JOURNAL OF CELLULAR PHYSIOLOGY 142449-457 (1990)
Adenine Nucleotides Modulate Phosphatidylcholine Metabolism in Aortic Endothelial Cells SABINE PIROTTON,* BERNARD ROBAYE, CHRISTIAN LAGNEAU, AND JEAN-MARIE BOEYNAEMS Institute of Interdisciplinary Research, School of Medicine, Free University of Brussels, Campus Erasme, Brussels, Belgium ATP and ADP, in concentrations ranging from 1-1 00 pM, increased the release of [3H]choline and [3H]phosphorylcholine (P-choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3Hlcholine. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATPyS and ADPPS) and adenosine 5’-(p,y imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5’-(a,P rnethy1ene)triphosphate (APCPP): these results are consistent with the involvement of P ,, receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 pM), the tumor promoter phorbol 12-myristate 13-acetate (PMA, 50 nM), and the calcium ionophore A231 87 (0.5 pM). Downregulation of protein kinase C, following a 24-hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P-choline, whereas the response to A231 87 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate bv a phospholipase C coupled to the P,, receptors (Pirotton et al., 1987a).
Signal transduction via the activation of specific phospholipases is now well established. The most obvious case is the hydrolysis of PIP, by a phospholipase C (PLC), leading to a n accumulation of two second messengers: inositol( 1,4,5)trisphosphate (InsP,), which can mobilize Ca2 from intracellular stores, and diacylglycerol (DAG), which stimulates PKC (Berridge, 1987). However, additional second messengers could be generated by phospholipases other than PLC, acting on various classes of phospholipids. A phospholipase D (PLD) hydrolyzing phosphatidylinositol in phosphatidic acid (PA) and inositol has very recently been identified in human neutrophils extracts (Balsinde et al., 1988). A direct coupling between receptors for hormones or neurotransmitters and a phospholipase A, releasing free arachidonic acid has been characterized in several systems: rod outer segments from bovine retina (Jelsema, 1987), MDCK canine kidney cells (Slivka and Insel, 1988a), and Swiss 3T3 fibroblasts (Burch and Axelrod, 1987). In the last few years, it has been observed that a n increase in the turnover of PC is a n early event in the action of diverse agonists, on a variety of cell types (for review: Pelech and Vance, 1989). Phosphatidylcholine can be hydrolyzed into either
DAG and P-choline by a PLC, or into PA and choline by a PLD. DAG is a known PKC activator, whereas PA might play a role as a mitogenic signal (Moolenaar et al., 1986). The activation of PC turnover can be as-
0 1990 WILEY-LISS. INC
Received April 6, 1989; accepted November 8, 1989. *To whom reprint requestskorrespondence should be addressed. Abbreviations used: ATPyS, adenosine 5’-0-(3-thio)triphosphate; ADPPS, adenosine 5‘-0-(2-thio)bisphosphate; APCPP, adenosine 5 ’ - ( a , p methy1ene)triphosphate; APPNP, adenosine 5 ’ 4 3 , ~imido)triphosphate; BAEC, bovine aortic endothelial cells; CT, cholera toxin; DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediamine tetraacetic acid; EGTA, ethyleneglycol bis(Paminoethylether N,N,N’,N‘)tetraaceticacid; GP-choline, glycerophosphorylcholine; HPLC, high-performance liquid chromatography; MDCK, Madin-Darby canine kidney; MEM, minimum essential medium; P-choline, phosphorylcholine; PC, phosphatidylcholine; PDGF, platelet-derived growth factor; PIP,, phosphatidylinositol bisphosphate; PKC, protein kinase C; PL A,,C,D, phospholipase A,,C,D; PMA, phorbol 12-myristate 13-acetate; PT, pertussis toxin; quin-2AM, quin-2 acetoxymethyl ester; TCA, trichloroacetic acid.
PIROTTON ET AL.
sessed by a n increased extracellular release and/or intracellular accumulation of water-soluble choline metabolites. Several agonists produce such a n increase in many systems: PDGF in 3T3-Ll pre-adipocytes (Besterman et al., 19861, bombesin in Swiss 3T3 fibroblasts (Muir and Murray, 1987), a,-adrenergic agonists in MDCK-D1 canine kidney cells (Slivka e t al., 1988b), ATP in rat hepatocyte membranes (Irving and Exton, 1987; Bocckino et al., 1987), insulin in BC3H-1 myocytes (Nair et al., 19881, vasopressin in REF52 rat embryo cells (Welsh et al., 1988), PMA in Swiss 3T3 fibroblasts (Takuwa et al., 1987; Muir and Murray, 19871, and NG108-15 neuroblastoma x glioma cells (Liscovitch et al., 1987). However, the respective role of a phospholipase C vs. D, a s well as the mechanism of phospholipase activation (role of PKC or of GTPbinding proteins), differed from one study to the other. The platelet secretory products ATP and ADP stimulate the release of prostacyclin (PGI,) (Pearson et al., 1983; Van Coevorden and Boeynaems, 1984) and of endothelium-derived relaxing factor (EDRF) (Burnstock and Kennedy, 1986; Houston et al., 1986) from aortic endothelial cells. They are also mitogenic for these cells (Van Coevorden et al., 1989). These actions might have a physiological importance in the interaction between platelets and the vascular endothelium: the release of PGI, will limit the extent of platelet aggregation following a lesion of the endothelium, whereas the mitogenic effect will accelerate the repair of that lesion. These effects of ATP are mediated by P,, receptors (Needham et al., 1987; Burnstock and Kennedy, 1986). We have previously shown that the P,, receptors of aortic endothelial cells are coupled to a phospholipase C hydrolyzing PIP, (Pirotton e t al., 1987a) and that this coupling involves a pertussis toxin-sensitive GTP-binding protein (Pirotton et al., 198713). In this study, we have evaluated the action of ATP on the turnover of PC in aortic endothelial cells by measuring the extracellular release and intracellular accumulation of water-soluble choline metabolites.
