Proc. Nati. Acad. Sci. USA Vol. 82, pp. 2623-2626, May 1985

1,2-Diacylglycerol and phorbol ester inhibit agonist-induced formation of phosphates in human platelets: Possible implications for negative feedback regulation of inositol hydrolysis (protein kinase C/inositol /thrombin/collagen/platelet-activating factor) STEPHEN P. WATSON AND EDUARDO G. LAPETINA* Department of Molecular Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709 Communicated by George H. Hitchings, December 26, 1984

ABSTRACT The present study has demonstrated that Samples (0.49 ml) were placed in aggregometer tubes and pretreatment of human platelets with either phorbol ester or left at 370C for 3 min. OAG and phorbol dibutyrate (PBt2) 1,2-diacylglycerol inhibits agonist-induced formation of inosi- were added and the tubes were left without stirring at 370C tol phosphates; this inhibition can be correlated with a de- for various times. [Similar results were obtained when plate- crease in the release of ATP and 5-hydroxytryptamine by lets were stirred in a Chrono-Log (Havertown, PA) aggre- thrombin. The mechanism of this action is not known, but a gometer.] The reaction was stopped by transferring the con- role for protein kinase C is suggested, as both phorbol ester tents to tubes containing 1.88 ml of CHCl3/MeOH/HCl and 1,2-diacylglycerol have in common the ability to activate (100:200:2) by means of a Pasteur pipette. Inositol phos- this enzyme. These results have important implications as a phates were analyzed by ion exchange chromatography, and possible negative feedback control over agonist-induced hy- inositol phospholipids were analyzed by thin-layer chroma- drolysis of inositol phospholipids. tography as described (3). For determinations of protein , however, the reaction was stopped by Platelet activation by agonists such as thrombin (1-3) and transferring aliquots (100 ul) to tubes containing 25 ,1l of 5 collagen (4) is mediated through the phosphodiesteric cleav- times concentrated Laemmli sample buffer and incubated at age of polyphosphoinositides and, possibly, phosphatidylin- 100°C for 10 min; proteins were then analyzed as described ositol. This hydrolysis generates two bioactive products, (14). 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Experiments involving the pretreatment of platelets with 1,2-Diacylglycerol activates protein kinase C, and this has PBt2 or OAG before addition of thrombin were carried out in been shown to phosphorylate a 40,000-dalton protein in the following manner. Platelets (0.49 ml) were incubated platelets of unknown function (5); phorbol esters can substi- with OAG (50 ,uM) or PBt2 (200 nM) in aggregometer tubes tute for 1,2-diacylglycerol in the activation of protein kinase for 9 min at 37°C without stirring. They/ were then trans- C (5). InsP3 mobilizes Ca2' from the endoplasmic reticulum ferred to a Chrono-Log aggregometer and, stirred for 1 min at of a number of tissues (6-9) and induces protein phosphoryl- 37°C. Thrombin, collagen, or platelet-activating factor was ation in platelets (10). then added and the reaction was terminated at various times Recently, it has been demonstrated that phorbol 12-myris- by transferring to CHCl3/MeOH/HCl (100:200:2) as de- tate 13-acetate (11, 12) and 1-oleoyl-2-acetyl-glycerol (OAG) scribed above. Light transmission (monitor of shape change (13) stimulate a rapid increase in the levels of [3 P]phosphati- and aggregation) and luciferase fluorescence (monitor of dylinositol 4-phosphate and [32P] 4,5- ATP release) were recorded continuously throughout the ex- bisphosphate in platelets. Moreover, Halenda and Feinstein periment. (12) further