Eur. J. Biochem. 171, 523-533 (1988) 0 FEBS 1988

Effects of guanosine 5'-[y-thioltriphosphate and thrombin on the phosphoinositide metabolism of electropermeabilized human platelets Martine CULTY', Monica M. L. DAVIDSONZ and Richard J. HASLAM'.

Departments of Biochemistry and Pathology, McMaster University, Hamilton, Ontario

(Received September 7,1987) - EJB 87 1013

Incubation of human platelets with my~-[~H]inositol in a low-glucose Tyrode's solution containing MnC12 enhanced the labelling of phosphoinositides about sevenfold and greatly facilitated the measurement of [3H]inositol phosphates formed by the activation of phospholipase C. Labelled platelets were permeabilized by high-voltage electric discharges and equilibrated at 0 "C with ATP, Ca2 + buffers and guanine nucleotides, before incubation in the absence or presence of thrombin.

Incubation of these platelets with ATP in the presence or absence of Ca2+ ions led to the conversion of [3H]phosphatidylinositol to [3H]phosphatidylino~itol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PtdInsP2). At a pCa of 6, addition of 100 pM GTP[yS] both prevented this accumulation of [3H]PtdInsP2 and stimulated its breakdown; the formation of [3H]inositol phosphates was increased ninefold. After 5 min these comprised 70% [3H]inositol monophosphate ([3H]InsP), 28% [3H]inositol bisphosphate ([3H]InsP2) and 2% [3H]inositol trisphosphate ([3H]InsP3). In shorter incubations higher percentages of [3H]InsP2 and [3H]InsP3 were found. In the absence of added Ca2+, the formation of [3H]inositol phosphates was decreased by over 90%.

Incubation of permeabilized platelets with GTP[yS] in the presence of 10 mM Li' decreased the accumulation of [3H]InsP and increased that of [3H]InsP2, without affecting [3H]InsP3 levels. Addition of unlabelled InsP, decreased the intracellular hydrolysis of exogenous [32P]InsP3 but did not trap additional [3H]InsP3. These results and the time course of [3H]inositol phosphate formation suggest that GTP[yS] stimulated the action of phospholipase C on a pool of [3H]phosphatidylinositol 4-phosphate that was otherwise converted to [3H]PtdInsP2 and that much less hydrolysis of [3H]phosphatidylinositol to [3H]InsP or of [3H]PtdInsP2 to [3H]InsP3 occurred.

At a pCa of 6, addition of thrombin ( 2 units/ml) to permeabilized platelets caused small increases in the formation of [3H]InsP and [3H]InsP2. This action of thrombin was enhanced twofold by 10- 100 pM GTP and much more potently by 4-40 pM GTP[yS]. In the presence of the latter, thrombin also increased [3H]InsP3. The total formation of [3H]inositol phosphates by permeabilized platelets incubated with thrombin and GTP[yS] was comparable with that observed on addition of thrombin alone to intact platelets. However, HPLC of the [3H]inositol phosphates formed indicated that about 75% of the [3H]InsP accumulating in permeabilized platelets was the 4-phosphate, whereas in intact platelets stimulated by thrombin, up to 80% was the 1-phosphate. With both preparations, the [3H]InsP2 formed coeluted with the 1,4-bisphosphate and [3H]InsP3 largely with the 1,3,4- trisphosphate. The results provide additional evidence that the activation of phospholipase C by thrombin in the platelet is mediated by a guanine-nucleotide-binding protein and indicate that, though PtdIns may be hydrolysed in intact platelets, it is not the major substrate of phospholipase C in permeabilized platelets at a pCa of 6. The formation of the 1,3,4-trisphosphate isomer of [3H]InsP3 in platelets suggests the presence of an InsP, pathway.

Correspondence to R. J. Haslam, Department of Pathology, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 325

Abbreviutions. Unless indicated otherwise, all inositol residues in phosphoinositides and inositol phosphates are assumed to be D-enantiomers of myo-inositol; PtdIns, phosphatidylinositol; PtdInsP, phosphatidylinositol4-phosphate; PtdInsP2, phosphatidyl- inositol 4,5-bisphosphate; InsP, InsP2, InsP3 and InsP,, inositol mono-, bis-, tris- and tetrakisphosphates with the positions of phos- phate residues not specified; Ins(l)P, Ins(3)P, Ins(4)P, Ins(1 ,4)P2, Ins(1 ,4,5)P3, Ins(1 ,3,4)P3, Ins(1 ,3,4,5)P4, corresponding inositol phosphates with the positions of phosphate residues specified; GTP[yS], guanosine 5'-[y-thioltnphosphate; pCa, -log [Ca2+]f,,,.

Enzymes. Phosphoinositide-specific phospholipase C (EC 3.1.4.1 0); phosphatidylinositol kinase (EC 2.7.1.67); phosphatidyl- inositol 4-phosphate kinase (EC 2.7.1.68); myo-inositol l-phospha- Case (EC 3.1.3.25); CDPdiacylg1ycerol:myo-inositol transferase (EC 2.7.8.11).

Physiological agonists that mobilize intracellular Ca2 +

ions act through receptors that stimulate the hydrolysis of phosphatidylinositol4,5-bisphosphate (PtdInsP2) by a phos- phoinositide-specific phospholipase C [l]. The products of this reaction, inositol trisphosphate (InsP3) that releases Ca2 +

ions from the endoplasmic reticulum [Z] and diacylglycerol that activates protein kinase C [3], both act as second messen- gers. PtdIns and PtdInsP can also be hydrolysed by phospholipase C and are therefore potential sources of diacylglycerol, though the kinetics of formation of InsP3, InsP2 and InsP have suggested that in many cells, PtdIns may be primarily utilized for the resynthesis of the poly- phosphoinositides [2, 4, 51. In the platelet the stimulation of aggregation and degranulation by thrombin is associated with a transient decrease in PtdInsP2 [6-81 and the rapid forma- tion of diacylglycerol[9] and inositol phosphates, particularly

524

lnsPz and InsP, [7, 101. However, thrombin also causes a large decrease in PtdIns that has been attributed to a direct action of phospholipase C, rather than to the regeneration of PtdInsP2 [9, 111. Thus, the relative importance of PtdIns, PtdInsP and PtdInsP2 as substrates of phospholipase C in the platelet remains uncertain, as does the ratio in which diacylglycerol and InsP3 are generated. In this context it is relevant that an increase in [CaZ+Ifree in the platelet cytosol may not be essential for platelet degranulation [12], though Ca2+ and diacylglycerol can act synergistically to promote secretion [3].

Analysis of the potential contributions of these signals to the secretory responses of the platelet has been facilitated by studies with platelets permeabilized by high-voltage electric discharges, in which the intracellular [Ca2+Ifree can be fixed experimentally [13 - 151. In this system, thrombin decreased the [Ca2'Ifree required for secretion of serotonin [13, 141, an effect that was associated with the enhanced formation of diacylglycerol, presumably as a result of the activation of phospholipase C [14]. Further studies of the factors affecting signal transduction in electropermeabilized platelets demon- strated that guanine nucleotides, particularly metabolically stable analogues of GTP such as GTP[yS], acted similarly [16 - 181. These investigations showed that guanine nucle- otides potentiated both the stimulation of secretion and for- mation of diacylglycerol induced by thrombin, so providing direct evidence of a role for a guanine-nucleotide-binding protein in the activation of phospholipase C by a physiological agonist. Experiments with a variety of isolated membrane systems [19 -231, including recent studies with platelet mem- branes [24,25], have now validated this concept. In the present study, we sought to extend work from this laboratory on the effects of guanine nucleotides on diacylglycerol formation [17] by measurement of the changes in [3H]phosphoinositides and [3H]inositol phosphates in platelets permeabilized after label- ling with [3H]inositol. Some of the results have been published in a preliminary form [26].

