Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts

Tet r ahydr obi opt er i n- dependent For mat i on of Ni t r i t e and Ni t r at e i n Mur i ne Fi br obl ast s By Gabr i el e Wer ner - Fel mayer , Ar no Hausen, Er nst R. Wer ner , Di et mar Fuchs, Gi l ber t Rei bnegger , and Hel mut Fr om t he Depar t ment of Medi cal Chemi st r y and Bi ochemi st r y, Wacht er Uni ver si t y of I nnsbr uck, 6020 I nnsbr uck, Aust r i a Summar y The pr esent st udy demonst r at es t hat mur i ne der mal f i br obl ast s pr oduce ni t r i t e ( N02 - ) and ni t r at e ( N03 - ) upon t r eat ment wi t h i nt er f er on y ( I FN- y) . Thi s f or mat i on i s dependent on L- ar gi ni ne and can be i nhi bi t ed by t he L- ar gi ni ne anal ogue NG- monomet hyl - L- ar gi ni ne. The ef f ect of I FN- y i s dr ast i cal l y i ncr eased by cot r eat ment wi t h t umor necr osi s f act or a ( TNF- a) , i nt er l euki n 1 ( I I r 1) , or l i popol ysacchar i de ( LPS) . The t est ed cyt oki nes al so i nduce f or mat i on of t et r ahydr obi opt er i n i n mur i ne f i br obl ast s . I nhi bi t i on of guanosi ne t r i phosphat e- cycl ohydr ol ase I , t he key enzyme of t et r ahydr obi opt er i n de novo synt hesi s wi t h 2, 4- di ami no- 6- hydr oxy- pyr i mi di ne, l eads t o decr eased f or mat i on of N02 - and N03 - . Thi s ef f ect can be r ever sed by addi t i on of sepi apt er i n, whi ch pr ovi des t et r ahydr obi opt er i n vi a a sal vage pat hway. Met hot r exat e, whi ch i nhi bi t s t he sal vage pat hway, bl ocks t he r est or at i on of N02 - and N03 - pr oduct i on by sepi apt er i n . The cyt ot oxi c ef f ect of combi nat i ons of I FN- a wi t h TNF- y, I W, or LPS i s at t enuat ed by i nhi bi t i on of t et r ahydr obi opt er i n synt hesi s . These r esul t s show t hat i nt r acel l ul ar concent r at i ons of t et r ahydr obi opt er i n cont r ol t he amount of N02 - and N03 - pr oduced i n si t u and suggest t hat t he r ol e of cyt oki ne- i nduced t et r ahydr obi opt er i n synt hesi s i s t o pr ovi de cel l s wi t h t he act i ve cof act or f or pr oduct i on of ni t r ogen oxi des. O ne of t he met abol i c act i ons of I FN- y on human cel l s i n vi t r o i s an up t o 100- f ol d st i mul at i on of guanosi ne t r i phosphat e ( GTP) t - cycl ohydr ol ase I ( EC 3 . 5 . 4 . 16) act i vi t y ( 1) . Thi s enzyme cl eaves GTP t o 7, 8- di hydr oneopt er i n t r i phosphat e, t hus i ni t i at i ng t he f or mat i on of 7, 8- di hydr oneopt er i n and 5, 6, 7, 8- t et r ahydr obi opt er i n ( 2) . Bot h compounds can be det ect ed i n cel l homogenat es of I FN- y t r eat ed human macr ophages, human f i br obl ast s, and a panel of human t umor cel l l i nes ( 1) . We showed pr evi ousl y t hat f ur t her enzyme act i vi t i es r equi r ed f or t he f or mat i on of t et r ahydr obi opt er i n, i . e . , 6- pyr uvoyl t et r ahydr opt er i n synt hase and sepi apt er i n r educt ase ( EC 1 . 1 . 1 . 153) , ar e not i nf l uenced by I FN- ' Y, but ar e const i t ut i vel y pr esent i n t he cel l s ( 3) . I FN- y- i nduced st i mul at i on of GTP- cycl ohydr ol ase I act i vi t y i s enhanced by cot r eat ment wi t h TNF- a, LPS, or dexamet hasone. Thi s was demonst r at ed f or human f i br obl ast s, human macr ophages and t he monocyt i c cel l l i ne THP- 1( 4, 5) . I n vi vo, cyt oki ne- i nduced st i mul at i on of GTP- cycl ohydr ol ase I i s i ndi cat ed by el evat ed neopt er i n excr et i on, whi ch i s r el at ed wi t h di seases chal l engi ng t he cel l - medi at ed i mmune syst em ( 6, 7) . Synt hesi s of t et r ahydr obi opt er i n was al so obser ved i n muAbbr evi at i ons used i n t hi s paper : DAHP, 2, 4- di ami no- 6- hydr oxypyr i mi di ne; GTP, guanosi ne t r i phosphat e ; NM, NG- monomet hyl - Lar gi ni ne; NOZ- , ni t r i t e ; NO3 - , ni t r at e . 1599 r i ne macr ophages ( 8, 9) . Fur t her , we f ound r ecent l y i n mouse der mal f i br obl ast s t hat t et r ahydr obi opt er i n synt hesi s i s st i mul at ed by I FN- y and even bet t er by TNF- a, or combi nat i on of I FN- y wi t h TNF- a ( Wer ner , E. R. , G. Wer ner - Fel mayer , D. Fuchs, A. Hausen, G. Rei bnegger , J. J . Yi m, and H. Wacht er , manuscr i pt submi t t ed f or publ i cat i on) . Al t hough cyt oki ne- i nduced pt er i di ne synt hesi s t hus seems t o be a gener al phenomenon i n mammal i an cel l s, no bi ol ogi cal f unct i on coul d so f ar be est abl i shed . Tet r abydr obi opt er i n i s an essent i al cof act or of phenyl al ani ne 4- , t yr osi ne 3- , and t r ypt ophan 5- monooxygenase ( 10) . However , we coul d not f i nd t hat t hese enzyme act i vi t i es wer e i nduced i n I FN- y- t r eat ed cel l s ( unpubl i shed dat a) . Mor eover , t her e i s no evi dence t hat oxi dat i on of et her l i pi ds, t he f our t h est abl i shed t et r ahydr obi opt er i n- dependent r eact i on ( 11) , i s i nf l uenced by I FN- y . Recent l y, a new t et r ahydr obi opt er i n- r equi r i ng r eact i on, t he f or mat i on of ni t r ogen oxi des f r om L- ar gi ni ne, was descr i bed f or cel l homogenat es of mouse macr ophages ( 8, 9) . Thi s oxi dat i on of L- ar gi ni ne i s i nduced i n mur i ne macr ophages by t r eat ment wi t h LPS ( 12) or l ymphoki nes, such as I FN- y ( 13) . The ef f ect of I FN- y can be f ur t her enhanced by cot r eat ment wi t h TNF- a, TNF- a, or LPS ( 14, 15) . Si nce pt er i di ne synt hesi s i s modul at ed by t he same f act or s, i t appear ed at t r act i ve t o hypot hesi ze t hat cyt oki nes i nduce f or mat i on of t et r ahydr obi opt er i n i n or der t o pr ovi de a cof act or f or t he gener at i on of J . Exp. Med . ® The Rockef el l er Uni ver si t y Pr ess - 0022- 1007/ 90/ 12/ 1599/ 09 $2 . 