showed that the increased incorporation of 32p Experiments studying the release of 5-[3H]hydroxytrypta- into these two was accompanied by an increase in their mine were carried out as described above except the reac- mass. The active concentration range of this action for both tion was terminated by transferring to tubes containing 0.5 agents was similar to that for phosphorylation of the 40,000- ml of 6% glutaraldehyde dissolved in 0.1 M sodium hydrox- dalton protein. These observations, therefore, suggest the ide. The tubes were then spun in a Vortex, centrifuged in a possibility that protein kinase C may exert some form of bench Microfuge for 30 sec, and aliquots (0.5 ml) of the su- feedback regulation over the receptor-induced hydrolysis of pernatant were counted by liquid scintillometry. polyphosphoinositides, and this possibility has been investi- Results are presented from one experiment performed in gated in the present study. triplicate or duplicate, which is representative of at least two others. OAG was synthesized as described (16). 5-[3H]Hy- METHODS droxytryptamine was from Amersham, platelet-activating factor was from Calbiochem-Behring, and collagen was Human platelets were isolated on the day of the experiment from Horm-Chemie (Munich, F.R.G.). All other reagents and labeled with [3H]inositol, [32P]orthophosphate, and 5- were from previously described sources (3, 4). [3H]hydroxytryptamine as described (refs. 3, 14, and 15, re- spectively). The platelets were finally suspended at a con- centration of 8 x 108 per ml in a modified Tyrode Hepes RESULTS buffer (134 mM NaCl/12 mM NaHCO3/2.9 mM KCl/0.36 Time Course of Phosphorylation of the Polyphosphoinosi- mM NaH2PO4/1 mM MgCl2/5 mM Hepes/5 mM glucose/1 tides and the 40,000-Dalton Protein by OAG and PBt2. Both mM EGTA, pH 7.4). OAG and PBt2 rapidly (within seconds) phosphorylate a

The publication costs of this article were defrayed in part by page charge Abbreviations: OAG, 1-oleoyl-2-acetyl-glycerol; PBt2, phorbol di- payment. This article must therefore be hereby marked "advertisement" butyrate; InsP3, inositol 1,4,5-trisphosphate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom all reprint requests should be addressed. 2623 Downloaded by guest on October 3, 2021 2624 Biochemistry: Watson and Lapetina Proc. NaM Acad Sci. USA 82 (1985) 40,000-dalton protein in human platelets (5), with maximal Table 1. Action of PBt2 and OAG on [3H] activity occurring at 50 ,tM (16) and 200 nM (14), respective- levels in human platelets ly. In the present study, we have observed that maximal 32p InsP phosphorylation of the 40,000-dalton protein by PBt2 (200 InsP2 InsP3 nM) is reached after 1 min and is maintained for at least 10 PBt2 102 ± 6 99 ± 11 63 ± 31 min (not shown). In contrast, de Chaffoy de Courcelles et al. OAG % ± 7 85 ± 3 110 ± 44 (11) reported that phosphorylation of the 40,000-dalton pro- PBt2 (200 nM) or OAG (50 ,uM) was incubated with platelets (no tein by OAG (10 ,uM) returns to basal levels within 4 min in stirring) for 10 min in the presence of EGTA (1 mM) and Ca2+ (2 human platelets. mM). The reaction was then stopped by transferring to CHCl3/ Phosphorylation of the polyphosphoinositides by OAG MeOH/HCl (100:200:2) and [3H]inositol phosphates were analyzed and PBt2 lagged behind the phosphorylation of the 40,000- as described. Results are expressed as % increase above basal and dalton protein and peaked after 1-3 min (not shown). The each point is the mean ± SEM of three experiments. InsP, inositol elevated levels of the polyphosphoinositides induced by monophosphate; InsP2, inositol bisphosphate. PBt2 (200 nM) remained