EXPERIMENTAL PROCEDURE

Materials

myo-[2-3H]Inositol (1 5 Ci/mmol) was from American Radiolabelled Chemicals (St Louis, MO, USA) and 32Pi (carrier-free) and [2-3H]Ins(4)P (1.5 Ci/mmol) were from Du Pont Canada (Markham, Ont.). L-rny~-[U-'~C]Ins(l)P ( 5 5 Ci/mol), a mixture of [2-3H]Ins(l)P, Ins(l,4)P2 and Ins(1,4,5)P3 (each 1 Ci/mmol) and ACS scintillant were from Amersham Canada Ltd (Oakville, Ont.). [32P]Ins(1,4,5)P3 was prepared from 32P-labelled red cells [27] and a mixture of [3H]Ins(l ,3,4)P3, [3H]Ins(1 ,4,5)P3 and [,H]Ins(l ,3,4,5)P4 was generously provided by Dr W. Schlegel of the University of Geneva, Switzerland. Ready-Solv HP/b scintillant was from Beckman Instruments Ltd (Toronto, Ont., Canada). GTP[yS] (tetralithium salt) was obtained from Boehringer Mannheim Canada Ltd (Dorval, Que.) and ATP (disodium salt, prepared by phosphorylation of adenosine), GTP, unlabelled Ins(1 ,4,5)P3, 2,3-bisphosphoglycerate and brain phosphoinositides were from Sigma (St Louis, MO, USA). Dowex-1 anion-exchange resin (AG 1-X8, 100 -200 mesh, formate form) and Dowex 50 cation-exchange resin (AG 50W- X8, 200-400 mesh, H' form) were obtained from Bio-Rad Laboratories (Canada) Ltd (Mississauga, Ont.). Silica gel TLC plates (Si250) were from the J.T. Baker Chemical Co. (Phillipsburg, NJ, USA) and 6-p-toluidino-2-naphthalene-

sulphonic acid from Eastman Kodak (Rochester, NY, USA). Apyrase was prepared according to Molnar and Lorand [28]. Human CI- thrombin (2700 units/mg) was kindly provided by Dr J. W. Fenton I1 of New York State Department of Health (Albany, NY, USA). Other materials were from sources listed elsewhere [ 3 41.

Labelling and perineabilization of human platelets

Fresh blood was collected by venepuncture into ACD anticoagulant [29] and centrifuged at 180 x g,, for 15 min to give platelet-rich plasma. The platelets were isolated at room temperature by centrifugation at 1500 x g,, for 15 min and resuspension in a modified Tyrode's solution at pH 6.5 con- taining 137 mM NaCl, 2.7 mM KCI, 11.9 mM NaHC03, 0.42 mM NaH2P04, 2 mM MgC12, 5.6 mM glucose, 5 mM Pipes, 3.5 mg bovine serum albumin/ml, 60 pg apyrase/ml and 50 units heparin/ml. After centrifugation of this suspension at 1000 x g:,, for 10 min, the platelets were usually resuspended at 2 x 109/ml in the same medium modified by omission of heparin and replacement of 2 mM MgClz and 5.6 mM glucose by 2 mM MnC12 and 0.56 mM glucose. This suspension was incubated for 2 h at 37°C with 20 pCi my~-[~H]inositol/rnl. In some early experiments, platelets were labelled in medium without heparin but containing 2 mM MgCI2 and 5.6 mM glucose. The labelled platelets were finally resuspended at room temperature at about 2 x 109/ml in the modified Tyrode's solution without apyrase or heparin. After addition of 5 mM EGTA, the platelets were permeabilized by high- voltage electric discharges and isolated by gel filtration on Sepharose CL4B, as described previously [14]. The elution medium (pH 7.4) contained 12.5 mM MgC12 and the potas- sium salts of glutamic acid (160 mM), Hepes (20 mM), EGTA (2.5 mM) and EDTA (2.5 mM) (buffer A). After addition of 5 mM ATP (Na' salt), the platelet suspension [(0.6- 1.0) x 109/ml] was stored at 0°C until used (within 3 h).

For experiments on intact platelets containing [3H]phos- phoinositides, labelling with [3H]inositol was carried out in the presence of 2 mM MnC12 and 0.56 mM glucose, as above. The platelets were then resuspended in Tyrode's solution con- taining 5 mM Hepes, pH 7.4, 3.5 mg bovine serum albumin/ ml and 6 pg apyrase/ml [30].

Incubation of permeabilized platelets

Samples (400 pl) of a suspension of permeabilized platelets were mixed with a total of 100 pl additions in buffer A, includ- ing the CaC12 required to give a particular pCa value (calculat- ed as in [31]) and any guanine nucleotide. The CaC12 solution contained sufficient KOH to give a final pH of 7.4. When the effects of Li+ were studied, 25 pl buffer A in which Li' re- placed K', was added. These mixtures were equilibrated for 15 min at 0°C and then incubated for up to 5 min at 25°C. Breakdown of InsP, was studied in samples equilibrated with [32P]InsP3 (1000-2000 cpm) in the presence and absence of 400 pM unlabelled InsP,. When the effect of thrombin was studied, it was added in 5 pl buffer A immediately before transfer of the samples to 25°C. All incubations were stopped by addition of 250 p1 30% (w/v) trichloroacetic acid. After 30 min at 0 ° C samples were centrifuged and 650 pl super- natant removed for analysis of [3H]inositol phosphates. The precipitate .was washed once with H 2 0 and resuspended in 1 ml HzO before extraction of phospholipids.

525

Analysis ofphosphoinositides

Resuspended pellets (1 ml) were extracted essentially ac- cording to Rittenhouse [32] by mixing with 3.75 ml chloroform/methanol/conc. HCl (100: 200: 3, by vol.) to give a one-phase system. After 30 min at room temperature, 1 ml chloroform and 1 ml 1.8 M KC1 were added and the mixture was vortexed. After centrifugation, the lower chloroform phase was removed and evaporated in a stream of N2. The lipids were redissolved in 80 pl chloroform/methanol(2: 1, by vol.) and mixed with 25 pg of carrier phosphoinositides. These samples were applied to silica gel TLC plates that had been impregnated with potassium oxalate and EDTA and were then chromatographed with chloroform/methanol/4 M NH40H (45 : 35 : 10, by vol.) as the solvent [6]. Lipids were visualized under ultraviolet light after spraying the plates with 1 mM 6-p-toluidino-2-naphthalenesulphonate in 50 mM Tris/HCl, pH 7.4 [33]. The phosphoinositides were scraped into vials, mixed with 500 p1 methanol/conc. HCl (100:4, by vol.) and 8 ml HP/b scintillant and counted for 3H (efficiency 25%). Results were expressed as dpm/109 platelets, for comparison with [3H]inositol phosphates formed in the same experiment.