MATERIALS AND METHODS Materials MEM D-valine, DMEM, Ham's F-12, glutamine, streptomycin, penicillin, amphotericin B, fetal calf serum were purchased from GIBCO Laboratories. Collagenase Type IA was from Cooper (Worthington Biochemical Corporation). Trypsin and phosphate-free MEM were obtained from Flow Laboratories. ATP, ADP, AMP, adenosine, adenosine 5'-(a,P methy1ene)triphosphate, adenosine 5 ' 4 3 , ~imido)triphosphate, bradykinin, phorbol 12-myristate 13-acetate, standards of phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine for TLC were obtained from Sigma Chemical Co. Adenosine 5'-0-(3-thio) triphosphate, adenosine 5'-0-(2-thio)bisphosphate, and A23187 were from Boehringer Mannheim. Glycine and sodium dodecyl sulfate were from BioRad, and all other electrophoresis reagents were obtained from Serva. The Silica Gel (p Porasil) column was from Waters Associates. Acetonitrile for HPLC was purchased from Alltech Associates Inc. The HPTLC Silica gel 60F,,, plates were from Merck. The radioactive groducts: [methyl-3H]choline (80 Ci/mmole), [methyl- Clphos-
phorylcholine (50 mCi/mmole), and [32Plphosphate were purchased from Amersham.
Cell culture BAEC were obtained by collagenase digestion of the aorta excised from a freshly slaughtered cow, as described previously (Booyse et al., 1975; Van Coevorden and Boeynaems, 1984). They were seeded in 100 mm Petri dishes and incubated a t 37" C in a humidified 95% air, 5% CO, incubator. The culture was started with a medium composed of MEM D-valine (80%, v/v), fetal calf serum (20%, v/v), 2 mM glutamine, 100 Uiml penicillin, 100 pg/ml streptomycin, 2.5 pg/ml amphotericin B, to prevent survival of contaminating smooth muscle cells (Marcum et al., 1986). The medium was changed the following day and later on every 3 days. When the primary culture formed a confluent monolayer the cells were detached by a short incubation in a Ca$+ and Mg2+ free Hanks' buffer containing 10 mg/ ml trypsin and 1mM EDTA and seeded in 35 mm Petri dishes. For the next passages, the culture medium was replaced by the following one: DMEM (60%, v/v), Ham's F-12 (20%, viv), fetal calf serum (20%, viv), antibiotics, amphotericin B, and glutamine a t the same concentrations. The experiments were performed with subconfluent cells ( 2 8 lo5 cells/dish) between passage 2 and 5. Assay of choline and phosphorylcholine formation and release BAEC were labelled during a 24-hour incubation in 1 ml of complete culture medium, containing 10 pCi [methyl-3H]choline (80 Ci/mmol). The cells then were washed twice with DMEM and incubated in 1 ml of DMEM in the presence of the tested agents. Aliquots of the incubation media (100 p1) were collected at various times for liquid scintillation counting. Intracellular hydrosoluble [3Hlcholine metabolites were extracted by the following procedure: The medium was rapidly re. cells were scraped placed by 1ml TCA (lo%, v / ~ )The and the dishes rinsed with TCA. Proteins were eliminated by centrifugation (20 minutes a t IOOOg, 4" C). TCA contained in these cell lysates was removed by four extractions in diethylether (4ml). A 100 pl aliquot of the aqueous phase then was counted in liquid scintillation. Lipid analysis The endothelial cells were labelled with t3H1choline and treated with ATP as described above. At the end of the incubation, the medium was removed and replaced by 1 ml of methanol. The cells were scraped and the dishes rinsed once with methanol. Lipids were extracted by the addition of 2 ml chlorofordconcentrated HCl (200:1, v:v) and 0.1 ml of EDTA 0.2M. After mixing, water (2 ml) and chloroform (2 ml) were added, and the two phases were separated by centrifugation (5 minutes, 1,OOOg). The lower phase was removed and dried under N, stream. The aqueous phase was reextracted twice with 2 ml chloroform, and the new organic phases were collected, combined with the first one, and dried. The dry lipid extracts were redissolved in chloroform/methanol (2:1, v:v) and applied on heatactivated Silica gel 6OF,,, plates. Separation of the phospholipids was performed using chloroform/metha-
ATP AND PC METABOLISM IN ENDOTHELIAL CELLS
nollacetic acidlwater (75:38:12:9, v:v:v:v) (Martin and Michaelis, 1988). After I, staining, the areas corresponding to phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine standards were scraped and the ['HI radioactivity measured by liquid scintillation counting.
