Vol.
166,
January
No. 30,
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
Aggregating
909-915
PIatelets Increase IntraeeIluIar Calcium in Endothelial Cells Through Release of Adenine Nucleotides
Roger C. Ashmore*, Richard F. O’Brien, ThomasJ. Stelzner, Ira M. Dauber, Lawrence D. Horwitz, Ivan F. McMurtry, and Karyl M. VanBenthuysen Division of Cardiology and CardiovascularPulmonary ResearchLaboratory, University of ColoradoHealth SciencesCenter, and the VeteransAdministration Medical Center; Denver, CO
Received
December
20,
1989
SUMMARY: Aggregating platelets relax isolated coronary arteries through the release of endothelium-derived relaxing factor (EDRF). Since releaseof EDRF may be calcium dependent, we tested if and how aggregating platelets stimulated a calcium responsein cultured endothelial cells. Aggregating platelets causeda transient increasein intracellular calcium in endothelial cells loaded with the fluorescent calcium indicator fura-2. The adeninenucleotidesADP and ATP, but not other platelet-derived mediators,mimicked the platelet-inducedcalcium response,and inhibition of adeninenucleotides impaired the responseto aggregatingplatelets. Thus, aggregatingplatelets releaseadeninenucleotidesand stimulatea rise in intracellular calcium in cultured endothelialcells. This calcium responsemay representthe intracellular transductionmechanismby which aggregating plateletsinduceendothelialreleaseof EDRF and subsequent relaxation of coronary arteries. o
1990
Academic
Press,
Inc.
The endothelium plays an important role in the modulation of vascular tone. Previous studies show that platelets, which contain and release numerous vasoactive substances, induce endothelium-dependentrelaxation of isolated coronary arteries (1). The endothelium-dependent relaxation is probably due to release of endothelium-derived relaxing factor (EDRF) (2). The intracellular transduction mechanismby which aggregating platelets induce production and/or releaseof EDRF by vascular endothelialcells, however, hasnot beenelucidated. There has been indirect evidence supporting a role of intracellular calcium in EDRF release. First, the calcium ionophore A23187 is a potent stimulatorof EDRF release(3). Second,relaxation of isolated vascular rings by endothelium-dependentvasodilators and production of EDRF by endothelial cells grown on microcarrier beads are impaired or prevented by the absenceof extracellular calcium (4). Third, severalendothelium-dependentvasodilatorsincreaseintracellular calcium in cultured endothelial cells (5-9). The effect of aggregating platelets on endothelial intracellular calcium, however, is unknown.
*To whomcorrespondence and reprint requests should be addressed at Univ. of Colorado Health Sci. Ctr., B130, 4200 E. 9th Ave., Denver, CO 80262. Abbreviations: EDRF, endothelium-derivedrelaxing factor; MEM, minimal essentialmedia; BSS, balanced salt solution; ADP, adenosine5’ diphosphate; ATP, adenosine5’ triphosphate; PAF, platelet-activating factor, EC& effective concentrationto elicit 50% of maximal response.
909
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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No.
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We hypothesized that aggregating platelets stimulate increased intracellular calcium in endothelial cells. To examine this possibility we measured intracellular calcium in cultured endothelial cells by using the fluorescent calcium indicator fura-2. The purpose of this study was twofold: 1) to examine the effect of aggregating platelets on endothelial intracellular calcium; and 2) to identify the specific platelet-derived mediator(s) responsible for the platelet-induced calcium response.
