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Effect of pertussis toxin on the phosphodiesteratic cleavage of the polyphosphoinositides by guanosine 5′-O-thiotriphosphate and thrombin in permeabilized human platelets
Biochimica et Biophvsica Acta 884 (1986) 219-224 Elsevier
219
BBA 22371
Effect of pertussis toxin on the phosphodiesteratic cleavage of the polyphosphoinositides by guanosine 5'-O-thiotriphosphate and t h r o m b i n in p e r m e a b i l i z e d h u m a n platelets E d u a r d o (3. L a p e t i n a Department of Molecular Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709 (U.S.A.) (Received 23 May 1986)
Key words: Phospholipase C: Inositol phosphate: GTP-binding protein; Pertussis toxin; Platelet stimulation
It has recently been shown in this laboratory that permeabilization of human platelets with 15-25 # g / m l saponin allows ADP-ribosylation by pertussis toxin of the ai-subunit of G i (N 0, a guanine nucleotide-binding regulatory protein. The same assay conditions have been used to determine phospholipase C in permeabilized platelets. Guanosine 5'-O-thiotriphosphate- (GTPIySI-) activated phospholipase C in permeabilized platelets whose inositol phospholipids were prelabeled with [3Hlinositol. Phospholipase C activity was measured by [3Hlpolyphosphoinositide decreases and formation of [3Hlinositol bisphosphate and [3H]inositol trisphosphate. Prostacyclin, cyclic AMP or pretreatment of permeabilized platelets with pertussis toxin did not alter this effect under conditions in which the ai-subunit was effectively ADP-ribosylated by pertussis toxin. This information indicated that ADP-ribosylation of Grprotein was not directly related to activation or inhibition of platelet phospholipase C by GTPITS]. Thrombin also activated phospholipase C in permeabilized platelets and, surprisingly, this action was enhanced by pertussis toxin pretreatment. This indicates that ADP-ribosylation of Gi-protein facilitates the action of thrombin on phospholipase C.
Introduction GTP, or GTP analogs such as GTP[yS] and Gpp[NH]p, can stimulate phospholipase C activity in platelets permeabilized by electrical stimulation [1]. GTP also activates phospholipase C'in plasma membranes obtained from human neutrophils, blowfly salivary gland and liver membranes [2-4]. These findings have led to the hypothesis that a GTP-binding protein is involved in the activation of the phospholipase C that causes
Correspondence address: Dr. E.G. Lapetina, Department of Molecular Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709 (U.S.A.)
degradation of the polyphosphoinositides [2]. These findings also indicated that this GTP-binding protein is susceptible to ADP-ribosylation, since pertussis toxin can inhibit the agonist-induced activation of phospholipase C in neutrophils, macrophages and LH60 cells [5-9]. However, it is now evident that in other cell types, pertussis toxin does not affect hormone-induced phosphoinositide turnover [9-11]. Platelets lack cell surface receptors for pertussis -toxin [12], but pertussis toxin produces ADPribosylation of the ai-subunit of Gi-protein in platelets permeabilized with saponin [13]. The same assay conditions have been used to stimulate phospholipase C with GTP[yS] and thrombin. It was shown that activation of phospholipase C by
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
220 the GTP[TS] is not affected by pretreatment with pertussis toxin that ADP-ribosylates the cq-subunit of the Gi-protein, but this pretreatment enhances the effect of thrombin on phospholipase C. Materials and Methods
Pertussis toxin was purchased from List Biological Laboratories, Campbell, CA. NAD was from Sigma Chemical Co., St. Louis, MO. Prostacyclin was supplied by The Wellcome Research Laboratories, Beckenham, Kent, U.K. Guanosine 5'-O-3-thiotriphosphate (GTP[TS]) and guanyl-5'yl imidodiphosphate (Gpp[NH]p) were from Boehringer-Mannheim, Indianapolis, IN. myo-[23H]Inositol was purchased from Amersham, Arlington Heights, IL. Precoated silica-gel LK 60 (layer thickness 0.25 mm), used for the separation of the inositol phospholipids, was purchased from Whatman, Clifton, NJ. Dowex 1 anion resin (100 200 mesh), used for the separation of inositol phosphates, was from Bio-Rad, Richmond, CA.
Preparation and labeling of human platelets with [ SH]inositol Blood (200 ml) was obtained from healthy human volunteers who had not received any medication in the previous 3 weeks. Blood was anticoagulated with 0.2 vol. of buffer (85 mM trisodium c i t r a t e / I l l mM dextrose/71 mM citric acid (pH 5.5)). Platelet rich plasma was obtained by centrifugation at 200 × g for 20 min and was then centrifuged at 250 × g for 15 min. The platelet pellet obtained was resuspended in 10 ml of a modified Tyrode-Hepes buffer (134 mM NaC1/12 mM NaHCO3/2.9 mM KC1/0.36 mM N a i l zPO4/1 mM MgC12/5 mM glucose/10 mM Hepes (pH 7.4)) containing 5 n g / m l prostacyclin, and the platelets were incubated at 37°C for 3 h with 0.5 m C i / m l [SH]inositol. Platelets were washed twice with 30 ml of the modified Tyrode-Hepes buffer containing 5 n g / m l prostacyclin and resuspended in the same buffer without prostacyclin. Platelet concentration was adjusted to 1.0- 109/ml using a Coulter Counter 2F (Coulter Electronic, U.S.A.).
