF by

endothelial cells, which is probably different from the for-mation of NO by most other cells in that it is an immediateresponse to cell activation and does not require enzymeinduction.

We are indebted to Miss E. G. Wood and Mrs. I. Vojnovic forproviding the cultured cells used in this study and to Prof. R.Gryglewski for helpful discussions. The William Harvey ResearchInstitute is supported by a grant from Glaxo Group Research Ltd.

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