THROMBOSIS RESEARCH 59; 475-487,199O 0049-3848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
RECEPTOR-DEPENDENT AND -INDEPENDENT PROTEIN PHOSPHORYLATIONIN PLATELETS OF SPONTANEOUSLYHYPERTENSIVE RATS.
Sophie Koutouzov, Isabelle INSERM U’I/CNRS UA 318, Department 161 rue de Sevres,
(Received
1.2.1990;
accepted
Limon, and Pierre Marche of Pharmacology, Hospital 75015 Paris, France
in revised form 15.5.1990
Necker,
by Editor M.C. Boffa)
ABSTRACT This study investigates the role of protein kinase C and of myosin light chain kinase in mediating platelet hyperresponsiveness spontaneously hypertensive rats (SHR). For this purpose, 32P-labeltdn washed platelets of both SHR and normotensive controls Wistar-Kyoto were challenged either with a receptor-mediated agonist (WKY) (thrombin) or with direct activators of myosin light chain kinase and protein kinase C. Such enzymatic activities were assessed by measuring changes in 32P-labeling of their respective target proteins, namely myosin light chain (20 KDa) and the 47 KDa protein. In resting platelets, the patterns of protein phosphorylation were similar between SHR and WKY, suggesting that the two cell types were in a comparable quiescent status. By contrast, in both dose-response and time-course studies, thrombin promoted a significantly greater phosphorylation of the 20- and 47 KDa proteins in platelets of SHR compared with that for WKY. Sensitivity of myosin light chain kinase to the calcium ionophore A23187 and of protein kinase C to both phorbol ester and dioctanoylglycerol was apparently not different between The data indicate that the exaggerated the two cell types. thrombin-induced protein phosphorylation observed for platelets of SHR is not linked to alterations in protein kinase C and/or myosin light chain kinase per se. These results therefore suggest that platelet hyperresponsiveness in SHR is likely to be related, at to abnormalities in receptor-mediated transmembrane least in part, signalling. INTRODUCTION Blood platelets genetic hypertension when abnormalities the features of
with of patients with essential hypertension and of rats SHR) display multiple (spontaneously hypertensive rats, The major controls. compared with cells of normotensive rate of are an enhanced modified platelet profile
Key words: Hypertension, platelet, protein kinase C, phospholipase
protein C 475
phosphorylation,
PROTEIN PHOSPHORYLATION
476
adhesion/aggregation stimuli (l-4). atherosclerosis coronary artery
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and an exaggerated release reaction in response to many These phenomena seem to be associated with progressive and a tendancy to thrombosis, which are involved in obstructive disease (5,6).
As with many other cell types, platelets respond to Ca2+-mobilizing agonists (e.g. thrombin, collagen, thromboxane) with a rapid phospholipase Cmediated hydrolysis of membrane inositol-containing phospholipids (7,8). This reaction generates a transient accumulation of two intracellular messengers, namely diacylglycerol (DG) and inositol trisphosphate (IPa) (7,9). These products have specific functions: DG activates protein kinase C (PKC) and IPs mobilizes Ca2+ from internal stores (10) leading to the activation of Ca2+ /calmodulin-dependent kinases such as myosin light chain kinase (MLCK) (11,12). Stimulation of this signal transduction pathway evokes phosphorylation of specific intracellular platelet proteins, - in particular of myosin light chain (20 KDa) and of the 47 KDa protein -, by myosin light chain kinase and protein kinase C, respectively (13-15). Many studies indicate that these phosphorylation processes are closely related to platelet functional responses: MLCK-dependent phosphorylation of the 20 KDa myosin light chain may trigger shape change whereas PKC-induced phosphorylation of the 47 KDa protein may regulate aggregation and release reactions (16-19). Recently, studies on the metabolism of phosphoinositides in platelets of SHR suggested that enhanced thrombin-induced functional responses may be linked to a receptor-mediated hypersensitivity of phospholipase C (20,211. On the other hand, the recent finding of an increased protein kinase C activity in platelets from SHR (22) has led to the suggestion that this enzyme may be involved in hypertension. In order to assess the relative contributions of protein kinase C and of myosin light chain kinase in mediating enhanced functional responses in between the hypertensive and compared, platelets of SHR, we have analysed the patterns of protein strain and Wistar-Kyoto (WKY) normotensive controls, phosphorylation obtained either with a receptor-mediated agonist (thrombin) or For the latter, a phorbol ester with direct activators of the protein kinases. (or a diacylglycerol analogue) and a Ca2+ionophore, which by-pass receptor have been used to probe the PKC-dependent pathway and the Ca2+ activation, pathway of platelet activation, respectively (14,23,24).