MATERIALS
AND METHODS
Prenaration of human ulatelet co ce trw Blood was drawn from the antecubital vein with a large bore needle and minimum tot&&et time from healthy nonsmoking male volunteers on no medications. Platelet concentrate was obtained as described by Cohen et al (1) and kept at room temperature until used within 2 hours. Endothelial cell culture and measurement of endothelial intracellular calcium: Bovine thoracic aortic endothelial cells were harvested and cultured as previously described (10). Confluent cultured endothelial cells (passage 2-5) were suspended with trypsin-EDTA, centrifuged into a pellet, resuspended in MEM (minimal essential media) containing 15% fetal bovine serum, and seeded on 13 mm quartz coverslips at a density of 3 x 105 cells / coverslip. Endothelial monolayers were used for experiments after reaching confluence, typically within 2-3 days. Endothelial intracellular calcium concentration was monitored by utilizing the fluorescent calcium indicator, fura-2. The endothelial monolayers were incubated with 5 pM fura-2,AM (aceto oxymethyl) / MEM for 30 min at 370 C. After loading, excess fura-2,AM was removed by washing the monolayer with MEM. The monolayers were then incubated for 20 min to allow further cleavage of fura-2,AM by intracellular esterases. The fura- loaded monolayers were placed diagonally in a cuvette containing 2.5 ml of balanced salt solution (BSS). Fluorescence was measured using a Perkin-Elmer 650-10s model fluorescence spectrophotometer. The 500 nm emission intensity at the two excitation wavelenths 345, and 380 nm, was measured every 5-10 set by manually shifting the monochronometer. Intracellular calcium concentrations were calculated as described by Grynkiewicz et al (11). ExDerimental nrotocol: After baseline resting intracellular calcium measurements were obtained, calcium responses to cumulative doses of platelet concentrate (final cone 200 to 40,000 platelets / mm3), ADP (lo-9 to 10-5 M), ATP (lo-8 to 10-4 M), serotonin (lo-8 to 10-3 M), U46619, a thromboxane A2 mimetic, (lo-l0 to 10-6 M), platelet-activating factor (PAF) (lo-8 to low4 M), or bradykinin (5 x 10-8 M) were recorded. Measurement of calcium responses to platelet-derived supematant was accomplished by allowing platelets to aggregate (final cone = 10,000 platelets / mm3) in a calcium containing BSS, centrifuging the suspension for 10 min at 3000 rpm, and adding the supematant to the fura- loaded endothelial monolayers. All cell culture experiments were performed at 370C. Thromboxane B2 assav: Platelet concentrate (final cone 1,000 to 20,000 platelets / mm3) was added to 2 ml of calcium containing BSS. The platelet suspension was centrifuged for 10 min at 3000 rpm and 1 ml of the supematant was withdrawn and stored at < 00 C. Thromboxane B2 (a stable thromboxane A2 metabolite released during platelet aggregation) levels in the supematant were determined using an enzyme immunoassay (12). Drugs and reagents: Sigma Chemical Co., St Louis, MO: apyrase (grade V), adenosine 5’ diphosphate (ADP), adenosine 5’ triphosphate (ATP), bradykinin, calcium ionophore A23 187, reactive blue-2 (basilen blue E-36), serotonin (5 hydroxytryptamine), platelet-activating factor (L-aphosphatidylcholine$-acetyl-y-o-alkyl). Molecular Probes Inc., Eugene, OR: fura-2,AM. The Upjohn Co., Kalamazoo, MI: U46619 (9,11-dideoxy-1 lol,9a-epoxymethano-prostaglandin F2a). Statistics: Data are expressed as means f SE, and n refers to the number of monolayers tested. Endothelial intracellular calcium concentration-responsecurves to aggregating platelets, adenine nucleotides, and PAF were plotted for each individual monolayer and were expressed as a 910
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percentage of maximal stimulated calcium responses. Graphic determination of EC50 (the concentration of agent required to elicit 50% of maximal stimulated calcium response) was obtained from each concentration-response curve. Comparisons among groups were made using a one-way analysis of variance with the Sheffe multiple comparison test. Significance was accepted at a 0.05 level.
RESULTS mtelet-induced calcmm responses in culm Aggregating platelets caused a rapid rise in endothelial intracellular calcium which returned to resting levels within 30-60 set (figure 1). The peak of this transient calcium response was dependent on the concentration of aggregating platelets (figure 1). The platelet vehicle, sodium citrate, had no effect on intracellular calcium (data not shown). To test the role of platelet-derived mediators and whether platelet adherence to the endothelium was necessary for the platelet-induced calcium response, the supematant from aggregating platelets was applied to endothelial monolayers. The peak endothelial calcium response stimulated by 10,000 platelets / mm3 was similar to that elicited by the supematant from the same number of aggregating platelets (127 IL 17 vs 146 rt 19 nM, respectively,
n = 6). Thus, aggregating platelets
stimulate a transient calcium response via a transferable secretory product, and platelet adherence to the endothelial monolayer is not required for this response. Effect of individual Dlatelet-derived mediators on endothelial calcium resbonsa:
To determine
individual platelet-derivedmediatorscould mimic the platelet-inducedcalciumresponse,the time and concentration-dependent effects of various platelet-derived vasoactive mediators on
whether
endothelial intracellular calcium were tested (figure 2). The adeninenucleotides ADP and ATP causeda transient andconcentration-dependentrisein intracellular calcium similarto the responseto aggregatingplatelets. In contrast, PAF causeda sustainedrise in intracellular calcium, and only at very high concentrations. Other platelet products such as serotonin (10-g to 10-3 M, n = 4) and a
0
30
60
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120
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PLATELETS (I mm3)
I. Effect of aggregating platelets on endothelial intracellular calcium. Aggregating platelets (10,000 platelets / mm3) caused a transient rise in endothelial intracellular calcium (left graft, results from a single endothelial monolayer). The concentration effect of aggregating platelets on peak intracellular calcium responses is shown on the right graft. Aggregating platelets (range 200 to 40,000 platelets / mm3) caused a concentration-dependent increase in the peak intracellular endothelial calcium response (n = 7). Figure
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Figure 2. Effect of individual platelet-derived mediators on endothelial intracellular calcium. ADP and ATP caused a transient rise in resting endothelial calcium (results from single monolayers) and a concentration-dependent increase in the peak intracellular endothelial calcium response (n = 7 ADP and ATP). Platelet activating factor (PAF) caused a sustained rise in resting endothelial intracellular calcium (results from a single monolayer), but only at highconcentrations (concentration-response n =4).