Measurement of phospholipase C activity These determinations were carried out in 0.5 ml that contained 0.375 ml of the platelet suspension. Assay conditions closely resembled those used for [32p]ADP-ribosylation with pertussis toxin in platelets permeabilized with saponin [13]. Final concentrations of the following constituents were as follows: saponin, 20 /~g/ml; NAD, 20 /~M; ATP, 1 mM; MgCI2, 1.5 mM; EDTA, 1 mM; dithiothreitol, 1 mM; thymidine, 10 mM; LiC1, 10 mM. GTP[TS] was usually added at a concentration of 200/~M; when pertussis toxin was present, its concentration was 5 /~g/ml. Concentration of thrombin was usually 1 unit/ml. Incubations were at 37°C in a shaking incubator bath for 1 to 30 rain. 15-min experiments were most common. In some experiments, platelets were incubated with pertussis toxin for 15 rain before addition of GTP[TS] or thrombin (1 unit/ml) for 15 min more. Reactions were stopped by addition of 1.88 ml of chloroform/methanol/concentrated HC1 (100:200:2, v/v). Phases were then separated by addition of 0.6 ml of chloroform and 0.6 ml of water. The organic phase was dried under a flow of N 2, and the inositides were separated on thinlayer plates (Whatman LK 6D impregnated with 1% potassium oxalate/2 mM EDTA) using chlor o f o r m / m e t h a n o l / 4 M NH4OH (45 : 35 : 10, v/v). The lipids were visualized by autoradiography [14]. The TLC plates were previously sprayed with EN3HANCE Spray (Dupont) in preparation for autoradiography of the phospholipids. Aqueous phases were analyzed for [3H]inositol monophosphate, [3H]inositol bisphosphate and [SH]inositol trisphosphate as recently described [15]. Triplicate determinations were carried out for each experiment. Results are expressed as percent of controls, because the radioactivity incorporated into inositol phospholipids varied from experiment to experiment as can be visualized in Tables I and II. The range of control values for inositoi phosphates in 10 experiments varied from 834 to 8203 dpm for inositol monophosphate, 482 to 5130 dpm for inositol bisphosphate and 210 to 2135 dpm for inositol trisphosphate. Results
Stimulation of phospholipase C by GTP[yS] in platelets permeabilized with saponin GTP[TS] (200 /~M) activated phospho|ipase C,
221
as reflected by the formation of [3H]inositol bisphosphate and [3H]inositol trisphosphate, in platelets that had been prelabeled with [3H]inositol and permeabilized with 20 ~g/ml saponin (Fig. 1). The formation of these inositol polyphosphates was evident after 1 min and was maximal between 15 and 30 rain (Fig. 1). Formation of the two [3H]inositol polyphosphates was greater than formation of [3 H]inositol monophosphate (Fig. 1). Similarly, 200 btM Gpp[NH]p stimulated phospholipase C, but it was less effective than GTP[~,S] (not shown). GTP (500 ttM) was minimally effective, and GDP (200 ~M) did not produce stimulation of phospholipase C (not shown). Pertussis toxin (5 ~g/ml) did not cause the formation of [3H]inositol phosphates under the same assay conditions (Fig. 1). Recently, it was shown [13] that pertussis toxin at 5 to 50 ~g/ml causes maximal ADP-ribosylation of the c~i-subunit of the Gi-protein. A concentration curve for the effect of GTP[TS]
200[,p Controlg
]00L ~
600 IP2
/ ~
indicated that 2 ~M GTP[3,S] effectively stimulated the formation of [3H]inositol polyphosphates. Maximal formation was observed at 20 to 500 #M GTP[TS] (not shown). All these experiments were carried out in the presence of 1 mM EDTA without the addition of Ca 2+. Therefore, the effect of GTP[TS] or Gpp[NH]p on phospholipase C was CaZ+-independent. Fig. 2 shows that GTP[TS] (200 #M) caused appreciable disappearance of platelet [3H]phosphatidylinositol monophosphate and a dramatic loss of [3H]phosphatidylinositol bisphosphate. Preincubation of platelets with prostacyclin (1 ~g/ml) or dibutyryl cyclic AMP (1 mM) did not affect the action of GTP[TS] on the disappearance of the polyphosphoinositides (Fig. 2). Increased formation of [3H]polyphosphoinositides was observed when ATP was allowed inside the platelet by saponin (Table I) but not when platelets were treated with saponin in the absence of ATP [15]. The decrease of radioactivity in [3H]phosphatidylinositol parelleled the increase in [3H]polyphosphoinositides. This reflects sequential phosphorylation of [3H]phosphatidylinositol to [3H]phosphatidylinositol monophosphate and [3H]phosphatidylinositol bisphosphate. Therefore, it seemed that GTPTS degraded a specific fraction of the polyphosphoinositides (Fig. 2,Table I). This
5O0 a: 400
TABLE I
Z ©
EFFECT OF GTP[?,S] AND Ca 2+ ON THE [3H]INOSITOL PHOSPHOLIPIDS OF PLATELETS PERMEABILIZED WITH SAPONIN
o 300 2O0
100
PT
100~- ~ i
0
Control
8
~m coat,o, 1
5
1'5
30
TIME, MIN Fig. 1. Time-course of the formation of inositol phosphates induced by GTP[TS] in platelet permeabilized with saponin. Platelets prelabeled with [3H]inositol were incubated either without additions (control), with 200 ~M GTP[TS] (GTP[TS]), or with 5 ~g/ml pertussis toxin (PT) for different time periods. IP, inositol monophosphate; IP2, inositol bisphosphate; IP3, inositol trisphosphate. Results are the mean_+ S.E. of five different experiments.