MATERIALS AND METHODS Animals Male SHR of the Okamoto strain and normotensive WKY were obtained from C.E.R.J. (St Berthevin, France) and were studied at 12-16 weeks of age. measured by the tail-cuff method was 202 + 10 Systolic arterial blood pressure, (n=6; mean + SEMI. and 132 i: 7 mm Hg for SHR and WKY, respectively Preparation
of
P-labeled
platelets.
Blood was withdrawn from the abdominal aorta of rats under light ether anaesthesia into 10% (v/v) of ACD-C (170 mM trisodium citrate, 130 mM citric acid and 4% dextrose) as anticoagulant. Platelet-rich plasma (PRP) was obtained by centrifugation at 230 g for 15 min at room temperature and was further centrifuged at 120 g for 8 min to sediment contaminating red and white cells. Platelets in PRP were labeled at 10’ cells/ml with 3 pCi 32P-orthophosphate/10s
PROTEIN PHOSPHORYLATION
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477
cells for 2 hours at 37OC ilmCi/ml, Amersham, Buckinghamshire, UK). Thereafter, all subsequent steps were performed at room temperature. Labeled platelets were isolated by centrifugation at 400 g for 15 min and resuspended in a Ca*+-f ree Tyrode’s buffer (NaCl, 137 mM; KCl, 2.6 mM; NaHCOs, 12 mM; MgS04, 0.9 mM; glucose, 5.5 mM; gelatin, 0.25%; pH 6.5). Platelets were washed once in this buffer and were finally resuspended in a Ca*+-free Tyrode’s / HEPES buffer (NaCl, 137 mM; KCl, 2.6 mM; MgSO,, 0.9 mM; glucose, 5.5 mM; HEPES, 5 mM; gelatin, 0.25%; pH 7.4). Platelet suspensions from SHR and WKY were adjusted to equivalent concentrations using a Coulter Counter (3-5 lo8 cells /ml) and were maintained for 30-40 min at 37OC before experimentation. Measurement
a@
separation
of
P-labeled
proteins.
Samples of 32P-labeled platelets (0.4 ml) were placed in aggregometer tubes and preincubated while stirring (1000 rpml for 1 min at 37’C in the presence of 1.3 mM CaC12. Then thrombin, phorbol 12-myristate 13-acetate (PMA), ionophore dioctanoylglycerol or calcium A23187 (all dissolved in dimethylsulfoxide except for thrombin) were added at concentrations and for periods indicated in the specific experiments. Incubations were terminated by addition of 0.2 ml of SDS-stop buffer (20% glycerol, 10% 2-mercaptoethanol and 0.004% bromophenol blue) and immediately thereafter, samples were placed in a boiling water bath for 3 min. Samples (40 pl aliquots, 25-35 ug protein) were electrophoresed on SDS-polyacrylamide (11%) gels (26) and after staining, destaining and drying procedures, the gels were autoradiographed using MP films integrator-linked (Amersham, UK), The autoradiographs were scanned with an 32P-incorporation into densitometer (Ultroscan XL-2200, LKB, Sweden) to assess specific proteins. Statistical
analysis
described Individual animals were used in the experiments Data are expressed as the measurements were performed in duplicate. and tests of significance were performed using the unpaired Student’s
above and means f SEM t test.
