European Jmtrnal of Phanmwoh*g; - Moire dar Pt arm woh~v Sccmm. 227 (P~'42)427- 43t '~ ltlq2 Elsevier Science Publishers B.V. All righls resetwcd ~q22 -Itlta/92/$05.t1~
-*27
[!J P M O L 9{1378
Effects of endothelins on the human megakars, oblastic cell line MEG-01 Sylvie D i o c h o t % D a n i e l V i t t e t ", M a r i e - N o i i l l e M a t h i e u ", J e a n - M a r i e L a u n a y h a n d C h m d e C h e v i H a r d " "
INSliRM U. 300. I:acultd tie Piutrma~t~'. ,Montpellwr. I"rame. a n d ¢' FP.A ~. laude Berm*rd '%~'¢#rt~chttm: de,, (
i*~lt~tt ~ati
!~ (~,,l]leh;g~, "
th'~pila! 5t~iltl l.oI~t~. Part~. I:rftll~"
Received 15 April Iqq2. revised MS received 2S ]ul~ 1902.accepted tS Augus~ I~C/2
Some cffec:.s of cndoflmlin-I (ET-I) were sludicd on the megaka~'ohiaslic celt line MEG4H. ET-] induced an cte;atkm of the i~:;,accllular !evels of Ca-" ~![Ca-" ~],) as measured ~ith lhe fluorcscem ind;ca~or indo-1. ~hich consists uf an. initial mmsiem increase and an ensuing sustained plaleau. The plateau phase was abolished b~ rcm~3~alof cx'r ,cclhflar ('a: ". In a~ddititm. ET-I induced a rapid (within 5 s) increase in the accumulation of inosilol !.4.5-trispho~0;;alc (h~s~,t.4.'~:' 0 ,rod more dctaycd increases in Ins{I.3.4)P 3 and Ins(I.3,4,5)P a, but did not modify cAMP levels. ET-1 i~o. 1.4/: NazlIPO~. 8; MgCt:, 0,5: Ca('l ,, 0.q: glucose. 5.55. It was adjusted at pit = 7.4. 2.2. Celt cultun" MEG-01 cells wcrc from Dr. 14. Saito, University of Nagoya. Japan. MEG-Ill suspension cultnres were rontinety pax~aged 3 limes weekly in RPM! I¢,.RI mediun~ supplemented with 2 mM glutl, m m c and 10G ( v / x ) heat-inactivcd l~.lal calf serum under a humidified atmosphere containing 5 r ; C O . at 370( ". 2.3. Meaxttrtvmvtl of [('a-'"
],
Cells were Imlvcsted 2-3 days after seediug, c.~ntrifugcd (18fl × g, 5 mint and washed lwiee ~illl ['BS. The washed cells "~e;c resuspended to ill" ccll,/nfl. incubated 2{) rain at 37°C lit tile dark with 5 p.M i Mo-I AM. Loading was termina~.ed by dilution with cold PBS followed by ccnlrifugation. The cell pellet ~ a s resuspcnded ill PBS and aliquots (1t.8-0.8 × 1(1" ce[[s'l werc transferred to a 1 ml quartz cuvcttc equipped for temperature conrail and magnetic stirring. Fluorescence signals (|:) obtained by agents were monitored with excitation and emission wavelengths of 332 aqd 411{t nm. respectively, using a Ktmtron SFM 25 spccImfluorimeter. The cells were subsequently lysed by adding 5() ;.:.M digit.omn m obtain the si,znat of the Ca 2" _~aturatcd dye (F~,.,,). k~,,,,, for the Ca-" "-trce form of tile dye ~ a s recorded by adding 10 mM E ( H A . p | l 8.2. The concentration of intraceiiular ionized calcm,n [('at e. ], was ca!cub|ted accord:.ng to (hynkicwiez el al. (19,R5). using the equation: [ C a : ' ], = K,dF--I:~,,,jF',,,,,- F). with K,~ = 25(! nM All agcnls x~crc added to the quarlz cuvettc in a volume .'ff !tl p t. 2.4. Mcasun'mcn[ it] ip,l~itol IrA- ~im t tetr,.gk&phoslgmh" MKG4)t ceils (8 × I0 ~ ce~ls/mlI were incubated t\w 24 h in culture medium containing ill , u C i / m l myo-12311]it:ositol. After washing v, ilh PBS, ce!!s (6 × ll~' celts pet" t u b O were equilibrated fiar 15 rain aa 37°(" and further incubated f~r various limes at 37':(" ~ithout