constant for at least 20 min, but for OAG (50 ,uM) they returned to basal levels by 15 min (not Effect of Pretreatment of Platelets with OAG and PBt2 on shown). The increase varied from 30% to 70% ofcontrol val- the Action of Thrombin. It is important to stress that these ues, and the effect on phosphatidylinositol 4-monophos- experiments have been carried out in Ca2+-free medium con- phate was always greater than on phosphatidylinositol 4,5- taining EGTA (1 mM). Under these conditions, neither OAG bisphosphate (e.g., see Fig. 1). This increase was occasional- (17) nor PBt2 (14) induces significant aggregation and re- ly accompanied by a slight (<10%) decrease in the levels of leases responses in platelets. This is in contrast to platelets phosphatidylinositol (see Fig. 1). Essentially similar results maintained in EGTA-free medium, which respond to both were obtained by de Chaffoy de Courcelles et al. (11, 13) and stimuli with significant aggregation and release responses Halenda and Feinstein (12). (18). Action of OAG and PBt2 on Inositol Phosphates and Inositol Pretreatment of platelets with OAG (50 ,uM) and PBt2 (200 Phospholipids. Exposure of platelets to OAG (50 uM) or nM) for 10 min inhibited formation of all three [3H]inositol PBt2 (200 nM) for times up to 10 min had no effect on the phosphates by thrombin (1 unit/ml) (Figs. 2 and 3). This in- levels of [3H]inositol phosphates in Ca2+ (2 mM)-containing hibitory action could be detected as early as 5 sec after expo- medium (Table 1). Previously, it had been reported that nei- sure to thrombin (1 unit/ml) (Fig. 2); e.g., the levels of ther OAG (50 ,uM) nor 1,2-didecanoylglycerol (50 ,uM) stim- ulated formation of [3H]inositol phosphates in platelets 12 r maintained in the absence of Ca2' and in the presence of EGTA (1 mM) for 2 min (16). These data thereby demon- 10 strate that neither PBt2 nor OAG induces the phosphodies- teric cleavage of inositol phospholipids. 8 6 150 150 0 4 r- ---O 130 x 0-4 Q4(n O r- E 120- l. *-, 0 110 m

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J O L 70 00.10.3 0.5 1.0 00.10.3 0.5 1.0 Thrombin, units/ml O00 5110 300 60 90 FIG. 1. Action of PBt2 on concentration curves for thrombin- Time, sec induced labeling of [32P]inositol phospholipids and [32P]. Platelets were treated with PBt2 (200 nM) for 10 min at 37°C FIG. 2. Effect of PBt2 and OAG on thrombin-induced formation and transferred to a Chrono-Log aggregometer. They were then of [3H]inositol phosphates. (A) PBt2. Platelets were pretreated with stirred for 1 min, and various concentrations of thrombin were add- PBt2 (200 nM) for 10 min at 37°C and then transferred to a Chrono- ed for 60 sec. The reaction was stopped by CHCl3/MeOH/HCI Log aggregometer. They were then stirred for 1 min and thrombin (1 (100:200:2) and phospholipids were analyzed as described. Each unit/ml) was added for various times. The reaction was stopped by point represents the mean of triplicates from one experiment. Two transferring to CHC13/MeOH/HCI (100:200:2) and [3H]inositol other experiments gave the same results. SEM values were consis- phosphates were analyzed as described. Each point is the mean of tently <5% of the mean. o, No PBt2; *, with PBt2. Ptdlns, phospha- duplicates from one experiment. Two other experiments gave the tidylinositol; PtdInsP, phosphatidylinositol 4-monophosphate; same results. o and *, inositol monophosphate; o and e, inositol PtdInsP2, phosphatidylinositol 4,5-bisphosphate; Ptd, phosphatidic bisphosphate; A and A, InsP3. (B) OAG. Conditions were as de- acid. scribed in A. Downloaded by guest on October 3, 2021 Biochemistry: Watson and Lapetina Proc. Natl. Acad. Sci USA 82 (1985) 2625

