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Leukotriene C4 ([3H] - LTC4) binding to membranes isolated from a hamster smooth muscle cell line (DDTIMF2)
L i f e Sciences, Vol. 35, pp. 441-448 Printed in the U.S.A.
Pergamon Press
LEUKOTRIENE C, ([3H] -LTC,) BINDING TO MEMBRANES ISOLATED FROM A H~J~STER SMOOT~ MUSCLE CELL LINE (DDTIMF2) Mike
A. C l a r k , M i c h a e l C o o k , S e y m o u r M o n g , G. Robert Shorr, Jeffrey S t a d e l and S t a n l e y
Kurt Hogaboom, T. C r o o k e
Smith Kline and French Laboratories 1500 Spring Garden Street Philadelphia, PA 19101 (Received in final form May 21, 1984) Summary Leukotriene C 4 (LTCA) has been demonstrated to induce contraction of the smooth muscle cell llne DDTI~IF2. A partially purified membrane fraction obtained from these cells exhibited a high affinity binding site for LTC 4. Binding of [-H~LTC 4 was saturable, specific and reversible with a dissociation constant (KA) of 21 + 4 nM. The maximum number of binding sites~(B m x ) was . 55 _+ 5 -pmol/m~ . . . . . . v of protezn. Speclflclty was demonstrated in competltzon studies3in which the Ki of LTC 4 against specifically bound [ II] -LTC. was 12 nM whereas Leukotriene D. (LTDA) and Leukgtriene E, (LTE 4) had a Ki of 38 $ A and 4.7 + 0.5 nM respectl~vely. A previously described antagonist of leukotriene-induced smooth musc1~ • contraction PFL 55712 had a K. of 23 + 2 nM as determined by competition binding experiments. Leukotrienes, a family of compounds that are metabolites of arachadonic acid, are believed to be important mediators of anaphylaxis (for review see ref i). The peptidoleukotrienes, leukotriene C A (LTC4) , leukotriene D, (LTD 4) and leukotriene E, (LTE.) are potent spasmogens in whole animal~ as 4 4 well as in organ bath systems. The potency of these compounds as well as the discovery of end organ antagonists implies that the physiological effects of leukotrienes are mediated by specific receptors. This concept is supported by the observations of Pong e t al. (2) in which specific LTC~ binding was observed in a membrane preparation obtained from rat lungs. -To date a cell culture system for studying the effect of leukotrienes has not been described. In this report we describe for the first time a cell culture system for studying the effects of LTC A on smooth muscle cells. In addition, a high affinity binding site for LTC. in this cell population is demonstrated and is 4 characterized with regard to molecular specificity, saturability, reversibility. Methods Cell Culture. The smooth muscle cell line DDTIMF2 was derived from a hamster leiomyosarcoma (3). These cells were generously provided by Dr. Jim Norris, University of Arkansas, Little Rock, AR. Cell Contraction. LTC 4 the silicone
In order to demonstrate the responsiveness of these cells to rubber substrate technique of Harris et al. (4) was used. 0024-3205/84 S3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
442
la, u k o t r i e n e
C. B i n d i n ~
Vol.
"35, .No. 4,
1984
Preparation o f .~lembranuH. I , n ~ > ~ r i t h y m i c a l l v :,~rowin~ c e l l s w e r e c o l ! c o t e d by centrilug:ltion a t 50~3 x ~ f o r I0 rain .~t 4 ° (:. Thv c e l l p e l l e t was t h e n lO .?,In,.] S n v b e a n t r v p s i n , i n h i b i t o r , 100 .g/n:1 B e n z a m i d i n e , O . l p e r c e n t 1)FISO, pl! 7.5 a t 4r~(" and h o m o g e n i z e d . A P o t t e r l ] ] v o h j e m hom¢~p,e n i z e r was used an(] the cells disrupted by I0 s t r o k e s . N u c l e ~ "i'~d i n t a c t (ells were: r e m o v e d by centrifugation ;it 900 x g f a r ]f) rain ;it 4 ° C . Ti,c .';UpL.rilatant was t h e n , ' e n t r i . f u g e d a t 2 9 . 0 0 0 x F~ f,~r 2(.) rain ;it 4 ° C . The r e s u l t i n g pellet was resuspended in 100 nFl X,iCl and I0 mN Iris pH 7.5 at a final protein concentration of 10 m~/ml. The membranes were quickly frozen using: liquid nitrogc, n and stored at -70o(: until used. The [ 'II]-L'IC, binding,, was stable for at least 2 months.