00 Vol ume 172 December 1990 1599- 1607 ni t r ogen oxi des . I f t hi s was t r ue, pt er i di ne- dependent f or mat i on of ni t r ogen oxi des shoul d not onl y occur i n macr ophages but al so i n ot her cel l s wi t h i nduci bl e pt er i di ne synt hesi s, e. g. , i n f i br obl ast s . I n t hi s r epor t , we show t hat mur i ne f i br obl ast s can be st i mul at ed by I FN- y t o r el ease ni t r i t e ( N02 - ) and ni t r at e ( N03 - ) . The ef f ect of I FN- y i s enhanced by TNF- ac, I L1, or LPS. Dat a obt ai ned by i nhi bi t i on and r est or at i on of pt er i di ne synt hesi s i ndi cat e t hat t et r ahydr obi opt er i n i s essent i al f or t he f or mat i on of N02 - and N03 - , and t hat gener at i on of ni t r ogen oxi des i s r el at ed t o cyt oki ne- medi at ed cyt ot oxi ci t y. Mat er i al s and Met hods Mat er i al s . Mouse r I FN- y ( pr oduced i n Chi nese hamst er ovar y cel l s) was obt ai ned f r om Hol l and Bi ot echnol ogy ( Lei den, The Net her l ands) and had a speci f i c act i vi t y of 10' U/ mg of pr ot ei n . Mouse f f NF- a ( 4 x 10' U/ mg pr ot ei n ; pr oduced i n Escher i chi a col i was a ki nd gi f t of Dr . G. R. Adol ph ( Bender Co. , Vi enna, Aust r i a) . Human r I I r l - a ( 10' U/ mg pr ot ei n ; expr essed i n E. col t ) was f r om Genzyme ( Bost on, MA) . LPS f r om E. col i 055: B5 ( phenol i c ext r act ) , 2, 4- di ami no- 6- hydr oxy- pyr i mi di ne ( DAHP) , and met hot r exat e wer e f r om Si gma Chemi cal Co. ( Muni ch, FRG) . N' G- monomet hyl - L- ar gi ni ne ( NMA) was f r om Cal bi ochem- Behr i ng Cor p. ( La Jol l a, CA) . Pt er i di nes wer e f r omDr . B. Schi r cks aona, Swi t zer l and) . Cel l Cul t ur e. Mur i ne f i br obl ast s wer e ki ndl y pr ovi ded by Dr . C. Heuf l er ( Depar t ment of Der mat ol ogy, Uni ver si t y of I nnsbr uck) . Cel l s wer e est abl i shed f r om ear der mi s expl ant s of 8- 10- wk- ol d BALB/ c mi ce, whi ch wer e cul t ur ed i n RPMI 1640 ( Bi ochr ome, Ber l i n, FRG) cont ai ni ng 10%heat - i nact i vat ed FCS ( Bi ochr ome) , 2 mML- gl ut ami ne, 100 U/ ml of peni ci l l i n, and 0. 1 ng/ ml of st r ept omyci n ( Si gma Chemi cal Co. ) . Al l cul t ur es wer e mai nt ai ned i n humi di f i ed ai r cont ai ni ng 5% C02 at 37°C wi t h a medi um change ever y week . Nonadher ent cel l s wer e r emoved by washi ng. Af t er 4 wk, cel l s gr own out f r om t he expl ant s wer e passaged by t r ypsi ni zat i on and f ur t her pr opagat ed by a weekl y t r ansf er of 1 : 3 . Cel l s wer e det er mi ned t o be f i br obl ast s due t o mor phol ogi cal f eat ur es, gr owt h char act er i st i cs, and MHC ant i gen expr essi on. The r esul t i ng cel l popul at i on, f or mi ng a monol ayer , consi st ed of mul t i pol ar cel l s, assumi ng a pavement - l i ke and par t l y spi ndl e- shaped appear ance when conf l uent , as i s char act er i st i c f or mouse f i br obl ast s . Cel l s ceased t o gr ow when conf l uent , and no f or mat i on of f oci was obser ved. For i sol at i on and cul t ur e of cel l s, no pr et r eat ment of sur f aces or suppl y wi t h gr owt h f act or s except FCS was pr ovi ded . Under t hese st andar d condi t i ons, onl y f i br obl ast s sur vi ve i n t he cul t ur e . MHCcl ass I ant i gen expr essi on of t hese cel l s was enhanced t wo- t o t hr eef ol d by I FN- y ( 5- 500 U/ ml ) as compar ed wi t h unt r eat ed cont r ol s and unt r eat ed cont r ol cel l s, and I FN- y- st i mul at ed cel l s wer e negat i ve f or MHC cl ass I I ant i gen. Thi s was r epor t ed pr evi ousl y f or 3T3 mouse f i br obl ast s ( 16) . MHC ant i gen expr essi on was det ect ed by ELI SA as per f or med pr evi ousl y ( 17) . Cel l s gr own i n 96- wel l pl at es wer e st ai ned wi t h ei t her undi l ut ed super nat ant f r om 34- 1- 2S hybr i doma ( Kd, Dd ; Amer i can Type Cul t ur e Col l ect i on, Rockvi l l e, MD) or wi t h 20 ng/ wel l of pur i f i ed ant i body r eact i ng wi t h I - Ad ( Bect on Di cki nson & Co. , Mount ai n Vi ew, CA) andwi t h a hor ser adi sh per oxi dase- conj ugat ed second ant i body t o mouse I gG whol e mol ecul e ( Si gma Chemi cal Co. ) . Cel l s of passages 10- 25 wer e used f or exper i ment s . For mat i on of NOz - and NOZ- + N0, , - . Fi br obl ast s wer e seeded at a densi t y of 1( 5 / ml i n 24- wel l cul t ur e pl at es ( Fal con Lab1600 war e, Oxnar d, CA) and gr own t o conf l uency wi t h one change of medi um on day 4 of cul t ur e, usi ng RPMI 1640 ( suppl ement ed as descr i bed above) . On day 8, cel l s wer e t r eat ed wi t h cyt oki nes and/ or ot her addi t i ves . Due t o i t s l ow cont ent of ni t r at e, al l i ncubat i ons wer e car r i ed out i n DMEM( Ser va, Hei del ber g, FRG) , suppl ement ed as descr i bed above. We t est ed di f f er ent doses of DAHP ( 1- 10 mM) , sepi apt er i n ( 50- 400 , AM) , and met hot r exat e ( 10- 100 uM) . Opt i mal doses wer e 5 mMf or DAHP, 100 AMf or sepi apt er i n, and 10 AM f or met hot r exat e, and wer e used t hr oughout al l f ur t her exper i ment s. Accor di ngl y, ot her pt er i di nes wer e used at a concent r at i on of 100 AM. I n t he case of met hot r exat e t r eat ment , t he medi umwas suppl ement ed wi t h 40 AMt hymi ne and 100 AMi nosi ne ( Fl uka, Buchs, Swi t zer l and) . Al l addi t i ves wer e di ssol vedi n compl et e DMEM, and, wher e necessar y, t he pH was adj ust ed t o 7. 4 . The addi t i ves wer e appl i ed t o cel l s j ust bef or e cyt oki nes. I n t he case of cyt oki ne combi nat i ons, TNF- a, I I A, or LPS wer e added i mmedi at el y af t er I FN- y ( i . e. , wi t hi n 1 mi n) , whi ch was essent i al f or opt i mal st i mul at i on. The f i nal vol ume per wel l was 1 ml . NMA was appl i ed t o cel l s 24 h bef or e st i mul at i on wi t h cyt oki nes. For some exper i ment s, conf l uent monol ayer s wer e t r eat ed f or 7 h wi t h cul t ur ed medi umwi t h or wi t hout L- ar gi ni ne ( MEM/ Ear l e, Sel ect Ami ne Ki t ; Gi bco Labor at or i es, Gr and I sl and, NY) , cont ai ni ng ser um, gl ut ami ne, and ant i bi ot i cs ( see above) . Af t er changi ng t he r espect i ve medi um once agai n, cel l s wer e st i mul at ed wi t h cyt oki nes . Af t er i ncubat i on f or 72 h, super nat ant s wer e har vest ed. Monol ayer s wer e washed once wi t h war mPBSand wer e l ysed wi t h 0. 05 MH3P04 f or pr ot ei n det er mi nat i on . N0 2 - was measur ed i n super nat ant s as descr i bed ( 18) , usi ng t he st abl e Gr i ess- I l osvay' s ni t r i t e r eagent obt ai ned f r om Mer ck ( Dar mst adt , FRG) . N03 was r educed enzymat i cal l y by 0. 125 Wmol / mi n of ni t r at e r educt ase ( EC1. 6 . 6 . 2) f r omAsper gi l l us sp. ( Ser va) , and2. 