Coinduction of Nitric Oxide Synthase and Argininosuccinate ...

Vol. 269,No.2, Issue of January 14,pp. 1257-1261, 1994 Pnnted in U.S.A.

THEJOURNAL OF B I ~ ~ C I C CHEMISTRY AL 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Coinduction of Nitric Oxide Synthase and Argininosuccinate Synthetase in a Murine Macrophage Cell Line IMPLICATIONS FOR REGULATION OF NITRIC OXIDE PRODUCTION* (Received forpublication, June 29, 1993, and in revised form, August 24, 1993)

Andreas K. NusslerS, Timothy R. BilliarS, Zhi-Ze LiuS, and Sidney M. Morris, Jr.8n From the mepartments of Surgery and §Molecular Geneticsand Biochemistry, University of Pittsburgh School of 'Medicine, Pittsburgh, Pennsyluania 15261 In macrophages and other cell types, bacterial lipo- membrane transport system (Bogle et al., 1992; Sat0 et al. 1992), fromintracellular protein degradation, or by endogenous polysaccharide and certain cytokines stimulate nitric synthesis of arginine. The major sites of arginine synthesis in oxide (NO) production via expression of the inducible (NOS).Citrulline, which terrestrial vertebrates are liver, where arginine generated in isoform of nitric oxide synthase is the coproduct of NOS-catalyzed metabolism of argithe urea cycle is rapidly converted to urea and ornithine by of argini- arginase, and kidney, where arginine is synthesized from cinine, can be recycled to arginine by the action nosuccinatesynthetaseandargininosuccinatelyase, trulline and released into the circulation (reviewed in Morris, which are present at high levels in hepatocytes and re- 1992). However, it has long been known that many other cell nal tubularcells but normally at very low levels in other types also contain low levels of argininosuccinate synthetase cell types such as macrophages. "he present study dem-and argininosuccinate lyase which together synthesize argionstrates that lipopolysaccharide and interferon-?, nine from citrulline (Ratner, 1973; Beaudet et al., 1986).More which induce NOS in the murine macrophage cell line recently, two NO-producing cell types, bovine aortic endothelial RAW 264.7, also coinduce activity and mFtNA for argininosuccinate synthetase, which is limiting for arginine cells (Hecker et al., 1990) and rat peritoneal macrophages (Wu synthesis. Argininosuccinate lyase activity and mRNA and Brosnan, 19921, were shown to synthesize arginine from abundance are unaffected. Induction of argininosucci- citrulline, thecoproduct of the NOS reaction. These investiganate synthetaseis not blocked byNG-monomethyl-L-ar- tors suggested that the ability to recycle citrulline to arginine ginine, a potent inhibitor of NOS, indicating that ar- might represent a mechanism for regulating NO production in these cells (Mitchell et al., 1990; Wu and Brosnan,1992). Moregininosuccinate synthetase induction is not the consequenceofdepletingcellularargininelevels by over, LPS-activated macrophages were %fold more active in NOS. Because plasma levels of arginine are limiting forsynthesis of arginine from citrulline than were unstimulated NO synthesis, enhanced cellular capacity to regeneratemacrophages, suggesting that NOS and arginine biosynthetic a significant role in activities werecoinduced (Wu and Brosnan,1992). Activities of arginine from citrulline could play regulating NO production, especially under conditions