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The Metabolism of L-Arginine and Its Significance for the Biosynthesis Of Proc. Nati. Acad. Sci. USA Vol. 87, pp. 8607-8611, November 1990 Physiology/Pharmacology The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: L-Glutamine inhibits the generation of L-arginine by cultured endothelial cells WILLIAM C. SESSA, MARKUS HECKER*, JANE A. MITCHELL, AND JOHN R. VANE The 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 The mechanism by which L-glutamine (L- rate-limiting for EDRF biosynthesis. While investigating the Gln) inhibits the release ofendothelium-derived relaxing factor mechanisms by which endothelial cells generate L-Arg (17), from bovine aortic cultured endothelial cells was investigated. we discovered a constituent of the culture medium inhibited The intracellular concentration of L-arginine (L-Arg) in Arg- both the generation of L-Arg and the release of EDRF (16, depleted endothelial cells was inversely related to the level of 18). This report describes the characterization of this factor, L-Gln. Removal of L-Gln from the culture medium (usually the amino acid L-glutamine (L-Gln), and suggests a biochem- containing L-Gln at 2 mM) abolished the inhibitory effect ofthe ical mechanism for its inhibitory action on EDRF biosynthe- culture medium on L-Arg generation. L-Gln (0.2 and 2 mM) but sis. not D-Gln inhibited the generation of L-Arg by both Arg- depleted and nondepleted endothelial cells. L-Gln did not interfere with the uptake of L-Arg or the metabolism of MATERIALS AND METHODS L-Arg-L-Phe to L-Arg but inhibited the formation ofL-Arg from Materials. The amino acids, indomethacin, adenosine di- L-citrulline (L-Cit), L-Cit-L-Phe, and N0-monomethyl-L- phosphate di(monocyclohexylammonium) salt (ADP), and arginine. L-Gln also inhibited the conversion of L-["4C]Cit to superoxide dismutase from bovine erythrocytes were pur- L-[14C]Arg by Arg-depleted endothelial cells. However, L-Gln chased from Sigma. L-[14C]Arg and L-[14C]citrulline (L-Cit) did not inhibit the conversion of L-argininosuccinic acid to were obtained from Amersham. Peptides were purchased L-Arg by endothelial cell homogenates. Thus, L-Gln interferes from Sigma or Bachem. Glyceryl trinitrate (Nitronal; nitro- with the conversion of L-Cit to L-Arg probably by acting on glycerin) was supplied by Lipha Pharmaceuticals Ltd. (West argininosuccinate synthetase rather than argininosuccinate Drayton, U.K.). 9,11-Dideoxy-lla,9a-epoxymethanopros- Iyase. L-Gln also inhibited the generation of L-Arg by the taglandin F2,a (U-46619) was a generous gift of J. Pike monocyte-macrophage cell line J774 but had no effect on the (Upjohn, Kalamazoo, MI). All other reagents and solvents conversion of L-Cit to L-Arg by these cells. As the release of were of the highest commercially available quality from endothelium-derived relaxing factor from cultured and non- Sigma or BDH. cultured endothelial cells is limited by the availability ofL-Arg, Cultured Bovine Aortic Endothelial Cells. Bovine aortic endogenous L-Gln may play a regulatory role in the biosyn- endothelial cells (19) were grown on Cytodex 3 microcarrier thesis of endothelium-derived relaxing factor. beads (Pharmacia) in Dulbecco's modified Eagle's medium (DMEM; Flow Laboratories) containing 0.6 mM L-Arg, 4 Vascular endothelial cells (1) and stimulated macrophages (2) mM L-Gln, and 10% (vol/vol) fetal calf serum (FCS). Endo- generate nitric oxide (NO) or a chemically related molecule thelial cells were identified as such by their cobblestone-like (3) through the oxidative metabolism of one of the guanidino appearance when seeded onto Petri dishes and by positive nitrogens ofthe amino acid L-arginine (L-Arg). The release of immunostaining for factor VIII. The confluent cells were NO derived from L-Arg accounts for the vasorelaxant prop- either used directly or transferred to Eagle's minimum es- erties ofendothelium-derived relaxing factor (EDRF) and the sential medium (MEM) containing 2 mM L-Gln without L-Arg cytotoxicity of activated macrophages (1, 4, 5). The avail- or FCS for 24 hr. ability of L-Arg analogues as NO synthase inhibitors and the Determination of EDRF Release by Bioassay. Approxi- unique pharmacologic profile of NO has enabled its biosyn- mately 6 x 107 endothelial cells were packed into a jacketed thesis from L-Arg to be demonstrated in many cell types chromatography column and perfused at 5 ml/min with including polymorphonuclear leukocytes (6), astrocytes (7), warmed (37°C) oxygenated (95% 02/5% CO2) Krebs' solu- hepatocytes (8), mast cells (9), platelets (10), adrenal (11) and tion containing 5.6 ,uM indomethacin and superoxide dismu- brain (12, 13) tissue(s), and