RESULTS
were
In unstimulated platelets, comparable between cells
of
the patterns SHR and WKY.
of basal
protein
phosphorylation
TABLE 1 Levels
of Protein
Phosphorylation 32P-20
in Unstimulated
Platelets 32P-47
KDa
of SHR and WKY. KDa
SHR (n=19)
0.32
f 0.03
(1.5
+ 0.1)
0.48
f 0.04
(2.1
? 0.11
WKY (n=20)
0.31
+ 0.04
(1.4
* 0.1)
0.47
f 0.06
(2.2
f 0.11
?-labeled platelet proteins were separated on 11% polyacrylamide gels. Labeled Values represent the means + SEM polypeptides were located by autoradiography. the densitometric measurements of the autoradiographic bands and are given of Values in brackets represent the percentage of in arbitrary units (OD x area). P-20 KDa and -47 KDa proteins versus the radioactivity incorporated in all protein bands of the platelet sample.
PROTEIN PHOSPHORYLATION
478
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there was no significant difference between the two rat strains In particular, with respect to the radioactivity associated with the 20- and 47 KDa proteins of the total radioactivity which represented 1.5 and 2.1%, respectively, incorporated into proteins (Table 1 & Fig lA, lanes 1 & 7). When platelets were challenged with thrombin (0.1 U/ml), the two proteins of molecular weight 20- and 47 KDa were rapidly phosphorylated within the first 20-30 set, followed by a plateau thereafter (Fig 1A). A comparative study of the kinetics of protein phosphorylation in response to thrombin indicated that 32P-incorporation into the 20 KDa protein was from 10 set and up to 90 set, in platelets from SHR than from WKY (Fig lA,lB). significantly greater of SHR exhibited a significantly Likewise, platelets during this period, than extent in the radioactivity associated into the 47 KDa protein greater cells of WKY (Fig lA,lC) .
PZO
-
---II
WKY
TIME (seconds)
._I
SHR
TIME (secondsI
P-labeled Time-course for thrombin-induced protein phosphorylation. Fisure 1. platelets from SHR (01 and WKY (01, were stimulated with thrombin (0.1 U/ml) for the designated times. Platelet samples were electrophoresed and protein (a): a phosphorylation was quantified as described in Materials and Methods. typical autoradiogram: the Mr values (x 10s3) determined from coelectrophoresed standards are indicated by the arrows. results are presented as Lower panel: the means _+ SEM (n=6-8) and express the percentage of 32P in 20 KDa (b) and 47 KDa (cl proteins in stimulated samples related to that in unstimulated samples (arbitrary taken as 100%). Asterisks indicate significant differences between SHR and WKY. *, p ( 0.05; +, p ( 0.02; **, p ( 0.01; ***, p ( 0.001.
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PROTEIN PHOSPHORYLATION
479
The thrombin-induced phosphorylation of both 20- and 47 KDa proteins also occured in a dose-dependent and saturable manner (Fig 2) with maximal increases 32P-incorporation being 3 to 3.5 fold above control values at high doses of iie agonist (0.3 U/ml) . From such dose-profiles, it was evident that, even at very low doses of thrombin (0.025-0.05 U/ml) and up to 0.1-0.2 U/ml, platelets from SHR displayed higher levels of 32P-incorporation (-, 1.3 fold greater) into both the 20- (Fig 2A) and 47 KDa (Fig 2B) proteins than platelets from WKY. However, the difference between the two rat strains was no longer significant for doses of thrombin ranging between 0.2 and 0.3 U/ml.
T
0
0.05
0.1
0.P
THROMBIN(U/ml)
0.8
0
0.1
0.3
0.2
THROMBIN
B
W/ml)
Figure 2. Dose-response for thrombin-induced protein phosphorylation. Platelets from SHR (01 and WKY (01 were prelabeled with 32P and challenged with the doses of thrombin for 20 sec. Proteins were separated by indicated and phosphorylation was assesed.as described in Materials and electrophoresis Data are presented as the means f SEM (n=5-11, except for O.lU/ml Methods. and express the percentage of 32P in 20 KDa (A) and 47 KDa (B) where n=25) relative to that in unstimulated controls stimulated samples proteins in Asterisks indicate significant differences between (arbitrary taken as 100%). t, p ( 0.02; **, p ( 0.01; ***, p ( 0.001; N.S., not SHR and WKY. *, p ( 0.05; significant.