argininosuccinate synthetase and argininosuccinate lyase were where the inducible isoform of NOS is expressed. not measured in the latter study; thus it was not clear whether increased argininebiosynthetic capacity reflectedincreased activity of these enzymes or increased uptake of extracellular Murine macrophages can be activated by agents such as citrulline. Because argininosuccinatesynthetase activity in bacterial lipopolysaccharide (LPW and certain cytokines to some cells varies in response to arginine concentration, an inexpress microbicidal and tumoricidal activity. The production creased capacity forarginine biosynthesis could be secondaryto of nitric oxide (NO), an important mediatorof these activities, depletion of intracellular arginine as NOS activity increased is stimulated following macrophageactivation, concomitant rather than being a direct response to LPS. The objectives of with increasedexpression of an inducible isoform of nitric oxide the present study were to determine whether endogenous arginine synthesis canprovide substrate for cellular NO producsynthase(NOS)(StuehrandMarletta, 1987; Nathanand tion, whether argininosuccinate synthetase or argininosucciHibbs, 1991; Xie et al., 1992; Lorsbach et al., 1993). Cellular NO production is absolutely dependent on availabil- nate lyase are coinduced with NOS in a murine macrophage ity of arginine, which donates the nitrogen atomfor the NOS- cell line, and, if so, whether argininosuccinate synthetase or dependent formationof NO. No other physiologic amino acids argininosuccinatelyase induction is secondary toincreased or guanidino-containing compounds can substitutefor arginine NOS activity. in this reaction (Iyengar et al., 1987; Granger et al., 1988). EXPERIMENTALPROCEDURES Arginine can be obtainedfrom exogenous sources via a plasma Materials-Cytokines used includedmurinerecombinant interferon-y (Genzyme,Cambridge, MA), human recombinant interleukin 1 * This work was supported in part by National Institutes of Health (Cistron, Pine Brook,NJ), and tumor necrosis factor-cu (Genzyme, CamGrants DK33144 (to S. M. M.), GM44100 (to T. R. B.), GM37753 (to R. bridge, MA). LPS (Escherichia coli lll:B4) was obtained from Sigma. L. Simmons), and by a George H.A. Clowes Memorial Career Development Award of the American College of Surgeons (to T. R. B.). The costs Radiochemicals were purchased from Dupont-New England Nuclear. of publication of this article were defrayed in part by the payment of P-monomethyl-L-arginine(NMA) was prepared by a modification of page charges. This article must therefore be hereby marked "advertise- the method of Corbine and Reporter (1974).All other reagentswere of the highest commercially available grade. ment" in accordance with 18 U.S.C. Section 1734 solely to indicate this Cell Culture-The RAW 264.7 murine macrophage cell line was obfact. 7 To whom correspondence should be addressed. Tel.: 412-648-9338; tained from the American Type Tissue Collection. Cells were maintained at 37 "C, 5% CO,, in Williams E medium supplemented with1PM Fax: 412-624-1401. The abbreviations used are: LPS, lipopolysaccharide; NO, nitric insulin, 15 mM HEPES, 2 mM L-glutamine,1%dialyzed calf serum (Life oxide;NOS, nitric oxide synthase; NMA, NG-monomethyl-L-arginine. Technologies, Inc.), and 10,000 unitslml penicillin + streptomycin until

