Enhancement of the endothelial production of prostacyclin by substituted derivatives of BAPTA-AM

European Journal of Pharmacology, 233 (1993) 13-20 © 1993 Elsewer Science Pubhshers B V All rights reserved 0014-2999/93/$06 00

13

EJP 52962

Enhancement of the endothelial production of prostacyclin by substituted derivatives of BAPTA-AM J e a n - M a n e B o e y n a e m s a, Sylwe H e d p o r n b, F a b t e n n e B r o e d e r s b a n d J e a n - C l a u d e B r a e k m a n b a

Instttute of Interdtsctphnary Research, School of Medlcme and b Laboratory of Bto-orgamc Chemtstry, Faculty of Sciences, Unwerslt~Ltbre de Bruxelles, Brussels, Belgtum

Recewed 25 June 1992, revised MS recewed 7 December 1992, accepted 15 December 1992

Our observation that loading of bovine aortic endothehal cells with qum 2 or 1,2-bls(O-ammophenoxy)ethane-N,N,N',N'-tetraaceuc acid (BAPTA) enhances their release of prostacychn (PGI 2) has been studied m detad The action of the acetoxymethyl ester (AM) of BAPTA (BAPTA-AM) was blphaslc at high concentrations (50/zM) and after prolonged exposure (60 mm or more), it behaved as an inhibitor instead of an amphfier Since mhlbmon could be related to calcmm chelaUon, we tested 5,5'-dlfluoro-BAPTA-AM (5FBAPTA-AM), 4,4'-dlfluoro-BAPTA-AM (4FBAPTA-AM) and 5,5'-dtbromo-BAPTA-AM (5BBAPTA-AM), whose correspondmg free acids have a reduced affinity for Ca 2+ these compounds were much more actwe m enhancing PGI 2 production than BAPTA-AM itself The effect of 5BBAPTA-AM was detectable at 0 5/zM and almost maramal at 5/~M (5-fold mcrease) 5BBAPTA-AM mcreased the release of free arachldonate induced by ATP but had no effect on the generation of inosltol phosphates, the release of choline metabohtes, and the accumulation of cAMP 5BBAPTA-AM had a cytotoxlc effect on the endothehal cells only after prolonged exposure to a high (50/zM) concentration 5BBAPTA-AM inhibited the platelet production of thromboxane sUmulated by a high (0 5 U/ml) concentraUon of thrombm and shghtly potentmted the effect of a low (0 005 U / m l ) concentration In conclusion, the effect of 5BBAPTA-AM on the production of PGI 2 seems to be rather specific to arachldonate metabohsm in endothehal cells Endothehal cells, Prostacychn, Ca 2+ indicators, Aorta (bovine), Arachldonate metabohsm

1. Introduction 1 , 2 - b t s ( O - a m m o p h e n o x y ) e t h a n e - N , N , N ',N '-tetraacetic acid (BAPTA) is the parent compound of a famdy of Ca 2÷ chelators and indicators (Tslen, 1980) The qum 2 and fura 2 derlvatwes of B A P T A have been widely used to measure the free Ca 2÷ concentration ([Ca2+]) in cells by fluorescence (Tslen et a l , 1982, Grynklewlcz et al, 1985), whereas 19F N M R determination can be performed with symmetrically substituted difluoro derwatwes (Smith et al, 1983) In general, cells can be loaded with these indicators by mcubatlon with the hpophdic acetoxymethyl esters (AM), which are readily hydrolyzed m the cytoplasm Few toxic or pharmacological actions of these compounds have been described (Arian et a l , 1985, Meldolesl et a l , 1987) As expected from their high buffering capacity for free Ca 2+, quln 2 and B A P T A are able to inhlbtt CaE+-dependent cellular processes, such as N a + / C a 2+