platelets were challenged with In a second series of experiments, activators of either protein kinase C or of myosin light chain kinase. In all the agents were dissolved in dimethylsulfoxide (DMSO) (0.2%, studies, concentration did not affect the level of concentration) ; such a solvent proteins in unstimulated controls. Activation of protein kinase C was first studied with a concentration sufficient to induce protein phosphorylation to phorbol (PMA, lo-*M)
direct these final 32Pof levels
400
PROTEIN PHOSPHORYLATION
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comparable to those observed with 0.1 U/ml thrombin (see above). Incorporation of 32P into the 47 KDa protein was time-dependent and after 2 min reached = 170% of control values (Fig 3A). Under such conditions, no significant increase in the extent of 32P-incorporation into the 20 KDa protein could be observed. This figure additionally shows that the time-dependent phosphorylation of the 47 KDa protein was comparable between SHR and WKY. Exposure of platelets to increasing concentrations of PMA (10-s - 5.10D7 M) for 20 set resulted in a dose-dependent, albeit differential, phosphorylation of both the 47- and 20 KDa proteins (Fig 3B). The maximum increase (4-5 fold above resting levels) of 32Pincorporation into the 47 KDa protein occured at concentration of PMA between 10’7 - 5.10-7M. The increase in the radioactivity associated to the 20 KDa protein was barely detectable at low concentrations of PMA but reached =: 150% values at maximum doses of this agonist. of, control These figures clearly demonstrate PMA-induced protein phosphorylation of each 20- and 47 KDa proteins to be similar for platelets of SHR and WKY.
*so -
I
0
10
20
30
00
TIME
(seconds)
120
I
10-a
1
I
I
10-7 [PMA(M)]
1
5.10-'
1
Figure 3. Effects of PMA on protein phosphorylation. Platelets from SHR (01 and WKY (01 were prelabeled with 32P and exposed to PMA (lo-sM) for the indicated times (Al or with the designated doses of PMA for 20 set (B). Data are given as the means ? SEM (n=3-51 and express the percentage phosphorylation of the 47 KDa protein (A) or of both 20- and 47 KDa proteins (B) in stimulated samples relative to that in 0.2% DMSO-containing unstimulated controls (arbitrary taken as 100%).
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PROTEIN PHOSPHORYLATION
481
Stimulation of protein kinase C by 100 pM of dioctanoylglycerol (a diacylglycerol analogue), evoked a rapid 32P-phosphorylation of the 47 KDa target protein, which peaked after 15-20 set at a level 4-5 fold above control values (Fig 4). Our data indicate, at all times studied, a comparable level of radioactivity into the 47 KDa protein, in both platelets of SHR and WKY.
TIME (seconds)
Ficrure 4. Kinetics of dioctanoylglycerol-induced protein phosphorylation. Platelets from SHR (0) and UKY (0) were prelabeled with 32P and challenged with dioctanoylglycerol (100 uM in DMSO, 0.2% final concentration) for the indicated times. Data are expressed as the means f SEM (n=3-5) and express the percentage phosphorylation of the 47 KDa protein in stimulated samples relative to that in DMSO-containing control samples (taken as 100%).
Activation of myosin light chain kinase through the exposure of 32Plabeled platelets to calcium ionophore A23187 (0.25-5 pM) for 30 set, promoted phosphorylation of the 20 KDa protein in a dose-dependent manner (Fig 5). The into the 20 KDa protein was obtained for 2 maximum extent of 32P- incorporation A23187 also evoked phosphorylation of the 47 M A23187 (160-170% of controls). KDa protein which was barely detectable at 0.25-0.5 uM and reached maximum levels (280% of control values) at 3 uM of the agent (not shown). Figure 5
PROTEIN PHOSPHORYLATION
482
indicates that and WKY, as it
the labeling was the case
of myosin light chain for the 47 KDa protein
I
0
1
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was not different (not shown).