[ 3HJ-l.ICA___Bin_d.in_k,~. LiRand binding assays were performed t,sin~ 5 ~ / m l of membrane prntein per nss,'p," tilt)(! unJess ot.herwi~;e staled. Rea('tion buffer contained: 80 m>! S e r i n e - B o r a t e , 10(1 mFl ".;aCl, 5 r.u~l ( ] v c i n e , 20 mM T r i s , pH 7.3 at 22°C. Serine-Borate was i n c l u d e d in t h e rea( t i o n b u f f e r to p r e v e n t ~ o n v e r s i o i ~ of I.TCa i n t o I.TD, and I.TE, ( 5 ) . ,\ t , ~ t a l r e a c t i o n v o l u m e ~,f ]00 ;1 w~s usvd in a l l oxperiplents~except a~ n o t e d f o r tile s a t u r n t i o n bin(linl,~. 'lhe [ - H ] - I , T C , by v a c u u m f i l t r a t i o n t h r o u g h What (;F/C ~ : l a s s f i b e r f i l t e r s . Each -4 . determinatt,m was d o n e in d u p l i c a t e . Filters we, r e i m m e d i a t e l y w a s h e d 3 t i m e s u s i n ~ 3 .ml o f i,:v t t , ! d b u f f e r c o n t a i n i n ~ 10(] nL*l NaCI and 10 mM T r l s , pll 7 . 5 . Saturati(m b i n d i n g wns d o n e a s d e s ( : r i b e d a b o v e e X ( c ' p t t h e r e a c t i o n v o l u b l e was r e d u c e d t o ( ) . l re]. Th(. r e a c t i o n t i m e was 30 rain and a l O 0 0 / f o l d exces.~ nf u n l a b e J e d LTC, was u s e d . The K. and B w e r e d e t e r m i n e d by t h e muth,,d ,~f ml X. Scatchard (6) ..\11 f i l t e r s wer e counte(t in n l i q u i d ~ i n t i l n t i o n c,)unter u s i n a Beckman Hp/B f l u o r . t l i ~ h P e r f o r m a n c e Liquid Chromato.uraj~hv ftlPI.C) Identification o f tile b o u n d l i g a n d in t h e me.mbrane p r e p a r a t i o n was d e t e r m i n e d by tlPI,C u s i n Z a 5 t.m OI)S A ! t e x c o l u m n ,'~t- 3 . 9 x 30 cm in s i z e . Tim m o b i l e p h a s e u s e d was c o m p o s e d o f ]0 mM Xa_PO pH 6 . 7 and a c e t o n i t r i l e . Initial c o n d i t i c m s w e r e 75 n e r c e n t • 3 4. _ !qlosp!~nte b u : f , , r ,'rod 2) p e r c e n t a c e t o n / t r L ! e f,~r 10 r a i n , f o l l o w e d by a ! i n e a r K r a d i e n t in which the acutonitrile , oncentration was i t : ~ r e a s e d t o 35 p e r c e n t o v e r a 10 rain p e r i o d . A m o b i l e p h a s e ~f 63 p e r c e n t p h o s p h a t e b u f f e r and 33 p e r c e n t a c u t o n i t r i l e was t h e n c o n t i n u e d 3 f o r an a d d i t i o n a l I0 rain. A flow r a t e ,,f 1 . 5 ml rn,tn was u s e d t h r o u ' , , h o u t . [ tt }-L1"(:4 was e x t r a c t e d from membrnnes usin~ methanol.