1257

1258

Coinduction of NO and Arginine Biosynthetic Enzymes 150

TABLEI Enhancement of cellular NO synthesis by supplemental citrulline to Williams E Just prior t o confluence,RAW 264.7 cells were switched medium containing 50 or 80 p~ arginine, with or without 0.5 mM citrulline or 0.5 rn ornithine, 100 unitdm1 interferon-y, 10 pg/ml LPS, orno added cytokines, as indicated. Cells wereincubated under the indicated conditions fora total of 18 h, with a medium changeat 6 h. Nitrite plus nitrate values represent the average accumulation in the culture medium from 6 to 18 h for duplicate plates. Results are shown for two independent experiments.

1

NO;

+ NO;

Stimulus Arginine Experiment Supplemental amino

150

1

80 50

Interferon-y

80 30.8 50

0

LPS Tine (h)

FIG.1. NO production in RAW 264.7 cells stimulated with interferon-y (ZFN-g,upperpanel) or LPS (lowerpanel).Cumulative NO production was measured as nitrate (NO,) plus nitrite (NO,) concentration in theculture medium forthe times and conditions indicated. Values represent means standard error of the mean ( n = 4).

semiconfluent.At the time of addition of LPS or cytokines, the cells were changed t o serum-free medium containing 0.4 mM arginine hydrochloride except when otherwise indicated. Where specified, cells were exposed to mediumcontaining 100 unitdm1 interferon-y, 10 pg/ml LPS, 5 unitdm1 interleukin-1, 500 unitdml tumor necrosis factor-a, or 0.5 rn NMA. Enzyme and Metabolite Assays-Culture supernatants were assayed for nitrate and nitrite by an automated procedure based on the Griess reaction (Green et al., 1982). Argininosuccinate synthetase activity in cell homogenates wasdetermined by measuring conversion of [ 14C]aspartate and citrulline into [14Clargininosuccinic acid(Su et al., 1981). Argininosuccinatelyase activity in cell homogenateswas determined by measuring formation of ['4Clargininosuccinic acid from arginine and P4Clfurnarate (OBrien and Barr, 1981).Protein concentration was determined by a commercially available Lowry assay kit (Sigma). Isolation and Analysis of RNA--Total RNA was isolated according to Chomczynski and Sacchi (1987) and electrophoresed on formaldehydeagarose gels as described (Morris et al., 1987). Following transfer to Genescreen (DuPont-New England Nuclear) membranes and furation by UV cross-linking,hybridizations were carried out as described (Morris et al., 1987). Relative mRNA abundance was quantified bytwodimensional densitometric analysis of autoradiograms using preflashed x-ray film (Laskey and Mills, 1975)and aBio-Rad model 620densitometer. ClonedcDNAs usedfor hybridization included those encoding mouse macrophage NOS (Lowenstein et al., 1992), rat argininosuccinate synthetase (Morris et al., 1989), and rat argininosuccinate lyase (Lambert et al., 1986). Hybridization to 18 S ribosomal RNA was performed with a labeled rat genomic DNA clone (Katz et al., 1983).

Citrulline Ornithine

(PI

None (control)

504

acid None

d

( p ~ )

80 63.5 54.8 50

I I1 I I1

5.3 10.1 7.3 5.0

8.8 8.0 7.3 4.6

I46.8 30.0 I1 57.844.5 I28.3 12.5 I1 25.3 11.3 I I1

50.0

I 37.0 I1 38.5 32.5 24.8

8.5 7.5 8.8 4.0 44.0 12.0 11.6

77.3 -

54.3 54.8 33.3

that cellular NO production requires the presence of arginine in the culture medium, NO-producing cells such as cultured endothelial cells (Heckeret al., 1990) and murine macrophages

(Wu and Brosnan, 1992) can convert citrulline to arginine, suggesting that the requirement forarginine might be met, at least in part, by citrulline. Although substitution of citrulline alone for arginine in a defined culture medium did not sustain NO et al., 1987), production by stimulated RAW 264.7 cells (Iyengar this experiment could not evaluate the potential role of endogenous arginine synthesis because the definedmedium contained no amino acids whichcould serve as amino group donors for synthesis of arginine from citrulline. Citrulline recycling would contribute little to NO production if the extracellular arginine concentration were sufficient to support maximalor near maximal rates of NO production.The apparent K,,, for arginine in NO synthesis by intact RAW 264.7 cells is 0.15 mM (Iyengar et al., 19871, whereas plasmaarginine concentrationsarenormallyinthe 80-110 p~ range (e.g. Clowes et al., 1980; Sax et al., 1988). Thus, cellular NO production should be substrate-limited at physiologic concentrations of arginine. Indeed, experimentsin this laboratory have determined that NO production by LPS- or interferon-y-stimulated RAW 264.7 cells cultured in 80 PM arginine is approximately in 1 mM half of that produced by these cells when cultured arginine. Thus, at physiologic levels of extracellular arginine, synthesis of arginine from citrullinecould contribute substrate for NO production. If so, one would predict that addition of citrulline to the culture medium should boost NO production. RESULTS AND DISCUSSION Table I shows the effects of citrulline on cellular NO production Previous work in other laboratories has established the RAW at two concentrations of arginine, 80 PM, typical of normal , of plasma arginine concentrations, and 50 p ~ representative 264.7 cell line as a model for murine macrophage function, reduced plasma arginine concentrations in sepsis (Clowes et including induction of NOS and NO production by LPS and cytokines (Stuehr and Marletta, 1987; Xie et al., 1992; Lors- al., 1980). The latter concentration was chosen because sepsis bach et al., 1993). The concentrations of LPS and interferon-y is a condition in which NOS expression is strongly induced. With the possible exceptionof LPS-stimulated cells culturedin used in the present study have been shown to maximally inand Marletta, 50 PM arginine, addition of citrulline to the culture medium duce NO production in this cell line (Stuehr consistently stimulates NO productionin activated RAW 264.7 1987). Following a brief lag, NO production as measured by cells (Table I), clearly demonstrating the presence of an arginitrate plus nitrite production increased in LPS- and interin these cells which can be functionferon-y-stimulated RAW 264.7 cells, confirming previous re- nine biosynthetic pathway ally linked to NO synthesis. However, addition of citrulline ports (Stuehr and Marletta, 1987); this increase was largely blocked by0.5 mM NMA (Fig. 1). Althoughit is well established does not eliminate the differences in NO production between

