Biochimica et Biophysics Acta, 1124 (1992) 273-278 0 1992 Elsevier Science Publishers B.V. All rights reserved
Evidence for phosphatidylcholine hydrolysis by phospholipase in rat platelets
Edward Randell, Hemendra Mulye, Sailen Mookerjea and Arun Nagpurkar Department of Biochemistry, Memorial Unicersity of Newfoundland, St. John’s, Newfoundland (Canada) (Received
Production of [3H11,2-dipalmitoylglycerol (13HIDAG) from l-palmitoyl-2-~9,10-3H1palmitoyl-sn-glycero-3-phosphocholine and [3H1phosphorylcholine from 1,2-dipalmitoyl-sn-glycero-3-[Me-3H1phosphocholine was studied using sonicated rat platelets. The formation of 13HIDAG and [3H1phosphorylcholine occurred at a comparable rate. [3H1Phosphorylcholine formation was dependent on the concentration of the substrate, platelet sonicates and calcium in the incubation medium. The [3Hlphosphorylcholine formation increased in presence of 0.01% deoxycholate and 0.01% Triton X-100. The phosphatidylcholine-phospholipase C (PC-PLC) in the platelet sonicates was recovered in both the supernatant and particulate fractions obtained after ultracentrifugation at 105 000 X g for 1 h. The PC-PLC activity in both fractions was inhibited by 2 mM EDTA. In the presence of 0.01% deoxycholate and 0.01% Triton X-100 the activity in the particulate fraction increased compared to the activity in the supernatant, which was inhibited by 0.01% Triton X-100. The pH optima for PC-PLC in both fractions was between pH 7.2 and 7.6. PC-PLC activity was also found in rabbit and human platelet sonicates, but the activity was significantly lower than in rat platelet sonicates. There was no evidence to suggest presence of phosphatidylcholine-specific phospholipase D activity in rat sonicated platelets. This data, therefore, provides direct evidence for the presence of PC-PLC activity in rat platelets. Introduction
Agonist-induced stimulation of platelet membrane receptors result in platelet shape change, aggregation and secretion. These responses are often initiated by receptor-mediated activation of certain phospholipases [l]. The role of phospholipase A, in platelet function has been documented [l-3]. In platelets, phosphatidylinositol-specific phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate leads to generation of two signalling substances, inosito1 1,4,5-triphosphate and 1,2-diacylglycerol (DAG) . In presence of calcium, DAG activates protein kinase C that results in the phosphorylation of a 47 kDa protein involved in the control of platelet cellular responses [1,51. Recent evidence suggesting agonist-induced DAG generation from phosphatidylcholine in a variety of tissues, has challenged the classical scheme involving inositol lipids as the only source of DAG during signal transduction (for review see Refs. 6,7). DAG can be
Correspondence: A. Nagpurkar, Department of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada AlB 3X9.
produced from phosphatidylcholine by the action of phosphatidylcholine-phospholipase C (PC-PLC), or by a combined action of phosphatidylcholine-phospholipase D (PC-PLD) and phosphatidic acid phosphohydrolase [6,7]. Recent studies [8-121 have reported the formation of DAG and phosphatidic acid (PA) from phosphatidylcholine in activated platelets. However, these studies have not conclusively shown the presence of PC-PLC enzyme in platelets. The purpose of the present study was to obtain evidence that PC-PLC activity is indeed present in rat platelets. Materials
Sodium deoxycholate was from Fisher Scientific (New Jersey, USA); Triton X-100, oleic acid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), lpalmitoyl-2-lyso-sn-glycero-3-phosphocholine, glyceryl3-phosphocholine, palmitic acid, CDP-choline, 1,2-dipalmitoyl-sn-glycero-3-phosphate (PA), phospholipase C (C. welchii; 10.6 U/mg protein), 1,2-dipalmitoyl-snglycerol and phosphorylcholine chloride (Calcium salt) were from Sigma (St. Louis, MO, USA); choline chloride was from J.T. Baker Chemical (New Jersey, USA); l-palmitoyl-2-[9,10-3H]palmitoyl-sn-glycero-3-phospho-