Reagents. Leukotrienes C,, D~ and E, were prepared by total synthesis and were "~ 98 p e r c e n t p u r e as d e t e r m i n e d by I!}LC. b o t h t h e LI'C and I.TE. w e r e r acem] ( : m i x t u r e s c o n t a i n i n p , t h e 5R,6S a n d t h e 5S,6R ~ s o m e r s . ~he FPL 5~712 (7) was o b t a i n e d from Fisot~s P h a r m a c e u t i c a l s . The [ "H ]-L'I'C; was o b t a i n e d f r o m New l : n F ] a n d N u t ] e a r and had a s p e c i t ic a c t i v i t y o f 35 C~/rm"I.
Results
Ce]lular Contraction. Peptidoleukotrienes induce contraction in tissue strips. In o r d e r to d e m onstrate a physiological r e s p o n s e by t h e s e c e i l s t o l.TCz t h e silicone rubber substrate t e c h n i q u e was u s e d . Prior to the addition o~ LTC 4 , few ~ r i n k t e s w e r e o b s e r v e d in t h e s u b s t r a t e ( F i g 1A). The a d d i t i o n o f LTC, ( 1 0 - ~ ) t o t h e medium r e s u l t e d in many w r i n k l e s a p p e a r i n g w h i c h r a d i a t e d fr(~m the cells (Fig IB). This concentration o f LTC 4 a p p e a r e d t o he t h e minimum concentration necessary to elicit sufficient contractile force to wrinkle the s u b- ) s t r a t e , l'retreatment of the cells f o r 15 m i n p r i o r t o a d d i t i o n o f LTC 4 w i t h 10 M FPL 55712 ( F i g IC) i n t d b i t e d the contractile i n r e s p o n s e t o LTC 4 .
Vol. 35, No. 4, 1984
C
Leukotriene C, Bindin?.
443
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The s i l i c o n e r u b b e r s u b s t r a t e was u s e d t o d e t e c t contraction of i n d i v i d u a l DDTIMF2 c e l l s . Control culture~ exhibit relatively few w r i n k l e s i n t h e s u b s t r a t e ( F i g 1A). A d d i t i o n o f 10-'" M LTC,, r e s u l t e d i n many w r i n k l e s a p p e a r ing in the s u b s t r a t e (~lg IB). P r e t n c u b a t t o n w t t h 10 M of t h e l e u k o t r i e n e a n t a g o n i s t FPL 55712 ( F i g IC) a p p e a r e d to inhibit contraction in r e s p o n s e t o LTC~. •
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444
Leukotriene
C~ Binding
Vol.
35, No. 4, 1984
Receptor Bindin 8. The binding of [3H]-LTC4 to membranes prepared from DDTIMF2 cells was rapid and reached steady state in 10 min. When equilibrium was established it was stable for at least 30 min (Fig 2). The reversibility of the binding was demonstrated by adding a 1000 fold excess un~abe3ed I.TC4 after i0 min of reaction time. ~ e resulting dissociation of [ H ] - L T C was 90 percent complete in 6 min (Fig 2). Furthermore, it was demonstrate~ that the amount of [ H]-LTC A bound was dependent on the amount of membrane protein added to the reaction (Fig 3). The slope observed in this experiment was ~ 1 indicating that the membrane protein was the limiting factor in the binding experiments. Scatchard analysis of specific LTC 4 binding in four separate experiments indicated a single high affinity site wlth a K, of 21 + 4 ~i and a B of 55 + 5 pmol/mg of protein ", . -m x -(errors indlcated are the standard deviations~ (Fi~ 4)
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The kinetics of the binding of [3H]-LTC, (5 nM) t~ DDTIMF2 membranes. Non-specific binding ~ o ) of [JH]-LTC 4 was defined as that binding which occurred in the presence of i000 fold excess unlabeled LTC.. The specific binding ( N ) was defined as the difference between the total binding ( m ) and non-specific binding. When a ~000 fold excess of unlabeled LTC. was added 10 min 4 after [JH]-LTC 4 ( • ) the labeled ligand was displaced.