Coinduction of NO and Arginine Biosynthetic Enzymes

1259

TARLE I1 Eflects of interferon-y and LPS on activities ofargininosuccinate synthetase and argininosuccinate lyase in RAW 264.7 cells Cells were incubated under the indicated conditions for 24 h, and enzyme activities in fresh cell homogenates were determined as described under "Experimental Procedures." Results in each experiment are averages ofduplicate determinations for each condition. Results are shown for two independent experiments.

-

""""""""

Specific activity Additions to mediumlyase

Argininosuccinate Argininosuccinate synthetase

I

I1

I

*c

NOS

&

I1

- 28s - 18s

Inmollminlmgpmtein)

None (control) +0.5 mM NMA

0.09 0.10

0.25 0.22

4.40

5.36

3.57

5.28

Interferon-y Interferon-y

0.47

0.52

0.50

0.70

4.00 4.19

4.72 5.12

0.21 0.29

0.41 0.42

3.19

4.68 4.66

+ 0.5 mM NMA

LPS LPS + 0.5 mM NMA

3.67

stimulated cells cultured in 50 versus 80 PM arginine, indicating that the rates of endogenous arginine synthesis under these conditions are not sufficient to generate amounts of arginine which would saturate the NOS enzyme. In contrast to added - 28s -0 " citrulline, added ornithine,which can be converted to citrulline NOS by reaction with carbamyl phosphate in liver and small intes- 18s tine, hasno effect, indicating thatRAW 264.7 cells lack significant carbamyl phosphate synthetase I and/or ornithine transcarbamylase activity. 18s An enhanced cellular capacity to regenerate arginine from FIG.2.Induction of NOS mRNA in RAW 284.7 cells stimulated citrulline could improve the ability to maintainNO production, with interferon-y (IFN-g, u p p e r p a n e l ) or LPS (lower panel ). particularly in low arginine environments. It was therefore of Northern blot analysis of totill 11SA was as descnhcd undrr ' E x p n mental Procedures." Equal loadlng of samples in each lane was coninterest to determine whether cellular arginine biosynthetic firmed by hybridizing the same mrmhranes with a laheled DNA pmhc activity is increased wheneverNO production is induced. This for 18 S ribosomal RNA. possibility was raised by a recent report that LPS-stimulated rat macrophages have increased capacity to convert citrulline similar to that of NOS mRNA (Figs. 3 and 4). In contrast, to arginine (Wu and Brosnan, 1992). However, it was not clear argininosuccinate lyase mRNA abundance was unaffected by whether the elevated arginine biosynthetic capacity reflected either interferon-yor LPS (Fig.3). demonstrating thespecifican increased capacity for uptake of extracellular citrulline or ity of these agents for NOS and argininosuccinate synthetase whether activities of arginine biosynthetic enzymes also inmRNA induction. Induction of argininosuccinate synthetase mRNA a t 6h in several experiments averaged 3.4-5.7-fold creased. Thus, enzyme activity measurements were carried out to determine whether activities of argininosuccinate syntheincreases over control levels of mRNA for LPS and interferontase and argininosuccinate lyase inRAW 264.7 cells change in y. respectively. These increases in mRNA abundance are suffiresponse to LPS or interferon-y. The activity of argininosucci- cient to accountfor the increased argininosuccinate synthetase activity. The similarities in time courses of induction of argininate synthetase increased approximately 2-fold and 2-5-fold 24 h following exposure to LPSandinterferon-y, respectively nosuccinate synthetase andNOS mRNAs further suggesL9 that (Table 11). In contrast, the activity of argininosuccinate lyase expression of these mRNAs may be coreplated via the same was largely unaffected by either agent (Table 11). Inclusion of signal transduction pathways. The fact that argininosuccinate NMA in the medium had no significant effect on activities of synthetase activity in several cell lines increases when extrathese enzymes. The specific activities of argininosuccinate syn- cellular argininelevels are low (Schimke, 1964; I r r and Jacoby, thetaseandargininosuccinatelyaseinunstimulated RAW 1978; Suet al., O'Brien, 1981)suggested that argininosuccinate 264.7 cell homogenates are virtually identical to those reported synthetase could be induced in stimulated RAW 264.7 cells a s for rat alveolar andbone marrow macrophages (Hofmann al., et increased NO production depleted cellular argininepools. How1978). Comparisonof the specific activities of these enzymes in ever, induction of argininosuccinate s-ynthetase mRNA was not RAW 264.7 cell homogenates indicates that argininosuccinate blocked by 0.5 mM NMA, a potent inhibitor of NOS activity, synthetase activity is probably limiting for arginine synthesis indicating that argininosuccinate synthetaseinduction was not in RAW 264.7 cells. This could explain why only argininosuc- the consequence of reductions in cellular arginine levels nor of cinate synthetase is coinduced with NOS. increases in citrulline or NO levels as NOS activity increased Total RNA was isolated from RAW 264.7 cells a t various (Figs. 3 and 4). Interestingly, NMA plus LPS or interferon-y times after exposure to LPS or interferon-y andused in North- largely blocked the usual decline in NOS mRNA abundance at e m blot analyses to determine whether increases in arginino- 24h (Fig. 2), suggesting that NO production is inhibitory for succinate synthetase activity reflected increases in argininocontinued NOS expression. In contrast, NMA had no effect on succinatesynthetase mRNA abundance. For comparison, argininosuccinate synthetase mRNA levels in LPS- or interinduction of NOS mRNA also was examined. NOS mRNA was feron-y-stimulated cells a t 24 h (Fig. 3). Although the mecharapidly induced by both agents, with maximal induction occur- nism for increased argininosuccinate synthetasemRNA abunring by 6-10 h (Fig. 2). Induction of argininosuccinate synthe- dance was not determined in this study, it most likely reflects tase mRNA by LPS and interferon-y followed a time course increasedtranscription of theargininosuccinate s-ynthetase "