(control) or with Ill0 nM ET-I. The reaction was stopped by addition of perchloric acid (SG v / v final concenlration) and the samples were centrifuged. T h e s u p e m a t a n t s were neutralized by 3 M KOII-150 m M HFPES. High-performance liquid chromatography (HPLC) was carried out ,..'-n an ankm-,rxchangc column (Partisil SAX-II)). lno,dtol phosphates were separated using a muhilinear gradient from water to 3 M ammoniunl formate (adjusted It) ptI 3.7 with i-lsPO~). After injection of the sample. ,he co~ .;m was washed Ibr 5 nun (flow late 1.2 , d / , ~ , m ) ~ith water. The grzMicnt of a m m o n i n m fl)rma,.c used w-;:,: 0-0.6 M tot 8 rain: then 0.6 M for 3 rain: 0.6-1 M for 7 min: 1 M over 5 min: 1-2 M fi~r 7 rain; 2 M fi~r 5 rain: finally 2 - 3 M for 7 rain and 3 M for 3 min. Eluales werc collected ever3, 0.5 min and a.~sayed for radioactivity content. Ins( 1,3.4)P~. Ins( 1.4,5)P3 and Ins( 1,3.4.5)P~ were cluted from the c o h m m between 27.5-29JI. 29.5-31.1). and 39.5-41.5 min. respectively. Tritiated derivatives were uscd as standards for the calihralion of the gradient. 2.5. ('yclic A M P assay hltr:|ce]luJar c A M P accumnlatknl was 0,~ernlined hi intact cells by measuring [~H]cAMP Uion after prclabcling the endogenous ATP poe.,!'-. ~4::~G-()l cells (approximately 5 × I( c e l l s / m . ) were labeled by incu-bafion in the culture medium with I /.tCi/ml of [2.8;H]adenine fl~r 4 h at 37°C in lhc cell incubator. Unincorporated adenine ~ a s removed by centrifugalion ( I 8 0 x g . 5 mini. The loaded cells were then washed twice with PBS and rcsuspended te 2 x lip c d i s / m l in the incuhation med.'h.ml i fresh PBS containing 1 m M isobutyl-methylxanthine (IBMX). an inhibitor of c A M P - d e p e n d e n t pnosphodicstcrases). Cells (48,0 /.tit wcrc distributed in lest tubes and prcincubated for I0 rain at 370( ` bcflwc tile addition of agonist 120/.tit. Incnhations were performed for I(! miu at 37°C and terminated by adding 500 /.t[ Ill(?,; ice-cold trichloroacetlc acid t l t A~. After lhI.Ht~Ugii III;,~iiig. ""'t,L~ suspensions ~ e r e kept in an ice bath for lit min l~rior to ccnlrifugation at 4500 × g for 5 rain all 4%'. [ ~H]cAMP was isolated from other labeled substances using tile sequential chro*:latography method of Sa!onion e| ill. t,g'74). Briefly. the s u p e r n a t a n t was app!icd to catkin-exchange columns (Dowcx 50 WX8. 20()-4(Ri mesh. H fi~rm) and the ~H-lahelcd precursor nucleot]de~ ~ c r c ctuted with distilled water. After further elutMn of the Dowex eohnlln col:tents into neutral alumina columns. [~HlcAMP was etuted from alumina with 0.1 M im,idazolc at pH 7.4. Recovcpy evalualed with labeled cAMP was better than 5(Fi and there was no detectable contamination by labeled ATP. Radioactivity present in l=,ofll ahmiina eluatcs and ~H-labeled precursor fractions was determined by liquid scinliltalion counting. "i he fin:d results arc expressed as the
4Ell (At
400
I
(B)
10o
2.
%
•
u
(C)
ee
10o I
I t m~n
Fig. i. El'feels o f c n d o t h c l i n - ! l E T - I : l!t;t h M ) oil [ ( ' a : " j, ill indo-1l o a d e d M E G - I l l cells. A: E x l m c e l l t i l a r [('a 2 " ] = 03J a i M ; B: [ E G T A ] = 3 raM; (': c r o s s - d e s e n s i t i z a t i o n bclv, e e n F.T-I (101) n M l a n d sarafi~loxin ilb ( I n I i n M ) a n d hick o f dcscn~,itization bctv,.ccn ET-I arul l h m r i l b i n (5 U / r o l E l : 1-t ~as added ai ilic tiinc i n d i c a l c d b)' arrows, sarafoloxin h b IS} and IhlFoml~in ( T ) w c r c a d d e d at the |llllUS i n t l i c a l c d by alrm~hcads.
fraction o f 3H incorporated into the cells that was then coL,ecrtcd to [3H]cAMP.
3. Results In the presence of (t.9 mM CaCI, in the medium. 100 nM ET-I induced an immediate transient increase of [Ca :+ ]i- the duration of which was about 30 s. This peak was followed by a sustained [Ca2-]i increa.-,c lasting at ie:~,st 5 min. When the measurement was carried out aficr prior 2 rain treatment of the cclls by 3
!