Results are expressed in dpm_+S.E, of triplicate determinations. Qualitatively similar results were obtained in three other experiments. PI, phosphatidylinositol; PIP, phosphatidylinositol monophosphate; PIP2, phosphatidylinositol bisphosphate. Saponin was used at 20 ~g/ml, GTP[TS] was 200 ~M and Ca 2+ was 1 mM. It should be noted that saponin is always added together with the other components of the assay system such as ATP, NAD, EDTA. MgCI2, dithiothreitol, thymidine and LiCI.
No saponin Saponin Saponin, GTP[ 7 S] Saponin, Ca 2+ Saponin, Ca 2+, GTP[TS]
PI
PIP
PIP2
21608_+247 12888-+ 33 13335_+ 15 23313_+113
2595_+38 8095-+78 4983_+54 1203-+16
1218_+31 2448-+64 933-+27 428-+13
24208+__482 1370-+27
433_+ 4
222
•
PI
.
LPI
•
PIP
-
PIP2
PI LPI
Il-
Md
L
PIP PIP2
-
I CONTROL GTPyS
PGI2
PGI2 GTPyS
cAMP cAMP I GTPyS
Fig. 2. Prostacyclin and dibutyryl cAMP do not affect the GTP[ y S]-induced degradation of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. Autoradiography shows the thin-layer chromatographic separation of [3H]inositol-labeled phosphatidylinositol (PI), lysophosphatidylinositol (LPI), phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2). PGI 2, prostacyclin (1 ~g/ml); cAMP, dibutyryl cAMP (1 mM); GTP[yS] (200 I~M). All assay contained saponin (20 p,g/ml). Areas corresponding to origin and solvent front are not shown.
indicated that increases in [3 H]inositol monophosphate might be derived from [3H]inositol bisphosphate by the action of an inositol phosphatase. Table I also shows the effect of Ca 2+ on the metabolism of the inositol phospholipids in cells permeabilized in the presence of ATP. Ca 2+ inhibited increased formation of the polyphosphoinositides; this might reflect an action on phosphatidylinositol kinase or polyphosphoinositide phosphomonesterases. Furthermore, GTP[yS] did not stimulate phospholipase C in the presence of Ca 2+.
A DP-ribosylation induced by pertussis toxin in platelets permeabilized with saponin did not affect the activation of phospholipase C by GTP[7S] In a recent study [13], it was shown that pertussis toxin ADP-ribosylates the cq-subunit (41000 molecular weight) of the Gi-protein in platelets that are permeabilized with saponin. Pertussis toxin has now been used - in saponin-treated platelets under the same assay conditions in which the ai-subunit is ADP-ribosylated - to assess the importance of this action on the GTP[yS] activation of phospholipase C (Table II and Fig. 3). Pertussis toxin slightly decreased the breakdown of the polyphosphoinositides (Table II) and did
not affect the action of GTP[yS] on phospholipase C (Table II and Fig. 3). The same conclusion was obtained when Gpp[NH]p was used to stimulate platelet phospholipase C in platelets permeabilized with saponin (not shown).
TABLE II EFFECT OF PERTUSSIS TOXIN AND GTP[yS] ON [3H]INOSITOL PHOSPHOLIPIDS OF PLATELETS PERMEABILIZED WITH SAPONIN Results are expressed in dpm_+ S.E. of triplicate determinations. Qualitatively similar results were obtained in four other experiments. PI, phosphatidylinositol; PIP, phosphatidylinositol monophosphate; PIP2, phosphatidylinositol bisphopshate. Saponin was used at 20 t*g/ml, GTP[yS] was 200 #M and Ca ,-+ was 1 mM. All assays contained saponin and the other components of the assay system as described in Table I.
Control GTP[yS], 200 p,M Pertussis toxin, 5/*g/ml Pertussis toxin, GTP[TS]
PI
PIP
PIP2
15833_+248 15333_+412
10413_+ 44 7225_+ 93
3133_+48 1948_+23
16460_+284
9578_+284
2678_+77
14930_+ 92
6908_+ 73
1568_+23
223 IP3
IP_.23
500j-
400,
300 "
200
-
100 -
GTpyS
THROMBIN
THROMBIN +
GTPrS
Fig. 3. Pertussis toxin does not affect the formation of [3H]inositol phosphates induced by the action of GTP[TS] but potentiates the action of thrombin. Platelets prelabeled with [3H]inositol were incubated for 30 min. Pertussis toxin (PT, 5 / z g / m l ) was added at time zero and GTP[yS] (200 /~M) a n d / o r thrombin (1 u n i t / m l ) were added during the last 15 min. Abbreviations and other details are as in Fig. 1. Pertussis toxin had no effect on control values for IP, IP2 and IP3. Results are the m e a n + S.E. of three different experiments.