Correspondence to J M Boeynaems, Budding C, Campus Erasme, 808 Route de Lenmk, 1070 Brussels, Belgmm

exchange (Allen et al, 1985), actwatlon of the N a + / H + antlport (Mltsuashl and Ives, 1988), platelet aggregation (Rao et a l , 1986), and the histamine-reduced release of von Wlllebrand factor from endothelial cells (Hamilton and Sims, 1987) In neurons lacking the Ca2+-blndlng protems parvalbumm and calbmdln, loading with B A P T A protects the cells from the Ca 2+mediated degeneration induced by prolonged stlmulatton (Scharfman and Schwartzkroln, 1989) Paradoxically, m a few systems, qum 2-AM and B A P T A - A M have been shown to induce or amphfy CaE+-medlated cellular responses They enhance the turnover of phosphatldyllnosltol and mduce a mltogenlc response m mouse thymocytes and pig lymphocytes it has been suggested that these responses are mediated by an increase m [Ca2+] 1 (Hesketh et al, 1983) Incubation of washed h u m a n platelets with quln 2-AM (10 /zM) or fura 2-AM (1 /zM) for 30 m m increases their aggregatory and secretory responses to several agomsts (Lanza et a l , 1987) As indicated above, higher concentrations of quln 2-AM are mhtbltory (Rao et al, 1986, Lanza et a l , 1987) We have previously reported that qum 2-AM and B A P T A - A M enhance the release of prostacychn

14 (PGI 2) from aortic endothelial cells stimulated by A T P or bradyklnln (Rasp6 et al, 1989) In the pre~ent study, we investigated th~s achon ot B A P T A - A M m detail and compared the activity of several substituted derivatives The results indicated that there is an inverse relationship between the enhancement of PGI z release and the affinity for Ca 2+ The B A P T A - A M derivatives had some selectivity for arachldonate metabolism in endothelial cells and were not cytotoxlc

2. Materials and methods

2 1 Culture of endothelial cells Bovine aortic endothelial cells were obtained by collagenase digestion of an aorta excised from a freshly slaughtered cow, as described previously (Van Coevorden and Boeynaems, 1984) The cells were seeded in 100-mm Petrl dishes and incubated at 37°C under an atmosphere of 5% C O 2 / 9 5 % air in the following medium 80% ( v / v ) minimal essential medium (MEM) with D-vahne, 20% ( v / v ) fetal calf serum (FCS), 2 m M glutamlne, 100 u n l t s / m l penicillin, 1 0 0 / z g / m l streptomycin, and 2 5 / z g / m I amphoterlcln B The m e d m m was changed the next day and then every 3 days After 4 or 5 days, the primary cultures formed confluent monolayers and could be subcultured The ceils were detached by incubation with Ca 2+- and Mg2+-free Hanks buffer containing trypsin (100 n g / m l ) and E D T A (1 mM) for 5 mln Thereafter, the cells were seeded m 35-ram Petrl dishes or In 16-mm wells of 24-well plastic plates and cultured in 60% ( v / v ) Dulbecco-mo&fied essential medium (DMEM), 20% ( v / v ) H a m ' s F12, 20% ( v / v ) FCS, with the same concentrations of penicillin, streptomycin and amphoterlcm B as mentioned above Experiments were performed with confluent monolayers between passage 2 and 5

2 2 Preparatton and mcubatton of washed human platelets

pernatants were collected ior the assay of thromboxane B2 (TxBx)

2 3 Measurement o] 6-keto-PGFl~, arachldont~ actd releave

TrB~ and Jlee

For the m e a s u r e m e n t of 6-keto-prostaglandln F~. (6-keto-PGFl.) release, bovine aortic endothelial cells, cultured in the presence of B A P T A - A M derlvanves or dlmethylsulfoxlde ( D M S O 0 5% v / v ) as control, were washed twice with D M E M and incubated at 37°C for 20 m m in D M E M with A T P 6-keto-PGF~. was measured in the lncubanon medium by radaolmmunoassay (Van Coevorden and Boeynaems, 1984) For the measurement of arachldonate release, the incubation with A T P was performed in D M E M containing fatty acidpoor bovine serum albumin (BSA 1 m g / m l ) and mdomethacln (1 / z g / m l ) Arachldonate was determined by gas chromatography with electron capture detection as described previously (Van Coevorden and Boeynaems, 1984) The statistical significance ot results was determined with Student's t-test TxB. released from platelets was measured by radlolmmunoassay (Boeynaems et al, 1987)