Separation of [3H]cholinemetabolites by HPLC The separation of choline metabolites was performed by HPLC using a protocol described by Liscovitch et al. (1985) with some modifications. Aliquots of incubation media or cellular extracts were injected directly on a Silica Gel (p Porasil) column (3.9 mm x 30 cm, 10 pm particles). Elution was performed with a mixture of acetonitrile, water, ethanol, acetic acid, and 0.83 M sodium acetate in water. The composition of this mixture was 800/127/68/2/3 (v/v) in initial conditions (A) and 4001400168153179 (vlv) in final conditions (B). After 5 minutes in initial conditions, a linear gradient from 0 to 100% of B was developed within 30 minutes; final conditions then were maintained for 15 minutes. The flow rate was 2.7 ml/minute, and fractions of 2.7 ml were collected for liquid scintillation counting. The injector (U6K), the pumps (6000A), and the gradient programmer (660) were from Waters Associates. The retention times of choline and P-choline were determined using radioactive standards. Protein phosphorylation BAE cells were washed twice with phosphate-free MEM and incubated for 6 hours'in this medium, supplemented with 500 pCi1ml of [''Plphosphate and 8.5 pM KH,PO,. The tested agents then were added for 10 minutes and the incubations stopped by replacing the medium with 200 p1 of the following lysis buffer: 9.5 M urea, 2% (w/v) 3-(3-cholamidopropyl) dimethyl-ammonio-1-propane sulfonate (Chaps), 1.6% (vlv) Servalyt pH 5-7, 0.4% (vlv) Servalyt pH 2-11, 5% (vlv) P-mercaptoethanol, supplemented with protease inhibitors. The samples were immediately frozen in liquid nitrogen. The phosphorylated proteins were analysed by two-dimensional gel electrophoresis, a s previously described (Lecocq et al., 1979; Demolle et, al., 1988). They were first separated by isoelectric focusing on cylindrical gels (pH 5-7) and secondly according to molecular weight on linear gradient (6-16%) polyacrylamide slab gels containing 0.1% sodium dodecyl sulfate (SDS). After protein fixation in methanol 30%, acetic acid 10% (vlv), the gels were dried and exposed to pmax Amersham Corporation films at room temperature for a period calculated to reach a total of 4.10' disintegrations. The radioactivity associated to the proteins was determined using the Siekevitz assay (Siekevitz, 1952). RESULTS Release of ['Hlcholine metabolites BAEC were labelled during a 24-hour incubation in complete culture medium containing 10 pCi1ml of [3H]choline. At the end of this period, 22.2 6.5% of the radioactivity was incorporated into the cells (mean 2 SD of four independent experiments). Of the radioactivity incorporated into the lipidic fraction, 87.8 & 0.2% was in PC; a smaller amount (10.9 2 1.1%) comigrated with a standard of sphingomyelin on thin-layer
T i m e . min
Fig. 1. Effect of ATP on ['Hlcholine metabolites release from BAEC. 13Hlcholine-prelabelled BAEC were incubated in DMEM in absence (A-A) or in the presence of 100 p M ATP (*-*I. At various times, aliquots of the incubation medium were collected and counted in liquid scintillation. The results are expressed as the total amount of L3H] radioactivity (CPM x lo-") releasedhl at a given time (mean f SD of triplicate determinations in one representative experiment).
chromatography (TLC) plates (mean 2 range of two experiments). When [3Hlcholine-labelled endothelial cells were incubated for up to 60 minutes, without added stimulus, a basal release of radioactive material was observed (Fig. 1). After 40 minutes of incubation, it represented 11.8 & 3.5% of the total ['HI radioactivity in the cellular pool of water-soluble metabolites (mean SD of three separate experiments). The addition of ATP stimulated the release of ['HI compounds as shown on Figure 1 and Table 1.The action of ATP was significant 5 minutes after its addition and was sustained for 40 minutes (Fig. 1). At this time, the amount of r3H] radioactivity in the medium represented 198 & 37% of the basal release (mean 5 SEM of 23 independent experiments). In contrast, ATP (100 pM) did not affect the total amount of intracellular choline metabolites (Table 1).As shown in Figure 2, the ATP-induced release of [3Hl products was concentration-dependent: a significant effect was obtained with a concentration of ATP as low as 1 pM. The purinergic receptors involved in a n action of ATP can be characterized using different adenine nucleotides and nucleosides (Burnstock and Kennedy, 1986). As illustrated in Table 2a, ADP was as potent a s ATP, whereas AMP and adenosine had little effect on the release of ['Hlcholine metabolites. The P,,-selective agonist, adenosine 5'-(a,p methylene) triphosphate (APCPP),was not more active than AMP or adenosine. These results are consistent with the involvement of PZyreceptors in the action of ATP. To exclude t h a t the action of the adenine nucleotides involved a phosphorylation reaction, we tested their phosphorothioate analogs (ATPyS and ADPpS, ~ respectively) as well as adenosine 5 ' 4 3 , imido)triphos-
PIROTTON ET AL
TABLE 1. Effect of ATP on the level of [3Hlcholine derived radioactivity in incubation medium and aqueous extracts of BAEC'
TABLE 2. Effect of different adenine nucleotides and their analogs on QHlcholine metabolites release from BAEC' ~~~
[3Hlcholine metabolites CPM ( x 10-3)/dish Control ATP (100 uM) Incubation medium 5 Minutes 10 Minutes Cell aqueous extracts 5 Minutes 10 Minutes
361t06 478 f 2 9
438 f 4 427 i 57
431 41 437 t- 6
'BAEC prelabelled with 13Hlcholine were incubated for 5 or 10 minutes in the absence or presence of 100 pM ATP The L3H1 radioactivity was quantitated in the incubation medium and in cell aqueous extracts (see Materials and Methods) by liquid scintillation counting The results are expressed as mean f SD of duplicate determinations in one representative experiment
'BAEC were labelled with [3Hlcholine and the incubated for 40 minutes in the presence of ATP, ADP, AMP, adenosine, APCPP, ATPyS, ADPpS, APPNP. all a t a 100 pM concentration After this penod, the amount of L3H] radioactivity released in the incubation medium was determined by liquid scintillation counting The results are expressed as mean t- SD of triplicate determinations in one representative experiment of two
150 - 1
ATP c o n c e n t r a t i o n . uM
Fig. 2. Concentration-dependency of the ATP-induced release of [3Hlcholine metabolites from BAEC. BAEC were labelled with L3H1choline and then incubated for 40 minutes in the presence of increasing concentrations of ATP. The C3H1 radioactivity released in the medium at the end of the incubation was quantitated by liquid scintillation counting. The results are expressed as the PHI radioactivity releasedim1 (CPM x mean t SD of triplicate determinations in one representative experiment of two).
phate (APPNP), which is known to be much less hydrolyzable than ATP and ADP (Cusack et al., 1983). All of these analogs reproduced the effects of ATP and ADP on the release of choline metabolites (Table 2b, c).