Pertussis toxin increased the actioation of phospholipase C induced by thrombin but not that caused by GTP[ySI Fig. 3 shows that GTP[TS] and thrombin induced formation of [3H]inositol phosphates. The effect of GTP[yS] is not altered by previous treatment of the saponized platelets with pertussis toxin. Surprisingly, pertussis toxin-induced ADPribosylation caused an increase in the effect of thrombin on the formation of the [3H]inositol phosphates (Fig. 3). Discussion
The evidence presented in this study indicates that platelet phospholipase C stimulated by GTP[7S] is not inhibited by the previous ADPribosylation of a GTP-binding protein by pertussis toxin. However, pretreatment with pertussis toxin enhances thrombin-induced activation of phospholipase C. This differs from the effect of pertussis toxin in inhibiting the agonist-induced stimulation of phospholipase C in various cell systems [5-9] or not inhibiting hormone-induced phosphoinositide turnover in other cell types
[9-11], and it might indicate a mechanism that is unique for certain agonists. Thrombin decreases the availability of Gi-protein susceptible to ADPribosylation by pertussis toxin [13]; this correlates with activation of phospholipase C in human platelets. This effect of thrombin is similar to that of trypsin. Trypsin stimulates phospholipase C [16], and at the same time it produces proteolysis of the cq-subunit of the Gi-protein that is ADP-ribosylated by pertussis toxin in platelets [13]. These results suggest that removal of the ai-subunit might correlate with activation of phospholipase C. Our results also show that GTP[yS] preferentially produces the phosphodiesteratic cleavage of the polyphosphoinositides. The accumulation of inositol bisphosphate and inositol trisphosphate is sustained for 15 min, with a concomitant decrease of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. During this period, there is not much accumulation of inositol monophosphate or degradation of phosphatidylinositol. Subsequent formation of inositol monophosphate might be derived by the sequential action of inositol trisphosphatase and inositol bisphosphatase on inositol trisphosphate and inositol bisphosphate, respectively. The GTP[TS]-induced breakdown of the polyphosphoinositides is not affected by preincubation of intact platelets with prostacyclin or cyclic AMP, which cause the activation of cyclic AMP-dependent protein kinase and the phosphorylation of specific proteins in platelets [17,18]. The phosphorylation of these proteins does not seem to be directly related to the inhibition of the phosphodiesterase that cleaves the polyphosphoinositides. Instead, it seems that the effect of cyclic AMP is related to reactions that precede the activation of phospholipase C [19]. When platelets are permeabilized with saponin in the presence of ATP, as has been carried out for the experiments reported herein, an appreciable amount of phosphatidylinositol is sequentially phosphorylated to polyphosphoinositides. C a 2+ blocks the phosphorylation of phosphatidylinositol to phosphatidylinositol monophosphate or, alternatively, activates inositide phosphomonesterases. Under those conditions, GTP[yS] does not produce the phosphodiesteratic
224
cleavage of phosphatidylinositol. This may indicate that under normal conditions of platelet stimulation [19], the initial effect of the agonists is on the inositide kinases to produce a sensitive fraction of polyphosphoinositides that is then cleaved by a polyphosphoinositide-specific phospholipase C. Conversely, when platelets are saponized in the absence of ATP, most of the cellular ATP is lost [20], and the Ca2+-induced loss of polyphosphoinositides with accumulation of phosphatidylinositol may be due to activation of inositide-phosphomonoesterases [15,20]. This study appears to indicate that a GTPbinding protein different than G i is related to the stimulation of platelet phospholipase C. However, it should also be considered that GTP[TS] may still affect the enzymatic activity of phospholipase C through G i, even after it has been ADP-ribosylated. Moreover, the ADP-ribosylation of G i by pertussis toxin seems to facilitate the activation of phospholipase C by thrombin. This suggests.that under normal conditions, G i could have an inhibitory action on phospholipase C, in a similar fashion to the action on adenylate cyclase. Acknowledgments I am most grateful to Bryan Reep for excellent technical help, to Allen Jones for editing the manuscript and to Tonya Beasley and Marian Goodwin for preparing the manuscript.
References 1 Haslam, R.J, and Davidson, M.M.L. (1984) J. Receptor Res. 4, 605-629 2 Cockcroft, S. and Gomperts, B.D. 91985) Nature 314, 534 536 3 Litosch, I., Wallis, C. and Fain, J.N. (1985) J. Biol. Chem. 260, 5464-5471 4 Wallace, M.A. and Fain, J.N. 91985) J. Biol. Chem. 260, 9527-9530 5 Volpi, M., Naccache, P.H., Molski, T.F.P., Shefcyk, J., Huang, C.K., Marsh, M.L., Munoz, J., Becker, E.L. and Sha'afi, R.I. (1985) Proc. Natl. Acad. Sci. USA 82, 2708-2712 6 Brandt, S.J., Dougherty, R.W., Lapetina, E.G. and Niedel, J.E. (1985) Proc. Natl. Acad. Sci. USA 82, 3277-3280 7 Verghese, M.W., Smith, C.D. and Snyderman, R. (1985) Biochem. Biophys. Res. Commun. 127, 450-457 80kajima, F., Katada, T. and Ui, M. (1985) J. Biol. Chem. 260, 6761-6768 9 Murayama, T. and Ui, M. (1985) J. Biol. Chem. 260, 7226-7233 10 Masters, S.B., Martin, M.W., Harden, T.K. and Brown, J.H. (1985) Biochem. J. 227, 933-937 11 Uhing, R.J., Prpic, V., Jiang, H. and Exton, J.H. (1986) J. Biol. Chem. 261, 2140-2146 12 Ui, M. (1984) Trends Pharmac. Sci. 5, 277 279 13 Lapetina, E.G., Reep, B, and Chang, K.-J. (1986) Proc. Natl. Acad. Sci. USA 83, 5880-5883 14 Billah, M.M. and Lapetina. E.G. (1982) J. Biol. Chem. 257, 12705-12708 15 Lapetina, E.G., Silio, J. and Ruggiero, M. (1985) J. Biol. Chem. 260, 7078 7083 16 Ruggiero, M. and Lapetina, E.G. (1985) Biochem. Biophys. Res. Commun. 131, 1198-1205 17 Nishizuka, Y. (1984) Nature 308,693-698 18 Lapetina. E.G., Watson, S.P. and Cuatrecasas, P. (1984) Proc. Natl. Acad. Sci. USA 81, 7431-7435 19 Watson, S.P., McConnell, R.T. and Lapetina, E.G. (1984) J. Biol. Chem. 259, 13199-13203 20 Ruggiero, M., Zimmerman, T.P. and Lapetina, E.G. (1985) Biochem. Biophys. Res. Commun. 131,620-627