274 choline ([3H]palmitoyl-DPPC) (32.9 Ci/mmol) and 1,2-dipalmitoyl-sn-glycero-3-[ Me-3H]phosphocholine ([ “HIcholine-DPPC) (37 Ci/mmol) was from DuPont, Canada; [Me- “C]choline (55 mCi/mmol) was from Amersham; Male Sprague-Dawley rats (200-300 g) were from Charles River. Canada. Preparation of platelet Sonicates Rat blood was collected via the abdominal aorta of ether anaesthetized rats into plastic syringes containing 0.1 volume of 3.6% citrate. Rabbit blood was collected from the central ear artery into plastic syringes containing 0.1 volume of 3.6% citrate. Human blood was collected from healthy volunteers into evacuated tubes containing 0.1 volume of 3.8% citrate. Platelet rich plasma was prepared by centrifuging blood (220 X g for 8 min). Platelets were washed with 12 mM Tris-HCl (pH 7.4) containing 1.5 mM EDTA, 135 mM NaCI, 4 mM o-glucose t_ 2 mM phosphorylcholine as described . Washed platelets (5. 10’ platelets/ml; 0.3 mg protein/ml) were resuspended in 25 mM Tris-HCl (pH 7.41, containing 125 mM NaCI, 2.5 mM CaCl, and sonicated on ice, 3 x for 30 s, using Branson Sonifier fitted with a microtip (setting 60 Watts) . Assay for PC-PLC acticity The standard incubation mixture contained 25 mM Tris-HCl (pH 7.41, 125 mM NaCl, 2.5 mM CaCl,, tritium-labelled substrates (18 PM; 10 000-30 000 dpm/pM) and platelet sonicates, as specified, in a final volume of 400 ~1. Incubations were carried out for 40 min at 37°C in a water bath. Reactions were stopped by adding 1.87 ml of a mixture of chloroform/ methanol containing 2% acetic acid (1: 2, v/v). This was immediately followed by the addition of 100 ~1 of phosphorylcholine/ choline (20 mg/ml each) as internal standards. The assays were extracted by the Bligh and Dyer method  and centrifuged at 1000 x g for 15 min to separate aqueous and non-aqueous layers. Aliquots of aqueous layers containing [ 3H]phosphorylcholine were added to 10 ml Readysafe Scintillation Cocktail (Beckman) and counted for radioactivity in a Wallac 1209 Rackbeta Liquid Scintillation Counter. In certain cases, aqueous layers (100 ~1 aliquots) were applied to Whatman K5 silica gel 150A TLC plates (layer thickness of 250 km> and developed in methanol/ 0.9% NaCl/ammonium hydroxide (50 : 50 : 5, v/v> (system A) as previously described [161. After air drying the plates, the products were, visualized by staining with iodine vapour. Bands corresponding to choline and phosphorylcholine were scraped and counted for radioactivity. Authentic standards of DPPC (5 pg), l-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (5 pug>,glycero-3-phosphocholine (20 pg), CDP-choline (20 pug), phosphorylcholine (20 pg), choline chloride (20 pg), were chromatographed with the aqueous ex-
tracts from the assays to confirm adequate separation of the choline metabolites. In assays using [ “Hlpalmitoyl-DPPC as substrate, the non-aqueous layer (100 ~1 aliquots) was removed and applied to two silica gel 60 (Merck) TLC plates (layer thickness of 250 pm>. One plate was developed in hexane/diethyl ether/90% formic acid (60: 40: 1, v/v) (system B) to isolate [“HIDAG. The second plate was developed in chloroform/ pyridine/ 70% formic acid (50 : 2.5: 7, v/v) (system C> to separate PA [ 171 that may have formed as a result of phospholipase D activity. Authentic standards consisting of DPPC (5 pg), 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (5 Fg), palmitic acid (10 kg), PA (10 pg), and 1,2-dipalmitoylsn-glycerol (5 cl.g) were included in each plate which was stained with iodine. The stained lipids were scraped from the plate and counted for radioactivity. Distribution of phospholipase C activity in the supernatant and particulate fractions of platelet sonicates To determine the distribution of PC-PLC activity, rat platelets (5 . 