thromboxane analogue, U46619, (lo- 10 to 10-6 M, n = 5) did not alter endothelial intracellular calcium (data not shown). Fffect of adeninenucleotide inhibitors on endoscalciumonses: To furthur test the role of adeninenucleotidesin platelet-inducedcalcium signaling,the effects of adeninenucleotideinhibitors on endothelialcalcium responsesto aggregatingplatelets were studied. The inhibitors testedwere apyrase,an enzyme which degradesADP and ATP (13), and reactive blue-2, a putative purinergic receptor antagonist (14). Both apyrase and reactive blue-2 significantly impaired the calcium responsesto aggregatingplatelets (figure 3). The inhibitory effect of apyrasewasdiminishedat the higher concentration of platelets. The effects of the adeninenucleotide inhibitors on the calcium 912
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platelets platelets
+ apyrase
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+ reactive
COMMUNICATIONS
blue-2
PLATELETS (/mm3)
3. Effect of adeninenucleotide inhibitors on platelet-inducedpeak intracellular calcium responses. The platelet-induced peak intracellular calcium responses (same data as shown in figure 2) were significantly impaired by apyrase (.67 u /ml) and by reactive blue-2 (2 x IO-5 M) (platelets = closed circles, EC50 = 6.4 k 0.6 x 1000 platelets / mm3, n = 5; platelets + apyrase = open circles, EC50 = 15.4 + 1.9 x 1000 platelets / mm3, n = 5; and platelets + reactive blue-2 = closed triangles, EC50 = 12.3 f 1.5 x 1000 platelets /mm 3, n = 5; p c 0.05 platelets vs platelets + apyrase and platelets + reactive blue-2). Figure
response to platelet-derived supematant were also tested. Both apyrase and reactive blue-2 markedly impaired the calcium response to the supematant from aggregating platelets (supematant from 10,000 platelets / mm3 = 100 + 19 nM, n = 7; supematant + apyrase = 15 f 15 nM, n = 3; and supematant + reactive blue-2 = 7 f 4 nM, n = 3; p < 0.05 supematant vs supematant + apyrase and supematant + reactive blue-2). These data suggest that adenine nucleotides are the transferable mediator responsible for the platelet-induced calcium response. The effect of the inhibitors apyrase and reactive blue-2 was tested on the ADP-stimulated calcium response in order to confii adenine nucleotide inhibition. The endothelial calcium response to cumulative concentrations of ADP was significantly impaired by apyrase and reactive blue-2 (ADP EC50 = 2.2 rf: 0.4 x 10-7 M, n = 8; ADP + apyrase EC50 = 20.4 f 3.0 x 10e7 M, n = 6; and ADP + reactive blue-2 EC50 = 17.6 + 8.3 x 10-7 M, n = 4; p < 0.05 for ADP vs ADP + apyrase and ADP + reactive blue-2). The effects of apyrase and reactive blue-2 on the non-purinergic
calcium
stimulating agent bradykinin were tested to exclude non-specific inhibition of endothelial calcium responses. Bradykinin
(5 x lo-8 M) produced a peak intracellular calcium response comparable to
that achieved with 10,000 platelets / mm 3. This response was not affected by apyrase or reactive blue-2 (bradykinin EC50 = 132 f 39 nM, n = 8; bradykinin + apyrase EC50 = 125 f 21 nM, n = 7; and bradykinin + reactive blue-2 EC50 = 158 I!I 33 nM, n = 6; p = NS). Thromboxane B? determinations: To test whether the platelets were aggregating and whether the adenine nucleotide inhibitors impaired platelet aggregation, we measured thromboxane B2 levels. When added to calcium containing BSS, platelets released concentration-dependent increases in thromboxane B2 (1,000 platelets = 916 pg/ml; 10,000 platelets = 2473 pgAnl; 20,000 platelets = 3545 pgjml). The thromboxane B2 release was markedly blunted if the platelet concentrate (10,000 platelets / mm3) was placed in the antiaggregatory sodium citrate solution (10,000 platelets + NaCit = 452 pg/ml). Reactive blue-2 did not significantly impair thromboxane B2 release from platelet concentrate (data not shown).