Identification of the [3Hl products Choline metabolites were separated by HPLC on a Silica Gel (pPorasil) column as described by Liscovitch et al. (1985). Resting cells released both [3Hlcholine and L3H]P-choline (Fig. 3A): 81 k 3% of the total radioactivity released corresponded to choline (mean SD of 18 determinations from three different experiments). When the endothelial cells were incubated in the presence of 100 pM ATP, the release of both metabolites was stimulated. Forty minutes after ATP addition, choline and P-choline levels in the medium were 40% of the basal respectively 210 k 50% and 250 levels (mean SD of three independent experiments).
Conditions a Control ATP ADP AMP Adenosine APCPP b Control ATP ATPyS ADP ADPpS c Control ATP APPNP
[3Hlmetabolites release CPM r3Hl ( x lO-'Vdish 37 -+ 2 101 t 5 85 lr 6 51 r 3 44 2 4 53 t 12 31 f 3 82 5 10 82 If- 5 69 2 3 84 2 13 38 r 5 82 ? 1 74 t 3
The time courses of choline and P-choline releases in response to ATP were similar (Fig. 3B, C). A characteristic HPLC profile of endothelial cell extracts is illustrated on Figure 4A. It shows that Pcholine was the major metabolite in cell lysates (more than 90% of the total radioactivity detected). A third minor peak is present on the HPLC chromatogram: according to Liscovitch et al. (1985), the retention time of that peak is comparable with that of glycerophosphorylcholine (GP-choline). ATP (100 pM)yoduced a significant and reproducible increase of the [ Hlcholine level, whereas a slight increase of P-choline level could be observed only in some experiments (Fig. 4A, 4C). ATP had no effect on GP-choline level (Fig. 4A). As soon as 30 seconds after the addition of ATP, the accumulation of choline was significantly increased; the maximal effect of ATP was obtained a t 2 minutes, and it was maintained for at least 20 minutes (Fig. 4B). At this time, ATP induced a threefold increase of the intracellular choline level (2.8 5 0.3, mean & SD of three different experiments). By comparison, the level of Pcholine in ATP-stimulated cells was 102 t 4% of its level in control cells (mean k SD of three experiments). The [3H]cholinedetected in the incubation medium might have been generated from released P-choline by a membrane-bound phosphatase. To exclude this possibility, BAEC were incubated with [l4C1 labelled Pcholine for up to 40 minutes. The medium then was analysed by HPLC: no degradation of the added Pcholine could be observed even after 40 minutes (two experiments). On the other hand, we determined that a reuptake of the released choline might have occurred during our experiments. Therefore, [3H]choline was added to endothelial cells: no significant decrease of the radioactivity in the medium was detected after 40 minutes (data not shown). Bradykinin, phorbol 12-myristate 13-acetate (PMA), and the calcium ionophore A23187 also stimulated the release of [3H]choline and P-choline from prelabelled endothelial cells (Fig. 5). Bradykinin was slightly less effective than ATP: 40 minutes after its addition, the release of C3H] metabolites was 164 k 15% of the con-
ATP AND PC METABOLISM IN ENDOTHELIAL CELLS A:
Cho 1 ine 40
10 30 20
i _. .
20 30 40
I O L 10 20 30 40 0 10 20 30 40
Retention time, min
indicated by the arrows. B, C : The labelled cells were incubated in the absence (&--A)or presence ( . a ) of ATP 100 pM for various periods. The choline and P-choline released in the medium then were separated by HPLC as in A. The results are expressed as CPM of ['Hlcholine (B) or of ['HIP-choline (C) released /ml a t a given time (mean f range of duplicate determinations in one representative experiment of two).
Fig. 3. Release of choline and P-choline from ATP-stimulated BAEC: HPLC characterization and time course. A: [3Hlcholine labelled BAEC were incubated in DMEM in the absence (-----I or presence 1-( of ATP 100 pM for 40 minutes; 800 p1 of the incubation medium (Iml total) was injected on a Silica gel p Porasil column. The [3Hlcholine metabolites were separated as described in Materials and Methods. The retention times of choline and P-choline standards are
+ 1 I F,
- 3. X
10 20 30 40 Retention time, min 0
Fig. 4. Intracellular accumulation of choline in ATP-stimulated BAEC: HPLC characterization and time course. A BAEC were labelled with ['Hlcholine and then incubated in the absence (------) or presence (-) of 100 pM ATP for 20 minutes; 100 ~1 of the cellular extracts (1.5 ml total) were injected on a Silica gel p Porasil column and the r3H1choline metabolites analysed as described in Materials and Methods. The arrows indicate the retention times of choline and
12 16 20
trol value (mean SD of three experiments) vs. 198 37% for ATP '(as said above). On the other hand, A23187 and PMA both were more active than ATP: the stimulated release of [3H]products represented, respectively, 228 -C 91% (seven experiments) and 215 k 42% (13 experiments) of the basal release, 40 minutes after the addition of the agonists. The HPLC profiles were identical for ATP and bradykinin, with an almost equal stimulation of choline and P-choline.
8 12 16 20
P-choline standards. B, C: Labelled endothelial cells were incubated in the absence ( A - 4 or presence ( L O )of ATP 100 pM for various periods. The cells extracts were analysed as in A. The results are expressed as CPM of L3H1choline (B) or P-choline (C) in 100 p1 of cell extracts (mean ? range of duplicate determination in a representative experiment).
Role of Ca2+,G proteins, and PKC in the action of ATP
To probe the role of G proteins in the stimulatory action of ATP on the release of choline metabolites, we used bacterial toxins such as cholera toxin (CT) and pertussis toxin (PT). None of these compounds had a significant effect on the basal release of metabolites nor on its increase in response to ATP (Table 3). Neg-
PIROTTON ET AL.