2 4 Measurement of lnosttol phosphates Bovine aortxc endothelial ceils were cultured for 48 h in a mixture of lnosltol-free D M E M (67 5%), H a m ' s F12 ( 2 2 5 % ) and FCS (10%), supplemented with anhblotlcs and containing myo[2-3H]lnosltol (10/J.C1/ml) The medium was then removed and the cells were washed twice with D M E M and incubated for 30 m m in D M E M containing LlC1 (10 mM), with 5 B B A P T A - A M or D M S O (0 5%) A T P was added and the incubation was stopped by rapid removal of the medium and addition of 2 ml perchlorlc acid (3%) The extraction and separation of lnosltol phosphates on Dowex AG1X8 columns was performed as described by Plrotton et al (1987)

2 5 Measurement of chohne metabohtes release Blood from volunteers who had not taken antllnflammatory drugs during the last 2 weeks was collected with 1/10 volumes E D T A (0 077 M) and centrifuged for 15 mln at 200 × g at room temperature The resultlng platelet rich plasma was centrifuged at 100 × g for 15 mln The platelet pellet was resuspended in buffer (NaC1 139 mM, Trls HCI p H 7 4 10 mM, glucose 5 mM, E D T A 1 5 raM) The platelets were washed once with the same buffer and were then resuspended in a medium containing NaCl 139 raM, T n s HC1 p H 7 4 10 raM, glucose 5 m M The platelet count was about 800000//xl Incubations were performed at 37°C Thereafter the platelets were rapidly centrifuged (30 s in an E p p e n d o r f centrifuge model 5412), and the su-

Bowne aortic endothelial cells were incubated for 24 h m the complete culture medium containing [methyl3H]chohne ( 1 0 / z C 1 / m l ) as described by Plrotton et al (1990) The cells were then washed twice with D M E M and incubated in D M E M in the presence of the test agents Ahquots of incubation medium (100 /xl) were collected at various times for hquld scintillation countmg

2 6 Measurement of cAMP Bovine aortic endothelial cells, approximately 400000 cells in 300 /zl complete culture medium con-

15 taming 20% FCS, were seeded on 10 5 × 22 mm plastic covershps (Thermanox TM, Lux) After 24 h, the covershps were rinsed and eqmhbrated for 60 mm at 37°C m a tube containing 2 5 ml Krebs-Rlnger hepes buffer (124 mM NaCI, 5 mM KC1, 1 25 mM MgSO 4, 1 45 mM CaCI2, 1 25 mM KH2PO 4, 25 mM HEPES buffer, pH 7 4, 8 mM glucose) and the phosphodlesterase inhibitor rohpram (10 ~M) and 5 BBAPTA-AM The cells were transferred to fresh medmm and incubated for 10 mln m the presence of forskohn or histamine At the end of this incubation, the covershps were plunged into 4 ml boiling water for 4 mln, to stop all reactions and to extract cAMP cAMP was assayed by radlolmmunoassay after acetylatlon, as described previously (Heklmmn et al, 1992)

2 7 Cytotoxlctty assays The cytotoxtcity of BAPTA-AM derwatwes was evaluated by the release of radloactwlty from bovine aortic endothelial cells labelled with either [3H]thymldine or [S1Cr]chromate Bovine aortic endothehal cells were incubated with [methyl-3H]thymldme (5/zCl/ml) for 24 h in complete culture medmm or with [S1Cr]sodlum chromate (10/zCl/ml) for 3 h in serumfree culture medmm They were then washed twice with DMEM and incubated in DMEM Ahquots of the medium were collected at various times to count the radloactwlty

28 Synthesis of 1,2-bts[2-(N-(acetoxymethoxycarbonyl)methyOmethylammophenoxy]ethane (1) and 1-[2-(N(acetoxymethoxycarbonyl)methyl)methylammophenoxy]2-[2-bts(acetoxymethoxy-carbonyl)rnethyl) ammophenoxy]ethane (2)