Vol. 35, No. 4, 1984
Leukotriene CL, Binding
445
Inte~rlty of Bound LTC.. LTC. can ben converted to LTD. and LTE., therefore 4 4 ~ 4 it was necessary to demonstrate that the labeled material bound to the membrane under these conditions was LTC 4. To confirm this, HPLC was used to identify the bound material. Fractions were collected at 0.~ mln intervals. A~ least 3 ~ractions were collected between the elutlon of [°H]-LTC. and [ jH]_LTD4 " " 4 Competition Analysis. The ability of various compounds to inhibit binding o-f [~H]-LTC 4 to DDTIMF2 membranes is shown in Fig 6. The results illustrated are the averages of three separate experiments.
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ProtOn f~c:/~ FIC. 3 The total amount of specific binding observed with [3H]-LTC. increased linearly with respect to the amount 4 of membrane protein added to the reaction.
Discussion The recent work of Pong et al. (2) has described a binding system which allows the physical characterization of an LTC A high affinity binding site from tissues. We have adapted these procedures to define a high affinity binding site in a cell culture system. A smooth muscle cell line was chosen because of the large amount of evidence which suggests that leukotrienes induce smooth muscle contraction. The data demonstrate that the DDTIMF2 cells contract in response to I0- M LTC 4. In addition, evidence is presented which initiates the physical and pharmacological characterization of an LTC4 binding site. The results suggest that the K d of the binding site is 21 + 4 nM. It was interest-
4&h
lt,ukotricne C. Binding
Vol.
35, No.
4.
1984
ing that c o n t r a c t i o n of these cells was induced by a L o n c e n t r a t i o n ~,f L'[CI that was similar to tile K d concentrar ion. "lhe existence of a hiEin ,qffinity, saturable b i n d i n g site in this ct~!l line is observt.d. The binding is roversible and maybe inhibited by a p h a r n a c o l o ~ ical antagonist. Tilese d,lta are consistent with the h>'pothes[s that the biological e f f e c t s of LTC 4 may be mediatc'd by specific receptors on smooth muscle c e i l s .
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Vol. 35, No. 4, 1084
leukotri~n~ (i. Bindin~
447
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10 20 30 Retention Time imin~
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FIG. 5 HPLC of bound [311]-LTC.. Approximate]v 1 ug of each ]eukotriene standar~ was added to the sample.
448
Leukotrlene C,, Binding
Vol. 35, No. 4, 1984
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Competition bindin~ experitrent. The contraction 3 of [ Ill -LTC 4 used in this experiment was 5 nM. The reaction was i n i t i a t e d by t h e a d d i t i o n ~,t t h e m e m b r a n e s t o the, r e a c t i o n t u b e and t e r m i n a t c . d ( a f t e r 20 n i n <>f l n c u b a t i ~ m a t 22°C) by v a c u u m f i [ t r n t : o n .
References 1. "2. 3. 4. 5. 6. 7.
R.H. GRt:I:N and P . F . I,~MBETIt, T e t r a h e d r e n 39 ] 6 8 7 - 1 7 2 1 ( 1 9 8 ~ ) S . S . PONG+ R.N. DEtIAVKX, F . A . KUCHL:\ and R.W. ECAN. .T. B i o l . Chem. 9616-9619 (1983). ! . . t ' . CORNETT and J . S . NORRIS, .:. B i o l . Chem. 257 ~ 9 ~ - 6 9 7 ( 1 9 8 2 ) . A.K. tIARRIS, P. W:]I) and I). S T O P , ~ , S ( ' i e n c ' e 208 ] 7 7 - [ 7 9 ( 1 9 8 0 ) . 1. OP~XING and S. HA>DiARSTROM, .:. F.i<:l. Che,!. 255 8 0 2 3 - 8 0 2 6 ( 1 9 8 0 ) . G. SCATCHARD, A - n . N.Y. <\cad. S c i . 51 6 6 0 - 6 7 ? ( 1 9 : 9 ) . R.D. KRELI., B . S . '[SAT, A. BERDOUI,AY, M. BARONI-I and R.l!. (,ILl:S, !>rosta~',landins 25 171-178 (1983).