219
BBA 22371
Effect of pertussis toxin on the phosphodiesteratic cleavage of the polyphosphoinositides by guanosine 5'-O-thiotriphosphate and t h r o m b i n in p e r m e a b i l i z e d h u m a n platelets E d u a r d o (3. L a p e t i n a Department of Molecular Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709 (U.S.A.) (Received 23 May 1986)
Key words: Phospholipase C: Inositol phosphate: GTP-binding protein; Pertussis toxin; Platelet stimulation
It has recently been shown in this laboratory that permeabilization of human platelets with 15-25 # g / m l saponin allows ADP-ribosylation by pertussis toxin of the ai-subunit of G i (N 0, a guanine nucleotide-binding regulatory protein. The same assay conditions have been used to determine phospholipase C in permeabilized platelets. Guanosine 5'-O-thiotriphosphate- (GTPIySI-) activated phospholipase C in permeabilized platelets whose inositol phospholipids were prelabeled with [3Hlinositol. Phospholipase C activity was measured by [3Hlpolyphosphoinositide decreases and formation of [3Hlinositol bisphosphate and [3H]inositol trisphosphate. Prostacyclin, cyclic AMP or pretreatment of permeabilized platelets with pertussis toxin did not alter this effect under conditions in which the ai-subunit was effectively ADP-ribosylated by pertussis toxin. This information indicated that ADP-ribosylation of Grprotein was not directly related to activation or inhibition of platelet phospholipase C by GTPITS]. Thrombin also activated phospholipase C in permeabilized platelets and, surprisingly, this action was enhanced by pertussis toxin pretreatment. This indicates that ADP-ribosylation of Gi-protein facilitates the action of thrombin on phospholipase C.
Introduction GTP, or GTP analogs such as GTP[yS] and Gpp[NH]p, can stimulate phospholipase C activity in platelets permeabilized by electrical stimulation [1]. GTP also activates phospholipase C'in plasma membranes obtained from human neutrophils, blowfly salivary gland and liver membranes [2-4]. These findings have led to the hypothesis that a GTP-binding protein is involved in the activation of the phospholipase C that causes
Correspondence address: Dr. E.G. Lapetina, Department of Molecular Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709 (U.S.A.)
degradation of the polyphosphoinositides [2]. These findings also indicated that this GTP-binding protein is susceptible to ADP-ribosylation, since pertussis toxin can inhibit the agonist-induced activation of phospholipase C in neutrophils, macrophages and LH60 cells [5-9]. However, it is now evident that in other cell types, pertussis toxin does not affect hormone-induced phosphoinositide turnover [9-11]. Platelets lack cell surface receptors for pertussis -toxin [12], but pertussis toxin produces ADPribosylation of the ai-subunit of Gi-protein in platelets permeabilized with saponin [13]. The same assay conditions have been used to stimulate phospholipase C with GTP[yS] and thrombin. It was shown that activation of phospholipase C by
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
220 the GTP[TS] is not affected by pretreatment with pertussis toxin that ADP-ribosylates the cq-subunit of the Gi-protein, but this pretreatment enhances the effect of thrombin on phospholipase C. Materials and Methods
Pertussis toxin was purchased from List Biological Laboratories, Campbell, CA. NAD was from Sigma Chemical Co., St. Louis, MO. Prostacyclin was supplied by The Wellcome Research Laboratories, Beckenham, Kent, U.K. Guanosine 5'-O-3-thiotriphosphate (GTP[TS]) and guanyl-5'yl imidodiphosphate (Gpp[NH]p) were from Boehringer-Mannheim, Indianapolis, IN. myo-[23H]Inositol was purchased from Amersham, Arlington Heights, IL. Precoated silica-gel LK 60 (layer thickness 0.25 mm), used for the separation of the inositol phospholipids, was purchased from Whatman, Clifton, NJ. Dowex 1 anion resin (100 200 mesh), used for the separation of inositol phosphates, was from Bio-Rad, Richmond, CA.
Preparation and labeling of human platelets with [ SH]inositol Blood (200 ml) was obtained from healthy human volunteers who had not received any medication in the previous 3 weeks. Blood was anticoagulated with 0.2 vol. of buffer (85 mM trisodium c i t r a t e / I l l mM dextrose/71 mM citric acid (pH 5.5)). Platelet rich plasma was obtained by centrifugation at 200 × g for 20 min and was then centrifuged at 250 × g for 15 min. The platelet pellet obtained was resuspended in 10 ml of a modified Tyrode-Hepes buffer (134 mM NaC1/12 mM NaHCO3/2.9 mM KC1/0.36 mM N a i l zPO4/1 mM MgC12/5 mM glucose/10 mM Hepes (pH 7.4)) containing 5 n g / m l prostacyclin, and the platelets were incubated at 37°C for 3 h with 0.5 m C i / m l [SH]inositol. Platelets were washed twice with 30 ml of the modified Tyrode-Hepes buffer containing 5 n g / m l prostacyclin and resuspended in the same buffer without prostacyclin. Platelet concentration was adjusted to 1.0- 109/ml using a Coulter Counter 2F (Coulter Electronic, U.S.A.).