M r at m / z 244, IR (KBr) 3381 cm -l, 1H NMR (CDC13, 250 MHz) 3 82 (4H,broad s), 4 36 (4H,s), 6 68-6 89 (8H,m) The N-methylatton of 3 (1 g) was performed according to the method of Krtsnamurthy (1982) After workup, the product was flash-chromatographed on sthca gel (hexane/ethylacetate 9 1), yieldmg 812 mg of 1,2-bls(2-N-methylammophenoxy) ethane (4, 73% yield) mp 52-53°C, ElMS M r at m / z 272, IR (KBr) 3400 cm-l, 1H NMR (CDC13, 250 MHz) 2 82 (6H,s), 4 28 (2H,broad s), 4 34 (4H,s), 6 58-6 96 (8H,m) For one of our trials we used half the quantity of the acetic formic anhydride Under these con&tlons and after the usual workup, a mtxture of 1,2-bls(2-N-methylammophenoxy)ethane (4, 32%), 1-(2-N-methylammophenoxy)-2-(2-ammophenoxy) ethane (5, 14%) and starting material (16%) was isolated and separated by flash-chromatography on slhca gel (hexane/ ethylacetate 9 1) 1-(2-N-Methylamlnophenoxy)-2-(2ammophenoxy) ethane (5) mp 77-80°C, ElMS M r at m / z 258, IR (KBr) 3400 cm -1, 1H NMR (CDC13, 250 MHz) 2 74 (3H,s), 3 88 (3H,broad s), 4 27 (4H,s), 6 51688 (8H,m) The N-(ethoxycarbonyl-methyl)methyl derwatwes, their hydrolysis products, and the N(acetoxymethylester) derwatwes 1 (dIAM) and 2 (triAM) were prepared starting from the amines 4 and 5, respectively, according to the procedure described by Tslen (1980) 1 (38%) oil, ElMS M r at m / z 532, IR (NaC1 film) 1766 cm -1, 1H NMR (CDC13, 250 MHz) 2 03 (6H,s), 2.94 (6H,s), 4 05 (4H,s), 4 33 (4H,s), 5 51 (4H,s), 6 89-6 99 (8H,m) 2 (22%) oil, ElMS M r at m / z 648, IR (NaC1 film) 1766, 1760, 1733 cm 1, IH NMR (CDCI3, 250 MHz) 2 03 (3H,s), 2 09 (6H,s), 2 94 (3H,s), 4 07 (2H,s), 4 19 (4H,s), 4 31 (4H,m), 5 51 (2H,s), 5 62 (4H,s), 6 84-7 02 (8H,m) (fig 1)

29Ma~nab 1,2-Bls(2-aminophenoxy) ethane (3) was obtained by treatment of 2 53 g of 1,2-bls(2-mtrophenoxy) ethane (1) with hydrazme hydrate in ethanol m the presence of P d / C The crude amine was recrystalhzed from ethanol to gwe 1 9 g of white crystals (93%) mp 130-131°C, electron impact mass spectrometry (ELMS)

R

R

Dulbecco's Modified Eagle's Medmm (DMEM), lnosltol free DMEM, Ham's F12, penlcllhn, streptomycin, amphotertcin B and glutamlne were obtamed from Flow Laboratory, collagenase type II was purchased from Cooper (Worthington), Minimal Essential

R

R

L..N..I HaC-..N,/I

Compound

H3C.. N/J

Compound !

R = -COOCHzOCOCH 3

Fig 1 Structuresof compounds 1 and 2

R

16

Medium (MEM) with D-vahne replacmg L-vahne (MEM-D-Val) and fetal calf serum (FCS) were from Glbco ATP and human thrombm were obtamed from Sigma Chem Co BAPTA-AM and its derivatives were purchased from Molecular Probes (Eugene, Oregon) 6-keto-PGF1. and TxB 2 were obtained from Upjohn Diagnostics (Kalamazoo, Michigan) [3H]6-keto-PGFl,,, [3H]TxB2, myo[2-aH]lnositol, [methyl-3H]chohne, [methyl-3H]thymldlne and [51Cr]sodlum chromate were purchased from Amersham Corp