Because apyrase interfered with the assay, the effect of apyrase on 913
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thromboxane B2 release was unclear. However, clearing of the turbid suspensions suggested that aggregation occured in the presence of apyrase.
DISCUSSION Our studies showed that aggregating platelets stimulated an increase in intracellular calcium in cultured endothelial cells. The calcium response was transient, concentration-dependent, and reproduced by the supernatant of aggregating platelets. Therefore, a platelet-derived transferable mediator was responsible for the calcium response, and platelet adhesion to the endothelial monolayer was not required. We tested several platelet-derived products for evidence that they were the mediator. The adenine nucleotides ADP and ATP mimicked the platelet induced calcium response. A similar increase in intracellular calcium in cultured endothelial cells with adenine nucleotides has been reported by Luckhoff and Busse (8). We also found that inhibitors of adenine nucleotides impaired the platelet-, platelet-derived supematant-, and ADP-induced calcium responses. Although PAF also induced an increase in intracellular calcium, the response was sustained (instead of transient) and only produced with very high concentrations (10s4 M). Serotonin and the thromboxane A2 mimetic U46619 had no effect on endothelial intracellular calcium levels. Although, previous studies show that aggregating platelets cause endothelium-dependent relaxation of isolated coronary arteries (l), probably through the release of EDRF (2), and that the release and/or production EDRF is calcium-dependent (15), the effect of aggregating platelets on endothelial intracellular calcium was unknown. This study suggests that adenine nucleotides, and not the other platelet-derived vasoactive mediators tested, stimulate the platelet-induced calcium response in endothelial cells. These data are consistent with previous studies which show that adenine nucleotides released from aggregating platelets are probably responsible for the relaxation of isolated coronary arteries (13). The adenine nucleotide-induced calcium response may represent the intracellular transduction mechanism by which aggregating platelets stimulate endothelial release of EDRF and subsequent relaxation of isolated coronary arteries. However, the effects of platelet-induced calcium signaling are not necessarily limited to the release of EDRF and may represent a common mechanism for the release of other endothelium-derived anti-thrombotic and vasoactive substances, such as tissue plasminogen activator, prostacyclin, and endothelin. Improved understanding of the link between calcium signaling and endothelial release of anti-thrombotic and vasoactive substances may provide therapeutic applications in the management of numerous cardiovascular diseases.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the expert technical assistance of Ms. Holly CoUer, Ms. Karen Johnston, Ms. Julie Peach, and Ms. Sandra Walchak. We wish to thank Dr. Jay Westcott for his advice and support in thromboxane B2 measurements. This work was supported by grants HL41661 and HL07459 from the National Institutes of Health, the Veterans Administration Research Service, and the Corporate Heart Fund. 914
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REFERENCES 1. Cohen, R.A., Shephard,J.T., Vanhoutte, P.M. (1983) Science ; 221:273-274. 2. Furchgott, R.F. (1983) Circ. Res.; 53 (5):557-573. 3. Furchgott, R.F. (1981) Trends Pharmacol. Sci.; 2:173-176. 4. Peach, M.J., Singer, H.A., Izzo, N.J., Loeb, A.L. (1987) Am. J. Cardiol.; 59:35A-43A. 5. Jaffe, E.A., Grulich, J., Weksler, B.B., Hampel, G., Watanabe, K. (1987) J. Biol. Chem.; 262 (18):8557-8565. 6. Johns, A., Lategan, T.W., Lodge, N.J., Ryan, U.S., VanBreeman, C., Adams, D.J. (1987) TissueCell ; 19 (6):733-745. 7. Colden-Stanfield, M., Schilling, W.P., Ritchie, A.K., Eskin, S.G., Navarro, L.T., Kunze, D.L. (1987) Circ. Res.; 61:632&O. 8. Luckhoff, A., Busse, R. (1986) J. Cell. Physiol.; 126:414-420. 9. Danthuluri, N.R., Cybulsky, M.I., Brock, T.A.. (1988) Am. J. Physiol.; 255:H1549-H1553. lO.O’Brien, R.F., Robbins, R.J., McMurtry, I.F. (1987) J. Cell. Physiol.; 132:263-270. 1l.Grynkiewicz, G., Poenie, M., Tsien, R.Y. (1985) J. Biol. Chem.; 260 (6):3440-3450. 12Pradelles, P., Grassi ,J., Maclouf, J. (1985) Anal. Chem.; 57:1170-1173. 13.Houston, D.S., Shephard, J.T., Vanhoutte, P.M. (1986) J. Clin. Invest.; 78:539-544. 14.Houston, D.S., Bumstock, G., Vanhoutte, P.M. (1987) J. Pharmacol. Exp. Ther. 241(2):501-506. 15.Rubanyi, G.M., Vanhoutte, P.M. (1988) Ann. N. Y. Acad. Sci.; 522:226-233.