mM EGTA, the initial !rln~sient peak of tCa:" ii still occurred while the sustained increase was no more presenl. Fig. IA and I3 shows typical recordings of these E'F-1 effects. Similar responses, i.e. initial transient and sustained seconda~ increase in [Ca::']:, as well as a similar behavior in the presence of EGTA, also occurred for ET-2. ET-3, vasointestinat constrictor and sarah)toxin 6h (not shown). We aim observed that a first Ireatmenl by one of the ET isoforms completely prevented the (a-" mobilizing effect of the same or another analog. For cxampic, fig. iC ..,huws that sara~ozoxin 5b could no !,anger elicit a Ca ~,~ response after one treatment by ET-I. We have observed that all possible combinations gave the same result. However. such a phenomenon was not observed when the second stimulation was eticitcd by a non-structurally related agonist, such as thrombin (fig. I
"l
Fig. 2 presents increase in [Ca:" ], elicited by diffcrcnt concentrations of ET-I. ET-2, ET-3. vasointeslinal constriclor and saraf(mrxin (~h. From these cur~,es it can be observed thai the different isoforms display concentration-effect curves whose maximal responses arc in the same range, whercas the order of potency is sarafotoxin 6b > ET-I > ET-2 = vaso;ntesfina{ conrtrictor > ET-3. Tbc effects of ET-t on phosphoinositide breakdown arc shown in fig. 3 which r e p , a ~ the tim,_" e_)_,rse of the tritiated inositol phosphate accumulatkm following ET-t (100 eM) slimulaikm of MEG-0i c~,,.'-"° tns(1.4.5tP~ increased rapidly to 450Ci :~bove control leveis within the first 5 s then sligi~tiy decreased 'hereafter whereas Ins( t.3.4)P~ ami lnst !.3.4.5)P: increm.ed more slowly, reaching their r~aximal even 15{R'~ ard 200G above control levels, respcct[vely) between 30 and (~tt s. ET-i (t(lii nMt neither affected cAMP format:,on in MEG-01 cells nor modified the increases in cAMP
50.0 i 3"
~.~.~.~_.~.....,.~
In~! ~.~ 4~ P~
,'r
T T.;;~' . // t
100~
I
10 ~e
/
/
~lO~
i t ~/ /!
//[i
~0 a
I
:" 8
/
10 ~
z
•~0-e
E't'-amll(~Ts, M TIME !~g!
Fig. 2. Do~c-r~sponsc curves for ET-homologuc-induccd increase i~
l('a 2~ J~: each point r~prcsents the mca~ of 4--5 cxp::t-imcnls. Ear~ rcprescnl S~I:._M. ( Ill I ~T-I. ( a ) [~ |-2. (1) El-3. ( ~ l va~nlc~linal con~:rlcloL ( >- ) s;~rafotoxifl fib.
{ ~ ! ] ] ] n , { L 3 A . 5 1 P a ~,c~mali,a~
in
[5]"-[-~limu]a~cd
Mi:(;-(]!
cdN
431/ "FABLE I Effect o f
endt~lhclin-I{FT-I) on cAMP Ic,,cls in MEG-Ill cells.
Incubationsv.cr¢ pcrfiumcd for I11 rain as described in Malcrial', ;rod melhods, m th, presence of I mM IBMX. Data arc means ~S.E. t~f three separate cxpcrilrllent!,. : P , 11.1)5. significanllydilfcrc+~l from COlllrol non-lrealcd cons. II, rig Studcnl's I-IcM. 'IljcAMP, ~ of Iolal ~l I illt'ol-por~l|cd
Commb ET-I I lllOnMl F'~rskolin( 111/zM1 Pl_il:~1I(~ nMI E'I'-t + tor~,kolin
~kl-'~ i lL{12 0.15 ~ I).IM 11.411+ tl. 12 ~ t.1t3 -*0.40 1136+ 11.1)0 ~
ET-i + P(;~
Illr
,
l! e~, ~
accumulation due to either forskolin or prostaglandin E~ (PGE~) (table I).
4. Discussion
T h e present study demonstrates that ETs arc ; increase [Ca-"|, in MEG-Ill cells. This incrca~,~ , pears t¢~ cesult I¥om b,~ih mobilization of an intraceiiular p~)l of Ca -~. . . ,~.,~ .. entry of cxtraecllular calcium. Indeed. the. C a : ' initial transients elicited by all the ET analogs ~,.crc maintained when cxtraccllular Ca -'~ was chel;~+:3 by prior addition of E G T A into the medium. ~ ~',,.r,,as the secondaD' suslaincd increase was no longer Frcscnt. Therefore, at least one part of the calcium response elicited by ET analogs results from the mobilization of Ca-" ' from intraccl!ular stores. Since lns(l.43)P~ is known to mobilize C a : ' from these stores (Berridge and Ire'inc. 1989). we have studied the effects o f ET-I on ~l~e production o f inositol phosphates. Results show thai ET-I increased rapidly. ,,,;th;,, . . . r ~ aucnl~lUhi. . . . . . . . . . .c,. ,: fol!,,wing stimJj-?Ai:m. "[ ~.%( .! .4, tion in MEG-01 cells. This tirnc course is therefore consistent, with the po~tulaied C a " mobilizing effect of Ins( 1,4.51P s. In addition, the oilier trisphosphate isomer Ins(!,3,4)P s accumulated ntorc slowly than lns(l,4.5'fP. We also ohser'~cd a la~e accumulation of 1ns(I,3.4.511~ lollowit~,~ V ! ' . !ilnulalion of MEG-01 cells: the timecourse : :,~ of this tetrakisphosphate derivative b ¢!3 its implication in the exlracellu. lar ( ; ~owing E T stimulation. 7%,: ~ I , ~ : ~ ~i~zt~ ~.hc Ca-" peak can no longer be e l i d t e d by an ET anak)g after a first ET stimulation could mean I~a~ the intcrnal stores are emptied or could indicz~e a desensitization at the ET receptor level. Since a non-related agonist, such as thrombin was still able to elicit a C a : " response after ET. it