10X/ml) were sonicated and centrifuged at 105 000 X g for 60 min at 4°C. The supernatant was removed (soluble form) and the pellet was resuspended (particulate form) in a volume equal to the volume of the supernatant, by agitation and sonication for three 5 s intervals. Aliquots (40 ~1) of each fraction was assayed for PC-PLC activity as described earlier. Choline Kinase activity To determine whether the [“Hlphosphorylcholine was formed by the action of choline kinase on [3H]choline, choline kinase activity was determined in platelet sonicates. Platelet sonicates were added to standard incubation mixture containing [Me“C]choline (0.6 PM), as substrate, in a final volume of 400 ~1 at 37°C. Reaction was stopped after 20 min by adding 1.87 ml of chloroform/ methanol containing 2% acetic acid (1:2, v/v) and 100 ~1 of a mixture of phosphorylcholine/ choline (20 mg/ml each). The assays were extracted and the aqueous layer chromatographed. Bands corresponding to phosphorylcholine and choline were scraped and counted as described for PC-PLC assay. Results Sonicated rat platelets (5 . 10’ cells/ml) when incubated with [3H]choline-DPPC resulted in the formation of [3H]phosphorylcholine. Identification of this metabolite was made by TLC in system A, using authentic standards (Fig. 1A). The identification of [ 3H]phosphorylcholine was also confirmed by comparing the R, of [ 3H]phosphorylcholine formed by platelet sonicate PC-PLC with the R, of [“H]phosphoryl-
C Phosphorylcholine (R,=O.i39)
200 .--o--n\ 0 --,-n0.0
60 80 Time (min)
Fig. 2. Time-course of the formation of [“H]DAG and [‘H]phosphorylcholine by rat platelet sonicates. Platelet sonicates (5.10’ cells/ml) were incubated with [sH]palmitoyl-DPPC (18 @M) or [‘HIcholine-DPPC (18 PM). The reaction was stopped at the speci-
600 400 -
fied times and the aqueous and non-aqueous layers of extracted assays were analyzed as described in Materials and Methods. Data points are the mean of two determinations for [‘HIcholine-DPPC as substrate and three determinations for [sH]palmitoyl-DPPC.
Rf Fig. 1. TLC of aqueous metabolites of [“H]choline-DPPC. [sH]Choline-DPPC (18 yM) was incubated for 40 min with: (A) rat platelet sonicates (5.10’ cells/ml); or (B) bacterial phospholipase C (0.02 U/ml). An aliquot (100 ~1) of aqueous layer was applied to the plate and developed to separate the choline metabolites. Each lane was divided into 1 cm segments which were scraped and counted. (C) shows the profile in the absence of any enzyme. TLC profiles shown are typical of at least five profiles giving similar results.
choline formed by the action of bacterial (C. we/&ii) phospholipase C on [3H]choline-DPPC (Fig. 1B). Fig. 1C shows the TLC profile from control incubations without any enzyme. Based on these results, the product of the [3H]choline-DPPC hydrolysis by platelet sonicates was identified as [ 3H]phosphorylcholine. Formation of [“HIcholine was not detected. When [ ‘Hlpalmitoyl-DPPC and [ ‘HIcholine-DPPC were used as substrates with platelet sonicates the [3H]DAG and [ 3H]phosphorylcholine formed were monitored as a function of time (Fig. 2). The production of [“Hlphosphorylcholine was dependent on the concentrations of [3H]choline-DPPC and platelet sonicates (Fig. 3A and B). [3Hlphosphorylcholine production increased with increasing concentrations of [“HI choline-DPPC (Fig. 3A). Transformation of this data produced a linear double reciprocal plot (Fig. 3A, inset) which gave a K, of approx. 100 PM. Fig. 3B shows a linear increase of [3H]phosphorylcholine formation with increasing platelet sonicate concentrations up to the equivalent of 10’ platelets/ml. Fig. 4 shows the platelet sonicate PC-PLC activity as a function of increasing calcium concentrations. The enzyme was active over the entire range of calcium concentrations (25 nM-25 mM). However, the enzyme activity increased by about 60% around 2.5 mM Ca*+.
Fig. 3. Formation of [‘H]phosphorylcholine as a function of [3H]choline-DPPC and rat platelet sonicate concentration. (A) Platelet sonicates (5.10’ cells/ml) were incubated with increasing concentrations of [3H]choline-DPPC (O-45 FM). Inset is a double reciprocal plot of the substrate dose curve data. (B) Increasing amounts of rat platelet sonicates (0-1.10’ cells/ml) were incubated with 18 PM [3H]choline-DPPC. Production of [3H]phosphorylcholine was mea-
sured as described in Materials and Methods. Data points are the mean + S.E. of three determinations.