0 A23187 500 nM i
,~~ P-Chol I n e
PMA 50 nM l
were difficult to interpret because EGTA, per se, increased the release of choline metabolites (data not shown). To investigate the role of kinase C in the action of ATP, we exposed endothelial cells to PMA (500 nM) for 24 hours, a procedure known to induce a downregulation of this enzyme. The effectiveness of the procedure was checked by monitoring the phosphorylation of a 36 kDa protein, which is stimulated by PMA more effectively than by A23187 (Demolle et al., 1988). As shown in Figure 6, the acute effect of PMA on this phosphorylation was abolished following a 24-hour exposure to the phorbol ester, indicating that this treatment reduced the PKC activity in endothelial cells. This observation is in complete agreement with the results of Uratsuji and DiCorleto (1988), who measured PKC activity, after such a treatment, in cultured porcine aortic endothelial cells. The exposure of [3Hlcholine-prelabelled endothelial cells to 500 nM PMA during 24 hours inhibited by 93 k 20% the release of choline metabolites induced by ATP and by 94 ? 21% that observed in response to PMA (mean SD of four independent experiments). On the contrary, this treatment had no effect on the action of the calcium ionophore (Fig. 7).
DISCUSSION We have shown that ATP and ADP enhance the release of [3Hlcholine and [3HlP-choline from prelabelled BAEC and increase the intracellular accumulation of [3H]choline. ADP and ATP were equiactive, whereas 0 10 20 30 40 50 AMP and adenosine had no effect. The action of ATP Retention Time. rnin Retention Time rnin was mimicked by nonhydrolyzable analogs, excluding Fig. 5. Comparison of the effects of ATP, bradykinin, A23187, and that a phosphorylation reaction has a role in this rePMA on the release of choline and P-choline from BAEC. Endothelial sponse. APCPP, which is selective for PZx receptors, cells were labelled with [3H]choline and then incubated in DMEM containing the tested agents: ATP (100 pM), bradykinin (1 pM), behaved as a poor agonist. This particular agonist specificity is thus comparable to that characterizing other A23187 (500 nM), PMA (50 nM) for 40 minutes. The media were collected, and the choline metabolites were analyzed by HPLC on a p effects elicited by adenine nucleotides in endothelial Porasil column as described in Materials and Methods. The conditions cells: stimulation of inositol phosphates accumulation were tested in duplicate. The arrows indicate the retention times of choline and P-choline standards. (-----) control, (-1 after stimula- (Forsberg et al., 1987; Pirotton et al., 1987a1, increase of intracellular free calcium concentration (Hallam tion. and Pearson, 1986; Luckhoff and Busse, 1986; Pirotton et al., 1987a1, and stimulation of prostacyclin release TABLE 3. Lack of CT and PT effect on the release of L3H] (Pearson et al., 1983; Van Coevorden and Boeynaems, metabolites induced by ATP' 1984). It is consistent with the involvement of P,,purinoceptors (Burnstock and Kennedy, 1986; Need[3Hlmetabolites release ham et al., 1987). CPM ( x lO-'))idish The effects of bradykinin on aortic endothelial cells Control ATP (100uM) are quite similar to those of ATP and involve similar 58 2 4 No toxin 27 t 5 transduction mechanisms: bradykinin increases inosiCT (5 pgiml) 28 t 1 62 t- 7 to1 phosphates formation (Derian and Moskowitz, 1986; 57 t- 10 32 i 3 PT (200 neiml) Lambert et al., 19861, and cytosolic Ca" (Luckhoff, 'BAEC were labelled with L3H]choline over 24 hours. FT ' (200 ngiml) was added 1986; Morgan-Boyd et al., 1987) induces the release of for the last 20 hours and CT (5 Wgirnl) for the last 4 hours. Cells then were washed and incubated in the absence or presence of ATP (100 wM) for 40 minPGI, (Hong, 1980), opens Ca2+-activatedK + channels utes. The amount of I3HJradioactivity released in the medium was quantitated (Gordon and Martin, 1983; Colden-Stanfield et al., by liquid scintillation counting. Results are expressed as mean 2 SD of triplicate determinations in one representative experiment of three. 1987), and produces the same modifications in the pattern of protein phosphorylation as ATP (Demolle et al., 1988). It is therefore not surprising that bradykinin ative results were also obtained with the NaF/AlCl, increased the release of choline and P-choline from BAEC. Furthermore, i t was recently shown that bradycombination (two experiments, data not shown). Calcium-clamping experiments were performed to kinin produces a sustained increase of intracellular block the Ca2+ transients induced by ATP (Ives and choline in bovine pulmonary artery endothelial cells Daniel, 1987; Rasp6 et al., 1989). Endothelial cells (Martin and Michaelis, 1988). Our data cannot clearly establish the enzymatic were exposed to EGTA (4 mM) and quin-2 AM (120 pM) for 40 minutes prior to the ATP challenge. Results pathway that was activated by ATP to induce the ac-
ATP AND PC METABOLISM IN ENDOTHELIAL CELLS
Fig. 6. Downregulation of PKC following a prolonged exposure of BAEC to PMA. Endothelial cells were incubated in the absence (A) or in the presence (B) of 500 nM PMA first during 18 hours in complete culture medium without serum and then for another 6-hour period in phosphate-free MEM containing 500 FCi/ml of [32Plphosphate.After
PMA p r e t r e a t .
Fig. 7. Effect of PKC downregulation on the release of 13Hlcholine metabolites induced by ATP, PMA, and A23187. BAEC were labelled with ['Hlcholine and then incubated with 0 or without 0 500 nM PMA for 24 hours. Cells were washed and exposed to ATP (100 pM), PMA (50 nM), or A23187 (500 nM). After 40 minutes of incubation, the L3H] radioactivity released in the medium was determined by liquid scintillation counting. Results are expressed a s CPM of ['HI releasediml (mean 2 SD of triplicate determinations in one representative experiment of three).
cumulation and release of choline metabolites in BAEC. The release of choline and P-choline could be explained by the activation of PC specific PLC and PLD by ATP. Both enzymes were identified in pulmonary
washing, the cells were challenged with PMA (50 nM) for 10 minutes. Cell lysis and [32P] labelled proteins analysis by two-dimensional electrophoresis were performed as described in Materials and Methods. All conditions were tested in duplicate.