Analysis of inositol phosphates The acid supernatants were neutralized with NaOH using

bromthymol blue as an indicator, diluted to 5 ml with H 2 0 and applied to columns containing 1.25 ml Dowex-1 resin. Inositol phosphates were isolated by stepwise elution, as de- scribed by Berridge et al. [34]. [3H]Inositol was first eluted with 3 x 5 ml H 2 0 , followed by [3H]glycerophosphoinositol with 2 x 5 ml 60 mM sodium formate/5 mM disodium tetra- borate. [3H]InsP and 32Pi were then eluted with 4 x 5 ml 0.2 M ammonium formate in 0.1 M formic acid, [3H]InsP2 and [32P]InsP2 with 4 x 5 ml 0.4 M ammonium formate in 0.1 M formic acid and [3H]InsP3, [32P]InsP3 and [3H]InsP4 with 3 x 5 ml 1 M ammonium formate in 0.1 M formic acid. Individual 5-ml fractions were adjusted to contain 1 M am- monium formate, mixed with 15 ml ACS scintillant and counted for 3H (efficiency 14%) and, when present, 32P. Values for 3H were corrected for crossover of any 32P into the 3H channel (< 0.5%) and expressed as dpm/109 platelets.

In some experiments, the identities of the [3H]inositol phos- phates collected from Dowex-1 columns were provisionally established by comparison with authentic standards during HPLC on a Whatman Partisil 10 SAX column (0.46 cm- x 25 cm) protected by a Whatman pre-column. Dowex-1 frac- tions containing [3H]InsP, [3H]InsP2 or [3H]InsP3 were first desalted on columns containing excess Dowex-50 resin (H' form) and dried using a rotary evaporator. The residues from three identical incubation mixtures were dissolved in water (0.3 -0.5 ml) and samples were analysed by HPLC after mixing with internal standards consisting of AMP, ADP and ATP (50 nmol each) and [14C]Ins(l)P or [32P]Ins(1,4,5)P3. The combined losses of [3H]inositol phosphates during des- alting and HPLC were less than 20% and the amount of each injected approximated that found in a single incubation mixture. Inositol phosphates were eluted from the Partisil 10 SAX column with linear gradients of ammonium formate buffered to pH 3.7 with phosphoric acid (flow rate 1.25 ml/ min) [35, 361. For samples containing [3H]InsP or [3H]InsP2, the ammonium formate concentrations used were: 0 - 2 min, none; 2-5min, 0-0.1 M; 5-20min, 0.1-0.2M; 20- 35 min, 0.2-0.4 M; 35-50 min, 0.4-1.0 M; 50-55 min, 1.0 M. Fractions (0.5 ml) were counted in 5 ml ACS

scintillant. However, for samples containing [3H]InsP3 and [3H]InsP4, the following ammonium formate concentrations were used: 0-20 min, 0-0.75 M; 20-22 min, 0.75 M; 22- 32 min, 0.75-1.0 M; 32-37 min, 1.0 M; 37-47 min, 1.0- 1.7 M; 47- 53 min, 1.7 M. Fractions (0.375 ml) were col- lected during the steps in which compounds of interest were eluted and mixed with 1 ml methanol/H20 (1 : 1, by vol.) and 5 ml ACS scintillant for counting [35].

RESULTS

Optimal conditions for labelling platelets with (3H]inositol In preliminary experiments, platelets were labelled with

[3H]inositol by incubation for 2 h at 37°C in a modified Tyrode's solution at pH 6.5 containing 2 mM MgC12 and 5.6 mM glucose. However, the total incorporation of 3H into the phosphoinositides amounted to only 0.10% of the added [3H]inositol (mean of two expts). We then replaced the MgC12 by MnClz and decreased the glucose concentration to 0.56mM. As a result, incorporation of 3H into phos- phoinositides after 2 h was increased to 0.77 f 0.03% of the added [3H]inositol (mean f SEM, five expts). In these exper- iments the distribution of 3H between the phosphoinositides of the intact platelets was 79.6% in [3H]PtdIns, 12.8% in [3H]PtdInsP and 7.6% in [3H]PtdInsP2, when MgC12 was used, and 85.3 f 1.2% in [3H]PtdIns, 9.5 f 1.2% in [3H]PtdInsP and 5.2 f 0.6% in [3H]PtdInsP2, when labelling was carried out in the MnC12/low-glucose medium. Further experiments indicated that the major factor responsible for the increased incorporation of [3H]inositol was the replacement of MgC12 by MnC12, though the decrease in glucose concen- tration enhanced this effect (results not shown). Permeabiliza- tion of platelets labelled by these two methods yielded prep- arations with comparable distributions of 3H between [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2, but with about sevenfold greater incorporation of 3H into these phosphoinositides when MnC12 was used (Table 1). As the yield of [3H]inositol phosphates on incubation of these permeabilized platelets with 100 pM GTP[yS] at a pCa of 6 was increased four to sixfold (e.g. Table l), we concluded that the relative labelling of the GTP[yS]-sensitive and insensi- tive phosphoinositide pools differed only slightly in the two preparations of permeabilized platelets and that we could use the MnC12/low-glucose labelling procedure with advantage in subsequent experiments.

ESfects of GTP(yS] on (3H]phosphoinositide metabolism in permeabilized platelets

Incubation of permeabilized platelets with ATP in the absence of Ca2+ ions or GTP[yS] led to a substantial fall in their [3H]PtdIns content, which was accounted for by in- creases in [3H]PtdInsP and to a lesser extent [3H]PtdInsP2, without any formation of [3H]inositol phosphates. Similar results were obtained whether the platelets were labelled in the presence of MgC12 or MnC12 (Table 1). In the latter case the permeabilized platelets contained 75.6 f 3.4% [3H]PtdIns, 18.6 f 2.5% [3H]PtdInsP and 5.8 _+ 1.0% [3H]PtdInsP2 be- fore incubation, whereas after 5 min at 25°C they contained 58.1 f 5.4% [3H]PtdIns, 34.5 f 4.3% [3H]PtdInsP and 7.4 f 1.3% [3H]PtdInsP2 (mean values f SEM, five expts). The increases in [3H]PtdInsP (90 f 13%) and [3H]PtdInsP2 (28 f go/,) were both significant ( P < 0.05, paired t-tests), in-

526

Table 1. Effects of GTP[yS] on the metabolism of [3H]phosphoinositides in platelets permeabilized after labelling with [3H]inositol in different media Platelets from the same blood were labelled with [3H]inositol for 2 h at 37°C in medium containing either 2 mM MgC12 and 5.6 mM glucose (I) or 2 mM MnClz and 0.56 mM glucose (11), as described under Experimental Procedure. After permeabilization and gel filtration of the platelets and addition of ATP, samples were equilibrated at 0°C for 15 min with no additions, with Caz+ buffer giving a pCa of 6 or with Ca2+ buffer and 100 pM GTP[yS], as indicated. The samples were incubated for 0 or 5 min at 25°C before addition of trichloroacetic acid and analysis of [3H]phosphoinositides and [3H]inositol phosphates. Results are means f SEM from three identical incubation mixtures

Labelling Additions Incubation x 3H present medium period

PtdIns PtdInsP PtdInsPz InsP InsP2 InsP3

min

I none 0 none 5 Ca2 + 5 CaZ+ + GTP[yS] 5

I1 none 0 none 5 CaZ + 5 CaZ+ + GTP[yS] 5

dpm/109 platelets

19.13k 1.14 5.74k0.59 12.02k 0.19 33.01+0.40 9.37k 0.25 12.43 k0.32 9.83k 0.26 11.52+0.12