possibly smooth muscle cells (14). tase at 10 units/ml. The Krebs' solution (pH 7.4) consisted of Some information is available regarding the metabolism of 118 mM NaCl, 4.7 mM KCI, 1.2 mM KH2PO4, 1.17 mM L-Arg by intact cells or semipurified preparations from en- MgSO4, 2.5 mM CaC12, 25 mM NaHCO3, and 5.6 mM dothelial cells (15), cytotoxic macrophages (2), or adrenal (11) glucose. The effluent from the column superfused a cascade or brain (12) tissues, but little is known about the regulation (20) of four spirally cut rabbit aortic strips (RbAs), which and sources of intracellular L-Arg required for NO biosyn- were denuded of endothelium and preconstricted with 30 nM thesis. U-46619. The lengths of the RbAs were monitored with We have demonstrated (16) that cultured endothelial cells, auxotonic levers and displayed on a Wantanabe WR3101 deprived of extracellular L-Arg for 24 hr, when transferred to recorder. Glyceryl trinitrate was used to calibrate the re- Krebs' solution, release increasing quantities of EDRF con- sponse of the detector tissues that were electronically comitant with the generation of L-Arg from an intracellular adjusted to give similar relaxations to a standard dose. source, implying that the availability of intracellular L-Arg is Abbreviations: EDRF, endothelium-derived relaxing factor; Cit, citrulline; NO, nitric oxide; FCS, fetal calf serum; RbA, rabbit aortic The publication costs of this article were defrayed in part by page charge strips; MeArg, NG-monomethyl-L-arginine; Argsucc, argininosucci- payment. This article must therefore be hereby marked "advertisement" nate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom correspondence should be addressed. 8607 Downloaded by guest on October 2, 2021 8608 Physiology/Pharmacology: Sessa et al. Proc. Natl. Acad. Sci. USA 87 (1990) HPLC/Fluorescence Detection Analysis of L-Arginine and cances by an unpaired Student's t test with P < 0.05 taken as L-Citrulline Levels in Endothelial Cells. HPLC analysis and significant. quantification of the intracellular amino acid concentrations were performed as described in ref. 17. Briefly, confluent endothelial cells on beads were washed 5-10 times with 9 vol RESULTS of warmed (370C) oxygenated (95% 02/5% C02) Krebs' bi- L-Gln Inhibits the Release of EDRF from Cultured Endo- carbonate buffer. A portion ofthe cells (100 ,ul corresponding thelial Cells. Fig. 1 depicts the inhibitory effect ofL-Gln on the to 3.5 x 106 endothelial cells) was directly extracted by release of EDRF from endothelial cells grown in the presence adding 5 vol of methanol for determination of basal amino of L-Arg, as determined by the cascade bioassay technique. acid levels. The remaining cells (200 Al) were incubated in Infusions of L-Gln (0.1-2.0 mM) over the assay tissues (OT) Krebs' buffer (total volume, 1 ml) in the absence or presence did not interfere with the detection ofEDRF. However, when of various amino acids for 60 min at 37TC. Incubations were infused through the column of endothelial cells (TC), L-Gln terminated by transferring 100 dul of the cells into ice-cold significantly reduced the release of EDRF induced by ADP. methanol (500 ,1). The samples were left for 10 min at 0-40C The inhibition produced by L-Gln was maximal at 0.2 mM (to facilitate deproteinization) and centrifuged at 10,000 x g (46.5 + 5.5%; n = 8), for L-Gln had no additional inhibitory for 10 min, and the supernatants were stored at -200C prior effect at 2 mM (48.1 ± 7.2%, n = 3). L-Gln also caused a to HPLC analysis. graded increase in the tone of the detector tissues, indicative TLC Analysis of L-['4CJCit Conversion to L-[14C]Arg by of inhibition of basal EDRF release. The inhibitory effects of Endothelial Cells. L-[carbamoyl-_4C]Cit (18.4 4M; specific L-Gln (0.2 mM) on stimulated and basal EDRF release were activity, 54.5 Ci/mol; 1 Ci = 37 GBq) was incubated with fully reversed by coinfusions of L-Arg (0.1 mM; n = 6) but not Arg-depleted endothelial cells (200 ,1) in 1 ml of Krebs' buffer D-Arg (0.1 mM; n = 3). L-Gln (0.02-2 mM) exerted a similar in the absence or presence of L-Gln (200 uM) for 60 min at inhibitory effect on the release of EDRF from Arg-depleted 37°C. Incubations were terminated by placing 100 ,ul of the endothelial cells (18). D-Gln (2 mM) had no effect on EDRF cells into 500 ,l of ice-cold methanol. The samples were release (n = 4). Similarly, L-glutamic acid (L-Glu; 2 mM; n = centrifuged (10,000 x g for 10 min) and 50 ul of the super- 4) or ammonium chloride (1 mM; n = 5), products of the natant was spotted onto TLC plates (Whatman silica gel metabolism of L-Gln by endothelial cells (23), did not affect 150A).
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