artery endothelial cells (Martin et al., 1987; Martin, 1988). The fact that ATP mostly increased the cellular level of choline suggests that PLD activation is the major event; the release of P-choline might therefore be due to a modification of the plasma membrane permeability in response to ATP. Consistent with the activation of a PLD is the observation that bradykinin produced a parallel increase in intracellular choline and PA in bovine pulmonary artery endothelial cells, while having a minimal effect on diacylglycerol (Martin and Michaelis, 1988). The sustained activation of PC hydrolysis seems to be a general second-phase response occurring after the hydrolysis of PIP, (Welsh et al., 1988; for review: Pelech and Vance, 1989). The relative importance of PLC and PLD activation is a function of the cell type and/or the agonist. For example, activation of PC hydrolysis by a PLC has been observed in 3T3-Ll pre-adipocytes, in Swiss 3T3 fibroblasts, and in MDCK canine kidney cells, in which a n increased production of DAG and/or P-choline has been measured in response to PDGF, PMA, and serum (Besterman et al., 1986); PMA and bombesin (Takuwa et al., 1987; Muir and Murray, 1987); and a,-adrenergic agonists (Slivka et al., 1988b). In other cell types, the same or related agonists activated a PLD: PMA in the NG108-15 neuroblastoma x glioma cell line (Liscovitch et al., 1987), PMA, vasopressin, and serum in REF52 r a t embryo cells (Cabot et al., 1988a, b), formylated chemotactic peptide in HL-60 granulocytes (Pai et al., 1988). In r a t hepatocyte membranes, ATP stimulated the hydrolysis of PC by both PLC and PLD (Irving and Exton, 1987; Bocckino et al., 1987).
PIROTTON ET AL
Stimulation of PLC or PLD might result from their direct coupling to receptors via a specific GTP-binding protein(s). Alternatively, it might be the indirect consequence of PKC activation. Consistent with the first theory is the observation that ATP and vasopressin induced the release of choline and P-choline from r a t hepatocyte membranes only in the presence of GTP-yS (Irving and Exton, 1987; Bocckino et al., 1987). It was also suggested that activation of PLC by a,-adrenergic agonists in MDCK cells is a primary event after receptor occupancy, as it was not blocked by kinase C antagonists or when PIP, hydrolysis was inhibited (Slivka et al., 1988b). On the other hand, PMA appears as a ubiquitous activator of PLC and PLD (Liscovitch et al., 1987; Cabot et al., 1988a, b). Furthermore, the downregulation of PKC partially inhibited stimulation of PLC by PDGF in 3T3-Ll cells (Besterman et al., 1986) and abolished the response to bombesin in 3T3 fibroblasts (Muir and Murray, 1988). In BAEC, the stimulatory effect of ATP on the release of choline and P-choline was not affected by pertussis and cholera toxins or by the NaF-A1C1, combination: these data do not exclude the involvement of a G protein, because the same agents were inactive on hepatocyte membranes where the requirement for GTPyS is consistent with the role of such a protein (Irving and Exton, 1987; Bocckino et al., 1987). However, the reproduction of the ATP action by PMA and its abolition in endothelial cells depleted in PKC activity strongly suggest that PKC activation constitutes a link between P, receptors and PLD stimulation. As noticed by Irving and Exton (19871, it is unlikely that either choline or P-choline would operate as intracellular mediators, as their cellular concentration is in the mM range, but a role of intercellular messenger cannot be excluded for P-choline.’ The physiological role of the agonist-induced PC hydrolysis by a PLD is more likely the generation of PA. PA might be involved in the sustained influx of Ca2+,which follows its transient mobilization by inositol trisphosphate in response to ATP (Hallam and Pearson, 1986; Carter et al., 1988), although the hypothesis that PA is a Ca2+ ionophore (Putney Jr. et al., 1980; Harris et al., 1981; Serhan et al., 1982) is controversial (Holmes and Yoss, 1983). Alternatively, its growth-factor-like action (Moolenaar e t al., 1986) might contribute to the mitogenic effect of ATP on BAEC (Van Coevorden et al., 1989). In conclusion, our results, indicate that in aortic endothelial cells binding of ATP to P,, receptors produces a sustained stimulation of a phospholipase D hydrolyzing PC, probably as a consequence of PKC activation.
‘P-choline (1to 100 FM) had no effect on t h e basal release of PGI, from BAEC nor on its stimulation by ATP (unpublished data). ‘During the revision of this manuscript, a work about coupling between P,-purinergic receptors and P C hydrolysis by a PLD in bovine pulmonary artery endothelial cells was published by Martin and Michaelis (1989). Their results are quite similar to those t h a t we have obtained in BAEC.