132.84k 10.54 43.07k2.32 92.70 k 4.64 97.25 k4.08 90.32+ 3.82 96.26k3.54 97.80+ 2.52 93.30k1.31

2.54 & 0.1 5 3.1 5f0.28 3.1 1 k0.25 1.47 f 0.07

16.91 fO.80 21.58 f 2.44 22.54k 2.12 11 .61 k0.65

0.53k0.10 0.38+0.03 0.83 k0.14 3.11 k0.23 1.04f0.07 0.82 k 0.02 2.04 f 0.07

16.87k0.34

0.35 k0.06 0.47 k0.09 0.55 k0.15 2.00 f 0.22 0.52 k0.05 0.66 f0.03 1.28 kO.10 7.56 k0.09

0.55 f 0.07 0.51 k0.07 0.53 k0.14 0.50 f 0.08 0.35k0.01 0.40 k0.04 0.39+0.04 0.78 k 0.03

dicating that PtdIns and PtdInsP kinases were both active in the permeabilized platelets. The changes in [3H]phos- phoinositide levels after equilibration and incubation with sufficient CaC12 to give a pCa of 6 were not significantly different, though small amounts of [3H]inositol phosphates, equivalent to 0.7 ? 0.1% of the total [3H]phosphoinositides (mean f SEM, seven expts), were formed. However, when the platelets were equilibrated and incubated with 100 pM GTP[yS] at a pCa of 6, the normal increase in [3H]PtdInsP2 was abolished and, in five out of seven experiments, a decrease in [3H]PtdInsP2 was observed relative to unincubated as well as incubated controls (e. g. Table 1). GTP[yS] also prevented the accumulation of [3H]PtdInsP2 at a pCa of 7 but had no effect on [3H]PtdInsP2 levels in the absence of added Ca2+ ions (not shown). With respect to the values reached in incu- bations with Ca2+ alone, GTP[yS] caused a decrease in [3H]PtdInsP2 of 39 f 4% at a pCa of 6 (mean f SEM, seven expts) and of 30+2% at a pCa of 7 (mean+ SEM, three expts). These results indicate that GTP[yS] either prevented the formation of [3H]PtdInsP2 or stimulated its breakdown (or both). Overall, incubation with GTP[yS] caused a small but significant decrease in [3H]PtdInsP (6 & 1%, P < 0.02, seven expts) but no significant change in [3H]PtdIns relative to samples containing Ca2+ alone (e. g. Table 1). In the pres- ence of 100 pM GTP[yS], additional [3H]inositol phosphates equivalent to 7.7 _+ 1.5% of the initial [3H]phosphoinositides accumulated after 5 min (mean SEM, seven expts). The changes in the [3H]PtdInsP2 and [3H]PtdInsP contents of platelets caused by incubation with GTP[yS] at a pCa of 6 accounted for 72+6% (mean+ SEM, seven expts) of the total [3H]inositol phosphates that accumulated. The remain- der are presumably derived from changes in [3H]PtdIns that were too small in percentage terms to reach statistical signifi- cance. Results qualitatively similar to those described above were obtained when platelets were labelled with [3H]inositol in the presence of 2 mM MgC12 and 5.6 mM glucose (Table 1).

At a pCa of 6, 100 pM GTP[yS] increased the total [3H]inositol phosphates formed after 5 min by (9.3 f 0.9)- fold (mean f SEM, 13 expts). These [3H]inositol phosphates comprised 70 2% [3H]InsP, 28 f 2% [3H]InsP2 and 2 f 0% [3H]InsP3 (mean values + SEM). Although the increases in

[3H]InsP3 caused by 100 pM GTP[yS] were very small, they were statistically significant both in individual experiments (e. g. Table 1) and in aggregate (13 expts; P < 0.005, paired t- test). The effects of 100 pM GTP[yS] on the accumulation of [3H]inositol phosphates were also studied at a pCa of 7 and in the absence of added CaC12 (pCa > 8) (Fig. 1). In the for- mer case the formation of [3H]inositol phosphates with GTP[yS] closely approached that observed at a pCa of 6, but in the latter the additional [3H]inositol phosphates amounted to only 8% of those seen at a pCa of 6 (mean of two expts), indicating a requirement for Ca2 + ions. The proportions in which the [3H]inositol phosphates accumulated at pCa values of 7 or > 8 were similar to those observed at a pCa of 6 (Fig. 1).

The effects of increasing concentrations of GTP[yS] on the accumulation of [3H]inositol phosphates over 5 rnin was studied at a pCa of 6 (Fig. 2). A concentration of 4 pM in- creased [3HlInsP and [3H]InsP2, whereas LO pM was usually required to increase [3H]InsP3. Higher concentrations up to 100 pM caused progressively larger increases in the accumu- lation of all three [3H]inositol phosphates. The time course of formation of the [3H]inositol phosphates in the presence and absence of 100 pM GTP[yS] was studied at pCa values of 7 (not shown) and 6 (Fig. 3), with almost identical results. Dur- ing the first 0.5 rnin of incubation with CTP[yS], [3H]InsP2 formation was equal to or greater than that of [3H]InsP, whereas at later time intervals [3H]InsP accumulation exceed- ed that of [3H]InsP2. After 5 min (not shown), [3H]InsP con- tinued to accumulate though the level of [3H]InsP2 ap- proached a plateau. Little change in the accumulation of [3H]InsP3 occurred after 0.5 min, when the amount present represented 6% of the total [3H]inositol phosphates formed.

Effects of Li' and unlabelled InsP3 on the accumulation of [3H]inositol phosphates

To clarify the metabolic pathways leading to the formation of [3H]InsP2 and [3H]InsP in permeabilized platelets, exper- iments were carried out using Li+, an inhibitor of InsP phos- phatase [37] Equilibration and incubation of the platelets with 10 mM Li' caused a small decrease rather than an increase

527

PCa

Fig. 1. Caz+ dependence of the effects of GTP[yS] on [3H]inositol phosphate formation in permeabilizedplatelets. Platelets were labelled with [3H]inositol in an MnCl,/low-glucose medium and permeabilized, as described in Experimental Procedure. After gel filtration of the platelets and addition of ATP, samples were equilibrated for 15 rnin at 0°C either without CaC1, (pCa > 8) or with CaZ+ buffers giving the indicated pCa values, in each case in the presence and absence of 100 pM GTP[yS]. The samples were then incubated for 5 rnin at 25°C and [3H]inositol phosphates determined. A (InsP); B (InsP,); C (InsP3). Values are means f SEM from three identical incubation mixtures. Mean values for [3H]InsP, [3H]insPz and [3H]InsP3 prior to equilibration of the samples were 1.04 x lo3 dpm, 0.29 x lo3 dpm and 0.20 x lo3 dpm/lOg platelets, respectively. (Q) No GTP[yS]; ( 8 ) with 100 pM GTP[yS]