-

b ~ r ~ ~ # e ~ o O o O

Coinduction of NO and Arginine Biosynthetic Enzymes

1260

*NO )/cos Arginine

4

""""""-"-AS

Citrulline

/I\f ArglnlnSuccinate

a-Keto Aspartate

Amino acid

-

Fumarate

AL

4 acid

Malate

FIG.5. The citrulline-NO cycle. Glutamate is the most common amino group donor in the transamination of oxaloacetate to aspartate.

dothelial and neuronal cells (Bredt and Snyder, 1990, 19911. Changes in levels of the inducible NOS isoform in response to various stimuli have been demonstrated for a variety of cell - - types ( e g . Billiar et al., 1990; Galea et al., 1992; Lowenstein et al., 1992; Geller et al., 1993), as has the dependence of NO production on synthesis of the NOS cofactor tetrahydrobiop0 3 hr 6 hr 10 hr 24 hr terin (Werner et al., 1991; Gross et al., 1991). LPS-stimulated FIG.3. Induction of argininosuccinate synthetase (AS)mRNA murine macrophages andcell lines have an increased arginine i n RAW 264.7 cells stimulated with interferon-y (IFN-g, upper transport capacity (Bogle et al., 1992; Sato et al., 1992). reprep a n e l ) or LPS (lowerpanel).The membrane usedin the upper panel senting an additional mechanism for regulating NO production was stripped and reprobed for argininosuccinate lyase ( A L ) mRNA. via substrate availability. The present study indicates thatrecycling of citrulline to arginine via the citrulline-NOcycle f Fig. 5) may be an important regulatory mechanism for determining rates of cellular NO production in vivo, as previously suggested (Mitchell et al., 1990; Wu and Brosnan. 1992).Like arpnase in the urea cycle, NOS can be viewed a s a component of a citrulline-NO cycle whereby transamination reactions feed nitrogen atoms onto a carrier molecule to generate arginine, which ulNOS timately provides the nitrogen atom for NO (Fig. 5). Because AS plasma arginine levels in both normal and septic rats or humans ( e . g Carey et al., 1987; Clowes et af., 1980; Ochoa et af., AL 1991; Sax et al., 1988)are below the apparent K,,, for cellular FIG.4. Coordinate responses of NOS and a M n i n o s u c c i n a t e NO synthesis, the potential impact of endogenous arginine synsynthetase mRNAa to interferon-y, interleukin-1 ( I C 1 ), tumor necrosis factor-a(7°F). a n d NMA. RAW 264.7 cells were incubated thesis on cellular NO production is likely to be widespread and not limited merely to specialized environments such a s wounds for 6 h with the indicated agents at the concentrations listed under "Experimental Procedures." Total RNA was isolated and subjected to where concentratims of extracellular arginine may be unusuNorthern blot analysis as described. ally low (Currie et al., 1979; Albina et al., 1988). Indeed,coinduction of NOS and argininosuccinate s-ynthetase may be a gene as has been shown for the NOS gene in this cell line (Xie widespread adaptive response, a s we have observed this phenomenon also in other cell types.2 Thus, because of its influence et al., 1992; Lorsbach et al., 1993). to produce on NO