TABLE 2 Effect of BAPTA-AM on the release of PGI 2 from endothelial cells comparison with analogs bearing 3 and 2 AM ester functions Bovine aortic endothehal cells were exposed to BAPTA-AM, compound 1 or compound 2 for the last 60 man of the culture period The medmm was replaced by DMEM and the cells were incubated for 20 rain in the presence of ATP (5 /xM) Results represent the amount of 6-keto-PGFi. accumulated m the medium at the end of this incubation, In ng/well (mean ± S D of tnphcate wells in one representative experiment out ot four) The control value In the absence of ATP was 0 9 ± 0 3 ng/well ~ P < 0 0 5 b p > 0 1 , c o m p a r e d w , t h the control value I0/xM

3. Results We have prevtously reported that BAPTA-AM is able to enhance the release of PGI 2 from aortic endothehal cells stimulated with either ATP or bradyklnln (Rasp6 et al, 1989) In this study the action of BAPTA-AM was investigated in greater detail As shown in tables 1 and 2, the effect of BAPTA-AM was blphaslc the enhancement of PGI 2 release observed at 10 /zM was lost at 50 /zM or even replaced by a time-dependent lnhtbltlOn An analog of BAPTA-AM contalnmg only 3 AM ester functmns (compound 2) exhtblted a similar blphaslc action, whereas a compound with only 2 AM ester functions (compound 1) was purely mhlbitory (table 2) Since the inhibitory component in the actton of these compounds might be due to chelation of cytoplasmic Ca 2+, several symmetrically substituted derivatives of BAPTA-AM were tested 5,5'-d~methyl-BAPTA-AM (5Me BAPTA-AM), 5,5'-dIfluoro-BAPTA-AM (5FBAPTA-AM), 4,4'-dtf l u o r o - B A P T A - A M ( 4 F B A P T A - A M ) and 5,5'-dlbromo-BAPTA-AM (5BBAPTA-AM) As shown in table 3, substitution by halogen atoms greatly increased the capacity to enhance PGI 2 release as compared to BAPTA-AM ~tself or its dImethyl derivative There was an approxtmately inverse correlation between the el-

TABLE 1 Concentration and time dependence of the action of BAPTA-AM on the release of 6-keto-PGF1, ~ from endothehal cells BAPTA-AM (10 or 50 /xM) was added to the culture medmm of bovine aorhc endothelial cells for the last 120, 60 or 30 mln of the culture period The medium was removed and replaced by D M E M containing 5 / z M ATP, for 20 mln Results represent the amount of 6-keto-PGF1, ~ accumulated in the medium at the end of this incubation, m ng/well (mean _+S D of trlphcate wells in one representative experiment out of four) The control release in the absence of ATP was 1 3 + 0 l ng/well d p < 0 05, b p > 0 1, compared with the control value Time

BAPTA-AM

(mm)

10 # M

0 30 60 120

21+01

-

50/zM 17±102

22±02 d 30±04 ~ 42±03 d

18±06 b 14±03 b 13±01 a

BAPTA-AM Compound 2 Compound 1

50/zM

3 0+05 a 4 0_+ 0 4 a 20+02 b

05_+0 1 a 13± 0 2 a 1 1+0 1 d

fect on PGI 2 and the affinity of the corresponding free carboxyhc acids for Ca 2+ (table 3) (Smith et al, 1983) Most of the following experiments were performed with 5BBAPTA-AM A 30 rain prelncubaUon of bovine aortic endothelial cells with 10 /zM 5BBAPTA-AM increased the ATP-lnduced release of PGI 2 to 500 _+ 77% of the value measured with untreated cells (mean _+ S E , n = 20, range 150-1484%) 5BBAPTA itself had no effect (data not shown) The action of 5BBAPTA-AM was concentration dependent (figs 2 and 3) at 5 /zM, it produced an almost maximal enhancement of the ATP-lnduced release of PGI 2 with a minimal effect on the basal release of PGI 2 Figure 3 illustrates the time dependence of the 5BBAPTA-AM effect, which became detectable after a few mln delay and progressively increased wlth the time of exposure a plateau was reached after 60 rain (not shown) The enhancing effect of 5BBAPTA-AM was slowly reversible and had completely vanished 24 h after 5BBAPTA-AM withdrawal (table 4) ReaddltlOn of 5BBAPTA-AM at that time induced a new response which had a magnitude comparable to the response of control cells (table 4) TABLE 3 Enhancement of endothehal 6-keto-PGFl, ~ release by substituted derwatlves of BAPTA-AM comparison with the affimty for Ca 2÷ The pK Ca 2÷ values of the substituted derivatives of BAPTA are derived from Tsxen (1980) and Smith et al (1983) The increase In 6-keto-PGF1, ~ release is expressed as a percentage of the ATP-lnduced release of 6-keto-PGF1, ~ In treated and control cells (mean of five experiments)