Measurement of phospholipase C activity These determinations were carried out in 0.5 ml that contained 0.375 ml of the platelet suspension. Assay conditions closely resembled those used for [32p]ADP-ribosylation with pertussis toxin in platelets permeabilized with saponin [13]. Final concentrations of the following constituents were as follows: saponin, 20 /~g/ml; NAD, 20 /~M; ATP, 1 mM; MgCI2, 1.5 mM; EDTA, 1 mM; dithiothreitol, 1 mM; thymidine, 10 mM; LiC1, 10 mM. GTP[TS] was usually added at a concentration of 200/~M; when pertussis toxin was present, its concentration was 5 /~g/ml. Concentration of thrombin was usually 1 unit/ml. Incubations were at 37°C in a shaking incubator bath for 1 to 30 rain. 15-min experiments were most common. In some experiments, platelets were incubated with pertussis toxin for 15 rain before addition of GTP[TS] or thrombin (1 unit/ml) for 15 min more. Reactions were stopped by addition of 1.88 ml of chloroform/methanol/concentrated HC1 (100:200:2, v/v). Phases were then separated by addition of 0.6 ml of chloroform and 0.6 ml of water. The organic phase was dried under a flow of N 2, and the inositides were separated on thinlayer plates (Whatman LK 6D impregnated with 1% potassium oxalate/2 mM EDTA) using chlor o f o r m / m e t h a n o l / 4 M NH4OH (45 : 35 : 10, v/v). The lipids were visualized by autoradiography [14]. The TLC plates were previously sprayed with EN3HANCE Spray (Dupont) in preparation for autoradiography of the phospholipids. Aqueous phases were analyzed for [3H]inositol monophosphate, [3H]inositol bisphosphate and [SH]inositol trisphosphate as recently described [15]. Triplicate determinations were carried out for each experiment. Results are expressed as percent of controls, because the radioactivity incorporated into inositol phospholipids varied from experiment to experiment as can be visualized in Tables I and II. The range of control values for inositoi phosphates in 10 experiments varied from 834 to 8203 dpm for inositol monophosphate, 482 to 5130 dpm for inositol bisphosphate and 210 to 2135 dpm for inositol trisphosphate. Results
Stimulation of phospholipase C by GTP[yS] in platelets permeabilized with saponin GTP[TS] (200 /~M) activated phospho|ipase C,
221
as reflected by the formation of [3H]inositol bisphosphate and [3H]inositol trisphosphate, in platelets that had been prelabeled with [3H]inositol and permeabilized with 20 ~g/ml saponin (Fig. 1). The formation of these inositol polyphosphates was evident after 1 min and was maximal between 15 and 30 rain (Fig. 1). Formation of the two [3H]inositol polyphosphates was greater than formation of [3 H]inositol monophosphate (Fig. 1). Similarly, 200 btM Gpp[NH]p stimulated phospholipase C, but it was less effective than GTP[~,S] (not shown). GTP (500 ttM) was minimally effective, and GDP (200 ~M) did not produce stimulation of phospholipase C (not shown). Pertussis toxin (5 ~g/ml) did not cause the formation of [3H]inositol phosphates under the same assay conditions (Fig. 1). Recently, it was shown [13] that pertussis toxin at 5 to 50 ~g/ml causes maximal ADP-ribosylation of the c~i-subunit of the Gi-protein. A concentration curve for the effect of GTP[TS]
200[,p Controlg
]00L ~
600 IP2
/ ~
indicated that 2 ~M GTP[3,S] effectively stimulated the formation of [3H]inositol polyphosphates. Maximal formation was observed at 20 to 500 #M GTP[TS] (not shown). All these experiments were carried out in the presence of 1 mM EDTA without the addition of Ca 2+. Therefore, the effect of GTP[TS] or Gpp[NH]p on phospholipase C was CaZ+-independent. Fig. 2 shows that GTP[TS] (200 #M) caused appreciable disappearance of platelet [3H]phosphatidylinositol monophosphate and a dramatic loss of [3H]phosphatidylinositol bisphosphate. Preincubation of platelets with prostacyclin (1 ~g/ml) or dibutyryl cyclic AMP (1 mM) did not affect the action of GTP[TS] on the disappearance of the polyphosphoinositides (Fig. 2). Increased formation of [3H]polyphosphoinositides was observed when ATP was allowed inside the platelet by saponin (Table I) but not when platelets were treated with saponin in the absence of ATP [15]. The decrease of radioactivity in [3H]phosphatidylinositol parelleled the increase in [3H]polyphosphoinositides. This reflects sequential phosphorylation of [3H]phosphatidylinositol to [3H]phosphatidylinositol monophosphate and [3H]phosphatidylinositol bisphosphate. Therefore, it seemed that GTPTS degraded a specific fraction of the polyphosphoinositides (Fig. 2,Table I). This
5O0 a: 400
TABLE I
Z ©
EFFECT OF GTP[?,S] AND Ca 2+ ON THE [3H]INOSITOL PHOSPHOLIPIDS OF PLATELETS PERMEABILIZED WITH SAPONIN
o 300 2O0
100
PT
100~- ~ i
0
Control
8
~m coat,o, 1
5
1'5
30
TIME, MIN Fig. 1. Time-course of the formation of inositol phosphates induced by GTP[TS] in platelet permeabilized with saponin. Platelets prelabeled with [3H]inositol were incubated either without additions (control), with 200 ~M GTP[TS] (GTP[TS]), or with 5 ~g/ml pertussis toxin (PT) for different time periods. IP, inositol monophosphate; IP2, inositol bisphosphate; IP3, inositol trisphosphate. Results are the mean_+ S.E. of five different experiments.
Results are expressed in dpm_+S.E, of triplicate determinations. Qualitatively similar results were obtained in three other experiments. PI, phosphatidylinositol; PIP, phosphatidylinositol monophosphate; PIP2, phosphatidylinositol bisphosphate. Saponin was used at 20 ~g/ml, GTP[TS] was 200 ~M and Ca 2+ was 1 mM. It should be noted that saponin is always added together with the other components of the assay system such as ATP, NAD, EDTA. MgCI2, dithiothreitol, thymidine and LiCI.