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FIG. 3. Effect of PBt2 and OAG on concentration curve for 0.0 0.01 0.03 0.10 0.30 1.0 3.0 thrombin formation of [3H]inositol phosphates. (A) Inositol mono- log thrombin concentration, units/mi phosphate; (B) inositol bisphosphate; (C) InsP3. Platelets were incu-

bated with various concentrations of thrombin for 60 sec. Condi- FIG. 4. Effect of PBt2 and OAG on concentration curve for tions were as described in Fig. 2. *, Control; e, OAG (50 IxM); *, thrombin release of 5-[3H]hydroxytryptamine and ATP. (A) 5- PBt2 (200 nM). [3H]Hydroxytryptamine. Conditions were as described in Fig. 2 ex- cept the reaction was stopped by using 0.5 ml of 6% glutaraldehyde [3H]InsP3 in OAG- and PBt2-treated platelets after this time in 0.1 M sodium hydroxide. Platelets were then centrifuged, and 5- [3H]hydroxytryptamine release was estimated by counting aliquots were 33.5% ± 6.0% (n = 3) and 26.5% ± 10.8% (n = 3) of of the supernatant fluid. A maximally effective dose of thrombin controls, respectively. This inhibitory action of OAG and released between 60%o and 70%t of the tissues stores of 5-[3H]hy- PBt2 appeared to result from a decrease in the maximum, droxytryptamine. (B) ATP. ATP release was measured during the rather than a shift to the right in the concentration curve for experiment by luciferase fluorescence. For other conditions see thrombin formation of [3H]inositol phosphates (Fig. 3). above. *, Control;e*, QAG; A, PBt2. Similar results were seen for the concentration curve of thrombin-induced formation of [32P]phosphatidic acid (re- DISCUSSION flection of phospholipase C activity and phosphorylation of the resultant 1,2-diacylglycerol by 1,2-diacylglycerol kinase) In agreement with de Chaffoy de Courcelles et a!. (11, 13) in platelets pretreated with PBt2 (Fig. 1). In addition, this and Halenda and Feinstein (12), we have observed that phor- bol esters and 1,2-diacyiglycerols rapidly increase the levels decreased formation of [32P]phosphatidic acid by thrombin was associated with increased levels of [32P]polyphosphoin- of polyphosphoinositides in platelets. The mechanism of this indicat- ositides but a decreased level of [32P]phosphatidylinositol action is not known, but a role for protein kinase C is relative to thrombin-stimulated controls (Fig. 1). The decreased formation of [3H]inositol phosphates by Table 2. Action of PBt2 on formation of inositol phosphates by thrombin was accompanied by a 2- to 3-fold shift to the right various stimuli in the dose response for thrombin-induced release of 5- InsP InsP2 InsP3 [3H]hydroxytryptamine and ATP (Fig. 4). On their own, Basal 2534 ± 72 992 ± 78 42 ± 4 both OAG and PBt2 caused little release of 5-[3H]hydroxy- Thrombin (1 unit/ml) 4488 ± 284 4455 ± 173 1116 ± 157 tryptamine (Fig. 4), but, in contrast, PBt2 caused a signifi- Thrombin/PBt2 3294 ± 146 2532 ± 210 452 ± 111 cant release of ATP, which approached =30% of the amount PAF (10-7 M) 3012 ± 112 1222 ± 123 134 ± 43 released by thrombin (Fig. 4B). In addition, OAG, but not PAF/PBt2 2358 ± 143 825 ± 114 48 ± 30 PBt2, caused a decreased maximal release of ATP by throm- Collagen (20 /ig/ml) 3878 ± 267 2870 ± 310 548 ± 58 bin (Fig. 4). Collagen/PBt2 3292 ± 329 2223 ± 367 444 ± 98 Action of PBt2 on Inositol Phosphate Formation by Other Agonists. Table 2 shows that PBt2 (200 nM) also partially in- Platelets were preincubated at 370C with or without PBt2 (200 nM) for 9 min and then transferred to a Chrono-Log aggregometer and hibited formation of the three [3H]inositol phosphates by stirred for 1 min. Agonists were then added and