5 mMni cot i nami de adeni ne di nucl eot i de phosphat e, r educed f or m, and was t hen measur ed as N02- ( desi gnat ed NOz - + N03 - ) . Backgr ound l evel s wer e det er mi ned i n cel l - f r ee cont r ol s of cul t ur e medi um wi t h or wi t hout addi t i ves and wer e subt r act ed f or cal cul at i on of t he t ot al amount of ni t r ogen oxi des f or med . Dat a ar e pr esent ed as mi cr omol es per l i t er . The det ect i on l i mi t of N02 - was 1 Amol / l i t er . Appr oxi mat e val ues i n nanomol es per mi l l i gr am of t ot al cel l pr ot ei n or nanomol es per 105 cel l s can be cal cul at ed f r om t he pr ot ei n val ues gi ven bel ow. Det er mi nat i on of I nt r acel l ul ar Bi opt er i n . Fi br obl ast s wer e seeded i n 25- CM2 cul t ur e f l asks ( Fal con Labwar e) at a densi t y of 105 / ml i n 5 ml of compl et e RPMI 1640 and gr own t o conf l uency wi t h one medi umchange . On day 8, cel l s wer e t r eat ed wi t h cyt oki nes, DAHP, met hot r exat e, and sepi apt er i n i n a f i nal vol ume of 5 ml of compl et e DMEM( f or det ai l s, see above) . Af t er 72 h, super nat ant s wer e col l ect ed f or det ect i on of N02 - and N0 2 - + N03( see above) . Absol ut e val ues of ni t r ogen oxi des pr esent ed i n Tabl e 2 ( see Resul t s) ar e hi gher t han i n t he ot her exper i ment s shown . Thi s was due t o t he use of cul t ur e f l asks ( 25 cmz/ 5 ml ) i nst ead of 24- wel l pl at es ( 2 cmz/ ml ) , r esul t i ng i n a di f f er ent cel l t o super nat ant r at i o. Cel l s wer e har vest ed by t r ypsi ni zat i on and washed wi t h PBS. Pel l et s wer e r esuspended i n 250 Al of di st i l l ed wat er , r api dl y f r ozen i n l i qui d ni t r ogen, and kept at - 20°C unt i l oxi dat i on, whi ch was st ar t ed wi t hi n 1 h af t er f r eezi ng. Sampl es wer e t hawed one by one, cent r i f uged f or 2 mi n at 10, 000, g, and subj ect ed t o oxi dat i on wi t h i odi ne at aci di c or al kal i ne pH ( 19) . Br i ef l y, 100 Al of cel l ext r act s wer e mi xed wi t h 5 A1 of 1 MHCl and 5 Al of 0. 1 MK1/ 12 i n or der t o det er mi ne t he t ot al amount of bi opt er i n, i ncl udi ng 5, 6, 7, 8t et r ahydr o and 7, 8- di hydr o der i vat i ves . Anot her 100 Al of cel l ext r act was mi xed wi t h 5 AI of 1 MNaOH and 5 141 of 0. 1 MKI / I z Bi opt er i n- dependent For mat i on of Ni t r ogen Oxi des i n Fi br obl ast s f or dest r oyi ng t he t et r ahydr o der i vat i ve, t hus det ect i ng t he sum of 7, 8- di hydr obi opt er i n and bi opt er i n . Af t er i ncubat i on f or 1 h i n t he dar k at r oomt emper at ur e, sampl es wer e cent r i f uged f or 2 mi n at 10, 000 g, and t he super nat ant was mi xed wi t h 10 Al of 0 . 1 M ascor bi c aci d . Sampl es wer e st or ed at - 20°C unt i l HPLCanal yses ( LC 5500; Var i an Associ at es, I nc . , No Al t o, CA) . Sampl es wer e t hen ext r act ed wi t h AASPSCX car t r i dges ( Anal yt i chem I nt er nat i onal , I nc . , Har bor Ci t y, CA) , and pt er i di nes wer e di r ect l y el ut ed ont o a r ever sed- phase HPLC col umn ( Li chr osor b, RP- 18, 7- , ur n par t i cl e si ze, Mer ck) and quant i f i ed by f l uor escence det ect i on ( LS 4, Per ki n- El mer Cor p. , Beaconsf i el d, UK) , as pr evi ousl y descr i bed ( 20) . Val ues ar e pr esent ed as pi comol es per mi l l i gr am of t ot al cel l pr ot ei n . Cel l Vi abi l i t y Assay. Fi br obl ast s ( 105 / ml ) wer e seeded i n 100 Al / wel l i n 96- wel l pl at es ( Fal con Labwar e) and gr own t o conf l uency, usi ng RPMI 1640 wi t h suppl ement s. We used conf l uent monol ayer s i n or der t o l i mi t possi bl e ef f ect s of addi t i ves on cel l gr owt h. On day 8 of cul t ur e, monol ayer s wer e t r eat ed wi t h cyt oki nes, DAHP, and pt er i di nes i n DMEM( see above) i n a f i nal vol ume of 100 Al / wel l f or 48- 96 h . I n some exper i ment s, t he i nf l uence of L- ar gi ni nedef i ci ent medi um, of 250 AmNMA, of 10 MML- or D- ar gi ni ne, and of NMA pl us 10 MM. L- or D- ar gi ni ne was t est ed . For t hi s pur pose, conf l uent monol ayer s wer e pr et r eat ed wi t h t he ment i oned addi t i ves 48 h bef or e addi t i on of cyt oki nes i n t he r espect i ve cul t ur e medi um. Af t er 48- 96 h, medi um was r emoved, and cel l s wer e washed once wi t h 100 pl / wel l of war mPBS and f i xed t hen wi t h 5% gl ut ar al dehyde i n PBS ( 100 A1/ wel l ) f or 10 mi n . Pl at es wer e washed t hr ee t i mes wi t h PBS. Fi xed cel l s wer e st ai ned wi t h 0. 05% met hyl ene bl ue ( Mer ck) f or 5 mi n and washed f i ve t i mes wi t h PBS. Met hyl ene bl ue, t r apped i n f i xed cel l s, was sol ubi l i zed wi t h 100 Al / wel l of 0 . 33 MHCI , and OD at 620 nmwas measur ed usi ng a 96- wel l pl at e r eader ( Ant hos ; Labt ec, Sal zbur g, Aust r i a) . Rel at i ve cel l vi abi l i t y i s expr essed as per cent of unt r eat ed cont r ol cel l s . For each t est ed combi nat i on of f act or s, ei ght wel l s per pl at e wer e t r eat ed i n par al l el . I n compar i son wi t h 24- wel l pl at es ( 2 cmz/ ml ) used i n t he ot her exper i ment s, cyt ot oxi c ef f ect s coul d be obser ved ear l i er ( i . e . , af t er 72- %h) i n %- wel l pl at es ( 0. 32 cmz/ 100 Al ) due t o a di f f er ent cel l t o super nat ant r at i o . N02 - was det er mi ned i n super nat ant s of i ndi vi dual wel l s as descr i bed above. Pr ot ei n Det er mi nat i on. Pr ot ei n cont ent of cel l ext r act s was det er mi ned accor di ng t o Br adf or d ( 21) , modi f i ed f or 96- wel l pl at es, usi ng t he pr ot ei n dye r eagent f r om Bi o- Rad Labor at or i es ( Ri chmond, CA) and BSA ( Ser va) as pr ot ei n st andar d . I ndi vi dual wel l s of cont r ol cel l s cont ai ned 76 . 6 t 7. 7 pg ( mean of 12 cul t ur es ± SD) of pr ot ei n, whi ch was not r educed >10%by cyt oki ne t r eat ment f or 72 h usi ng 24- wel l pl at es and 1 ml of cul t ur e medi um. Pr ot ei n cont ent of 10 5 cel l s was 25 . 4 ± 2 . 5 Wg ( mean of 10 cul t ur es ± SD) , so t hat t he f i nal cel l number per wel l was N3 x 105 . Resul t s I nduct i on of N02 - and NO., - For mat i on i n Mur i ne Fi br obl ast s by Cyt oki nes. Mur i ne f i br obl ast s pr oduced N02 - and N03 - upon t r eat ment wi t h I FN- - y i n a dose- dependent manner ( Fi g. 1) . The ef f ect of I FN- y was about t wo- t o t hr eef ol d enhanced by cot r eat ment wi t h TNF- a, I Ll , or LPS . N02 - r epr esent ed 58 . 2 ± 6 . 3% ( mean of 36 cul t ur es ± SD) of t he t ot al amount of N02 - + N03 - , as cal cul at ed f r om t he val ues gi ven i n Fi g . 1 . TNF- a, I L1, or LPS wer e near l y i nef f ect i ve as si ngl e st i mul i . However , i n t he case of I Ll , 10 U/ ml was suf f i ci ent t o enhance t he I FN- ' y ef f ect , and 100 U/ ml had onl y a sl i ght l y bet t er cost i mul at or y ef f ect ( Fi g . 