No saponin Saponin Saponin, GTP[ 7 S] Saponin, Ca 2+ Saponin, Ca 2+, GTP[TS]
PI
PIP
PIP2
21608_+247 12888-+ 33 13335_+ 15 23313_+113
2595_+38 8095-+78 4983_+54 1203-+16
1218_+31 2448-+64 933-+27 428-+13
24208+__482 1370-+27
433_+ 4
222
•
PI
.
LPI
•
PIP
-
PIP2
PI LPI
Il-
Md
L
PIP PIP2
-
I CONTROL GTPyS
PGI2
PGI2 GTPyS
cAMP cAMP I GTPyS
Fig. 2. Prostacyclin and dibutyryl cAMP do not affect the GTP[ y S]-induced degradation of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. Autoradiography shows the thin-layer chromatographic separation of [3H]inositol-labeled phosphatidylinositol (PI), lysophosphatidylinositol (LPI), phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2). PGI 2, prostacyclin (1 ~g/ml); cAMP, dibutyryl cAMP (1 mM); GTP[yS] (200 I~M). All assay contained saponin (20 p,g/ml). Areas corresponding to origin and solvent front are not shown.
indicated that increases in [3 H]inositol monophosphate might be derived from [3H]inositol bisphosphate by the action of an inositol phosphatase. Table I also shows the effect of Ca 2+ on the metabolism of the inositol phospholipids in cells permeabilized in the presence of ATP. Ca 2+ inhibited increased formation of the polyphosphoinositides; this might reflect an action on phosphatidylinositol kinase or polyphosphoinositide phosphomonesterases. Furthermore, GTP[yS] did not stimulate phospholipase C in the presence of Ca 2+.
A DP-ribosylation induced by pertussis toxin in platelets permeabilized with saponin did not affect the activation of phospholipase C by GTP[7S] In a recent study [13], it was shown that pertussis toxin ADP-ribosylates the cq-subunit (41000 molecular weight) of the Gi-protein in platelets that are permeabilized with saponin. Pertussis toxin has now been used - in saponin-treated platelets under the same assay conditions in which the ai-subunit is ADP-ribosylated - to assess the importance of this action on the GTP[yS] activation of phospholipase C (Table II and Fig. 3). Pertussis toxin slightly decreased the breakdown of the polyphosphoinositides (Table II) and did
not affect the action of GTP[yS] on phospholipase C (Table II and Fig. 3). The same conclusion was obtained when Gpp[NH]p was used to stimulate platelet phospholipase C in platelets permeabilized with saponin (not shown).
TABLE II EFFECT OF PERTUSSIS TOXIN AND GTP[yS] ON [3H]INOSITOL PHOSPHOLIPIDS OF PLATELETS PERMEABILIZED WITH SAPONIN Results are expressed in dpm_+ S.E. of triplicate determinations. Qualitatively similar results were obtained in four other experiments. PI, phosphatidylinositol; PIP, phosphatidylinositol monophosphate; PIP2, phosphatidylinositol bisphopshate. Saponin was used at 20 t*g/ml, GTP[yS] was 200 #M and Ca ,-+ was 1 mM. All assays contained saponin and the other components of the assay system as described in Table I.
Control GTP[yS], 200 p,M Pertussis toxin, 5/*g/ml Pertussis toxin, GTP[TS]
PI
PIP
PIP2
15833_+248 15333_+412
10413_+ 44 7225_+ 93
3133_+48 1948_+23
16460_+284
9578_+284
2678_+77
14930_+ 92
6908_+ 73
1568_+23
223 IP3
IP_.23
500j-
400,
300 "
200
-
100 -
GTpyS
THROMBIN
THROMBIN +
GTPrS
Fig. 3. Pertussis toxin does not affect the formation of [3H]inositol phosphates induced by the action of GTP[TS] but potentiates the action of thrombin. Platelets prelabeled with [3H]inositol were incubated for 30 min. Pertussis toxin (PT, 5 / z g / m l ) was added at time zero and GTP[yS] (200 /~M) a n d / o r thrombin (1 u n i t / m l ) were added during the last 15 min. Abbreviations and other details are as in Fig. 1. Pertussis toxin had no effect on control values for IP, IP2 and IP3. Results are the m e a n + S.E. of three different experiments.