915
166,
January
No. 30,
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
Aggregating
909-915
PIatelets Increase IntraeeIluIar Calcium in Endothelial Cells Through Release of Adenine Nucleotides
Roger C. Ashmore*, Richard F. O’Brien, ThomasJ. Stelzner, Ira M. Dauber, Lawrence D. Horwitz, Ivan F. McMurtry, and Karyl M. VanBenthuysen Division of Cardiology and CardiovascularPulmonary ResearchLaboratory, University of ColoradoHealth SciencesCenter, and the VeteransAdministration Medical Center; Denver, CO
Received
December
20,
1989
SUMMARY: Aggregating platelets relax isolated coronary arteries through the release of endothelium-derived relaxing factor (EDRF). Since releaseof EDRF may be calcium dependent, we tested if and how aggregating platelets stimulated a calcium responsein cultured endothelial cells. Aggregating platelets causeda transient increasein intracellular calcium in endothelial cells loaded with the fluorescent calcium indicator fura-2. The adeninenucleotidesADP and ATP, but not other platelet-derived mediators,mimicked the platelet-inducedcalcium response,and inhibition of adeninenucleotides impaired the responseto aggregatingplatelets. Thus, aggregatingplatelets releaseadeninenucleotidesand stimulatea rise in intracellular calcium in cultured endothelialcells. This calcium responsemay representthe intracellular transductionmechanismby which aggregating plateletsinduceendothelialreleaseof EDRF and subsequent relaxation of coronary arteries. o
1990
Academic
Press,
Inc.
The endothelium plays an important role in the modulation of vascular tone. Previous studies show that platelets, which contain and release numerous vasoactive substances, induce endothelium-dependentrelaxation of isolated coronary arteries (1). The endothelium-dependent relaxation is probably due to release of endothelium-derived relaxing factor (EDRF) (2). The intracellular transduction mechanismby which aggregating platelets induce production and/or releaseof EDRF by vascular endothelialcells, however, hasnot beenelucidated. There has been indirect evidence supporting a role of intracellular calcium in EDRF release. First, the calcium ionophore A23187 is a potent stimulatorof EDRF release(3). Second,relaxation of isolated vascular rings by endothelium-dependentvasodilators and production of EDRF by endothelial cells grown on microcarrier beads are impaired or prevented by the absenceof extracellular calcium (4). Third, severalendothelium-dependentvasodilatorsincreaseintracellular calcium in cultured endothelial cells (5-9). The effect of aggregating platelets on endothelial intracellular calcium, however, is unknown.
*To whomcorrespondence and reprint requests should be addressed at Univ. of Colorado Health Sci. Ctr., B130, 4200 E. 9th Ave., Denver, CO 80262. Abbreviations: EDRF, endothelium-derivedrelaxing factor; MEM, minimal essentialmedia; BSS, balanced salt solution; ADP, adenosine5’ diphosphate; ATP, adenosine5’ triphosphate; PAF, platelet-activating factor, EC& effective concentrationto elicit 50% of maximal response.
909
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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We hypothesized that aggregating platelets stimulate increased intracellular calcium in endothelial cells. To examine this possibility we measured intracellular calcium in cultured endothelial cells by using the fluorescent calcium indicator fura-2. The purpose of this study was twofold: 1) to examine the effect of aggregating platelets on endothelial intracellular calcium; and 2) to identify the specific platelet-derived mediator(s) responsible for the platelet-induced calcium response.