1) . For LPS, opt i mal cost i mul at i on of 50 U/ ml of I FN- y was achi eved at 100 ng/ ml . LPS doses hi gher t han 100 ng/ ml di d not f ur t her enhance t he ef f ect of I FN- y . Maxi mal pr oduct i on of N02 - was obser ved upon t r eat ment of cel l s wi t h 50 U/ ml I FN- - y i n combi nat i on wi t h 500 U/ ml of TNF- a ( Fi g. 1) . Thi s cyt oki ne combi nat i on was t her ef or e used i n f ur t her exper i ment s. Fi g . 2 shows t he t i me cour se of N02 - and N02 - + N03 - f or mat i on upon st i mul at i on wi t h 50 U/ ml of I FN- y i n combi nat i on wi t h ei t her 500 U/ ml TNF- a, 100 U/ ml I Ll , or 10 Ag/ ml LPS. Despi t e t he cyt ot oxi c ef f ect s of t hese cyt oki nes ( see bel ow) , whi ch can be obser ved af t er >72 h, t her e i s a st eady i ncr ease of N02 - and N02 - + N03 - i n t he super nat ant s over 144 h. Pr ot ei n cont ent of cont r ol cel l s was 82 . 0 t 4. 0 Ag ( mean of t hr ee cul t ur es ± SD) at t he begi nni ng of t he exper i ment , and 80 . 6 t 5 . 0 j i g ( t hr ee cul t ur es) af t er 144 h . Pr ot ei n val ues wer e not decr eased by t r eat ment wi t h cyt oki nes f or 72 h ( I FN- y/ TNF- a, 82 . 0 ± 5 . 3 wg ; I FN- y/ l l r l , 77 . 3 ± 1 . 1 Ag; I FN- y/ LPS, 78 . 0 ± 2 . 0 100 80 0 E 60 z° 40 N z° 20 1601 Wemer - Fel mayer et al . Fi gur e 1 . For mat i on of N02 - ( A) and N02 - + N03 - ( BI by mur i ne f i br obl ast s as a f unct i on of I FN- y concent r at i on and enhancement of t he I FN- - y ef f ect by TNF- a, I Ll and LPS. Conf l uent monol Vmwer e t r eat ed wi t h var i ous doses of I FNJy ei t her al one ( 0) or i n combi nat i on wi t h 500 U/ ml of TNF- a ( zyxwvutsrqponml " ) , 10 U/kjim hgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA l ( 0) , or 100- U/ ml ( 0) of I b1, or 100 ng/ ml of LPS ( A) f or 72 h . The r esul t s shown ar e means ± SD of t r i pl i cat e cul t ur es of one of t hr ee si mi l ar exper i ment s i n mi cr omol es per l i t er . N03 - was enzymat i cal l y r educed t o N02 - , whi ch was det ect ed by t he Gr i ess- r eact i on ( see Mat er i al s and Met hods) . t 140 t 120 t 80 t 60 40 t Ag [ t hr ee cul t ur es] ) as compar ed wi t h cont r ol s. Af t er 144 h, an r v20% r educt i on of pr ot ei n cont ent coul d be obser ved i n cyt oki ne- t r eat ed cel l s ( I FN- y/ TNF- a, 62. 6 ± 2 . 3 j i g; I FN- y/ I I , l , 63 . 3 ± 1. 1 Et g; I FN- y/ LPS, 62 . 6 ± 3 . 0 Ag [ t hr ee cul t ur es] ) . Agai n, NO2 - was f or med t o 59 ± 11% ( mean of 48 cul t ur es ± SD) of N02 - + N03 - ( Fi g. 2) . Dependence of t he For mat i on of N02- and NO., - on L- ar gi ni ne. W hen f i br obl ast s wer e st i mul at ed wi t h 5 U/ ml of I FN- y pl us 500 U/ ml of TNF- a i n cul t ur e medi umwi t hout L- ar gi ni ne ( see Mat er i al s and Met hods) , f or mat i on of N02 was r educed t o 1 . 8 ± 0 . 8 Amol / l i t er . I n t he pr esence of L- ar gi ni ne ( 2 . 4 mM) 38 . 5 ± 4 . 0 Amol / l i t er ( mean of si x cul t ur es ± SD) of N02 - was f or med . NO2 - + N03 - was r educed f r om 59 . 3 ± 5 . 1 wmol / l i t er i n t he pr esence of L- ar gi ni ne t o 5 . 5 ± 2 . 7 p, mol / l i t er when st i mul at ed wi t hout L- ar gi ni ne ( mean of si x cul t ur es ± SD, p < 0 . 0001, st udent ' s t t est ) . The L- ar gi ni ne anal ogue NMAi nhi bi t ed cyt oki ne- i nduced f or mat i on of N02 - and N02 - + N03 - i n a dosedependent way ( Fi g. 3) . Al r eady, 25 14M of NMA was suf f i ci ent t o decr ease t he amount of N02 - si gni f i cant l y f r om38 . 4 ± 1. 1 Amol / l i t er t o 28 . 2 ± 0. 7 p, mol / l i t er ( mean of t hr ee cul t ur es ± SD, p < 0 . 001) . Pr oduct i on of N02 + N03 - was i mpai r ed i n par al l el . About 60 AMNMAwas necessar y f or a 50% i nhi bi t i on of ni t r ogen oxi de f or mat i on ( Fi g. 3) . Ef f ect of Modul at i on of Pt er i di ne Synt hesi s on For mat i on of 20 0 Z 0 Fi gur e 2 . Ti me cour se of N02 ( l ef t ) and N02 - + N03 - ( r i ght ) i nduct i on by mur i ne f i br obl ast s t r eat ed wi t h cyt oki ne combi nat i ons . Conf l uent monol ayer s wer e st i mul at ed wi t h 50 U/ ml of I FN- y i n combi nat i on wi t h ei t her 500 U/ ml of TNF- a ( 0) , 100 U/ ml of I bl ( 0) , or 10 wg/ ml of LPS ( 0) . Super nat ant s wer e har vest ed at i ndi cat ed t i me poi nt s, and N02 - and N02 - + N03 - wer e det er mi ned as descr i bed i n Mat er i al s and Met hods, usi ng t he Gr i ess r eagent . Val ues ar e means of t r i pl i cat e cul t ur es t SD f r om one of t wo si mi l ar exper i ment s . r ever sed t he ef f ect of DAHP. Fur t her , when gi ven wi t hout DAHP, sepi apt er i n enhanced t he ef f ect of cyt oki ne combi nat i ons on N02 - f or mat i on by 20- 40%. I n t he case of I FN- y al one, no such st i mul at i on was obser ved ( Tabl e 1) . Unconj ugat ed pt er i di nes not yi el di ng t et r ahydr obi opt er i n ( 2) , such as pt er i n 6- car boxyl i c aci d and i soxant hopt er i n, di d not i nf l uence cyt oki ne- i nduced N02- f or mat i on or t he ef f ect s of DAHP ( Tabl e 1) . Tabl e 2 r el at es t hese f i ndi ngs t o i nt r acel l ul ar bi opt er i n concent r at i ons . Tr eat ment of f i br obl ast s wi t h I FN- y/ TNF- a r esul t ed i n an about t hr eef ol d i ncr ease of i nt r acel l ul ar t et r ahydr obi opt er i n l evel s. Sepi apt er i n was conver t ed i nt o t et r ahydr obi opt er i n i n unt r eat ed cont r ol cel l s as wel l as i n cel l s t r eat ed wi t h I FN- y/ TNF- a . Thi s conver si on occur s due t o sepi apt er i n r educt ase and f ur t her r educt i on of 7, 8- di hydr o- 0 s E 0 E 0 z 0 Z 0 Z N02 - and N02- + N03 - . Dat a gi ven i n Tabl e 1 demonst r at e t hat f or mat i on of N02 - upon t r eat ment wi t h I FN- y al one or i n combi nat i on wi t h TNF- ci , I I Jl , or LPS was si gni f i cant l y decr eased by t r eat ment of cel l s wi t h DAHP Thi s compound i nhi bi t s GTP- cycl ohydr ol ase I , t he key enzyme of t et r ahydr obi opt er i n synt hesi s ( 22) . The ef f ect of I FN- y on N02 - f or mat i on was t her eby decr eased t o 22% ( p < 0 . 0001) , t he ef f ect of combi ned st i mul i was r educed t o 130- 50% ( p < 0. 001 f or I FN- y/ I I r 1 and I FN- y/ LPS; p < 0 . 