25.8
Pergamon Press
LEUKOTRIENE C, ([3H] -LTC,) BINDING TO MEMBRANES ISOLATED FROM A H~J~STER SMOOT~ MUSCLE CELL LINE (DDTIMF2) Mike
A. C l a r k , M i c h a e l C o o k , S e y m o u r M o n g , G. Robert Shorr, Jeffrey S t a d e l and S t a n l e y
Kurt Hogaboom, T. C r o o k e
Smith Kline and French Laboratories 1500 Spring Garden Street Philadelphia, PA 19101 (Received in final form May 21, 1984) Summary Leukotriene C 4 (LTCA) has been demonstrated to induce contraction of the smooth muscle cell llne DDTI~IF2. A partially purified membrane fraction obtained from these cells exhibited a high affinity binding site for LTC 4. Binding of [-H~LTC 4 was saturable, specific and reversible with a dissociation constant (KA) of 21 + 4 nM. The maximum number of binding sites~(B m x ) was . 55 _+ 5 -pmol/m~ . . . . . . v of protezn. Speclflclty was demonstrated in competltzon studies3in which the Ki of LTC 4 against specifically bound [ II] -LTC. was 12 nM whereas Leukotriene D. (LTDA) and Leukgtriene E, (LTE 4) had a Ki of 38 $ A and 4.7 + 0.5 nM respectl~vely. A previously described antagonist of leukotriene-induced smooth musc1~ • contraction PFL 55712 had a K. of 23 + 2 nM as determined by competition binding experiments. Leukotrienes, a family of compounds that are metabolites of arachadonic acid, are believed to be important mediators of anaphylaxis (for review see ref i). The peptidoleukotrienes, leukotriene C A (LTC4) , leukotriene D, (LTD 4) and leukotriene E, (LTE.) are potent spasmogens in whole animal~ as 4 4 well as in organ bath systems. The potency of these compounds as well as the discovery of end organ antagonists implies that the physiological effects of leukotrienes are mediated by specific receptors. This concept is supported by the observations of Pong e t al. (2) in which specific LTC~ binding was observed in a membrane preparation obtained from rat lungs. -To date a cell culture system for studying the effect of leukotrienes has not been described. In this report we describe for the first time a cell culture system for studying the effects of LTC A on smooth muscle cells. In addition, a high affinity binding site for LTC. in this cell population is demonstrated and is 4 characterized with regard to molecular specificity, saturability, reversibility. Methods Cell Culture. The smooth muscle cell line DDTIMF2 was derived from a hamster leiomyosarcoma (3). These cells were generously provided by Dr. Jim Norris, University of Arkansas, Little Rock, AR. Cell Contraction. LTC 4 the silicone
In order to demonstrate the responsiveness of these cells to rubber substrate technique of Harris et al. (4) was used. 0024-3205/84 S3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
442
la, u k o t r i e n e
C. B i n d i n ~
Vol.
"35, .No. 4,
1984
Preparation o f .~lembranuH. I , n ~ > ~ r i t h y m i c a l l v :,~rowin~ c e l l s w e r e c o l ! c o t e d by centrilug:ltion a t 50~3 x ~ f o r I0 rain .~t 4 ° (:. Thv c e l l p e l l e t was t h e n lO .?,In,.] S n v b e a n t r v p s i n , i n h i b i t o r , 100 .g/n:1 B e n z a m i d i n e , O . l p e r c e n t 1)FISO, pl! 7.5 a t 4r~(" and h o m o g e n i z e d . A P o t t e r l ] ] v o h j e m hom¢~p,e n i z e r was used an(] the cells disrupted by I0 s t r o k e s . N u c l e ~ "i'~d i n t a c t (ells were: r e m o v e d by centrifugation ;it 900 x g f a r ]f) rain ;it 4 ° C . Ti,c .';UpL.rilatant was t h e n , ' e n t r i . f u g e d a t 2 9 . 0 0 0 x F~ f,~r 2(.) rain ;it 4 ° C . The r e s u l t i n g pellet was resuspended in 100 nFl X,iCl and I0 mN Iris pH 7.5 at a final protein concentration of 10 m~/ml. The membranes were quickly frozen using: liquid nitrogc, n and stored at -70o(: until used. The [ 'II]-L'IC, binding,, was stable for at least 2 months.