Fig. 2. Effects of different GTP(yS] concentrations on the formation of [3H]inositol phosphates in permeabilized platelets. Platelets were labelled with [3H]inositol in an MnCl,/low-glucose medium and permeabilized, as described in Experimental Procedure. After gel filtration of the platelets and addition of ATP, samples were equili- brated for 15 rnin at 0°C with Ca2+ buffer giving a pCa of 6 and the indicated concentrations of GTP[yS]. Samples were then incubated for 5 rnin at 25 "C and the [3H]inositol phosphates determined. Results are means SEM from three identical incubation mixtures. Mean values for [3H]InsP, [3H]InsPz and [3H]InsP3 prior to equilibration of the samples were 0.56 x lo3 dpm, 0.20 x 3 O3 dpm and 0.09 x lo3 dpm/ 1 O9 platelets, respectively. InsP (0, 0) ; InsP, ( A , A); InsP3 (0, m)

in the total [3H]inositol phosphates that accumulated in the presence of 100 pM GTP[yS] at a pCa of 6 (-15 f 5%; mean SEM from three expts). Higher Li' concentrations were more inhibitory. Thus, [3H]InsP was not broken down by a Li +-sensitive phosphatase in these experiments. However, in the presence of GTP[yS], Li' increased the accumulation of [3H]InsP2 at the expense of t3H]InsP, with the result that

0 1 2 3 4 5 Incubation period (rnin)

Fig. 3. Time course of the formation of (3H]inositol phosphates in perrneabilized platelets in the presence of GTP[yS]. Platelets were labelled with [3H]inositol in an MnCIz/low-glucose medium and permeabilized, as described in Experimental Procedure. After gel filtration of the platelets and addition of ATP, samples were equili- brated for 15 min at 0°C with Ca2+ buffer giving a pCa of 6 in the absence (0, A, 0) or presence (0 , A, m) of 100 pM GTP[yS]. The samples were then incubated at 25 "C for the periods of time indicated before determination of [3H]inositol phosphates. Values are means SEM from three identical incubation mixtures. InsP (0, 0 ) ; InsP2 ( A , A); InsP3 (0, m)

the amounts of the two compounds found after 5 rnin were roughly equal (Fig. 4). This result showed that at least one- third of the [3H]InsP present after 5 rnin was formed by hy- drolysis of [3H]InsP2. Li' had no significant effect on the accumulation of [3H]InsP3 (Fig. 4).

In an attempt to determine whether the [3H]InsP2 that accumulated was formed by hydrolysis of [3H]InsP3, we equi- librated and incubated permeabilized platelets with 400 pM unlabelled InsP3. However, no significant changes were seen in the accumulation of [3H]InsP, [3H]InsP2 or [3H]InsP3.

528

0 10 0 10

Li+ fmM)

r- 0.4

0.3

0.2

0.1

0 0 10

Fig. 4. Effects of Li+ on the accumulation of [3H]inositol phosphates in permeab,..zed platelets. Platelets were labelled with [3H]inos~tol in an MnC12/low-glucose medium and permeabilized, as described in Experimental Procedure. After gel filtration of the platelets and addition of ATP, samples were equilibrated for 15 rnin at 0°C with CaZ+ buffer giving a pCa of 6 in the absence or presence of 10 mM Li' and 100 pM GTP[yS], as indicated. The samples were then incubated at 25°C for 5 rnin and the [3H]inositol phosphates determined. A (InsP); B (Imp2); C (InsP3). Values are means k SEM from three identical incubation mixtures. Mean values for [3H]InsP, [3H]InsP2 and [3H]InsP3 in the ulatelet susuension urior to equilibration were 0.89 x lo3 dum. 0.16 x lo3 dpm and 0.07 x lo3 dpm/109 platelets, respectively. ( 0 ) No GTP[yS]; iffl) with I00 pM GTP[yS]

~

To determine whether the unlabelled InsP3 was effective in preventing the hydrolysis of [3H]InsP3 within the platelets, we added 7 pM [32P]InsP3 to monitor InsP3 breakdown. In 5-min incubations with 100 pM GTP[yS] at a pCa of 6, 400 pM unlabelled InsP3 decreased [32P]InsP3 breakdown by 50 f 7% (mean & SEM, three expts). In controls, the hydroly- sis of [32P]InsP3 by intact platelets suspended in buffer A containing ATP was also measured. Although incubation of these platelets with GTP[yS] had no effect on their content of [3H]inositol phosphates, significant [32P]InsP3 breakdown was detected. Calculation indicated that about 60% of the [32P]InsP3 hydrolysed by permeabilized platelets was broken down intracellularly and that this was inhibited by unlabelled InsP3 to the same extent as total [32P]InsP3 breakdown. These results indicate that little of the [3H]InsP2 was derived from [3H]InsP3. However, as breakdown of added InsP3 by the permeabilized platelets may have been limited to some extent by the diffusion of the compound into the platelets, accurate measurement of [3H]InsP3 formation and hydrolysis by this method is not possible. We carried out similar experiments with 2 mM and 10 mM 2,3-bisphosphoglycerate, which in- hibited [32P]InsP3 breakdown by 45% and 13% respectively. However, this compound decreased the formation of both [3H]InsP and [3H]InsP2 without increasing [3H]InsP3 levels, suggesting that it inhibited phospholipase C.

Effects of thrombin on theformation of [3H]inositolphosphates

Addition of a high concentration of thrombin (2 units/ml) to permeabilized platelets at a pCa of 6 caused significant but variable increases in the accumulation of [3H]InsP and [3H]InsPz amounting to 38-230%0 and 31 -168%, respec- tively, after 5 min (ranges from four expts similar to that shown in Table 2). No significant increases in [3H]InsP3 were detected. In the presence of 10- 100 pM GTP, which alone caused only small increases in [3H]inositol phosphates, the increases in [3H]InsP and [3H]InsP2 caused by thrombin were about twofold greater (Table 2). The action of thrombin was enhanced much more potently by GTP[yS] than GTP (Table 2). In the presence of 4 pM GTP[yS], the increases in [3H]InsP and [3H]InsP2 caused by thrombin were about threefold those

seen in the absence of this nucleotide, whereas in the presence of 40 pM G'TP[yS], the formation of [3H]InsP and of [3H]InsP2 caused by thrombin was enhanced 7 - 13-fold and 16 - 28- fold respectively. At the same time, thrombin increased the accumulation of [3H]InsP and [3H]InsP2 caused by 4-40 pM GTP[yS] by 2.5 - 3.0-fold. In the presence of GTP[yS], throm- bin caused a significant accumulation of [3H]InsP3 (Table 2). The proportions in which [3H]inositol phosphates accumulat- ed after incubation with thrombin and guanine nucleotides were similar to those observed with GTP[yS] alone.

Addition to thrombin alone at a pCa of 6 did not cause measurable decreases in the [3H]phosphoinositides present in permeabilized platelets. However, addition of both thrombin and GTP[yS] decreased [3H]PtdInsPz levels to 50% of those found in incubations with CaZ+ alone and to 62% of the preincubation values (means from two expts). Thus, thrombin enhanced the decreases in [3H]PtdInsP2 caused by GTP[yS] (e. g. Table 3 ) . However, in the presence of GTP[yS], thrombin caused in absolute terms much larger decreases in [3H]- PtdInsP, relative to the values found after incubation with Ca2+ alone. This could not be accounted for by decreased synthesis of [3H]PtdInsP from [3H]PtdIns (Table 3), indicating that [3H]PtdInsP was the principal source of the additional [3H]inositol phosphates that were formed in the presence of thrombin.