5MeBAPTA-AM BAPTA-AM 5FBAPTA-AM 5BBAPTA-AM 4FBAPTA-AM

pK Ca 2+

6-keto-PGF1. increase (%)

7 7 6 5 5

230 210 470 500 690

4 0 2 8 6

17 TABLE 4 Restlmulatlon of endothehal cells after a second exposure to 5BBAPTA-AM At time 0, 5BBAPTA-AM (10 p.M) was added to the culture medmm in half the wells ('prestlmulated cells') After 30 rain, this medmm was removed and replaced with fresh complete culture medmm 5BBAPTA-AM (10 ~M) was added 24 h later, as indicated m the table After 30 mm, the medmm was removed and the cells were washed twice They were then mcubated for 20 mm m DMEM, with or without ATP (5 /xM) The results represent the amount of 6-keto-PGF~,~ m the medmm at the end of the incubation, in ng/well (mean + S D of SLXdeterminations m two separate experiments) a p > 0 1, compared to the respectwe value in control cells

20

O')

U,.

~. 10

,~,+ 0

0'5

5'0

5B BAPTA-AM 50 pM

Fig 2 Enhancement of endothelial 6-keto-PGFl,~ release by 5BBAPTA-AM concentration response curve Various concentrations of 5BBAPTA-AM were added for the last 30 mm of the culture period The culture medmm was removed and the cells were washed twice with DMEM and incubated for 20 mm m DMEM with (o) or vathout (o) ATP (5 /zM) The results represent the amount of 6-keto-PGF1, ~ in the medium at the end of the incubation (mean + S D of triplicate determinations m one representative experiment out of seven)

ATP (5 p.M) 5BBAPTA-AM (10/zM) 5BBAPTA-AM (10/zM)+ ATP (5 p.M)

tratlon dependence ATP-induced

Prest~mulated cells

03+01 4 0+0 4 07+0 1

03+01 a 3 8+0 i a 1 3+05 a

86+10

105+11 a

of this action was quite similar to

that for the enhancement the

Control cells

of PGI 2 release In contrast,

generation

of inosltol phosphates

and release of water-soluble chohne metabohtes u n c h a n g e d in 5 B B A P T A - A M - t r e a t e d

Bovine

aortic

5BBAPTA-AM

endothelial

released

more

cells free

treated

with

arachldonate

in

r e s p o n s e t o A T P t h a n c o n t r o l c e l l s (fig 4) t h e c o n c e n -

d o t h e h a l cells ( d a t a n o t s h o w n )

was

bovine aortic en-

5BBAPTA-AM

also

h a d n o e f f e c t o n t h e a c c u m u l a t i o n o f c A M P in b o v i n e aortic e n d o t h e l i a l cells s t i m u l a t e d with e i t h e r f o r s k o h n or histamine (data not shown) Various protocols were

used to evaluate whether

B A P T A - A M d e r t v a t w e s w e r e toxac t o e n d o t h e l i a l c e l l s A 30-mm exposure of [3H]thymldlne-labelled bovine

75-

O)

=-~ 5-

100-

uS 6

0

l

0

0

05

50

50

5B BAPTA-AM MM

Fig 3 Enhancement of endothehal 6-keto-PGFl~ release by 5BBAPTA-AM time course 5BBAPTA-AM was added at various concentrations for the last 60 ( • ) or 5 ( [] ) mm of the culture per,od The culture medmm was removed and the cells were washed twice with DMEM and incubated for 20 mm in DMEM with (In, [], • ) or w,thout (12) ATP (5 tzM) The results represent the amount of 6-keto-PGFt,, ,n the medmm at the end of the incubation (mean + S D of tnphcate determinations ,n one representat,ve exper,ment out of six) Inset After removal of the culture medium and two wash steps, boxane aortic endothelial cells were incubated for 30 mm m DMEM ATP (5 /zM) was added 10 mm before the end of the ,ncubat,on 5BBAPTA-AM (10 ~M) was added at various times prior to ATP, as indicated on the X ares The results represent the amount of 6-keto-PGF~ in the medmm at the end of the incubation (mean 5: S D of tnphcate deternunatlons In one representatwe expenment out of three)

5o.