appears that a cross-desensitization between ET analogs at the receptor level occurred. Moreover. if the internal stores had been emptied by the first ET stimulation, at lea~:t the second ET-induced Ca -'+ response due to C a : ' eniD' would have been present, which was not the case. Thus. data arc consistent with a homologous eross-descnsiti/:;m~rl and indicate that all ET analogs act on the S;l!llC icceL0tor. R,:!dhqig:md binding ,~Iudies and a M n i | v cross-linking experiments have supported the existence of subtypes of e ndol he l i n receptors, and recently, c D N A s encoding two pharmaeok)gically distinct subtypes of the endothelin receptor have been cloned (for review Wcbb. 1991 ): the ETA endothelin receptor, which shows rank order of .ffinity H ' - I ~ ET-3, and the ET B cndothelin receptor having affinity rank order ET-I = ET-3. The orde r of notcncy of the different isoforms of ET in mobilizing ~ '" in MEG-01 cells was: sarafotoxin 6b > E T - I , ,-_-~ ~ - v a s o i n t e s t i n a l constrictor > ET-3. whk:h is eo:~,;,~,,-nt w i l h th,- im',~lvemen! of an ET A endotheiin receptor type. tl ap~c,tts therefore thal the pharmace, h;gy of ',he ET receptor site present on MEG-01 cells could bc different from that involved in atrial myocytc C a : ' response, in which the sequence is ET-1 > ET-2 > sarafotoxin 6b > ET-3 (Vigne et al.. 1990). Such a different pharmacology may be related to dif;crem ETx receptor subtypes. On the other hand, the higher potency of sarafotoxin 6b c ompa re d to ET-I and ET-2 could also bc related to differential enzymatic inactivation of the pcptides, it has indeed bccn d e m o n s t r a t e d that the ,ank of potency of ET homok;gucs could bc modified by pcptidasc inhibitors (Maggi el al.. 19901. In particular, it has been shown that sarafotoxins arc less sensitive than ETs to neutral c n d o p e p t i d a s c s (Sokolovsky ct al., 1990). In summaD,, wc have shown that ET-I induces on MEG-01 cells an increase in [Ca:*]~ which is due to both Ca -'* mobilization of internal stores and to Ca -'+ cntr.v, and theft ET-I activates phospholipasc C, leading to an increased m-oduction of lns(I,4,5)P~. Da!a show that the mcgaka~,oblastic cell line M E G - 0 i bears ET receptors and therctorc represents a new target for cndothclins. The subtype of receptor involved could bc ~ff an A type. on the basis of the higher potency of ET-t tov, ards ET-3. Nonetheless, the functional response elicited by endothclins on the MEG-(ll cell line remains to be determined.
Acknowledgements The anlhorn lhank Dr. II. Saito liar providing ME'G-I)I cells and Mrs. ('~.~ell¢ BeItc~z;ffde |bl her excellent editorial assistance. This work ~as ~upiy,~rivd by I,N S.E.|~.M. (]nsl~lul Nalit~fiai de ]a Sant~ ¢t
de k: l~.cchcrchc Mddicalc. France).
431
References Batlislini. B., J.G. Filep. F. ltcrman and P. Sirois, 1991LEndolheiin-I does not affect human platcle! aggrcgalion in ~..hole bita~d and platelct rich plasma, Thromb. Res. 61L 1115. Berridge, M.J. and R.F. Irvine, 198g, lnositol phosphales and cdi signaling. Nalure 341, 107. Grynkiewicz. G.. M. Poenic and R,Y. Tsien. 1985. A new gcnera}i~m ofCit -'~ indlcalorswilh grcally improved fluorescence prop,_'rlies. J. Biol. Chem. 2h6. 3440. Maggi, ( ' . \ . , S. Giu!iani, R Palacchini. P. Santiciolo. A. Giachelu ;rod A. Meli. l'4gil. Furlhcr sludies on tile response of the guinea-pig isolated bronchus to endothelins and sarafi~loxin S6b. Eu~. J. Phurmacol. 17!~, 1. Maisumoto. Y.. Y. Ozuki, T. Kariya and S. Kume. ig9!!. Polenliuling effects of endothelio on plalclel activulitm induced by epinephrine and ADP, Biochem, Pharmacol. 41L 911g. Ogura. M.. Y. Morishina. R. Ohno, Y. Kato, N. thrahayashi, H. Nagura and 11. Saito, lt,~85, Eslablishmem of a rowel megakaryoblastic leukemia cell line, MEG-0 t, ,,~ilh p,~silive Philadelphia chronu)some, Blood 66, !3~4. Ohlstein, EH.. B.L S~orcr. M (;kc~t and 1t. Lipplon. lgg0. Endothelin and platelel funclkm ',md~. Res. 57, 967.