0001 f or I FN- y/ TNF- a) . Addi t i on of 100 AMsepi apt er i n, whi ch pr ovi des t et r ahydr obi opt er i n vi a a sal vage pat hway ( 2) , 1602 NMA ( umol / 1) Fi gur e 3 . Ef f ect of NMA on N02 - ( O) and N02 - + N03 - ( 0) pr oduct i on i n cyt oki ne- t r eat ed mur i ne f i br obl ast s . Conf l uent monol ayer s wer e pr et r eat ed wi t h di f f er ent concent r at i ons of NMA f or 24 h and t hen st i mul at ed by 50 U/ ml of I FN- y pl us 500 U/ ml of TNF- a . Af t er 72 h, N02 - and N02 - + N03 - wer e det ect ed i n super nat ant s by t he Gr i ess r eact i on ( see Mat er i al s and Met hods) . One of t wo exper i ment s showi ng t he mean of t r i pl i cat e cul t ur es : t SD i s pr esent ed her e . Bi opt er i n- dependent For mat i on of Ni t r ogen Oxi des i n Fi br obl ast s I nf l uence of DAHP, Sepi apt er i n ( SP) , Pt er i n 6- car boxyl i c Aci d ( PCA) , and I soxant hopt er i n ( I X) on Cyt oki ne- i nduced For mat i on of N0 2 Tabl e 1 . N02- 50 U/ ml I FN- , y 50 U/ ml I FN- , y 50 U/ ml I FN- y I FN- y + 500 U/ ml TNF- a + 500 U/ ml I L- 1 + 10 hg/ ml LPS None 18 . 3±0 . 6 38 . 8±1 . 4 39 . 5±2 . 6 34 . 2±1 . 7 SP" 16 . 1± 1 . 0 57 . 9±2. 9 53 . 0±6 . 1 40. 8±1 . 3 4.1 ± 0. 5 12 . 0 ± 1 . 6 18 . 2 ± 1 . 0 16 . 6 ± 1 . 0 36. 9 t 500 U/ ml Addi t i ve Pmol / l i t er DAHP± 17. 0 ± 1 . 2 49 . 1 ± 0. 5 38 . 2 ± 3 . 1 PCAS ND 34 . 0 ± 0. 4 ND ND PCA + DAHP ND 13 . 1 t 1 . 3 ND ND I XI ND 34 . 9 ± 5 . 5 ND ND I X + DAHP ND 12 . 0 ± 0 . 5 ND ND SP + DAHP 1. 8 Conf l uent monol ayer s wer e t r eat ed wi t h DAHP, pt er i di nes, or bot h i n t he pr esence of I FN- ,y al one or i n combi nat i on wi t h TNF- a, I L- 1, or LPS f or 72 h . N02- was det er mi ned i n super nat ant s as det ai l ed i n Mat er i al s and Met hods . Val ues ar e means of t r i pl i cat e cul t ur es ± SD f r om one of t hr ee si mi l ar exper i ment s . Cont r ol cel l s di d not pr oduce any det ect abl e N02- when t r eat ed wi t h pt er i di nes or DAHP. ' 100 AM sepi apt er i n . t 5 mMDAHP. S 100 AMpt er i n 6- car boxyl i c aci d. n 100 PM i soxant hopt er i n . Tabl e 2. I nf l uence of DAHP, Met hot r exat e ( MTX) , and Sepi apt er i n ( SP) on Synt hesi s of Bi opt er i n and N0 2 i n Cyt oki ne- t r eat ed Mur i ne Fi br obl ast s Tr eat ment + N03- Tot al i nt r acel l ul ar bi opt er i n N02- + NO3i n super nat ant s pmol / mg amol / l i t er 18 . 7 ± 1 . 0 ( 96 . 1 t 5 . 7) ND SP" 5, 358 . 0 t 413 . 4 ( 94 . 0 t 0 . 5) ND DAHP# + SP 3, 801 . 3 t 149 . 6 ( 89 . 9 ± 0 . 1) ND 18 . 7 t 1 . 6 ( 56 . 9 ± 4 . 5) ND None MTXS I FN- y/ TNF- a 11 I FN- y/ TNF- a+SP 57 . 9 t 3 . 0 ( 89. 9 t 2 . 1) 127 . 2 t 11 . 6 1, 661 . 5 t 239 . 9 ( 95 . 5 ± 1 . 4) 197 . 8 t 17 . 3 24 . 4 t 4 . 3 ( >98) I FN- y / TNF- a+DAHP I FN- y/ TNF- a +DAHP+SP I FN- y/ TNF- a + MTX 72 . 7 t 8 . 1 1, 757 . 8 ± 541 . 2 ( 91 . 8 ± 4 . 0) 131 . 0 t 13 . 3 53 . 5 t 1 . 0 ( 73 . 5 ± 3 . 0) 124 . 2 t 15 . 2 70 . 0 t 15 . 9 153 . 4 t 27 . 7 ( 18 . 4 t 8 . 4) I FN- y / TNF- a+MTX+SP 16 . 0 t 5 . 5 ( 81 . 1 t 0 . 9) I FN- y/ TNF- a + DAHP + MTX 180. 3 ± 2 . 7 ( 14 . 0 t I FN- y/ TNF- a + DAHP + MTX+ SP 11 . 9) 62 . 6 ± 14 . 0 20 . 1 t 5 . 6 Conf l uent monol ayer s, gr own i n 25- cmz cul t ur e f l asks, wer e t r eat ed wi t h var i ous addi t i ves and cyt oki nes f or 72 h. Tot al i nt r acel l ul ar bi opt er i n was det er mi ned wi t h HPLC af t er i odi ne oxi dat i on at aci di c pH. The amount of bi opt er i n pl us 7, 8- di hydr obi opt er i n was assessed by i odi ne oxi dat i on at al kal i ne pH. Val ues i n par ant heses gi ve t he per cent age of t et r ahydr obi opt er i n, whi ch was cal cul at ed f r om t he r at i o of t ot al bi opt er i n t o bi opt er i n pl us 7, 8- di hydr obi opt er i n . Ni t r ogen oxi des wer e det er mi ned i n super nat ant s . For exper i ment al det ai l s, see Mat er i al s and Met hods . N02- i n super nat ant s was 47 t 10%of N02- + N03 - ( mean of 36 cul t ur es t SD) . Val ues ar e means of t r i pl i cat e cul t ur es ± SD f r om one of t wo exper i ment s . " 100 14M sepi apt er i n . t 5 mM DAHP. S 10 AMmet hot r exat e pl us 40 AM t hymi ne and 100 PM i nosi ne . 150 U/ ml I FNJy + 500 U/ ml TNF- a. 1603 Wer ner - Fel mayer et al . Tabl e 3. I nf l uence of DAHP, Sepi apt er i n, and Ar gi ni ne on Cel l Vi abi l i t y of Mur i ne Fi br obl ast s Tr eat ed wi t h Cyt oki nes Rel at i ve cel l vi abi l i t y af t er : Tr eat ment 48 h 72 h 96 h umol / l i t er 70 None NOZ - af t er 96 h 100 t 9 100±7 100± 13 3. 5±1 . 4 I FN- , y/ TNF- a' 86 t 8 73±4 45 t 6 57 . 4±3 . 9 I FN- y/ TNF- a + SP 80 t 6 75±10 50 t 6 83 . 0±6 . 7 I FN- y/ TNF- a + DAHPS 91 t 6 89 t 6 70 t 7 21 . 3±3. 2 I FN- y/ TNF- a + SP + DAHP 77±5 65±4 54 t 6 69 . 1±9 . 4 I FN- y/ TNF- a + NMAI I 81 ± 4 71 ±3 78 ± 7 15 . 0 ± 2. 3 I FN- y/ TNF- a + NMA + L- ARG 77±12 68 t 9 47 t 2 65 . 9±3 . 1 I FN- y/ TNF- a + NMA + D- ARG" 94 t 6 95±5 88 t 4 9 . 3±2 . 4 I FN- y/ TNF- a + L- ARG## 70 t 3 55 t 2 22 t 5 71 . 4±5 . 4 I FN- y/ TNF- a + D- ARGSS 94±6 94 t 5 89 t 3 8 . 8±1 . 2 I FN- y/ TNF- a wi t hout L- ARGI I I I 86 t 6 88 t 5 89 t 3 3. 9±1 . 2 Conf l uent monol ayer s of mur i ne f i br obl ast s wer e gr own i n 96- wel l pl at es and t r eat ed wi t h I FN- y/ TNF- a i n absence or pr esence of DAHP, sepi apt er i n ( SP) , hi gh doses of L- or D- ar gi ni ne wi t h or wi t hout NMA, and i n L- ar gi ni ne- def i ci ent medi um. At t he i ndi cat ed t i me poi nt s, cel l s wer e st ai ned wi t h met hyl ene bl ue, and OD at 620 nm was det er mi ned af t er sol ubi l i zi ng t he dye wi t h 0 . 33 MHCl ( see Mat er i al s and Met hods) . Val ues ar e expr essed as per cent of vi abl e cel l s r el at i ve t o unt r eat ed cont r ol s ( =100%a) and gi ve t he mean of ei ght wel l s ± SD det er mi ned i n one of t hr ee si mi l ar exper i ment s . Cont r ol s af t er 96 h wer e 112 t 7 f or sepi apt er i n, 108 ± 7 f or DAHP, 96 ± 7 f or NMA, 94 t 4 f or NMA pl us 10 MML- ar gi ni ne, 98 ± 7 f or NMA pl us 10 MMD- ar gi ni ne, 102 ± 6 f or 10 MML- ar gi ni ne, 96 ± 5 f or 10 MMD- ar gi ni ne, and 95 t 7 f or L- ar gi ni ne- def i ci ent medi um. N02 - i n t he r espect i ve wel l s was det er mi ned usi ng t he Gr i ess r eact i on . " 50 U/ ml I FN- y + 500 U/ ml TNF- a. # + 100 AM sepi apt er i n . S + 5 m M DAHP. II + 250 AMNMA. I + 250 AM NMA + 10 MM. L- ar gi ni ne. + 250 AMNMA + 10 mMD- ar gi ni ne . ## + 10 MM L- ar gi ni ne. SS + 10 MM D- ar gi ni ne . I I N St i mul at ed i n L- ar gi ni ne- def i ci ent medi um. bi opt er i n i nt o t et r ahydr obi opt er i n by di hydr of ol at e r educt ase ( EC 1 . 5 . 1 . 