Comparison of the effects of thrombin on intact and permeabilized platelets showed that the total of the [3H]inositol phosphates accumulating after incubation of in- tact platelets with thrombin for 5 rnin at 37°C was comparable with that found after incubation of permeabilized platelets with Ca2+ (pCa 6), 40 pM GTP[yS] and thrombin for 5 rnin at 25 "C (Table 2). However, [3H]InsP3 accounted for a larger fraction of the increase in total [3H]inositol phosphates ob- served in intact platelets (12.7 &0.5%0 after 1 min; mean f SEM from three expts; 3.9% after 5min, one expt). The time course of [3H]inositol phosphate formation in intact platelets showed that [3H]InsPz and [3H]InsP3 were formed first (ratio 3.6: 1 after 0.25 min) and that [3H]InsP accumulat- ed later (e. g. Table 2). Addition of GTP[yS] to intact platelets did not affect the formation of [3H]inositol phosphates (Table 2).

529

Table 2. Potentiation by guanine nucleotides of the effects of thrombin on the formation of [3H]inositol phosphates in perrneabilized platelets; comparison with the effects of thrombin on intact platelets Platelets were labelled with [3H]inositol in an MnCl,/low-glucose medium. One fraction was resuspended in medium for permeabilization of the platelets and the second in modified Tyrode's solution, pH 7.4, for investigation of the effect of thrombin on intact platelets. After gel filtration of the permeabilized platelets and addition of ATP, samples were equilibrated for 15 min at 0°C with Caz+ buffer giving a pCa of 6 and GTP[yS] or GTP, as indicated. These samples were then incubated 5 rnin at 25 "C in the absence or presence of 2 units thrombin/ml. Intact platelets were incubated from 0 to 5 min at 37°C with 2 units thrombin/ml or 100 pM GTP[yS]. Incubations were terminated by addition of trichloroacetic acid and the [3H]inositol phosphates determined. Results are means & SEM from three identical incubation mixtures

Preparation Additions Incubation x 3H present period

InsP InsPz InsP3

min dpm/lOg platelets

Permeabilized platelets none none thrombin GTP (10 pM) GTP (10 pM) + thrombin GTP (100 pM) GTP (100 pM) + thrombin

GTP[yS] (4 pM) + thrombin

GTP[yS] (40 pM) + thrombin

GTP[ySI (4 PM)

GTP[YS1(40 VM)

0 5 5 5 5 5 5

5 5 5 5

1.19f0.10 2.45f0.11 5.38k0.25 3.37k0.31 8.37k0.84 3.70k0.21 9.24+ 0.24

6.51 f0.41 15.96f0.30 17.49$0.17 40.40 k0.61

0.56 f 0.02 1 .10~0.10 2.01k0.11 1.66 k0.19 3.33k0.31 1.93 k0.17 3.55 k 0.27

2.78 k 0.24 6.1 3 f 0.20 6.78+0.11

24.05 f 1.99

0.21 f0.04 0.37 k 0.04 0.44 f 0.02

0.3750.03 0.53 f 0.04 0.31 k0.03 0.53 k0.02

0.56 k0.05 0.91 k0.03 0.66 f0.02 1.03f0.01

Intact platelets none thrombin

GTP[yS] (100 pM)

0.83 f0.03 0.68 f 0.09 0 12.07+0.24

0.25 13.08f0.72 3.87 k0.32 1.53f0.05 1.99k0.23 1 14.69 f0.72 6.63k0.58

5 43.18 f0.32 13.97 k 0.26 2.49 k0.06 0.75 kO.00 5 11.97 0.09 0.88 kO.01

Table 3. Effects of thrombin added without or with GTP[yS] on the metabolism of [3H]phosphoinositides in permeabilized platelets Platelets were labelled with [3H]inositol in an MnClz/low-glucose medium and permeabilized. After gel filtration of the platelets and addition of ATP, samples were equilibrated for 15 rnin at 0°C with no additions, with CaZ+ buffer giving a pCa of 6 or with Ca2+ buffer and 40 pM GTP[yS]. Samples were then incubated for 0 or 5 min at 25°C with or without thrombin (2 units/ml), as indicated. [3H]Phosphoinositides and [3H]inositol phosphates were determined as described in Experimental Procedure. Results are mean values f SEM from three identical incubation mixtures

Additions ~~~

Incubation 1O-j x 3H present period

PtdIns PtdInsP PtdInsP, InsP + InsP2 + InsP3 ~~

min dpm/109 platelets

None 0 189.9f6.2 37.8 f 0.8 13.4k0.5 1.5k0.2 CaZ + 5 141.6 f 2.5 90.6 k 3.9 17.7 f0 .2 3.4k0.6 Ca2+ + thrombin 5 143.1 f 1.3 91.9k1.4 17.4k 1 .O 4.1 50 .3 CaZ+ + GTP[yS] 5 130.6f2.3 81.2 f0.8 11.3 f0.9 16.3 50.7 Ca2+ + thrombin + GTP[yS] 5 132.1 f 1.9 66.7 k 0.6 9.3f0.2 41.7 5 1.2

Identification by HPLC of the f 3 Hjinositol phosphate isomers present in Dowex-1 fractions containing f3H]InsP, [3H]InsP2 or [3H]In~P3

[3H]InsP. As authentic ~-myo-[~H]Ins(l)P and L-myo- ['4C]Ins(l)P were eluted from Partisil 10 SAX columns in superimposed peaks immediately following AMP, the I4C- labelled compound was used as an internal standard to facili- tate identification of [3H]Ins(l)P in experimental samples. Commercial [3H]Ins(4)P was eluted immediately after

['4C]Ins(l)P (Fig. 5A). Because of the possibility of some overlap between platelet [3H]Ins(l)P and [3H]Ins(4)P, the composition of intermediate fractions was calculated from the 14C present, assuming that the ratio of 3H to 14C in the first Ins(1)P fraction reflects that of the pure compound. Using these methods, analysis of Dowex-I [3H]InsP fractions from experiments with permeabilized platelets that had been incu- bated for 5 min with both thrombin (2 units/ml) and GTP[yS] (40- 100 pM) at a pCa of 6 (e.g. Fig. 5B) indicated that the [3H]InsP present consisted of 73 -80% [3H]Ins(4)P and only 20-27% [3H]Ins(l)P. A similar result was obtained with

5 30

Fraction no

Fig. 5. Analysis of ( 3 H]InsP generated by stimulation of permeabilized and intact platelets. Dowex-1 fractions containing [3H]InsP, formed under specific incubation conditions in an experiment similar to that shown in Table 2, were desalted and analysed by HPLC, as described in Experimental Procedure. Only HPLC fractions in the InsP range are shown. (A) Elution profile of a mixture of standards comprising AMP, ['4C]Ins(l)P and [3H]Ins(4)P; (B) elution profile of [3H]InsP isolated from permeabilized platelets that had been incubated for 5 min at 25'-C with thrombin (2 units/ml) and GTP[yS] (40 pM) at a pCa of 6 [AMP and ['4C]Ins(l)P added as internal standards]; (C) elution profile of [3H]InsP isolated from intact platelets that had been incubated for 5 min at 37°C with thrombin (2 units/ml) [AMP and ['4C]lns(l)P added as internal standards]. Fractions (0.5 ml) were counted for 3H and I4C; values for [3H]InsP are corrected for crossover of I4C into the 3H channel

ATP

I Ub . . . . . . . , . . . . . . . .