0- ~ : " " ~ . 0 2

1'0

5B BAPTA-AM ~50 pM

Fig 4 Enhancement by 5BBAPTA-AM of the ATP-mduced release of arachidonate from boxane aortic endothehal cells 5BBAPTA-AM was added at various concentraUons for the last 30 mln of the culture period The medmm was removed and the cells were washed twice and incubated for 20 min in DMEM containing BSA (1 mg/ml) and mdomethacln (1/zg/ml), with (e) or without (o) ATP (10/~M) The results represent the amount of arachldonate m the medmm at the end of the incubation (mean 5: S D of tnphcate determinations, in one representative experiment out of two)

18

aortic endothehal cells to BAPTA-AM or 5FBAPTAAM mcreased the release of radioactivity over the next 24 h, whereas 5BBAPTA-AM had no effect (fig 5A) When the cells were exposed continuously to 50 /zM 5BBAPTA-AM over the 24-h test period, an mcreased release of radioactivity was detectable after 2 h, however, with lower concentrations (05 and 5 /zM), a significant effect was not observed even after 24 h (fig 5B) Similarly, these low concentrations of 5BBAPTAAM did not mcrease the release of 51Cr from prelabelled cells (data not shown) The effect of 5BBAPTA-AM on arachIdomc acid metabolism was tested In another cell type human platelets 5BBAPTA-AM mhtblted the release of TxB 2 from human platelets stimulated by a high (0 5 U / m l ) concentration of thrombm (fig 6 right) this inhibition increased w~th the duration of exposure to 5BBAPTAAM (not shown) Other BAPTA-AM derivatives mimicked this inhibitory effect, 5Me BAPTA-AM being the most effective (fig 6 right) When platelets were challenged with a low concentration of thrombm (0005 U / m l ) , 5BBAPTA-AM shghtly increased the release

THROMBIN 0 005 U/ml

THROMBIN 0 5 U/ml 10

400 I

I

~o

~ 200 ¸

ii I

m

I

10~

,-I iI i

< < < , <,

_ _

< 13_

o=<=<=<~ mm

to

to

83 to

:~ to

LL

to

• tO

Fig 6 Effect of BAPTA-AM derlvatwes on the production of TxB 2 in washed human platelets Right panel Washed human platelets, prepared as described m Methods, were incubated for 30 mm with BAPTA-AM or its derwatwes, at 1 ~ M Human thrombm (05 U / m D was added and the incubation was stopped 10 mln later Left panel The protocol was identical except for the concentration of thrombm, which was 0 005 U / m l For both panels, the results represent the concentration of TxB 2 in the supernatant (mean _+S D of triplicate determinations in one representatwe experiment out of

two)

of TxB 2 to 139 _+ 14% of the control value (mean _+ S D of three experiments) (fig 6 left) ~lS °

4. Discussion E

~

o~.~

,

Z

0

El

rn

,m

3 o

&

~2

o

1

j ,,~ 00~2 4 6~ 24 HOURS Fig 5 Effect of BAPTA-AM derwatwes on the release of [3H]thymldme from prelabelled bovine aortic endothehal cells Upper panel The agents (50 /zM) were added for the last 30 mm of the 24-h labelhng period The medium was removed and the cells were washed twice and incubated m D M E M for 24 h The results represent the radioactivity released Into the medium at the end of the incubation (mean_+ S D of triplicate determinations in one representatwe experiment out of three) Lowerpanel Bovine aortic endothehal cells label led with [3H]thymldme were washed twice and incubated in D M E M containing 5BBAPTA-AM at various concentrations Ahquots of the incubation medium were collected at various times for liquid scintillation counting The results represent the mean of triplicate determinations in one representatwe experiment ot.t of two (©) Control. (O) 5BBAPTA-AM (05 IzM), (©) 5BBAPTA-AM (5/~M), ( zx ) 5BBAPTA-AM (50 p,M)