MATERIALS
AND METHODS
Prenaration of human ulatelet co ce trw Blood was drawn from the antecubital vein with a large bore needle and minimum tot&&et time from healthy nonsmoking male volunteers on no medications. Platelet concentrate was obtained as described by Cohen et al (1) and kept at room temperature until used within 2 hours. Endothelial cell culture and measurement of endothelial intracellular calcium: Bovine thoracic aortic endothelial cells were harvested and cultured as previously described (10). Confluent cultured endothelial cells (passage 2-5) were suspended with trypsin-EDTA, centrifuged into a pellet, resuspended in MEM (minimal essential media) containing 15% fetal bovine serum, and seeded on 13 mm quartz coverslips at a density of 3 x 105 cells / coverslip. Endothelial monolayers were used for experiments after reaching confluence, typically within 2-3 days. Endothelial intracellular calcium concentration was monitored by utilizing the fluorescent calcium indicator, fura-2. The endothelial monolayers were incubated with 5 pM fura-2,AM (aceto oxymethyl) / MEM for 30 min at 370 C. After loading, excess fura-2,AM was removed by washing the monolayer with MEM. The monolayers were then incubated for 20 min to allow further cleavage of fura-2,AM by intracellular esterases. The fura- loaded monolayers were placed diagonally in a cuvette containing 2.5 ml of balanced salt solution (BSS). Fluorescence was measured using a Perkin-Elmer 650-10s model fluorescence spectrophotometer. The 500 nm emission intensity at the two excitation wavelenths 345, and 380 nm, was measured every 5-10 set by manually shifting the monochronometer. Intracellular calcium concentrations were calculated as described by Grynkiewicz et al (11). ExDerimental nrotocol: After baseline resting intracellular calcium measurements were obtained, calcium responses to cumulative doses of platelet concentrate (final cone 200 to 40,000 platelets / mm3), ADP (lo-9 to 10-5 M), ATP (lo-8 to 10-4 M), serotonin (lo-8 to 10-3 M), U46619, a thromboxane A2 mimetic, (lo-l0 to 10-6 M), platelet-activating factor (PAF) (lo-8 to low4 M), or bradykinin (5 x 10-8 M) were recorded. Measurement of calcium responses to platelet-derived supematant was accomplished by allowing platelets to aggregate (final cone = 10,000 platelets / mm3) in a calcium containing BSS, centrifuging the suspension for 10 min at 3000 rpm, and adding the supematant to the fura- loaded endothelial monolayers. All cell culture experiments were performed at 370C. Thromboxane B2 assav: Platelet concentrate (final cone 1,000 to 20,000 platelets / mm3) was added to 2 ml of calcium containing BSS. The platelet suspension was centrifuged for 10 min at 3000 rpm and 1 ml of the supematant was withdrawn and stored at < 00 C. Thromboxane B2 (a stable thromboxane A2 metabolite released during platelet aggregation) levels in the supematant were determined using an enzyme immunoassay (12). Drugs and reagents: Sigma Chemical Co., St Louis, MO: apyrase (grade V), adenosine 5’ diphosphate (ADP), adenosine 5’ triphosphate (ATP), bradykinin, calcium ionophore A23 187, reactive blue-2 (basilen blue E-36), serotonin (5 hydroxytryptamine), platelet-activating factor (L-aphosphatidylcholine$-acetyl-y-o-alkyl). Molecular Probes Inc., Eugene, OR: fura-2,AM. The Upjohn Co., Kalamazoo, MI: U46619 (9,11-dideoxy-1 lol,9a-epoxymethano-prostaglandin F2a). Statistics: Data are expressed as means f SE, and n refers to the number of monolayers tested. Endothelial intracellular calcium concentration-responsecurves to aggregating platelets, adenine nucleotides, and PAF were plotted for each individual monolayer and were expressed as a 910
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percentage of maximal stimulated calcium responses. Graphic determination of EC50 (the concentration of agent required to elicit 50% of maximal stimulated calcium response) was obtained from each concentration-response curve. Comparisons among groups were made using a one-way analysis of variance with the Sheffe multiple comparison test. Significance was accepted at a 0.05 level.