1~ah,.'Uin~< E M., R I:~ I,evcne. L L . K . Leung and R.L. ,'~aehman, 1981. ttuman megakaryocytes. ]i. Expression iff ~l,dctel proleins in earl3, marrow megakaP,.'ocyles, J. Exp. Mcd. 154, ~N. Saida. 14...y . Milsui and "4. Ishida. 19,~. A m,vel pc-pride, va~,aaclive intestinal constrictor c,f a new (emh;~hclin) family, J. ,'3ioL Chem. 2f~4. 14613. Salomon. Y.. ('. Londos and M. Rodbe;l, t~174. A highiy xensi~lvc adenylalc cyclase a,.say. Anal. Biochcm 5S, 541. Simonson, MS. and M J, Dunn, l¢)Oik (ellular signaling by pcplides of tile endolhelilz gene family. FASEB. J. 4. 298g. Sokolowsky. M. R. Galrem. Y. Kh~g, A. Bdolah. F.E Indig, S. Blumberg and G. F!emia~er lqgik Endolhelins arc more sensitive than sarafotoxins to neural endopeptidases: po~slble physiological significance. Proc. Natl. Acad. Sci. USA 87, 47¢~2 Vigne. P., J.P. Breittmaycr, R. Mamauh and C Fr-!ir< !c~,~0. Fr,dothelin mobilizes Ca: * from a caffeine- and ~'anodir, e-insen'-ilive inlracellular pl~t,i uf tal aiiial cells, J. Biol. Chem, 21~5.5-82. Wehb. D3., 1991, Endolhelin reeeplors cloned, endolhelin converling enzyme characterized and pz~thophystological roles for endolhclin proposed. Trends PharmacoL Sci. 12. 4? Yanagisawa. M. and T. Masaki. 1980. Endmhelin. it novel endothelium-derived peptide, Biochem. Pharmacol. 3~. I;'~77.
-*27
[!J P M O L 9{1378
Effects of endothelins on the human megakars, oblastic cell line MEG-01 Sylvie D i o c h o t % D a n i e l V i t t e t ", M a r i e - N o i i l l e M a t h i e u ", J e a n - M a r i e L a u n a y h a n d C h m d e C h e v i H a r d " "
INSliRM U. 300. I:acultd tie Piutrma~t~'. ,Montpellwr. I"rame. a n d ¢' FP.A ~. laude Berm*rd '%~'¢#rt~chttm: de,, (
i*~lt~tt ~ati
!~ (~,,l]leh;g~, "
th'~pila! 5t~iltl l.oI~t~. Part~. I:rftll~"
Received 15 April Iqq2. revised MS received 2S ]ul~ 1902.accepted tS Augus~ I~C/2
Some cffec:.s of cndoflmlin-I (ET-I) were sludicd on the megaka~'ohiaslic celt line MEG4H. ET-] induced an cte;atkm of the i~:;,accllular !evels of Ca-" ~![Ca-" ~],) as measured ~ith lhe fluorcscem ind;ca~or indo-1. ~hich consists uf an. initial mmsiem increase and an ensuing sustained plaleau. The plateau phase was abolished b~ rcm~3~alof cx'r ,cclhflar ('a: ". In a~ddititm. ET-I induced a rapid (within 5 s) increase in the accumulation of inosilol !.4.5-trispho~0;;alc (h~s~,t.4.'~:' 0 ,rod more dctaycd increases in Ins{I.3.4)P 3 and Ins(I.3,4,5)P a, but did not modify cAMP levels. ET-1 i~o. 1.4/: NazlIPO~. 8; MgCt:, 0,5: Ca('l ,, 0.q: glucose. 5.55. It was adjusted at pit = 7.4. 2.2. Celt cultun" MEG-01 cells wcrc from Dr. 14. Saito, University of Nagoya. Japan. MEG-Ill suspension cultnres were rontinety pax~aged 3 limes weekly in RPM! I¢,.RI mediun~ supplemented with 2 mM glutl, m m c and 10G ( v / x ) heat-inactivcd l~.lal calf serum under a humidified atmosphere containing 5 r ; C O . at 370( ". 2.3. Meaxttrtvmvtl of [('a-'"
],
Cells were Imlvcsted 2-3 days after seediug, c.~ntrifugcd (18fl × g, 5 mint and washed lwiee ~illl ['BS. The washed cells "~e;c resuspended to ill" ccll,/nfl. incubated 2{) rain at 37°C lit tile dark with 5 p.M i Mo-I AM. Loading was termina~.ed by dilution with cold PBS followed by ccnlrifugation. The cell pellet ~ a s resuspcnded ill PBS and aliquots (1t.8-0.8 × 1(1" ce[[s'l werc transferred to a 1 ml quartz cuvcttc equipped for temperature conrail and magnetic stirring. Fluorescence signals (|:) obtained by agents were monitored with excitation and emission wavelengths of 332 aqd 411{t nm. respectively, using a Ktmtron SFM 25 spccImfluorimeter. The cells were subsequently lysed by adding 5() ;.:.M digit.omn m obtain the si,znat of the Ca 2" _~aturatcd dye (F~,.,,). k~,,,,, for the Ca-" "-trce form of tile dye ~ a s recorded by adding 10 mM E ( H A . p | l 8.2. The concentration of intraceiiular ionized calcm,n [('at e. ], was ca!cub|ted accord:.ng to (hynkicwiez el al. (19,R5). using the equation: [ C a : ' ], = K,dF--I:~,,,jF',,,,,- F). with K,~ = 25(! nM All agcnls x~crc added to the quarlz cuvettc in a volume .'ff !tl p t. 2.4. Mcasun'mcn[ it] ip,l~itol IrA- ~im t tetr,.gk&phoslgmh" MKG4)t ceils (8 × I0 ~ ce~ls/mlI were incubated t\w 24 h in culture medium containing ill , u C i / m l myo-12311]it:ositol. After washing v, ilh PBS, ce!!s (6 × ll~' celts pet" t u b O were equilibrated fiar 15 rain aa 37°(" and further incubated f~r various limes at 37':(" ~ithout
(control) or with Ill0 nM ET-I. The reaction was stopped by addition of perchloric acid (SG v / v final concenlration) and the samples were centrifuged. T h e s u p e m a t a n t s were neutralized by 3 M KOII-150 m M HFPES. High-performance liquid chromatography (HPLC) was carried out ,..'-n an ankm-,rxchangc column (Partisil SAX-II)). lno,dtol phosphates were separated using a muhilinear gradient from water to 3 M ammoniunl formate (adjusted It) ptI 3.7 with i-lsPO~). After injection of the sample. ,he co~ .;m was washed Ibr 5 nun (flow late 1.2 , d / , ~ , m ) ~ith water. The grzMicnt of a m m o n i n m fl)rma,.c used w-;:,: 0-0.6 M tot 8 rain: then 0.6 M for 3 rain: 0.6-1 M for 7 min: 1 M over 5 min: 1-2 M fi~r 7 rain; 2 M fi~r 5 rain: finally 2 - 3 M for 7 rain and 3 M for 3 min. Eluales werc collected ever3, 0.5 min and a.~sayed for radioactivity content. Ins( 1,3.4)P~. Ins( 1.4,5)P3 and Ins( 1,3.4.5)P~ were cluted from the c o h m m between 27.5-29JI. 29.5-31.1). and 39.5-41.5 min. respectively. Tritiated derivatives were uscd as standards for the calihralion of the gradient. 2.5. ('yclic A M P assay hltr:|ce]luJar c A M P accumnlatknl was 0,~ernlined hi intact cells by measuring [~H]cAMP Uion after prclabcling the endogenous ATP poe.,!'-. ~4::~G-()l cells (approximately 5 × I( c e l l s / m . ) were labeled by incu-bafion in the culture medium with I /.tCi/ml of [2.8;H]adenine fl~r 4 h at 37°C in lhc cell incubator. Unincorporated adenine ~ a s removed by centrifugalion ( I 8 0 x g . 5 mini. The loaded cells were then washed twice with PBS and rcsuspended te 2 x lip c d i s / m l in the incuhation med.'h.ml i fresh PBS containing 1 m M isobutyl-methylxanthine (IBMX). an inhibitor of c A M P - d e p e n d e n t pnosphodicstcrases). Cells (48,0 /.tit wcrc distributed in lest tubes and prcincubated for I0 rain at 370( ` bcflwc tile addition of agonist 120/.tit. Incnhations were performed for I(! miu at 37°C and terminated by adding 500 /.t[ Ill(?,; ice-cold trichloroacetlc acid t l t A~. After lhI.Ht~Ugii III;,~iiig. ""'t,L~ suspensions ~ e r e kept in an ice bath for lit min l~rior to ccnlrifugation at 4500 × g for 5 rain all 4%'. [ ~H]cAMP was isolated from other labeled substances using tile sequential chro*:latography method of Sa!onion e| ill. t,g'74). Briefly. the s u p e r n a t a n t was app!icd to catkin-exchange columns (Dowcx 50 WX8. 20()-4(Ri mesh. H fi~rm) and the ~H-lahelcd precursor nucleot]de~ ~ c r c ctuted with distilled water. After further elutMn of the Dowex eohnlln col:tents into neutral alumina columns. [~HlcAMP was etuted from alumina with 0.1 M im,idazolc at pH 7.4. Recovcpy evalualed with labeled cAMP was better than 5(Fi and there was no detectable contamination by labeled ATP. Radioactivity present in l=,ofll ahmiina eluatcs and ~H-labeled precursor fractions was determined by liquid scinliltalion counting. "i he fin:d results arc expressed as the
4Ell (At
400
I
(B)
10o
2.