Pertussis toxin increased the actioation of phospholipase C induced by thrombin but not that caused by GTP[ySI Fig. 3 shows that GTP[TS] and thrombin induced formation of [3H]inositol phosphates. The effect of GTP[yS] is not altered by previous treatment of the saponized platelets with pertussis toxin. Surprisingly, pertussis toxin-induced ADPribosylation caused an increase in the effect of thrombin on the formation of the [3H]inositol phosphates (Fig. 3). Discussion
The evidence presented in this study indicates that platelet phospholipase C stimulated by GTP[7S] is not inhibited by the previous ADPribosylation of a GTP-binding protein by pertussis toxin. However, pretreatment with pertussis toxin enhances thrombin-induced activation of phospholipase C. This differs from the effect of pertussis toxin in inhibiting the agonist-induced stimulation of phospholipase C in various cell systems [5-9] or not inhibiting hormone-induced phosphoinositide turnover in other cell types
[9-11], and it might indicate a mechanism that is unique for certain agonists. Thrombin decreases the availability of Gi-protein susceptible to ADPribosylation by pertussis toxin [13]; this correlates with activation of phospholipase C in human platelets. This effect of thrombin is similar to that of trypsin. Trypsin stimulates phospholipase C [16], and at the same time it produces proteolysis of the cq-subunit of the Gi-protein that is ADP-ribosylated by pertussis toxin in platelets [13]. These results suggest that removal of the ai-subunit might correlate with activation of phospholipase C. Our results also show that GTP[yS] preferentially produces the phosphodiesteratic cleavage of the polyphosphoinositides. The accumulation of inositol bisphosphate and inositol trisphosphate is sustained for 15 min, with a concomitant decrease of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. During this period, there is not much accumulation of inositol monophosphate or degradation of phosphatidylinositol. Subsequent formation of inositol monophosphate might be derived by the sequential action of inositol trisphosphatase and inositol bisphosphatase on inositol trisphosphate and inositol bisphosphate, respectively. The GTP[TS]-induced breakdown of the polyphosphoinositides is not affected by preincubation of intact platelets with prostacyclin or cyclic AMP, which cause the activation of cyclic AMP-dependent protein kinase and the phosphorylation of specific proteins in platelets [17,18]. The phosphorylation of these proteins does not seem to be directly related to the inhibition of the phosphodiesterase that cleaves the polyphosphoinositides. Instead, it seems that the effect of cyclic AMP is related to reactions that precede the activation of phospholipase C [19]. When platelets are permeabilized with saponin in the presence of ATP, as has been carried out for the experiments reported herein, an appreciable amount of phosphatidylinositol is sequentially phosphorylated to polyphosphoinositides. C a 2+ blocks the phosphorylation of phosphatidylinositol to phosphatidylinositol monophosphate or, alternatively, activates inositide phosphomonesterases. Under those conditions, GTP[yS] does not produce the phosphodiesteratic
224
cleavage of phosphatidylinositol. This may indicate that under normal conditions of platelet stimulation [19], the initial effect of the agonists is on the inositide kinases to produce a sensitive fraction of polyphosphoinositides that is then cleaved by a polyphosphoinositide-specific phospholipase C. Conversely, when platelets are saponized in the absence of ATP, most of the cellular ATP is lost [20], and the Ca2+-induced loss of polyphosphoinositides with accumulation of phosphatidylinositol may be due to activation of inositide-phosphomonoesterases [15,20]. This study appears to indicate that a GTPbinding protein different than G i is related to the stimulation of platelet phospholipase C. However, it should also be considered that GTP[TS] may still affect the enzymatic activity of phospholipase C through G i, even after it has been ADP-ribosylated. Moreover, the ADP-ribosylation of G i by pertussis toxin seems to facilitate the activation of phospholipase C by thrombin. This suggests.that under normal conditions, G i could have an inhibitory action on phospholipase C, in a similar fashion to the action on adenylate cyclase. Acknowledgments I am most grateful to Bryan Reep for excellent technical help, to Allen Jones for editing the manuscript and to Tonya Beasley and Marian Goodwin for preparing the manuscript.
References 1 Haslam, R.J, and Davidson, M.M.L. (1984) J. Receptor Res. 4, 605-629 2 Cockcroft, S. and Gomperts, B.D. 91985) Nature 314, 534 536 3 Litosch, I., Wallis, C. and Fain, J.N. (1985) J. Biol. Chem. 260, 5464-5471 4 Wallace, M.A. and Fain, J.N. 91985) J. Biol. Chem. 260, 9527-9530 5 Volpi, M., Naccache, P.H., Molski, T.F.P., Shefcyk, J., Huang, C.K., Marsh, M.L., Munoz, J., Becker, E.L. and Sha'afi, R.I. (1985) Proc. Natl. Acad. Sci. USA 82, 2708-2712 6 Brandt, S.J., Dougherty, R.W., Lapetina, E.G. and Niedel, J.E. (1985) Proc. Natl. Acad. Sci. USA 82, 3277-3280 7 Verghese, M.W., Smith, C.D. and Snyderman, R. (1985) Biochem. Biophys. Res. Commun. 127, 450-457 80kajima, F., Katada, T. and Ui, M. (1985) J. Biol. Chem. 260, 6761-6768 9 Murayama, T. and Ui, M. (1985) J. Biol. Chem. 260, 7226-7233 10 Masters, S.B., Martin, M.W., Harden, T.K. and Brown, J.H. (1985) Biochem. J. 227, 933-937 11 Uhing, R.J., Prpic, V., Jiang, H. and Exton, J.H. (1986) J. Biol. Chem. 261, 2140-2146 12 Ui, M. (1984) Trends Pharmac. Sci. 5, 277 279 13 Lapetina, E.G., Reep, B, and Chang, K.-J. (1986) Proc. Natl. Acad. Sci. USA 83, 5880-5883 14 Billah, M.M. and Lapetina. E.G. (1982) J. Biol. Chem. 257, 12705-12708 15 Lapetina, E.G., Silio, J. and Ruggiero, M. (1985) J. Biol. Chem. 260, 7078 7083 16 Ruggiero, M. and Lapetina, E.G. (1985) Biochem. Biophys. Res. Commun. 131, 1198-1205 17 Nishizuka, Y. (1984) Nature 308,693-698 18 Lapetina. E.G., Watson, S.P. and Cuatrecasas, P. (1984) Proc. Natl. Acad. Sci. USA 81, 7431-7435 19 Watson, S.P., McConnell, R.T. and Lapetina, E.G. (1984) J. Biol. Chem. 259, 13199-13203 20 Ruggiero, M., Zimmerman, T.P. and Lapetina, E.G. (1985) Biochem. Biophys. Res. Commun. 131,620-627