RESULTS mtelet-induced calcmm responses in culm Aggregating platelets caused a rapid rise in endothelial intracellular calcium which returned to resting levels within 30-60 set (figure 1). The peak of this transient calcium response was dependent on the concentration of aggregating platelets (figure 1). The platelet vehicle, sodium citrate, had no effect on intracellular calcium (data not shown). To test the role of platelet-derived mediators and whether platelet adherence to the endothelium was necessary for the platelet-induced calcium response, the supematant from aggregating platelets was applied to endothelial monolayers. The peak endothelial calcium response stimulated by 10,000 platelets / mm3 was similar to that elicited by the supematant from the same number of aggregating platelets (127 IL 17 vs 146 rt 19 nM, respectively,
n = 6). Thus, aggregating platelets
stimulate a transient calcium response via a transferable secretory product, and platelet adherence to the endothelial monolayer is not required for this response. Effect of individual Dlatelet-derived mediators on endothelial calcium resbonsa:
To determine
individual platelet-derivedmediatorscould mimic the platelet-inducedcalciumresponse,the time and concentration-dependent effects of various platelet-derived vasoactive mediators on
whether
endothelial intracellular calcium were tested (figure 2). The adeninenucleotides ADP and ATP causeda transient andconcentration-dependentrisein intracellular calcium similarto the responseto aggregatingplatelets. In contrast, PAF causeda sustainedrise in intracellular calcium, and only at very high concentrations. Other platelet products such as serotonin (10-g to 10-3 M, n = 4) and a
0
30
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PLATELETS (I mm3)
I. Effect of aggregating platelets on endothelial intracellular calcium. Aggregating platelets (10,000 platelets / mm3) caused a transient rise in endothelial intracellular calcium (left graft, results from a single endothelial monolayer). The concentration effect of aggregating platelets on peak intracellular calcium responses is shown on the right graft. Aggregating platelets (range 200 to 40,000 platelets / mm3) caused a concentration-dependent increase in the peak intracellular endothelial calcium response (n = 7). Figure
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Figure 2. Effect of individual platelet-derived mediators on endothelial intracellular calcium. ADP and ATP caused a transient rise in resting endothelial calcium (results from single monolayers) and a concentration-dependent increase in the peak intracellular endothelial calcium response (n = 7 ADP and ATP). Platelet activating factor (PAF) caused a sustained rise in resting endothelial intracellular calcium (results from a single monolayer), but only at highconcentrations (concentration-response n =4).
thromboxane analogue, U46619, (lo- 10 to 10-6 M, n = 5) did not alter endothelial intracellular calcium (data not shown). Fffect of adeninenucleotide inhibitors on endoscalciumonses: To furthur test the role of adeninenucleotidesin platelet-inducedcalcium signaling,the effects of adeninenucleotideinhibitors on endothelialcalcium responsesto aggregatingplatelets were studied. The inhibitors testedwere apyrase,an enzyme which degradesADP and ATP (13), and reactive blue-2, a putative purinergic receptor antagonist (14). Both apyrase and reactive blue-2 significantly impaired the calcium responsesto aggregatingplatelets (figure 3). The inhibitory effect of apyrasewasdiminishedat the higher concentration of platelets. The effects of the adeninenucleotide inhibitors on the calcium 912
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PLATELETS (/mm3)
3. Effect of adeninenucleotide inhibitors on platelet-inducedpeak intracellular calcium responses. The platelet-induced peak intracellular calcium responses (same data as shown in figure 2) were significantly impaired by apyrase (.67 u /ml) and by reactive blue-2 (2 x IO-5 M) (platelets = closed circles, EC50 = 6.4 k 0.6 x 1000 platelets / mm3, n = 5; platelets + apyrase = open circles, EC50 = 15.4 + 1.9 x 1000 platelets / mm3, n = 5; and platelets + reactive blue-2 = closed triangles, EC50 = 12.3 f 1.5 x 1000 platelets /mm 3, n = 5; p c 0.05 platelets vs platelets + apyrase and platelets + reactive blue-2). Figure
response to platelet-derived supematant were also tested. Both apyrase and reactive blue-2 markedly impaired the calcium response to the supematant from aggregating platelets (supematant from 10,000 platelets / mm3 = 100 + 19 nM, n = 7; supematant + apyrase = 15 f 15 nM, n = 3; and supematant + reactive blue-2 = 7 f 4 nM, n = 3; p < 0.05 supematant vs supematant + apyrase and supematant + reactive blue-2). These data suggest that adenine nucleotides are the transferable mediator responsible for the platelet-induced calcium response. The effect of the inhibitors apyrase and reactive blue-2 was tested on the ADP-stimulated calcium response in order to confii adenine nucleotide inhibition. The endothelial calcium response to cumulative concentrations of ADP was significantly impaired by apyrase and reactive blue-2 (ADP EC50 = 2.2 rf: 0.4 x 10-7 M, n = 8; ADP + apyrase EC50 = 20.4 f 3.0 x 10e7 M, n = 6; and ADP + reactive blue-2 EC50 = 17.6 + 8.3 x 10-7 M, n = 4; p < 0.05 for ADP vs ADP + apyrase and ADP + reactive blue-2). The effects of apyrase and reactive blue-2 on the non-purinergic