We have previously reported our unexpected observation that loading of bovine aortic endothelial cells with quln 2 or B A P T A enhanced their capacity to release PGI 2 in response to A T P or bradyklnin (Rasp6 et al, 1989) These Ca 2÷ chelators are able to buffer mcreases in cytosohc Ca 2+ and therefore inhibit various CaE+-mediated responses (Allen et al, 1985, Mitsuashi et al, 1988, Rao et al, 1986, Hamilton and Sims, 1987) Elevation of [caa+], provides the major If not the exclusive intracellular pathway for the ATP-Induced release of PGI 2 (Carter et al, 1988) An inhibitory effect of quin 2 and B A P T A was therefore expected rather than a stimulatory effect The ATPmediated synthesis of PG1 z was indeed inhibited by the combmatlon of a high concentration (100 /zM) of quin 2-AM and EGTA, a procedure known to abolish the rise in [Ca2+], Induced by agents mobilizing lntracellular calcium (Rasp6 et al, 1989) Furthermore, as shown in this study, the effect of BAPTA-AM was blphaslc, enhancement being replaced by inhibition at high concentrations BAPTA-AM and related compounds also inhibited the production of TxB 2 in thrombln-stimulated platelets, a process mediated by an elevation I n [ C a 2+ ]~ Differences in drug distribution

19

and the size of calcium pools are probable explanations for the greater sensitivity to inhibition of platelets as compared to endothelial cells Several symmetrically substituted derlvattves of B A P T A differing in their affinity for Ca 2+ (Smtth et al, 1983) were used to show that the enhancement of PGI 2 production ts dissociated from the capactty to chelate Ca 2÷ The 5,5'-dlbromo, 5,5'-difluoro- and 4,4'-dlfluoro dertvatlves of BAPTA-AM, whose corresponding free acids have a low affinity for Ca 2+ (pK around 6), had a greater effect on PGI 2 production than BAPTA-AM or its dimethyl derivative (pK around 7) These data lndtcate that the abihty to enhance endothehal PGI 2 is inversely related to the capactty to chelate Ca 2+ In contrast, the cytotoxtcity, which was greater wtth BAPTA-AM than wtth 5BBAPTA-AM, probably results from changes in cellular calcium pools, related to the Ca2+-chelatmg properttes We also wished to determine if compounds of the BAPTA-AM famtly produced a general activation of endothelial cells or if their action was restricted to arachldonlc actd metabohsm The initial step in the action of ATP or bradykmln on endothehal cells is the activation of a phosphohpase C which generates lnOSltol phosphates from phosphatldyhnosltol btsphosphate (Pirotton et al, 1987) This ts followed by the activation of phosphatldylcholme hydrolysis by a phosphohpase D, which can be assessed by the release of water-soluble choline metabohtes (Ptrotton et al, 1990) 5BBAPTA-AM did not affect either the formatton of inosltol phosphates or the release of chohne metabohtes, in either resting or ATP-sttmulated cells It also had no detectable effect on cAMP levels In fact, 5 BBAPTA-AM enhanced the release of free arachldonate mduced by ATP, and this effect had a magnitude and a concentration dependence comparable to the actton on PGI 2 release It appears that, in endothelial cells, BAPTA-AM derlvattves specifically increase the availability of free arachldonlc actd without altermg the transductlon mechanisms of the PGI2-stimulating agonlsts Interestingly, it has been reported that quln 2-AM changes the relative mcorporation of arachldonate into phosphatldylchohne

versus phosphatidyhnosl-

tol (Pollock et al, 1986)

Acknowledgements We are grateful to N Galand and C Lagneau for their excellent techmcal assistance This work was performed under contract of the Mmlst~re de la Pohtlque Sclentlfique (Sciences de la Vie B10/04) and was supported by a grant from the Fonds de la Recherche Sclentlflque M6dlcale F Broeders was supported by a contract with the R6gmn Wallonne and the Umon Chlmtque Beige, within the framework of the FIRST programme

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