+ 0 01%
+ 2 mM EXITA
Fig. 4. Effect of calcium concentration on formation of [‘Hlphosphorylcholine from [“H]choline-DPPC by rat platelet sonicates. Platelet sonicates (5.10’ cells/ml) in calcium-free Tris buffered saline, were incubated with [~~~]choiine-DPPC (18 FM) in presence of increasing concentrations of calcium (2.5.10-‘-2.5. lo-’ MI. Formation of [“H]phosphorylcholine was measured as described in Materials and Methods. Data points are the mean&SE. of three determinations.
rj-iT-1 j 1 ] Fig. 6. Effect of Detergents and EDTA on fractionated platelet sonicates. Aliquots (40 ~1) of the soluble (A) and particutate iBf forms of PC-PLC from platelet sonicates were assayed using [3H]choline-DPPC (18 /*MI in presence or absence of 0.01% deoxycholate, 0.01% Triton X-100, or 2 mM EDTA in calcium-free incubation medium. After 40 min the reactions were stopped and extracted as described in Materials and Methods. Values shown are mean f SE. of three determinations.
The effect of deoxycholate, Triton X-100 and oleate on PC-PLC activity in rat platelet sonicates was studied and results are shown in Fig. 5. Deoxycholate (0.01%) increased PC-PLC activity by as much as 130%, while 0.01% Triton X-100 increased activity by 10 to 20%. Higher concentrations of deo~cholate, Triton X-100, or oleate inhibited the enzyme activity. PC-PLC activity associated with the rat platelet sonicates (0.775 f 0.041 PM [3H]phosphorylcholine formed after 20 min) was approx. 4-fold greater than the activity found in rabbit (0.140 i 0.005) and human (0.174 t_ 0.044) platelet sonicates. PC-PLC activity was assayed in platelet sonicates that were fractionated by ultracentrifugation as described in Materials and Methods. The activities in the
particulate and supernatant fractions were 0.288 + 0.150 and 0.310 i 0.080 PM [“Hlphosphorylcholine formed, respectively. The combined recovery of activities in the two fractions was greater than 75%. Both forms of the enzyme were inhibited by 2 mM EDTA when assayed in calcium free incubation medium (Fig. 6). The PC-PLC activity in the particulate fraction was increased by 0.01% deoxycholate and 0.01% Triton
Fig. 5. Effect of detergents on [‘H]phosphorylcholine formation by rat platelet sonicates. Platelet sonicates (5’ IO’ cells/ml) were incubated [~H]choiine-DPPC (18 PM) in presence or absence of deoxycholate, Triton X-100, or oleate. After 40 min the reactions were stopped extracted as described in Materials and Methods. Values are the mean rf-SE. of three determinations.
Fig. 7. Effect of pH on formation of water-soluble choline metabolites from [“H]choline-DPPC by whole and fractionated platelet sonicates. Platelet sonicates (5 *10’ cells/ml) (A) and 40 ~1 aliquots of soluble and particulate forms of PC-PLC (B and C) were incubated with increasing concentrations of [3HJcholine-DPP~ (I8 ,uM) for 40 min in SO mM Tris-Maleate (pH 5.2-8.4) containing 2.5 mM CaCI,. Reactions were stopped and extracted as described in Materials and Methods. Data points are the mean f S.E. of three determi nations.