calcium
stimulating agent bradykinin were tested to exclude non-specific inhibition of endothelial calcium responses. Bradykinin
(5 x lo-8 M) produced a peak intracellular calcium response comparable to
that achieved with 10,000 platelets / mm 3. This response was not affected by apyrase or reactive blue-2 (bradykinin EC50 = 132 f 39 nM, n = 8; bradykinin + apyrase EC50 = 125 f 21 nM, n = 7; and bradykinin + reactive blue-2 EC50 = 158 I!I 33 nM, n = 6; p = NS). Thromboxane B? determinations: To test whether the platelets were aggregating and whether the adenine nucleotide inhibitors impaired platelet aggregation, we measured thromboxane B2 levels. When added to calcium containing BSS, platelets released concentration-dependent increases in thromboxane B2 (1,000 platelets = 916 pg/ml; 10,000 platelets = 2473 pgAnl; 20,000 platelets = 3545 pgjml). The thromboxane B2 release was markedly blunted if the platelet concentrate (10,000 platelets / mm3) was placed in the antiaggregatory sodium citrate solution (10,000 platelets + NaCit = 452 pg/ml). Reactive blue-2 did not significantly impair thromboxane B2 release from platelet concentrate (data not shown).
Because apyrase interfered with the assay, the effect of apyrase on 913
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thromboxane B2 release was unclear. However, clearing of the turbid suspensions suggested that aggregation occured in the presence of apyrase.
DISCUSSION Our studies showed that aggregating platelets stimulated an increase in intracellular calcium in cultured endothelial cells. The calcium response was transient, concentration-dependent, and reproduced by the supernatant of aggregating platelets. Therefore, a platelet-derived transferable mediator was responsible for the calcium response, and platelet adhesion to the endothelial monolayer was not required. We tested several platelet-derived products for evidence that they were the mediator. The adenine nucleotides ADP and ATP mimicked the platelet induced calcium response. A similar increase in intracellular calcium in cultured endothelial cells with adenine nucleotides has been reported by Luckhoff and Busse (8). We also found that inhibitors of adenine nucleotides impaired the platelet-, platelet-derived supematant-, and ADP-induced calcium responses. Although PAF also induced an increase in intracellular calcium, the response was sustained (instead of transient) and only produced with very high concentrations (10s4 M). Serotonin and the thromboxane A2 mimetic U46619 had no effect on endothelial intracellular calcium levels. Although, previous studies show that aggregating platelets cause endothelium-dependent relaxation of isolated coronary arteries (l), probably through the release of EDRF (2), and that the release and/or production EDRF is calcium-dependent (15), the effect of aggregating platelets on endothelial intracellular calcium was unknown. This study suggests that adenine nucleotides, and not the other platelet-derived vasoactive mediators tested, stimulate the platelet-induced calcium response in endothelial cells. These data are consistent with previous studies which show that adenine nucleotides released from aggregating platelets are probably responsible for the relaxation of isolated coronary arteries (13). The adenine nucleotide-induced calcium response may represent the intracellular transduction mechanism by which aggregating platelets stimulate endothelial release of EDRF and subsequent relaxation of isolated coronary arteries. However, the effects of platelet-induced calcium signaling are not necessarily limited to the release of EDRF and may represent a common mechanism for the release of other endothelium-derived anti-thrombotic and vasoactive substances, such as tissue plasminogen activator, prostacyclin, and endothelin. Improved understanding of the link between calcium signaling and endothelial release of anti-thrombotic and vasoactive substances may provide therapeutic applications in the management of numerous cardiovascular diseases.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the expert technical assistance of Ms. Holly CoUer, Ms. Karen Johnston, Ms. Julie Peach, and Ms. Sandra Walchak. We wish to thank Dr. Jay Westcott for his advice and support in thromboxane B2 measurements. This work was supported by grants HL41661 and HL07459 from the National Institutes of Health, the Veterans Administration Research Service, and the Corporate Heart Fund. 914
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