3) ( 2) . DAHP r educed i nt r acel l ul ar t et r ahydr obi opt er i n l evel s by - 50%. Concur r ent addi t i on of DAHP and sepi apt er i n r esul t ed i n hi gh i nt r acel l ul ar t et r ahydr obi opt er i n l evel s, r est or i ng t he i nhi bi t i on of ni t r ogen oxi de f or mat i on by DAHP. Thi s r ever sal was i nhi bi t ed by met hot r exat e, whi ch i nt er f er es wi t h f or mat i on of t et r ahydr obi opt er i n f r om sepi apt er i n by i nhi bi t i on of di hydr of ol at e r educt ase ( 2) , t hus l eadi ng t o t he accumul at i on of di hydr obi opt er i n. I nt r acel l ul ar accumul at i on of di hydr obi opt er i n even had an i nhi bi t or y ef f ect on synt hesi s of N02 - and N02 - + N03 - , as can be seen f r om compar i son of t he ef f ect s of met hot r exat e al one or t oget her wi t h sepi apt er i n i n I FN- y/ TNF- t x- st i mul at ed cel l s ( p < 0 . 01) ( Tabl e 2) . Thi s may occur due t o i nhi bi t i on of GTP- cycl ohydr ol ase I by di hydr obi opt er i n ( 23) . As a consequence, t he combi nat i on of DAHP, met hot r exat e, and sepi apt er i n had t he hi ghest i nhi bi t i ng pot ency. Cel l Vi abi l i t y. Cyt oki ne- i nduced f or mat i on of ni t r ogen oxi des l eads t o i nhi bi t i on of aconi t ase ( EC 4 . 2 . 1 . 3) i n t umor 1604 cel l s ( 24) as wel l as i n act i vat ed macr ophages ( 15, 25) . Fur t her , i t was r epor t ed pr evi ousl y, t hat I FN- y al one or i n combi nat i on wi t h I I r l , TNF- ci , or LPS i s cyt ot oxi c f or mur i ne f i br obl ast s ( 26, 27) . We t her ef or e st udi ed t he ef f ect of I FNy/ TNF- a on cel l vi abi l i t y i n t he absence or pr esence of DAHP or sepi apt er i n ( Tabl e 3) . Af t er i ncubat i on f or 96 h wi t h I FNy/ TNF- a, a decr ease of cel l vi abi l i t y t o - 50%became appar ent . The same was obser ved wi t h I FN- y i n combi nat i on wi t h I L. 1 or LPS, and was l ess pr onounced wi t h I FN- y al one ( not shown) . Tabl e 3 shows t hat t he cyt ot oxi c ef f ect of I FN- y/ TNF- oc coul d be par t i al l y r ever sed by si mul t aneous t r eat ment wi t h DAHP ( p < 0 . 01) . Concur r ent t r eat ment wi t h sepi apt er i n r est or ed t he cyt ot oxi c ef f ect of I FN- y/ TNF- a, t hus par al l el i ng t he ef f ect s on N02 - f or mat i on . On cont r ol cel l s, nei t her DAHP nor sepi apt er i n coul d i nf l uence cel l vi abi l i t y ( Tabl e 3) . The cyt ot oxi c ef f ect of I FN- y/ TNF- ac was al so r educed by t r eat ment wi t h NMA or by wi t hdr awal of L- ar gi ni ne . Thi s ef f ect of NMA was r ever sed by addi t i on of L- ar gi ni ne but not by D- ar gi ni ne ( Tabl e 3) . Fur t her , addi t i on Bi opt er i n- dependent For mat i on of Ni t r ogen Oxi des i n Fi br obl ast s of 10 mML- ar gi ni ne but not of D- ar gi ni ne t o cyt oki ne- t r eat ed cel l s i n absence of NMA r esul t ed i n t he hi ghest cyt ot oxi c ef f ect ( Tabl e 3) ( p < 0. 0001 as compar ed wi t h NMA pl us 10 MML- ar gi ni ne) . Cel l vi abi l i t y was si gni f i cant l y cor r el at ed t o N02 - l evel s i n super nat ant s of t he r espect i ve wel l s ( Tabl e 3) , as det er mi ned by l i near r egr essi on anal ysi s ( R = - 0 . 78, p < 0. 000001 ; 144 sampl es) and Spear man Rank cor r el at i on ( R = - 0. 84, p < 0. 000001 ; 144 sampl es) . Di scussi on I n t hi s r epor t , we demonst r at e t hat mur i ne f i br obl ast s ar e capabl e t o pr oduce N02 - and N03 - f r om L- ar gi ni ne upon st i mul at i on wi t h I FN- y . Thi s ef f ect of I FN- y i s enhanced up t o t hr eef ol d by cost i mul at i on wi t h TNF- cr , I LI , or LPS. So f ar , cyt oki ne- i nduced f or mat i on of ni t r ogen oxi des has been obser ved i n mur i ne macr ophages, mur i ne macr ophage cel l l i nes ( 12- 15, 28) , r at Kupf f er cel l s, r at hepat ocyt es ( 29, 30) , and mur i ne endot hel i al cel l s ( 31) . As i n mur i ne macr ophages, N02 - i s f or med i n f i br obl ast s t o - 50% of t he t ot al l y det ect abl e N02 - + N03 - ( 13) . Fur t her , f i br obl ast s pr oduce N02 - and N03 - t o an ext ent compar abl e wi t h t hat f ound f or macr ophages ( 13) , macr ophage cel l l i nes ( 28) , and endot hel i al cel l s ( 31) . I n mur i ne macr ophages, t he ef f ect of I FN- y on f or mat i on of ni t r ogen oxi des was enhanced by TNF- a, TNF- f l , or LPS but , i n cont r ast t o our r esul t s wi t h f i br obl ast s, not by I M( 14, 15) . The si gni f i cance of t hi s di f f er ence i s uncl ear . N02 - and N03 - or i gi nat e f r om t he oxi dat i on of L- ar gi ni ne t o ci t r ul l i ne . Thi s r eact i on can be i nhi bi t ed by t he L- ar gi ni ne anal ogue NMA ( 32, 33) . The unst abl e i nt er medi at e f or med i ni t i al l y i s ni t r i c oxi de, whi ch i s conver t ed t o N02 and N03- i n t he pr esence of oxygen ( 34) . Ni t r i c oxi de has been i dent i f i ed as a cyt ot oxi c ef f ect or mol ecul e of act i vat ed mur i ne macr ophages ( 35, 36) . Recent l y, i t was shown i n pr epar at i ons f r om cyt osol of act i vat ed macr ophages t hat oxi dat i on of L- ar gi ni ne r equi r es t et r ahydr obi opt er i n ( 8, 9) . Tet r ahydr obi opt er i n was suggest ed t o t ake par t i n t he i ni t i al st ep of t he r eact i on, r esul t i ng i n N- hydr oxyl at i on of L- ar gi ni ne ( 8, 9) . I t was f ur t her shown t hat mur i ne macr ophages cont ai n t et r ahydr obi opt er i n ( 8, 9) . We show her e f or mur i ne f i br obl ast s t hat cyt oki ne- i nduced f or mat i on of N02 - and N03 - i s par al l el ed by i ncr eased i nt r acel l ul ar l evel s of t et r ahydr obi opt er i n . By i nhi bi t i ng t et r ahydr obi opt er i n de novo synt hesi s, usi ng t he GTP- cycl ohydr ol ase I i nhi bi t or DAHP ( 22) , we coul d demonst r at e t hat t he amount of i nt r acel l ul ar t et r ahydr obi opt er i n i s l i mi t i ng t he f or mat i on of N02 - and N03 - . Resuppl ement i ng t he cof act or by addi t i on of sepi apt er i n, whi ch i s conver t ed i nt o t et r ahydr obi opt er i n i n t he cel l s vi a a sal vage pat hway ( 2) , f ul l y r est or es t he f or mat i on of N02 - and N03 - . Met hot r exat e bl ocks t hi s r est or i ng ef f ect of sepi apt er i n by i nhi bi t i ng di hydr of ol at e r educt ase, whi ch conver t s di hydr obi opt er mf or med f r om sepi apt er i n t o t he t et r ahydr o- der i vat i ve ( 2) , t hus under l i ni ng t hat onl y t he t et r ahydr oder i vat i ve can ser ve as an act i ve cof act or . Whi l e