X-100, while, the PC-PLC activity in the supernatant was marginally increased by 0.01% deoxycholate, but was inhibited by 0.01% Triton x-100. Fig. 7 shows the pH profile of PC-PLC activity in platelet sonicates, particulate and supernatant fractions. The pH optima for PC-PLC activity in platelet sonicates and that associated with particulate and soluble forms were in the range of pH 7.2 to 7.6. Discussion
The present study demonstrates that rat platelet sonicates hydrolyse [3H]choIine-DPPC and [ 3H]palmitoyl-DPPC to produce [ 3H]phosphorylcholine and [ 3H]DAG, respectively, thus confirming the presence of PC-PLC activity in normal rat platelets. Furthermore, the activity of this enzyme, was dependent on time (Fig. 21, substrate (Fig. 3A) and platelet sonicate (Fig. 3B) concentrations. Analysis of the products of phosphatidylchohne hydrolysis, revealed phosphorylcholine to be the only labelled metabolite recov-
ered in the aqueous phase. DAG and palmitic acid were the only labelled products in the non-aqueous phase. The formation of DAG was a result of the action of PC-PLC, while palmitic acid was formed by phospholipase A, activity in the platelet sonicates . There was no choline and phosphatidic acid formation, or choline kinase activity in the platelet sonicates. This indicates that PC-PLC activity, and not PC-PLD, is responsible for the generation of phosphorylcholine and DAG. The PC-PLC activity in platelet sonicates was calcium-dependent, and was active over a wide range of calcium concentrations (25 nM-25 mM) (Fig. 4). This may suggest a role for this enzyme both in the platelet cytosol, where calcium levels are at 0.1 to 3 FM, and outside the platelet where calcium levels are above 1 mM. In addition to rat platelet sonicates, human and rabbit platelet sonicates also showed PCPLC activity, but the activity was less than 25% of that found in rat platelets. Platelet sonicates when fractionated by ultracentiifugation resulted in particulate and supernatant fractions. The PC-PLC in the supernatant was designated as the soluble form, while the PC-PLC in the resuspended pellet was designated as the particulate form of PC-PLC. Both the fractions contained almost equal PC-PLC activity which was inhibited by 2 mM EDTA (Fig. 6). The similarities in calcium requirement and substrate specificity for the two forms could indicate a common cellular origin. The effect of pH on the PC-PLC activity in ptatelet sonicates, and on the particulate and soluble forms of PC-PLC was determined (Fig. 7). The PC-PLC in all three cases was found to be optimally active between pH 7.2 and 7.6. It has been reported that phospholipase C with broad substrate specificity exist in lysosomes isolated from rat liver [18J. This Iysosomal enzyme was found to be optimally active below pH 5, and was inhibited by divalent cations. It is, therefore, unlikely that the PC-PLC activity associated with platelet sonicates is of Iysosomal origin considering its pH optimum and calcium-dependent activity. Membrane bound PC-PLD activity has been detected in most rat tissues in the presence of certain detergents [19-211. Deoxycholate and /I-octylglucoside have been used to measure PC-PLC activity [22,23]. The effect of deoxycholate, Triton X-100, and oleate on PC-PLC activity was, therefore, examined (Fig. 5). Of the three detergents used in this study, only 0.01% deoxycholate and 0.01% Triton X-100 increased the PC-PLC activity. These detergents appeared to have their greatest stimulatory effect on the particulate form of PC-PLC (Fig. 6). The activity of the soluble form of PC-PLC was only moderately increased by 0.01% deoxycholate but was inhibited by 0.01% Triton X-100. No PC-PLD activity was detected, in the platelet sonicates, in the presence of these detergents.
278 The presence of PC-PLC and/or PC-PLD, as active participants in signal transduction pathways, have been described for many tissues [6,7]. Although, a role for PC-PLC in signal transduction mechanisms has not been reported for platelets, a number of studies have indicated the possibility of such a role. In a recent study using platelets from spontaneously hypertensive rats, generation of phosphatidylcholine-derived DAG required for phosphatidylcholine may have been due to PC-PLC in rat platelets [lo]. It has been reported that DAG generated by the action of bacterial (C. welchii) phospholipase C on platelet plasma membrane phosphatidylcholine, resulted in the activation of protein kinase C, which phosphorylates a 47 kDa protein involved in platelet responses . This indicates that the generation of phosphatidylcholine derived DAG by phospholipase C can precede the onset of platelet activation and aggregation. A recent study, in support of this claim, has shown that stimulation of human platelets by HDL, results in generation of DAG from phosphatidylcholine, which activates protein kinase C [ll]. The present study confirms the presence of PCPLC activity in human platelets. Thus, the presence of PC-PLC in rat platelets, as well as, human and rabbit platelets may suggest a role for PC-PLC in platelet activation. Acknowledgements
This research was supported by grants from the Heart and Stroke Foundation of Canada. E.R. is a recipient of a traineeship award from the Heart and Stroke Foundation of Canada.
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