[ 3HJ-l.ICA___Bin_d.in_k,~. LiRand binding assays were performed t,sin~ 5 ~ / m l of membrane prntein per nss,'p," tilt)(! unJess ot.herwi~;e staled. Rea('tion buffer contained: 80 m>! S e r i n e - B o r a t e , 10(1 mFl ".;aCl, 5 r.u~l ( ] v c i n e , 20 mM T r i s , pH 7.3 at 22°C. Serine-Borate was i n c l u d e d in t h e rea( t i o n b u f f e r to p r e v e n t ~ o n v e r s i o i ~ of I.TCa i n t o I.TD, and I.TE, ( 5 ) . ,\ t , ~ t a l r e a c t i o n v o l u m e ~,f ]00 ;1 w~s usvd in a l l oxperiplents~except a~ n o t e d f o r tile s a t u r n t i o n bin(linl,~. 'lhe [ - H ] - I , T C , by v a c u u m f i l t r a t i o n t h r o u g h What (;F/C ~ : l a s s f i b e r f i l t e r s . Each -4 . determinatt,m was d o n e in d u p l i c a t e . Filters we, r e i m m e d i a t e l y w a s h e d 3 t i m e s u s i n ~ 3 .ml o f i,:v t t , ! d b u f f e r c o n t a i n i n ~ 10(] nL*l NaCI and 10 mM T r l s , pll 7 . 5 . Saturati(m b i n d i n g wns d o n e a s d e s ( : r i b e d a b o v e e X ( c ' p t t h e r e a c t i o n v o l u b l e was r e d u c e d t o ( ) . l re]. Th(. r e a c t i o n t i m e was 30 rain and a l O 0 0 / f o l d exces.~ nf u n l a b e J e d LTC, was u s e d . The K. and B w e r e d e t e r m i n e d by t h e muth,,d ,~f ml X. Scatchard (6) ..\11 f i l t e r s wer e counte(t in n l i q u i d ~ i n t i l n t i o n c,)unter u s i n a Beckman Hp/B f l u o r . t l i ~ h P e r f o r m a n c e Liquid Chromato.uraj~hv ftlPI.C) Identification o f tile b o u n d l i g a n d in t h e me.mbrane p r e p a r a t i o n was d e t e r m i n e d by tlPI,C u s i n Z a 5 t.m OI)S A ! t e x c o l u m n ,'~t- 3 . 9 x 30 cm in s i z e . Tim m o b i l e p h a s e u s e d was c o m p o s e d o f ]0 mM Xa_PO pH 6 . 7 and a c e t o n i t r i l e . Initial c o n d i t i c m s w e r e 75 n e r c e n t • 3 4. _ !qlosp!~nte b u : f , , r ,'rod 2) p e r c e n t a c e t o n / t r L ! e f,~r 10 r a i n , f o l l o w e d by a ! i n e a r K r a d i e n t in which the acutonitrile , oncentration was i t : ~ r e a s e d t o 35 p e r c e n t o v e r a 10 rain p e r i o d . A m o b i l e p h a s e ~f 63 p e r c e n t p h o s p h a t e b u f f e r and 33 p e r c e n t a c u t o n i t r i l e was t h e n c o n t i n u e d 3 f o r an a d d i t i o n a l I0 rain. A flow r a t e ,,f 1 . 5 ml rn,tn was u s e d t h r o u ' , , h o u t . [ tt }-L1"(:4 was e x t r a c t e d from membrnnes usin~ methanol.