ACKNOWLEDGMENTS The authors are grateful to Professor J.E. Dumont for his continuous interest and generous support. This work was supported in part by the “Ministere de la Politique Scientifique” (“Sciences de la vie B10104”) and by the “Fonds de la Recherche Scientifique Medicale.” B. Robaye is supported by the “Fondation Rose et Jean Hoguet” and S. Pirotton is a fellow of the “Institut #Encouragement pour la Recherche Scientifique dans 1’Industrie et 1’Agriculture.” LITERATURE CITED Balsinde, J., Diez, E., and Mollinedo, F. (1988) Phosphatidylinositolspecific phospholipase D: A pathway for generation of a second messenger. Biochem. Biophys. Res. Commun., 154:502-508. Berridge, M.J. (1987) Inositol trisphosphate and diacylglycerol: Two interacting second messengers. Annu. Rev. Biochem., 56:159-193. Besterman, J.M., Duronio, V., and Cuatrecasas, P. (1986) Rapid formation of diacylglycerol from phosphatidylcholine: A pathway for generation of a second messenger. Proc. Natl. Acad. Sci. U.S.A., 83:6785-6789. Bocckino. S.B., Blackmore. P.F.. Wilson. P.B.. and Exton, J.H. (1987) Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism. J. Biol. Chem., 262:15309-15315. Booyse, F.M., Sedlack, B.J., and Rafelson, M.E. (1975) Culture of arterial endothelial cells: characterization and growth of bovine aorta cells. Thromb. Diathes. Haemorrh., 34:825-839. Burch, R.M., and Axelrod, J . (1987) Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A,. Proc. Natl. Acad. Sci. U.S.A., 84:6374-6378. Burnstock, G., and Kennedy, C. (1986) A dual function for adenosine 5’-triphosphate in the regulation of vascular tone. Excitatory cotransmitter with noradrenaline from perivascular nerves and locally released inhibitory intravascular agent. Circ. Res., 58:319330. Cabot, M.C., Welsh, C.J., Zhang, Z., Cao, H., Chabbott, H., and Lebowitz, M. (1988a) Vasopressin, phorbol diesters and serum elicit choline glycerophospholipid hydrolysis and diacylglycerol formation in nontransformed cells: Transformed derivatives do not respond. Biochim. Biophys. Acta, 959t46-57. Cabot, M.C., Welsh, C.J., Cao, H., and Chabbott, H. (1988b)The phosphatidylcholine pathway of diacylglycerol formation stimulated by phorbol diesters occurs via phospholipase D activation. FEBS Lett., 233:153-157. Carter, T.D., Hallam, T.J., Cusack, N.J., and Pearson, J.D. (1988) Regulation of P,,-purinoceptors-mediated prostacyclin release from human endothelial cells by cytoplasmic calcium concentration. Br. J . Pharmacol., 95:1181-1190. Colden-Stanfield, M., Schilling, W.P., Ritchie, A.K., Eskin, S.G., Navarro, L.T., and Kunze, D.L. (1987) Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells. Circ. Res., 61:632-640. Cusack, N.J., Pearson, J.D., and Gordon, J.L. (1983) Stereoselectivity of ectonucleotidases on vascular endothelial cells. Biochem. J.,214: 975-981. Demolle. D.. Lecomte. M.. and Boevnaems. J.-M. (1988) Pattern of protein phosphorylation’in aortic endothe’lial cells. J. Biol. Chem., 263:18459-18465. Derian, C.K., and Moskowitz, M.A. (1986) Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent. J . Biol. Chem., 261:3831-3837. Forsbera E.J., Feuerstein. G., Shohami, E., and Pollard. H.B. (1987) Adenosine triphosphate’ stimulates inositol phospholipid metabolism and prostacyclin formation in adrenal medullary endothelial cells by means of P,-purinergic receptors. Proc. Natl. Acad. Sci. U.S.A., 84.5630-5634. Gordon, J.L., and Martin, W. (1983) Endothelium-dependent relaxation of the pig aorta relationship to stimulation of “Rb efflux from isolated endothelial cells. Br. J . Pharmacol., 79531-541. Hallam, T.J., and Pearson, J.D. (1986) Exogenous ATP raises cytoplasmic free calcium in fura-2 loaded piglet aortic endothelial cells. FEBS Lett., 207:95-99. Harris, R.A., Schmidt, J., Hitzemann, B.A., and Hitzemann, R.J. (1981) Phosphatidate as a molecular link between depolarization and neurotransmitter release in the brain. Science, 212:1290-1291.
ATP AND PC METABOLISM IN ENDOTHELIAL CELLS Holmes, R.P., and Yoss, N.L. (1983) Failure of phosphatidic acid to translocate Ca2 across phosphatidylcholine membranes. Nature, 305t637-638. Hong, S.L. (1980) Effect of bradykinin and thrombin on prostacyclin synthesis in endothelial cells from calf and pig aorta and human umbilical cord vein. Thromb. Res., 18t787-795. Houston, D.S., Shepherd, J.T., and Vanhoutte, P.M. (1986) Aggregating human platelets cause direct contraction and endothelium-dependent relaxation of isolated canine coronary arteries. Role of serotonin, thromboxane A, and adenine nucleotides. J . Clin. Invest., 78:539-544. Irving, H.R., and Exton, J.H. (1987) Phosphatidylcholine breakdown in rat liver plasma membranes. Roles of guanine nucleotides and P,-purinergic agonists. J . Biol. Chem., 262t3440-3443. Ives, H.E., and Daniel, T.O. (1987) Interrelationship between growth factor-induced pH changes and intracellular Ca2+. Proc. Natl. Acad. Sci. U.S.A., 84t1950-1954. Jelsema, C.L. (1987) Light activation of phospholipase A, in rod outer segments of bovine retina and its modulation by GTP-binding proteins. J. Biol. Chem., 262t163-168. Lambert, T.L., Kent, R.S., and Whorton, A.R. (1986) Bradykinin stimulation of inositol polyphosphate production in porcine aortic endothelial cells. J . Biol. Chem., 261t15288-15293. Lecocq, R., Lamy, F., and Dumont, J.E. (1979) Pattern of protein phosphorylation in intact stimulated cells: thyrotropin and dog thyroid. Eur. J . Biochem., 102t147-152. Liscovitch, M., Freese, A,, Blusztajn, J.K., and Wurtman, R.J. (1985) High-performance liquid chromatography of water-soluble choline matebolites. Anal. Biochem. 