i nhi bi t i on of t et r ahydr obi opt er i n concent r at i ons by DAHP si gni f i cant l y suppr esses ni t r ogen oxi de f or mat i on, a compar at i vel y hi gh amount of sepi apt er i n, and t hus a hi gh concent r at i on of t et r ahydr obi opt er i n, i s necessar y f or r est or at i on of ni t r ogen oxi de f or mat i on . A possi bl e expl anat i on f or t hi s phenomenon coul d be t hat t et r ahydr obi opt er i n gener at i on f r omexogenousl y suppl i ed sepi apt er i n may t ake pl ace i n a compar t ment di f f er ent f r om t he si t e of ar gi ni ne hydr oxyl at i on, wher eas endogenous t et r ahydr obi opt er i n synt hesi s may suppl y t et r ahydr obi opt er i n di r ect l y t o t he hydr oxyl at i on si t e. Ot her unconj ugat ed pt er i di nes t hat cannot be conver t ed t o t et r ahydr obi opt er i n due t o t he l ack of t he 1, 2' - di hydr oxypr opyl si de chai n at posi t i on 6 of t he pt er i n r i ng ( 2) coul d not i nf l uence f or mat i on of ni t r ogen oxi des and t he ef f ect of DAHP Sepi apt er i n al so enhanced t he ef f ect of I FN- y i n combi nat i on wi t h TNF- a, I M, or LPS, but not wi t h I FN- y al one. Thus, i t seems t hat i n pr esence of dual act i vat i on st i mul i , t he r eact i on f or mi ng N02 - i s not sat ur at ed wi t h t et r ahydr obi opt er i n, al t hough i t s synt hesi s i s enhanced i n par al l el . Taken t oget her , t he r esul t s obt ai ned wi t h DAHP, sepi apt er i n, and met hot r exat e gi ve evi dence t hat not onl y i n cyt osol pr epar at i ons ( 8, 9) , but al so i n i nt act cel l s, t et r ahydr obi opt er i n i s essent i al f or t he pr oduct i on of ni t r ogen oxi des . The cyt ot oxi c ef f ect of I FN- y i n combi nat i on wi t h TNFLoi , I Ll , or LPS on mur i ne f i br obl ast s can be at t enuat ed by r educi ng f or mat i on of ni t r ogen oxi des vi a i nhi bi t i on of L- ar gi ni ne hydr oxyl at i on wi t h NMA, vi a wi t hdr awal Of L- ar gi ni ne, or vi a i nhi bi t i on of t et r ahydr obi opt er i n synt hesi s wi t h DAHP Addi t i on of L- ar gi ni ne and sepi apt er i n, r espect i vel y, r est or es t he cyt ot oxi c pot ent i al of cyt oki ne t r eat ment . These dat a i ndi cat e t hat f or mat i on of ni t r ogen oxi des t akes par t i n t he pr evi ousl y r epor t ed cyt ot oxi c act i on of I FNJy wi t h TNF- oc, I L1, or LPS ( 26, 27) on mur i ne f i br obl ast s. Thus, i t i s not onl y possi bl e t o i nf l uence f or mat i on of N02 - and N03 - by modul at i on of pt er i di ne synt hesi s, but al so, t he bi ol ogi cal ef f ect of ni t r ogen oxi des on f i br obl ast s. The pr esent ed dat a suggest t hat i n mur i ne f i br obl ast s a maj or goal f or cyt oki ne- i nduced pt er i di ne synt hesi s i s t o pr ovi de cel l s wi t h an essent i al cof act or f or t he f or mat i on of ni t r ogen oxi des f r om L- ar gi ni ne. Ni t r i c oxi de, t he i ni t i al pr oduct of t he oxi dat i on of L- ar gi ni ne, exhi bi t s a mul t i t ude of bi ol ogi cal act i ons ( 37, 38) . I t r emai ns t o be seen whet her al so i n human cel l s cyt oki ne- i nduced pt er i di ne synt hesi s ( 1, 3- 7) i s r el at ed t o oxi dat i on of L- ar gi ni ne and f or mat i on of ni t r ogen oxi des. Thi s wor k was suppor t ed by t he Aust r i an Resear ch Funds " Zur For der ung der wi ssenschaf t l i chen For schung" ( P 6922) . 1605 Wer ner - Fel mayer et al . Addr ess cor r espondence t o Hel mut Wacht er , Depar t ment of Medi cal Chemi st r y andBi ochemi st r y Uni ver si t y of I nnsbr uck, Fr i t z- Pr egl - St r . 3, A- 6020 I nnsbr uck, Aust r i a . Recei ved f or publ i cat i on 14 May 1990 and i n r evi sed f or m 4 Sept ember 1990. Ref er ences 1 . Wer ner , E. R. , G. Wer ner - Fel mayer , D. Fuchs, A. Hausen, G. Rei bnegger , and H. Wacht er . 1989 . Par al l el i nduct i on of t et r ahydr obi opt er i n bi osynt hesi s and i ndol eami ne 2, 3- di oxygenase act i vi t y i n human cel l s and cel l l i nes by i nt er f er on- y . Bi ochem. J. 262: 861. 2. Ni chol , C. A. , G. K. Smi t h, and D.S. Duch. 1985 . Bi osynt hesi s and met abol i sm of t et r ahydr obi opt er i n and mol ybdopt er i n . Annu. Rev. Bi ochem. 54 : 729 . 3. Wer ner , E. R. , G. Wer ner - Fel mayer , D. Fuchs, A. Hausen, G. Pf l ei der er , and H. Wacht er . 1990 . Rei bnegger , J . J . Yi m, W Tet r ahydr obi opt er i n bi osynt het i c act i vi t i es i n human macr ophages, f i br obl ast s, THP- 1 and T- 24 cel l s . J. Bi ol . Chem. 265 : 3189 . 4. Wer ner - Fel mayer , G., E. R. Wer ner , D. Fuchs, A. Hausen, G. Rei bnegger , and H. Wacht er . 1989. Tumour necr osi s f act or al pha andl i popol ysacchar i de enhance i nt er f er on- i nduced t r ypt ophan degr adat i on and pt er i di ne synt hesi s i n human cel l s . Bi ol . Chem. Hoppe- Seyl er. 370: 1063 . 5. Wer ner - Fel mayer , G., E. R. Wer ner , D. Fuchs, A. Hausen, G. Rei bnegger , and H. Wacht er . 1990. Neopt er i n f or mat i on and t r ypt ophan degr adat i on by a human myel omonocyt i c cel l l i ne ( THP- 1) upon cyt oki ne t r eat ment . Cancer Res. 50 : 2863 . . P. 6. Fuchs, D., A. Hausen, G. Rei bnegger , E. R. Wer ner , M Di er i ch, and H. Wacht er . 1988 . Neopt er i n as a mar ker f or act i vat ed cel l - medi at ed i mmuni t y : appl i cat i on i n HI Vi nf ect i on. I mmunol . Today. 9: 150 . 7. Wacht er , H. , D. Fuchs, A. Hausen, G. Rei bnegger , and E. R. Wer ner . 1989 . Neopt er i n as mar ker f or act i vat i on of cel l ul ar i mmuni t y : i mmunol ogi c basi s and cl i ni cal appl i cat i on . Adv. Cl i n. Chem. 27 : 81. 8 . Tayeh, M. A. , and M. A. Mar l et t a. 1989 . Macr ophage oxi dat i on of I r ar gi ni ne t o ni t r i c oxi de, ni t r i t e, and ni t r at e. J. Bi ol . Chem. 264 : 19654 . 9. Kwon, N. S. , C. F. Nat han, and D. J. St uehr . 1989 . Reduced bi opt er i n as a cof act or i n t he gener at i on of ni t r ogen oxi des by mur i ne macr ophages. J. Bi ol . Chem. 264: 20496 . 10 . Kauf man, S. 1986 . The met abol i c r ol e of t et r abydr obi opt er i n . I n Chemi st r y and Bi ol ogy of Pt er i di nes . B. A. Cooper and V. M. Whi t ehead, edi t or s . Wal t er De Gr uyt er , Ber l i n, New Yor k . 185- 200 . 11 . Soodsma, J. F. , C. Pi ant adosi , and F. Snyder . 1972 . Par t i al char act er i zat i on of t he al kyl gl ycer ol cl eavage enzyme syst emof r at l i ver . J. Bi ol . Chem. 247: 3923 . 