Reagents. Leukotrienes C,, D~ and E, were prepared by total synthesis and were "~ 98 p e r c e n t p u r e as d e t e r m i n e d by I!}LC. b o t h t h e LI'C and I.TE. w e r e r acem] ( : m i x t u r e s c o n t a i n i n p , t h e 5R,6S a n d t h e 5S,6R ~ s o m e r s . ~he FPL 5~712 (7) was o b t a i n e d from Fisot~s P h a r m a c e u t i c a l s . The [ "H ]-L'I'C; was o b t a i n e d f r o m New l : n F ] a n d N u t ] e a r and had a s p e c i t ic a c t i v i t y o f 35 C~/rm"I.
Results
Ce]lular Contraction. Peptidoleukotrienes induce contraction in tissue strips. In o r d e r to d e m onstrate a physiological r e s p o n s e by t h e s e c e i l s t o l.TCz t h e silicone rubber substrate t e c h n i q u e was u s e d . Prior to the addition o~ LTC 4 , few ~ r i n k t e s w e r e o b s e r v e d in t h e s u b s t r a t e ( F i g 1A). The a d d i t i o n o f LTC, ( 1 0 - ~ ) t o t h e medium r e s u l t e d in many w r i n k l e s a p p e a r i n g w h i c h r a d i a t e d fr(~m the cells (Fig IB). This concentration o f LTC 4 a p p e a r e d t o he t h e minimum concentration necessary to elicit sufficient contractile force to wrinkle the s u b- ) s t r a t e , l'retreatment of the cells f o r 15 m i n p r i o r t o a d d i t i o n o f LTC 4 w i t h 10 M FPL 55712 ( F i g IC) i n t d b i t e d the contractile i n r e s p o n s e t o LTC 4 .
Vol. 35, No. 4, 1984
C
Leukotriene C, Bindin?.
443
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The s i l i c o n e r u b b e r s u b s t r a t e was u s e d t o d e t e c t contraction of i n d i v i d u a l DDTIMF2 c e l l s . Control culture~ exhibit relatively few w r i n k l e s i n t h e s u b s t r a t e ( F i g 1A). A d d i t i o n o f 10-'" M LTC,, r e s u l t e d i n many w r i n k l e s a p p e a r ing in the s u b s t r a t e (~lg IB). P r e t n c u b a t t o n w t t h 10 M of t h e l e u k o t r i e n e a n t a g o n i s t FPL 55712 ( F i g IC) a p p e a r e d to inhibit contraction in r e s p o n s e t o LTC~. •
.
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444
Leukotriene
C~ Binding
Vol.
35, No. 4, 1984
Receptor Bindin 8. The binding of [3H]-LTC4 to membranes prepared from DDTIMF2 cells was rapid and reached steady state in 10 min. When equilibrium was established it was stable for at least 30 min (Fig 2). The reversibility of the binding was demonstrated by adding a 1000 fold excess un~abe3ed I.TC4 after i0 min of reaction time. ~ e resulting dissociation of [ H ] - L T C was 90 percent complete in 6 min (Fig 2). Furthermore, it was demonstrate~ that the amount of [ H]-LTC A bound was dependent on the amount of membrane protein added to the reaction (Fig 3). The slope observed in this experiment was ~ 1 indicating that the membrane protein was the limiting factor in the binding experiments. Scatchard analysis of specific LTC 4 binding in four separate experiments indicated a single high affinity site wlth a K, of 21 + 4 ~i and a B of 55 + 5 pmol/mg of protein ", . -m x -(errors indlcated are the standard deviations~ (Fi~ 4)
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The kinetics of the binding of [3H]-LTC, (5 nM) t~ DDTIMF2 membranes. Non-specific binding ~ o ) of [JH]-LTC 4 was defined as that binding which occurred in the presence of i000 fold excess unlabeled LTC.. The specific binding ( N ) was defined as the difference between the total binding ( m ) and non-specific binding. When a ~000 fold excess of unlabeled LTC. was added 10 min 4 after [JH]-LTC 4 ( • ) the labeled ligand was displaced.