0 115 125 135 145

Fraction no.

Fig. 6. Analysis of [3H]lnsP3 generated by stimulation of intact platelets with thrombin. Intact [3H]inositol-labelled platelets were in- cubated with thrombin (2 units/ml) for 5 min at 37°C (as reported in Table 2). Dowex-1 fractions containing [3H]InsP3 were desalted and analysed by HPLC, as described in Experimental Procedure. ATP and ["P]Ins(l ,4,5)P3 were included as internal HPLC standards. Fractions (0.375 ml) were counted for 3H and 3zP under conditions in which no 3zP radioactivity appeared in the 'H channel

[3H]InsP from incubations of permeabilized platelets with either thrombin and 100 pM GTP or 40 pM GTP[yS] alone. In contrast, analysis of the [3H]InsP isomers present in intact platelets incubated for 5 min with thrombin (2 units/ml) gave mean values of 79% [3H]Ins(l)P and 21 'YO [3H]Ins(4)P (e. g. Fig. 2C). A slightly higher proportion of [3H]Ins(4)P (32%) was found in 0.25-min incubations. In all these studies a small amount of [3H]Ins(1,4)P2 (10- 15% of total 3H) was also found in the Dowex-1 [3H]InsP fraction.

[3H/In~P2. Authentic [3H]Ins(1,4)P2 was eluted from Partisil 10 SAX columns shortly after ADP in the same frac- tions as all the 3H present in Dowex-1 [3H]InsP2 from permeabilized platelets that had been incubated with either thrombin and GTP[yS] or GTP[yS] alone. Similarly, the

[3H]InsP2 isolated from intact platelets treated with thrombin appeared to be the 1,4 isomer.

[3H]ZnsP3. The Dowex-1 [3H]InsP3 fractions include any [3H]InsP4 that accumulated in the platelets and were therefore analysed on Partisil 10 SAX columns using ammonium for- mate gradients up to 1.7 M [36]. [3H]Ins(1,3,4)P3 and [3H]Ins(l ,4,5)P3 standards were eluted shortly after ATP but were well-separated; [3H]Ins(l ,3,4,5)P4 was eluted much later. ATP and [jLP]Ins( 1 ,4,5)P3 were routinely added to experimen- tal samples as internal standards (e.g. Fig. 6). Analysis of the small amounts of [3H]InsP3 (about 30 cpm) formed in permeabilized platelets incubated with both thrombin and GTP[yS] at a pCa of 6 (as in Table 2) demonstrated the presence of the 1,3,4 isomer. Trace amounts of [3H]- Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 also appeared to be pre- sent in some samples. The more highly labelled [3H]InsP3 fraction from intact platelets that had been incubated for 5 min with thrombin (2 units/ml) gave an easily detected peak of [3H]Ins(1,3,4)P3 (Fig. 6) that accounted for 83 k 3% of the total 3H recovered from the HPLC column (meanf SEM from three expts). The remaining 3H was found in Ins(1,4,5)P3; no [3H]Ins(1,3,4,5)P4 was detected. In 0.25-min incubations of intact platelets with thrombin, the [3H]InsP3 fraction contained a similar total amount of the 1,4,5 isomer but less than half as much of the 1,3,4 isomer, indicating that only the 1att.er accumulated in prolonged incubations.

DISCUSSION

Labelling of'platelets with f 3 Hlinositol To investigate changes in the metabolism of phospho-

inositides in permeabilized platelets, prior labelling with [3H]inositol is preferable to [3H]arachidonate, [3H]glycerol or 32Pi (e. g. [6 - 8]), since the labelled phosphoinositides and inositol phosphates can both be identified and quantified relatively easily. This approach has been applied to intact human platelets by several workers (e.g. [lo, 38, 39]), but the slow incorporation of [3H]inositol into phosphoinositides has necessitated use of very large amounts of [3H]inositol. In

531

the present study we utilized an MnC12/low-glucose medium, which enhanced the labelling of platelet phosphoinositides and inositol phosphates by about sevenfold and four to sixfold, respectively. This procedure was based upon the observations that Mn2 + promotes the incorporation of [3H]inositol into PtdIns by either CDP-diacylglycerol : myo-inositol transferase [40, 411 or the Ptd1ns:inositol exchange enzyme [40, 421 and that high concentrations of glucose inhibit inositol transport across membranes [43]. Others have also used MnCI2, which increased the accumulation of [3H]InsP2 and [3H]InsP3 in rat pancreatic acini labelled with [3H]inositol [44]. However, in brain preparations in contrast to platelets, MnC12 increased the labelling of an agonist-insensitive phosphoinositide pool by [3H]inositol [45, 461.

Effects of GTP[yS] and thrombin on phosphoinositide metabolism

In permeabilized platelets at pCa values of 6 or 7, which correspond to the physiological range [47], GTP[yS] prevented any increase in [3H]PtdInsP2 and caused the accumulation of [3H]InsP, [3H]InsP2 and small amounts of [3H]InsP3.. The formation of [3H]InsP2 can be explained by either the hydroly- sis of [3H]PtdInsP2 by phospholipase C and the rapid degra- dation of the [3H]InsP3 formed or by the hydrolysis of [3H]PtdInsP before it can be converted to [3H]PtdInsP2. These possibilities are not mutually exclusive. The accumu- lation of some [3H]InsP3 and the decrease, in the majority of experiments, of [3H]PtdInsP2 levels below the value found in unincubated controls indicate that [3H]PtdInsP2 can act as a substrate of phospholipase C in this system. However, the formation of much more [3H]InsP2 than [3H]InsP3 in short incubations and our failure to trap additional [3H]InsP3 with unlabelled InsP3 strongly suggest that [3H]PtdInsP is the major substrate. This conclusion is further supported by the markedly lower values for [3H]PtdInsP found after incu- bations with both GTP[yS] and thrombin.

Although Li' was initially described as an inhibitor of InsP phosphatase [37], it is known to be effective against at least one of the enzymes that degrades InsP2 [48, 491. How- ever, 10 mM Li' does not inhibit either the liver membrane or soluble platelet InsP3 5-phosphatases [49, 501. Consistent with these findings, it has been shown that in intact human platelets labelled with [3H]inositol, Lit enhances the accumu- lation of t3H]InsPand [3H]InsP2 but has much less or no effect on [3H]InsP3 levels [38,39]. Our finding that Lit increased the accumulation of [3H]InsP2 caused by addition of GTP[yS] to permeabilized platelets to a level equivalent to that of [3H]InsP indicates that at least half of the phospholipase C activity was directed against [3H]polyphosphoinositides. The correspond- ing decrease in [3H]InsP was unexpected and suggests that little breakdown of [3H]InsP occurred in this system, perhaps because diffusion of the compound from the permeabilized platelets greatly reduced its concentration. The [3H]InsP that was still formed in the presence of Li' could reflect either hydrolysis of [3H]InsP2 by a Li'-insensitive enzyme or the action of phospholipase C on PtdIns. However, the major decrease in [3H]PtdIns that we observed, occurred in the ab- sence of either Ca2' or GTP[yS] and was fully accounted for by the formation of [3H]PtdInsP. Thus, although a limited GTP[yS]-stimulated hydrolysis of PtdIns by phospholipase C was observed in occasional experiments (e. g. Table 3), there was no loss of PtdIns in permeabilized platelets comparable to that attributed to the action of phospholipase C in intact thrombin-stimulated platelets [9, 111.