after 30 sec (or for platelet-activating factor and collagen. These two agonists, collagen, 50 sec) the reaction was stopped by transferring to like thrombin, activate platelets through the phosphodies- CHCl3/MeOH/HCl (100:200:2). [3H]Inositol phosphates were then teric cleavage of inositol phospholipids (19). PBt2 also par- analyzed as described. Data are shown as mean dpm + SEM of tially inhibited the release of ATP by these two agonists (not triplicates from one experiment. Two other experiments gave simi- shown). lar results. PAF, platelet-activating factor. Downloaded by guest on October 3, 2021 2626 Biochemistry: Watson and Lapetina Proc. NatL Acad Sci. USA 82 (1985) ed as both of these agents activate this enzyme in the same phoinositides in this tissue (25), but it is not known whether concentrations that raise the levels of the polyphosphoinosi- platelet-activating factor induces this reaction and subse- tides (12). Conceivably, therefore, phosphatidylinositol 4- quent activation of protein kinase C; however, these results monophosphate and/or phosphatidylinositol may be directly certainly demonstrate the need for caution in extrapolating phosphorylated by protein kinase C or, alternatively, protein the present observations in platelets to other cells. kinase C may alter the activity (either directly or indirectly) of one of the enzymes involved in the production of inositol phospholipids (i.e., phosphatidylinositol-kinase, phosphati- We wish to thank Dr. Pedro Cuatrecasas for many useful discus- sions, Bryan Reep for expert technical assistance, Dr. R. M. Bell for dylinositol 4-monophosphate-kinase and their respective critical revision of this manuscript and the gift of OAG, and Cindy phosphatases). These results may, therefore, explain the McDowell and Tonya Beasley for the preparation of this manu- previous observations that thrombin (1, 20) and platelet-ac- script. tiving factor (21) produce an initial drop in the levels of poly- phosphoinositides in platelets, but that this is followed by a rapid resynthesis to above resting levels. 1. Billah, M. M. & Lapetina, E. G. (1982) J. Biol. Chem. 257, Surprisingly, the increased labeling of polyphosphoinosi- 12705-12708. tides produced by OAG or PBt2 was associated with a de- 2. Agranoff, B. W., Murthy, P. & Seguin, E. B. (1983) J. Biol. creased secretory response to thrombin. This inhibitory ef- Chem. 258, 2076-2078. fect was the consequence of impaired breakdown of inositol 3. Watson, S. P., McConnell, R. T. & Lapetina, E. G. (1984) J. Biol. Chem. 259, 13199-13203. phospholipids, as demonstrated by the decreased production 4. Watson, S. P., Reep, B., McConnell, R. T. & Lapetina, E. G. of inositol phosphates and phosphatidic acid. The mecha- (1985) Biochem. J., in press. nism of this effect is unclear, but it is not due to impaired 5. Nishizuka, Y. (1984) Nature (London) 308, 693-698. formation of polyphosphoinositides as their levels are in- 6. Streb, H., Irvine, R. F., Berridge, M. J. & Schulz, I. (1983) creased in thrombin-stimulated platelets relative to controls. Nature (London) 306, 67-69. Similarly, an inhibitory action at the level of the thrombin 7. Burgess, G. M., Godfrey, P. P., McKinney, J. S., Berridge, receptor can be ruled out, as PBt2 also decreased inositol M. J., Irvine, R. F. & Putney, J. W., Jr. (1984) Nature (Lon- phosphate formation by collagen and platelet-activating fac- don) 309, 63-66. tor. Therefore, it appears the inhibition takes place, either 8. Joseph, S. K., Thomas, A. P., Williams, R. J., Irvine, R. F. & Williamson, J. R. (1984) J. Biol. Chem. 259, 3077-3081. directly or indirectly, at the level of phospholipase