Vol. 35, No. 4, 1984
Leukotriene CL, Binding
445
Inte~rlty of Bound LTC.. LTC. can ben converted to LTD. and LTE., therefore 4 4 ~ 4 it was necessary to demonstrate that the labeled material bound to the membrane under these conditions was LTC 4. To confirm this, HPLC was used to identify the bound material. Fractions were collected at 0.~ mln intervals. A~ least 3 ~ractions were collected between the elutlon of [°H]-LTC. and [ jH]_LTD4 " " 4 Competition Analysis. The ability of various compounds to inhibit binding o-f [~H]-LTC 4 to DDTIMF2 membranes is shown in Fig 6. The results illustrated are the averages of three separate experiments.
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ProtOn f~c:/~ FIC. 3 The total amount of specific binding observed with [3H]-LTC. increased linearly with respect to the amount 4 of membrane protein added to the reaction.
Discussion The recent work of Pong et al. (2) has described a binding system which allows the physical characterization of an LTC A high affinity binding site from tissues. We have adapted these procedures to define a high affinity binding site in a cell culture system. A smooth muscle cell line was chosen because of the large amount of evidence which suggests that leukotrienes induce smooth muscle contraction. The data demonstrate that the DDTIMF2 cells contract in response to I0- M LTC 4. In addition, evidence is presented which initiates the physical and pharmacological characterization of an LTC4 binding site. The results suggest that the K d of the binding site is 21 + 4 nM. It was interest-
4&h
lt,ukotricne C. Binding
Vol.
35, No.
4.
1984
ing that c o n t r a c t i o n of these cells was induced by a L o n c e n t r a t i o n ~,f L'[CI that was similar to tile K d concentrar ion. "lhe existence of a hiEin ,qffinity, saturable b i n d i n g site in this ct~!l line is observt.d. The binding is roversible and maybe inhibited by a p h a r n a c o l o ~ ical antagonist. Tilese d,lta are consistent with the h>'pothes[s that the biological e f f e c t s of LTC 4 may be mediatc'd by specific receptors on smooth muscle c e i l s .
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FIC;. 4 S a t u r a t i o n b i n d i n g , and S c a t c h a r d a n a l y s i s . The raw data of a typical experiment is shown, the K, and B was calculated from the data obtained from max separate experiments.
Vol. 35, No. 4, 1084
leukotri~n~ (i. Bindin~
447
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10 20 30 Retention Time imin~
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FIG. 5 HPLC of bound [311]-LTC.. Approximate]v 1 ug of each ]eukotriene standar~ was added to the sample.
448
Leukotrlene C,, Binding
Vol. 35, No. 4, 1984
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Competition bindin~ experitrent. The contraction 3 of [ Ill -LTC 4 used in this experiment was 5 nM. The reaction was i n i t i a t e d by t h e a d d i t i o n ~,t t h e m e m b r a n e s t o the, r e a c t i o n t u b e and t e r m i n a t c . d ( a f t e r 20 n i n <>f l n c u b a t i ~ m a t 22°C) by v a c u u m f i [ t r n t : o n .
References 1. "2. 3. 4. 5. 6. 7.
R.H. GRt:I:N and P . F . I,~MBETIt, T e t r a h e d r e n 39 ] 6 8 7 - 1 7 2 1 ( 1 9 8 ~ ) S . S . PONG+ R.N. DEtIAVKX, F . A . KUCHL:\ and R.W. ECAN. .T. B i o l . Chem. 9616-9619 (1983). ! . . t ' . CORNETT and J . S . NORRIS, .:. B i o l . Chem. 257 ~ 9 ~ - 6 9 7 ( 1 9 8 2 ) . A.K. tIARRIS, P. W:]I) and I). S T O P , ~ , S ( ' i e n c ' e 208 ] 7 7 - [ 7 9 ( 1 9 8 0 ) . 1. OP~XING and S. HA>DiARSTROM, .:. F.i<:l. Che,!. 255 8 0 2 3 - 8 0 2 6 ( 1 9 8 0 ) . G. SCATCHARD, A - n . N.Y. <\cad. S c i . 51 6 6 0 - 6 7 ? ( 1 9 : 9 ) . R.D. KRELI., B . S . '[SAT, A. BERDOUI,AY, M. BARONI-I and R.l!. (,ILl:S, !>rosta~',landins 25 171-178 (1983).
25.8