151t182-187. Liscovitch, M., Blusztajn, J.K., Freese, A,, and Wurtman, R.J. (1987) Stimulation of choline release from NG108-15 cells by 12-0-tetradecanoylphorbol 13-acetate. Biochem. J., 241r81-86. Luckhoff, A. (1986) Measuring cytosolic free calcium concentration in endothelial cell with Indo-1: The pitfall of using the ratio of two fluorescence intensities recorded at different wavelengths. Cell Calcium, 7:233-248. Luckhoff, A., and Busse, R. (1986) Increased free calcium in endothelial cells under stimulation with adenine nucleotides. J . Cell. Physiol., 126;414-420. Marcum, J.A., Atha, D.H., Fritze, L., Nawroth, P., Stern, D., and Rosenberg, R.D. (1986) Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan. J. Biol. Chem., 261:7507-7517. Martin, T.W. (1988) Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells. Biochim. Biophys. Acta, 962t282-296. Martin, T.W., Wysolmerski, R.B., and Lagunoff, D. (1987) Phosphatidylcholine metabolism in endothelial cells: evidence for phospholiDase A and a novel Ca2+-indeDendent DhosDholiDase x ~ . . . C. Biochim. Biophys. Acta, 917t296-307. Martin, T.W., and Michaelis, K.C. (1988) Bradykinin stimulates phosphodiesteratic cleavage of phosphatidylcholine in cultured endothelial cells. Biochem. Biophys. Res. Commun., 157t1271-1279. Martin. T.W., and Michaelis, K.C. (1989) P,-puronergic aaonists stimulate' phosphodiesteratic cleavage of phosphatidykhofine in endothelial cells. Evidence for activation of a phospholipase D. J . Biol. Chem., 264t8847-8856. Moolenaar, W.H., Kruijer, W., Tilly, B.C., Verlaan, I., Bierman, A.J., and de Laat, S.W. (1986) Growth factor-like action of phosphatidic acid. Nature, 323:171-173. Morgan-Boyd, R., Stewart, J.M., Vavrek, R.J., and Hassid, A. (1987) Effects of bradykinin and angiotensin I1 on intracellular Ca2 dynamics in endothelial cells. Am. J. Physiol., 253:C588-C598. Muir, J.G., and Murray, A.W. (1987) Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: Evidence for a role of protein kinase C. J . Cell. Physiol., 13Ot382-391. +
Nair, G.P., Standaert, M.L., Pollet, R.J., Cooper, D.R., and Farese, R.V. (1988) Effects of insulin and phorbol esters on diacylglycerol generation and synthesis and hydrolysis of phosphatidylcholine in BC3H-1 myocytes. Biochem. Biophys. Res. Commun., 154r13451349. Needham, L., Cusack, N.J., Pearson, J.D., and Gordon, J.L. (1987) Characteristics of the P, purinoceptor that mediates prostacyclin production by pig aorta endothelial cells. Eur. J. Pharmacol., 134: 199-209. Pai, J.-K., Siegel, M.W., Egan, R.W., and Billah, M.M. (1988) Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes. J. Biol. Chem., 263t1247212477. Pearson, J.D., Slakey, L.L., and Gordon, J.L. (1983) Stimulation of prostaglandin production through purinoreceptors on cultured porcine endothelial cells. Biochem. J.,214t273-276. Pelech, S.L., and Vance, D.E. (1989) Signal transduction via phosphatidylcholine cycles. TIBS, 14t28-30. Pirotton, S., Raspe, E., Demolle, D., Erneux, C., and Boeynaems, J.-M. (1987a) Involvement of inositol 1,4,5-trisphosphate and calcium in the action of adenine nucleotides on aortic endothelial cells. J. Biol. Chem., 262t17461-17466. Pirotton, S., Erneux, C., and Boeynaems, J.-M. (1987133 Dual role of GTP-binding proteins in the control of endothelial prostacyclin. Biochem. Biophys. Res. Commun., 147t1113-1120. Putney Jr., J.W., Weiss, S.J., Van De Walle, C.M., and Haddas, R.A. (1980) Is phosphatidic acid a calcium ionophore under neurohumoral control? Nature, 284t345-347. Raspe, E., Ramboer, I., Galand, N., and Boeynaems, J.-M. (1989) Enhanced release of prostacyclin from quin 2-loaded endothelial cells. Eur. J . Pharmacol., 163t345-351. Serhan, C.N., Fridovich, J., Goetzl, E.J., Dunham, P.B., and Weissmann, G. (1982) Leukotriene B, and phosphatidic acid are calcium ionophores. Studies with arsenazo 111 in liposomes. J . Biol. Chem., 257r4746-4752. Siekevitz, P. (1952) Uptake of radioactive alanine in vitro into the proteins of rat liver fractions. J. Biol. Chem., 195549-565. Slivka, S.R., and Insel, P.A. (1988a) Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and phosphoinositide hydrolysis in Madin-Darby canine kidney cells. Evidence that bradykinin mediates noninterdependent activation of phospholipases A, and C. J. Biol. Chem., 263:14640-14647. Slivka, S.R., Meier, K.E., and Insel, P.A. (198813) a,-Adrenergic receptors promote phosphatidylcholine hydrolysis in MDCK-D1 cells. A mechanism for rapid activation of protein kinase C. J. Biol. Chem., 263:12242-12246. Takuwa, N., Takuwa, Y., and Rasmussen, H. (1987) A tumour promoter, 12-0-tetradecanoylphorbol13-acetate increases cellular 1,2diacylglycerol content through a mechanism other than phosphoinositide hydrolysis in Swiss-mouse 3T3 fibroblasts. Biochem. J . , 243t647-653. Uratsuji, Y., and DiCorleto, P.E. (1988) Growth-dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells. J . Cell. Physiol., 136:431-438. Van Coevorden, A,, and Boeynaems, J.-M. (1984) Physiological concentrations of ADP stimulate the release of prostacyclin from bovine aortic endothelial cells. Prostaglandins, 27:615-626. Van Coevorden, A,, Roger, P.P., Pirotton, S., and Boeynaems, J.-M. (1989) Mitogenic action of adenine nucleotides and nucleosides on endothelial cells. Thromb. Haemost., 62:190. Welsh, C.J., Cao, H., Chabbott, H., and Cabot, M.C. (1988) Vasopressin is the only component of serum-free medium t h a t stimulates DhosDhatidvlcholine hvdrolvsis and accumulation of diacvlglycerbl in'cultured REF52 ;ells. Biochem. Biophys. Res. Cornmu;., 152t565-572.