12 . St uehr , D.J . , and M.A. Mar l et t a. 1985 . Mammal i a n ni t r at e bi osynt hesi s : Mouse macr ophages pr oduce ni t r i t e and ni t r at e i n r esponse t o Escher i chi a col i l i popol ysacchar i de . Pr oc. Nat l . Acad. Sci . USA. 82 : 7738 . 13 . St uehr , D.J . , and M.A. Mar l et t t a. 1987 . I nduct i onof ni t r i t e/ ni t r at e synt hesi s i n mur i ne macr ophages by BCGi nf ect i on, l ymphoki nes or i nt er f er on- gamma . J. I mmunol . 139 : 518 . 14 . Di ng, A. H. , C. F. Nat han, and D. J . St uehr . 1988 . Rel eas e of r eact i ve ni t r ogen i nt er medi at es and r eact i ve oxygen i nt er medi at es f r om mouse per i t oneal macr ophages. J. I mmunol . 141 : 2407 . 1606 15 . Dr api er , J . C. , J . Wi et zer bi n, andj . B. Hi bbs. 1988 . I nt er f er on,y and t umor necr osi s f act or i nduce t he Dar gi ni ne- dependent cyt ot oxi c ef f ect or mechani smi n mur i ne macr ophages. Eur J. I mmunol . 18 : 1587. 16 . Wong, G. HW , I . Cl ar k- Lewi s, A. WHar r i s, andJW Schr ader. 1984 . Ef f ect of cl oned i nt er f er on- y on expr essi on of H- 2 and I a ant i gens on cel l l i nes of hemopoi et i c, l ymphoi d, epi t hel i al , f i br obl ast i c and neur onal or i gi n . Eur . J. I mmunol . 14: 52 . 17 . War r en, M . K. , and S. N. Vogel . 1985 . Bone mar r ow- der i ved macr ophages : devel opment and r egul at i on of di f f er ent i at i on mar ker s by col ony- st i mul at i ng f act or and i nt er f er ons. J. I mmunol . 134 : 982. 18 . Gr een, L. C. , D. A. Wagner , J . Gl ogowski , P. L . Ski pper , J . S. Wi shnok, and S. R. Tannenbaum. 1982 . Anal ysi s of ni t r at e, ni t r i t e and ( ' I N) ni t r at e i n bi ol ogi cal f l ui ds . Anal . Bi ochem. 126 : 131. 19 . Fukushi ma, T. , and J C. Ni xon . 1980 . Anal ysi s of r educed f or ms of bi opt er i n i n bi ol ogi cal t i ssues and f l ui ds . Anal . Bi ochem. 102 : 176 . 20 . Wer ner , E. R. , D. Fuchs, A. Hausen, G. Rei bnegger , and H. Wacht er . 1987 . Si mul t aneous det er mi nat i on of neopt er i n and cr eat i ni ne i n ser umwi t h sol i d phase ext r act i on and on- l i ne el ut i on l i qui d chr omat ogr aphy . Cl i n. Chem. 33 : 2028 . 21 . Br adf or d, M . M. 1976 . A r api d and sensi t i ve met hod f or t he quant i t at i on of mi cr ogr amquant i t i es of pr ot ei n ut i l i zi ng t he pr i nci pl e of pr ot ei n- dye bi ndi ng. Anal . Bi ochem. 72: 248 . 22 . Gi l , E. M. , J . M. Nel son, and A. D. Sher man . 1978 Bi opt er i n. I I I . Pur i f i cat i on and char act er i zat i on of enzymes i nvol ved i n t he cer ebr al synt hesi s of 7, 8- di hydr obi opt er i n . Neur ochem. Res. 3: 69. 23 . Shen, R. , A. Al am, and Y Zhang. 1988 . I nhi bi t i on of GTPcycl ohydr ol ase I by pt er i ns . Bi ochem. Bi ophys. Aci a . 965: 9 . 24 . Dr api er , J . C. , and J . B. Hi bbs . 1986 . Mur i n e cyt ot oxi c act i vat ed macr ophages i nhi bi t aconi t ase i n t umor cel l s . J. Cl i n. I nvest . 78: 790. 25 . Dr api er , J . C. , andJ . B. Hi bbs . 1988 . Di f f er ent i at i o n of mur i ne macr ophages t o expr ess nonspeci f i c cyt ot oxi ci t y f or t umor cel l s r esul t s i n Dar gi ni ne dependent i nhi bi t i on of mi t ochondr i al i r on sul f ur enzymes i n t he macr ophage ef f ect or cel l s . J. I mmunol . 140: 2829 . 26 . Di j kmans, R. , B. DeCock, H. Her emans, J. VanDamme, and A. Bi l l i au . 1989 . I nt er f er on- - y i s cyt ot oxi c f or nor mal mouse f i br obl ast s : enhancement by t umor necr osi s f act or and i nt er l euki n 1 . Lymphoki ne Res. 8: 25 . 27 . Di j kmans, R. , J . VanDamme, F. Cor net t e, H. Her emans, and A. Bi l l i au. 1990. Bact er i al l i popol ysacchar i de pot ent i at es gamma i nt er f er on- i nduced cyt ot oxi ci t y f or nor mal mouse and r at f i br obl ast s. I nf ect . I mmun . 58 : 32 . 28 . St uehr , D.J . , andM . A. Mar l et t a . 1987 . Synt hesi s of ni t r i t e and ni t r at e i n mur i ne macr ophage cel l l i nes . Cancer Res. 47 : 5590. 29 . Bi l l i ar , TR. , R. D. Cur r an, D.J . St uehr , M.A, West , B. G. Bent z, and R. L. Si mmons. 1989 . An L- ar gi ni ne- dependent mechani sm medi at es Kupf f er cel l i nhi bi t i on of hepat ocyt e pr ot ei n synt hesi s i n vi t r o . J. Exp Med. 169 : 1467 . Bi opt er i n- dependent For mat i on of Ni t r ogen Oxi des i n Fi br obl ast s 30. Cur r an, R. D. , T. R. Bi l l i ar , DJ . St uehr , K. Hof mann, and R. L . Si mmons . 1989. Hepat ocyt e s pr oduce ni t r ogen oxi des f r om L- ar gi ni ne i n r esponse t o i nf l ammat or y pr oduct s of Kupf f er cel l s . J. Ex p Med. 170 : 1769 . 31 . Ki l boum, R. G. , and P. Bel l oni . 1990 . Endot hel i al cel l pr oduct i on of ni t r ogen oxi des i n r esponse t o i nt er f er on- gamma i n combi nat i on wi t h t umor necr osi s f act or , i nt er l euki n- 1, or endot oxi n . J. Nad. Cancer I nst . 82 : 772 . 32 . Hi bbs, J . B. , R. R. Tai nt or , and Z. Vavr i n . 1987 . Macr ophage cyt ot oxi ci t y : Rol e f or I r ar gi ni ne dei mi nase and i mi no ni t r ogen oxi dat i on t o ni t r i t e. Sci ence ( Wash . DC) . 235: 473 . 33 . I yengar , R., D.J . St uehr , and M.A. Mar l et t a . 1987. Macr ophage synt hesi s of ni t r i t e, ni t r at e and N- ni t r osami nase : pr ecur sor s and r ol e of t he r espi r at or y bur st . Pr oc. Nad. Acad. Sci . USA. 84 : 6369 . 1607 Wer ner - Fel mayer et al . 34 . Mar l et t a, M.A. , P. S. Yoon, R. I yengar , C. D. Leaf , and J . S. Wi shnok . 1988 . Macr ophage oxi dat i on of Dar gi ni ne t o ni t r i t e and ni t r at e: ni t r i c oxi de i s an i nt er medi at e . Bi ochemi shy. 27 : 8706. 35 . Hi bbs, J. B. , R. R. Tai nt or , Z. Vavr i n, and E. M. Rachl i n . 1988 . Ni t r i c oxi de : a cyt ot oxi c act i vat ed macr ophage ef f ect or mol ecul e. Bi ochem. Bi ophys . Res. Commun . 157 : 87. 36 . St uehr , , D. J. , and C. F. Nat han . 1989 . Ni t r i c oxi de, a macr ophage pr oduct r esponsi bl e f or cyt ost asi s and r espi r at or y i nhi bi t i on of t umor t ar get cel l s. J. Ex p. Med. 169: 1543 . 37. Gar t hwai t e, J . , S. L . Char l es, and R. Chess- Wi l l i ams . 1988 . Endot hel i um- der i ved r el axi ng f act or r el ease on act i vat i on of NMDA r ecept or s suggest s r ol e as i nt er cel l ul ar messenger i n t he br ai n . Nat ur e ( Lond. ) . 336 : 385 . 38 . M.A. Mar l et t a . 1989 . Ni t r i c oxi de: bi osynt hesi s and bi ol ogi cal si gni f i cance . Tr ends Bi ochem. Sci . 14: 488 .