Further evidence that polyphosphoinositides rather than PtdIns are the major substrate of phospholipase C in permeabilized platelets is provided by the finding that most of the [3H]InsP that accumulated after incubation of the platelets with thrombin and GTP[yS] or GTP[yS] alone appeared to be [3H]Ins(4)P. This result contrasts with the effect of high thrombin concentrations on intact [3H]inositol-labelled platelets, in which two-thirds or more of the [3H]InsP formed coeluted with the 1-phosphate, as previously observed by Siess [38]. Although it is possible that differences in the relative rates of breakdown of Ins(l,4)P2 to Ins(1)P and Ins(4)P in permeabilized and intact platelets account for this discrep- ancy, vasopressin has been shown to stimulate the formation of a much higher proportion of Ins(4)P in intact platelets than thrombin [38]. This suggests that high thrombin concen- trations may cause the hydrolysis of PtdIns in intact platelets, perhaps because the cytosolic [Ca"] reaches a level above that obtained with vasopressin or studied in the present exper- iments with permeabilized platelets. However, we cannot rule out the possibility that some of the [3H]Ins(l)P we measured is the L enantiomer (identical to Ins(3)P) and is formed by breakdown of [3H]Ins(l,3,4)P3.

A number of observations in addition to those described in this report support the view that PtdInsP, rather than PtdIns or PtdInsPz may initially be the major substrate of phospholipase C in platelets. Thus, several reports [lo, 38, 391, in addition to this study, have shown that immediately after addition of thrombin to intact [3H]inositol-labelled platelets, the formation of [3H]InsP2 exceeds that of [3H]InsP3 by a factor of two to fourfold, whereas [3H]InsP tends to accumulate later. Moreover, in isolated rabbit platelet mem- branes containing [3H]polyphosphoinositides, GTP[yS] stimulated the hydrolysis of [3H]PtdInsP and [3H]PtdInsP2 by phospholipase C in a ratio of about 4:l [25] and exper- iments with purified soluble platelet phospholipase C have indicated that this enzyme can hydrolyse PtdInsP more rap- idly than PtdIns or PtdInsPz [51- 531. Failure to detect con- sistent decreases in PtdInsP in intact platelets stimulated with thrombin [6 - 81 could reflect resynthesis of the compound from PtdIns as rapidly as it is hydrolysed by phospholipase C. If a small pool of PtdInsP participates in these reactions in intact platelets, no marked change in labelling of the com- pound would be detected even in non-steady-state experiments [l I]. Although the available results are consistent with the hydrolysis of [3H]PtdInsP, as well as [3H]PtdInsP2, in intact as well as permeabilized platelets, the ratio of [3H]InsP2 to [3H]InsP3 formation was higher in the latter. This may partly reflect the higher ratio of [3H]PtdInsP to [3H]PtdInsP2 in permeabilized platelets, particularly after incubation with ATP. Hormonal stimulation of permeabilized GH3 cells [54] released [3H]inositol phosphates in very similar proportions to those we have described for the platelet. However, stimu- lation of permeabilized exocrine cells from the pancreas [55] yielded a much higher ratio of [3H]InsP3 to [3H]InsP2 (1 : 2 ) than we observed in permeabilized platelets. Utilization of PtdInsP in preference to PtdInsP2 as a substrate for phospholipase C in the platelet will generate a high ratio of diacylglycerol to InsP3 and is consistent with evidence that the former may be the more important second messenger in this cell [47].

Analysis of the [3H]InsP3 formed on stimulation of permeabilized or intact [3H]inositol-labelled platelets demon- strated that the principal isomer that accumulated coeluted with Ins(1,3,4)P3 and not Ins(1,4,5)P3. Previous studies [lo, 381 on the [3H]InsP3 found in thrombin-stimulated platelets

532

identified this material as the 1,4,5-isomer, which now appears to be only a minor component, at least after 0.25 min. More- over, it is possible that part of this [3H]Ins(l,4,5)P3 arose by acid hydrolysis of the 1,2-cyclic, 4,5-trisphosphate, which may also be formed in platelets incubated with thrombin [56]. Although significant amounts of [3H]Ins(1,3,4,5)P4 were not detected in platelets in the present study, the formation of [3H]Ins(1,3,4)P3 provides strong support for the existence in this cell of the Ins(1,4,5)P3 --f Ins(1,3,4,5)P4 --f Ins(1,3,4)P3 pathway [57]. Studies with 32P-labelled platelets have also suggested that Ins(1,3,4,5)P4 and 1ns(1,3,4)P3 are formed in thrombin-stimulated platelets [56, 581 and the conversion of exogenous [3H]Ins(l ,4,5)P3 to these compounds by platelet lysate has been demonstrated very recently [58].

Role of guanine nucleotides in the activation of phospholipase C

Previous results from this laboratory demonstrated that GTP[yS] enhances diacylglycerol formation in electropermea- bilized platelets [17]. Brass et al. [59] have extended this finding by showing that another GTP analogue, guanyl-5’-yl imidodiphosphate, stimulates both diacylglycerol and phosphatidic acid formation in saponin-permeabilized platelets. We have now found that GTP[yS] also causes a net breakdown of polyphosphoinositides with the formation of inositol phosphates in electropermeabilized platelets. This confirms that GTP[yS] activates platelet phospholipase C in this experimental system. Lapetina 1601 has reported some similar effects of GTP[yS] on [3H]phosphoinositide metab- olism of platelets permeabilized by saponin. However, in the latter study, the action of GTP[yS] was CaZ +-independent and much less [3H]InsP was formed relative to [3H]InsP2 and [3H]InsP3. We have also found that GTP or GTP[yS] potentiates the formation of [3H]inositol phosphates induced by thrombin in electropermeabilized platelets, indicating that the guanine-nucleotide-binding protein that mediates the ef- fects of GTP[yS] may participate in signal transduction from the thrombin receptor to phospholipase C. This conclusion is supported by the ability of guanosine 5’-[fl-thio]diphosphate to inhibit the effect of thrombin on diacylglycerol formation in saponin-permeabilized platelets [59] and by the recent dem- onstration of a GTP-dependent activation of phospholipase C by thrombin in isolated platelet membranes [24,25]. Exper- iments on the effects of many other CaZ+-mobilizing hor- mones on cell membranes are consistent with this model [19 - 231. However, it has also been reported that GTP and GTP[yS] can stimulate the activity of phospholipase C in platelet cytosol, utilizing as substrates polyphosphoinositides in either platelet membranes [24] or lipid vesicles [24, 61, 621. Such a mechanism could partly account for the effects of GTP[yS] on phosphoinositide metabolism in permeabilized platelets, but is less likely to be involved in the guanine-nucleotide-depen- dent activation of phospholipase C by thrombin, since treat- ment of platelets with thrombin did not increase the GTP or GTP[yS]-stimulated phospholipase C activity of platelet cytosol [61]. Our results provide a biochemical basis for the synergistic effects of guanine nucleotides and thrombin on the degranulation of electropermeabilized platelets [16, IS].

This work was supported by grants from the Heart and Stroke Foundation of Ontario (T443) and the Medical Research Council of Canada (MT-5626).

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