C. 9. Prentki, M., Biden, T. J., Janjic, D., Irvine, R. F., Berridge, The ability of both OAG and PBt2 to cause inhibition sug- M. J. & Wollheim, C. B. (1984) Nature (London) 309, 562-564. gests an involvement of protein kinase C. If this is the case, 10. Lapetina, E. G., Watson, S. P. & Cuatrecasas, P. C. (1984) the inhibitory action would appear to serve as a negative Proc. Natl. Acad. Sci. USA 81, 7431-7435. feedback control over receptor-induced hydrolysis of inosi- 11. de Chaffoy de Courcelles, D., Roevens, P. & van Belle, H. tol phospholipids. Thus, receptor activation generates 1,2- (1984) FEBS Lett. 173, 389-394. diacylglycerol, which stimulates protein kinase C; protein ki- 12. Halenda, S. P. & Feinstein, M. B. (1984) Biochem. Biophys. nase C then partially inhibits further breakdown of inositol Res. Commun. 124, 507-513. The present appear some- 13. de Chaffoy de Courcelles, D., Roevens, P. & van Belle, H. phospholipids. results, therefore, (1984) Biochem. Biophys. Res. Commun. 123, 589-595. what contradictory. Why should protein kinase C activation 14. Lapetina, E. G. (1984) Biochem. Biophys. Res. Commun. 120, lead to an increase in the levels ofpolyphosphoinositides but 3744. a decrease in their rate of hydrolysis? Are these two actions 15. Packham, M. A., Guccione, M. A., Greenberg, J. P., Kin- serving different functions within cells? For example, inosi- lough-Rathbone, R. L. & Mustard, J. F. (1977) Blood 50, 915- tol phospholipid hydrolysis is obviously involved in short- 926. term responses through the generation of the two second 16. Watson, S. P., Ganong, B. R., Bell, R. M. & Lapetina, E. G. messengers 1,2-diacylglycerol and InsP3, while the increase (1984b) Biochem. Biophys. Res. Commun. 121, 386-391. in the levels of polyphosphoinositides may be involved in 17. Lapetina, E. G. & Watson, S. P. (1984) in IUPHAR Ninth In- it is to ternational Congress ofPharmacology, Proceedings, eds. Pa- longer term responses. In this respect, interesting ton, W., Mitchell, J. & Turner, P. (Macmillan, New York), pp. note that a variety of growth factors and tumor promoters 127-131. have in common the ability to increase the levels of poly- 18. Rink, T. J., Sanchez, A. & Hallam, T. J. (1983) Nature (Lon- phosphoinositides within a variety of tissues (for review see don) 305, 317-319. ref. 22). 19. Lapetina, E. G. & Siess, W. (1983) Life Sci. 33, 1011-1018. In accordance with the present results, Labarca et al. (23) 20. Broekman, M. J. (1984) Biochem. Biophys. Res. Commun. reported that phorbol esters inhibit both carbachol- and nor- 120, 226-231. epinephrine-induced accumulation of [3H]inositol 1-phos- 21. Billah, M. M. & Lapetina, E. G. (1982) Proc. Natl. Acad. Sci. phate in hippocampal slices; this inhibition was manifested USA 80, 965-968. of 22. Berridge, M. J. (1984) BiolTechnology 2, 541-545. as a decrease in the maximal response the dose-response 23. Labarca, R., Janowsky, A., Patel, J. & Paul, S. M. (1984) Bio- curve rather than a shift to the right. In contrast, O'Flaherty chem. Biophys. Res. Commun. 123, 703-709. et al. (24) showed that, although phorbol ester and OAG 24. O'Flaherty, J. T., Schmitt, J. D., McCall, C. E. & Wykle, alone cause only slight release of lysozyme from polymor- R. L. (1984) Biochem. Biophys. Res. Commun. 123, 64-70; phonuclear leukocytes, they markedly potentiate the ability 25. Volpi, M., Yassin, R., Tao, W., Molski, T. F. P., Naccache, of platelet-activating factor and leukotriene B4 to do this. P. H. & Sha'afi, R. I. (1984) Proc. Natl. Acad. Sci. USA 81, Leukotriene B4 does not induce the hydrolysis of polyphos- 5966-5969. Downloaded by guest on October 3, 2021