%
•
u
(C)
ee
10o I
I t m~n
Fig. i. El'feels o f c n d o t h c l i n - ! l E T - I : l!t;t h M ) oil [ ( ' a : " j, ill indo-1l o a d e d M E G - I l l cells. A: E x l m c e l l t i l a r [('a 2 " ] = 03J a i M ; B: [ E G T A ] = 3 raM; (': c r o s s - d e s e n s i t i z a t i o n bclv, e e n F.T-I (101) n M l a n d sarafi~loxin ilb ( I n I i n M ) a n d hick o f dcscn~,itization bctv,.ccn ET-I arul l h m r i l b i n (5 U / r o l E l : 1-t ~as added ai ilic tiinc i n d i c a l c d b)' arrows, sarafoloxin h b IS} and IhlFoml~in ( T ) w c r c a d d e d at the |llllUS i n t l i c a l c d by alrm~hcads.
fraction o f 3H incorporated into the cells that was then coL,ecrtcd to [3H]cAMP.
3. Results In the presence of (t.9 mM CaCI, in the medium. 100 nM ET-I induced an immediate transient increase of [Ca :+ ]i- the duration of which was about 30 s. This peak was followed by a sustained [Ca2-]i increa.-,c lasting at ie:~,st 5 min. When the measurement was carried out aficr prior 2 rain treatment of the cclls by 3
!
mM EGTA, the initial !rln~sient peak of tCa:" ii still occurred while the sustained increase was no more presenl. Fig. IA and I3 shows typical recordings of these E'F-1 effects. Similar responses, i.e. initial transient and sustained seconda~ increase in [Ca::']:, as well as a similar behavior in the presence of EGTA, also occurred for ET-2. ET-3, vasointestinat constrictor and sarah)toxin 6h (not shown). We aim observed that a first Ireatmenl by one of the ET isoforms completely prevented the (a-" mobilizing effect of the same or another analog. For cxampic, fig. iC ..,huws that sara~ozoxin 5b could no !,anger elicit a Ca ~,~ response after one treatment by ET-I. We have observed that all possible combinations gave the same result. However. such a phenomenon was not observed when the second stimulation was eticitcd by a non-structurally related agonist, such as thrombin (fig. I
"l
Fig. 2 presents increase in [Ca:" ], elicited by diffcrcnt concentrations of ET-I. ET-2, ET-3. vasointeslinal constriclor and saraf(mrxin (~h. From these cur~,es it can be observed thai the different isoforms display concentration-effect curves whose maximal responses arc in the same range, whercas the order of potency is sarafotoxin 6b > ET-I > ET-2 = vaso;ntesfina{ conrtrictor > ET-3. Tbc effects of ET-t on phosphoinositide breakdown arc shown in fig. 3 which r e p , a ~ the tim,_" e_)_,rse of the tritiated inositol phosphate accumulatkm following ET-t (100 eM) slimulaikm of MEG-0i c~,,.'-"° tns(1.4.5tP~ increased rapidly to 450Ci :~bove control leveis within the first 5 s then sligi~tiy decreased 'hereafter whereas Ins( t.3.4)P~ ami lnst !.3.4.5)P: increm.ed more slowly, reaching their r~aximal even 15{R'~ ard 200G above control levels, respcct[vely) between 30 and (~tt s. ET-i (t(lii nMt neither affected cAMP format:,on in MEG-01 cells nor modified the increases in cAMP
50.0 i 3"
~.~.~.~_.~.....,.~
In~! ~.~ 4~ P~
,'r
T T.;;~' . // t
100~
I
10 ~e
/
/
~lO~
i t ~/ /!
//[i
~0 a
I
:" 8
/
10 ~
z
•~0-e
E't'-amll(~Ts, M TIME !~g!
Fig. 2. Do~c-r~sponsc curves for ET-homologuc-induccd increase i~
l('a 2~ J~: each point r~prcsents the mca~ of 4--5 cxp::t-imcnls. Ear~ rcprescnl S~I:._M. ( Ill I ~T-I. ( a ) [~ |-2. (1) El-3. ( ~ l va~nlc~linal con~:rlcloL ( >- ) s;~rafotoxifl fib.
{ ~ ! ] ] ] n , { L 3 A . 5 1 P a ~,c~mali,a~
in
[5]"-[-~limu]a~cd
Mi:(;-(]!
cdN
431/ "FABLE I Effect o f
endt~lhclin-I{FT-I) on cAMP Ic,,cls in MEG-Ill cells.
Incubationsv.cr¢ pcrfiumcd for I11 rain as described in Malcrial', ;rod melhods, m th, presence of I mM IBMX. Data arc means ~S.E. t~f three separate cxpcrilrllent!,. : P , 11.1)5. significanllydilfcrc+~l from COlllrol non-lrealcd cons. II, rig Studcnl's I-IcM. 'IljcAMP, ~ of Iolal ~l I illt'ol-por~l|cd
Commb ET-I I lllOnMl F'~rskolin( 111/zM1 Pl_il:~1I(~ nMI E'I'-t + tor~,kolin
~kl-'~ i lL{12 0.15 ~ I).IM 11.411+ tl. 12 ~ t.1t3 -*0.40 1136+ 11.1)0 ~
ET-i + P(;~
Illr
,
l! e~, ~
accumulation due to either forskolin or prostaglandin E~ (PGE~) (table I).