production, regulationof endogenous arginine synthesis LPS and interferon-y also stimulate macrophages interleukin-1 and tumor necrosis factor-a (Collartet al., 1986; may play an important role in diverse physiologic processes Burchett et al., 1988). raising the possibility that these cyto- such a s cell signaling, host defense mechanisms, maintenance kines could act in an autocrine fashion to regulate macrophage of vascular tone, autoimmune disorders, and inflammatoryreNOS or argininosuccinate synthetase expression. Neither of sponses. these agents alone induced NOS or argininosuccinate syntheAcknowledgments-We are grateful to Dr. Richard L. Simmons for tase mRNAs (Fig. 4), in agreement with previous reports that his continuing support and encouragement. We thank Theresa Toczek. these cytokines do not induce NO production in macrophages Diane Lenhart. Ken Dorko, and Debra Williams for excellent technical (Ding et al., 1988). Thus, interleukin-1 and tumor necrosis fac- assistance. tor-a do not appear to mediate induction of NOS or argininoREFERENCES succinate synthetase mRNA by LPS or interferon-y in RAW Albina. J. E.. Mills. C. D.. Rarhu1.A.. Thirkill. C. E.. H c n v . W. L.. Mantrnfrnnccsco. 264.7 cells. R.. and Caldwell. M. D. (19881Am. J . I'h.varol. 254. E4SB-E467 Cellular NO production is determined by NOS activity, in- Benudet.A. L.. O'Rrien, W. E.. Bock. H.-G. 0 , FrcytnR. S 0.. nnd Su. T.-S.( 1 9 f 6 1 tracellular arginine concentration, availability of the various Ad?. Human Grnpf. 15, 161-196 cofactors required for the NOS reaction, and cell responsive- Rilliar, T. R.. Curran. R. I).. Stuehr. D. J . . Went. Y. A.. B m t z . R. G . . and Simmons. R. L. (19R9) J . Exp. Mrd. 169. 1467-1472 ness to specific cytokines and other stimuli. Thus, there are Billiar, T. R.. Curran. R. I).. Stut-hr. I). .J. Stndler. J . . Simmona. R. 1. .. and . M u ~ ~ R ? . S. A. ( 1 9 9 0 1 Biorhem. Riophva. RPS.Commun. 1BR. 1034-1040 multiple potential sites whereby NO production may be reguRogle. R. C . . Raydoun. A. K.. Pranon. J. D.. Moncndn. S . . nnd Mnnn. C . E. I19921 lated, and the actual regulated steps may vary for different cell Rrochem. J . 2R4. I.S-1R types. Because NO production by intact cells has infrequently Bredt. D. S..and Snyder. S. H. (19901 P m . .Val/. Arad. S r r . (1. S A. A7. M 2 4 S D. S..Hwang, I? H.. Glatt, C.. Lowcnntein. C.. Rrrd. R. R.. and Snyder. S H been compared to actual levels of NOS, the contributions of Rredt. (1991)Nafurr 351.714-718 additional regulated steps have rarely been determined. The Burchrtt. S. K . Wr-aver,W. M.. Wentall. J. A,. 1.amn.A.. Kmnheim. S I and Wilron. fact that changesin NO production do notalways reflect changes in NOS enzyme levels is best exemplified by calcium D. K. Nakayama. S. M. Morris. Jr.. S.Morrow. P. Davies. R. R. Pitt, activation of the pre-existing constitutive NOS isoform in en- R. L. Simmons, and T. R. Rilliar. manuscript in preparation.

e

+

+

+

+

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