4. Discussion
T h e present study demonstrates that ETs arc ; increase [Ca-"|, in MEG-Ill cells. This incrca~,~ , pears t¢~ cesult I¥om b,~ih mobilization of an intraceiiular p~)l of Ca -~. . . ,~.,~ .. entry of cxtraecllular calcium. Indeed. the. C a : ' initial transients elicited by all the ET analogs ~,.crc maintained when cxtraccllular Ca -'~ was chel;~+:3 by prior addition of E G T A into the medium. ~ ~',,.r,,as the secondaD' suslaincd increase was no longer Frcscnt. Therefore, at least one part of the calcium response elicited by ET analogs results from the mobilization of Ca-" ' from intraccl!ular stores. Since lns(l.43)P~ is known to mobilize C a : ' from these stores (Berridge and Ire'inc. 1989). we have studied the effects o f ET-I on ~l~e production o f inositol phosphates. Results show thai ET-I increased rapidly. ,,,;th;,, . . . r ~ aucnl~lUhi. . . . . . . . . . .c,. ,: fol!,,wing stimJj-?Ai:m. "[ ~.%( .! .4, tion in MEG-01 cells. This tirnc course is therefore consistent, with the po~tulaied C a " mobilizing effect of Ins( 1,4.51P s. In addition, the oilier trisphosphate isomer Ins(!,3,4)P s accumulated ntorc slowly than lns(l,4.5'fP. We also ohser'~cd a la~e accumulation of 1ns(I,3.4.511~ lollowit~,~ V ! ' . !ilnulalion of MEG-01 cells: the timecourse : :,~ of this tetrakisphosphate derivative b ¢!3 its implication in the exlracellu. lar ( ; ~owing E T stimulation. 7%,: ~ I , ~ : ~ ~i~zt~ ~.hc Ca-" peak can no longer be e l i d t e d by an ET anak)g after a first ET stimulation could mean I~a~ the intcrnal stores are emptied or could indicz~e a desensitization at the ET receptor level. Since a non-related agonist, such as thrombin was still able to elicit a C a : " response after ET. it
appears that a cross-desensitization between ET analogs at the receptor level occurred. Moreover. if the internal stores had been emptied by the first ET stimulation, at lea~:t the second ET-induced Ca -'+ response due to C a : ' eniD' would have been present, which was not the case. Thus. data arc consistent with a homologous eross-descnsiti/:;m~rl and indicate that all ET analogs act on the S;l!llC icceL0tor. R,:!dhqig:md binding ,~Iudies and a M n i | v cross-linking experiments have supported the existence of subtypes of e ndol he l i n receptors, and recently, c D N A s encoding two pharmaeok)gically distinct subtypes of the endothelin receptor have been cloned (for review Wcbb. 1991 ): the ETA endothelin receptor, which shows rank order of .ffinity H ' - I ~ ET-3, and the ET B cndothelin receptor having affinity rank order ET-I = ET-3. The orde r of notcncy of the different isoforms of ET in mobilizing ~ '" in MEG-01 cells was: sarafotoxin 6b > E T - I , ,-_-~ ~ - v a s o i n t e s t i n a l constrictor > ET-3. whk:h is eo:~,;,~,,-nt w i l h th,- im',~lvemen! of an ET A endotheiin receptor type. tl ap~c,tts therefore thal the pharmace, h;gy of ',he ET receptor site present on MEG-01 cells could bc different from that involved in atrial myocytc C a : ' response, in which the sequence is ET-1 > ET-2 > sarafotoxin 6b > ET-3 (Vigne et al.. 1990). Such a different pharmacology may be related to dif;crem ETx receptor subtypes. On the other hand, the higher potency of sarafotoxin 6b c ompa re d to ET-I and ET-2 could also bc related to differential enzymatic inactivation of the pcptides, it has indeed bccn d e m o n s t r a t e d that the ,ank of potency of ET homok;gucs could bc modified by pcptidasc inhibitors (Maggi el al.. 19901. In particular, it has been shown that sarafotoxins arc less sensitive than ETs to neutral c n d o p e p t i d a s c s (Sokolovsky ct al., 1990). In summaD,, wc have shown that ET-I induces on MEG-01 cells an increase in [Ca:*]~ which is due to both Ca -'* mobilization of internal stores and to Ca -'+ cntr.v, and theft ET-I activates phospholipasc C, leading to an increased m-oduction of lns(I,4,5)P~. Da!a show that the mcgaka~,oblastic cell line M E G - 0 i bears ET receptors and therctorc represents a new target for cndothclins. The subtype of receptor involved could bc ~ff an A type. on the basis of the higher potency of ET-t tov, ards ET-3. Nonetheless, the functional response elicited by endothclins on the MEG-(ll cell line remains to be determined.
Acknowledgements The anlhorn lhank Dr. II. Saito liar providing ME'G-I)I cells and Mrs. ('~.~ell¢ BeItc~z;ffde |bl her excellent editorial assistance. This work ~as ~upiy,~rivd by I,N S.E.|~.M. (]nsl~lul Nalit~fiai de ]a Sant~ ¢t
de k: l~.cchcrchc Mddicalc. France).
431
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