I
I
I
I
2
3
4
5
A23187
between
SHR
I
(phi)
Figure 5. Dose-response for A23187-induced protein phosphorylation. 32 P-labeled platelets from SHR (01 and WKY (01 were exposed to various concentrations of A23187 (dissolved in 0.2% DMSO, final concentration) for 30 sec. Results are the means + SD (n=3) and express the percentage of 32P associated to the 20 KDa protein in stimulated samples relative to DMSO-containing unstimulated controls (taken as 100%).
DISCUSSION This study was designated to investigate the biochemical pathways involved in the thrombin-induced hyperresponsiveness displayed by platelets from the hypertensive strain SHR. In this respect, we have prepared and isolated 32Plabeled washed platelets under conditions previously described (21) in which enhanced thrombin-induced aggregation and release of serotonin were observed in the hypertensive-derived cells. Our results indicate that, the extent in 32P-labeling of myosin light chain (20 KDal and of the 47 KDa protein was found to be similar in platelets at rest from SHR and WKY, suggesting that before stimulation, the two cell types were in a comparable quiescent status. Our data also show, both in time-course and dose-response under stimulation of platelets with thrombin, phosphorylation chain (20 KDal and of the 47 KDa protein was markedly enhanced to WKY (Fig l-2).
studies, that of myosin light in SHR compared
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PROTEIN PHOSPHORYLATION
Our results confirm and extend those reported by Huzoor-Hakbar and Anwer since we provide in this work evidence for a leftward shift, at concentrations of thrombin, of the dose-response for protein phosphorylation stimulated platelets of SHR.
483
(20) low in
The enhanced thrombin-induced phosphorylation of the 20- and 41 KDa protein that we observe in the hypertensive-derived cells could possibly result from alterations in intrinsic activities of myosin light chain kinase and/or of protein kinase C. Nevertheless, our data indicate that A23187, which activate platelets from intracellular stores (14,18,24) through Ca2+ influx and Ca2+ mobilization evoked phosphorylation of myosin light chain (20 KDa) to a similar extent in platelets of SHR and WKY. Furthermore, the Ca2+-mediated phosphorylation of the 47 KDa protein (27) was also found to be similar between the two cell types. These results therefore suggest that sensitivity of both myosin light chain kinase and protein kinase C to Ca2+ ions seems to be unaltered in platelets of the hypertensive strain. intrinsic protein kinase C activity was probed On the other hand, by agents which by-pass the receptor-mediated activation of the enzyme and act in the capacity of diacylglycerol mimetics (23,281. Since many studies suggest that tumor-promoting phorbol diesters and DG analogues may not be fully equivalent activators of protein kinase C, partly owing to the rapid conversion of DG into the phosphatidic acid derivative and to the differential effects of both types of compounds upon intracellular Ca2+ mobilization (29-321, we have used both PMA and dioctanoylglycerol to assess and compare the intrinsic ability of PKC to be activated in platelets of SHR and WKY. Our data (Fig 3,4) indicate that regardless of the agonist used, activity of the endogenous protein kinase C was similar between platelets of both substrains. These are at variance with those of Takaori et al (22) who used an in vitro results assay for the enzymatic measurement of platelet protein kinase C activity in this activity was determined using SHR and WKY. However, high-speed supernatants, in which Ca2+- and phospholipid independent kinases, phosphatases and perhaps inhibitors may be present (33,34) in differing amounts in SHR and WKY. Our findings nevertheless do concur with recent studies which reported comparable protein kinase C activities in cultured smooth muscle cells from SHR and WKY (35,361. Taken together, our results strongly suggest that the exaggerated thrombin-induced phosphorylation of platelet proteins in SHR may be ascribed to an impaired receptor-mediated activation of both myosin light chain kinase and could reflect altered thrombin receptors protein kinase C. This abnormality However, the fact that enhanced cell functional properties in SHR platelets. responses have been observed with different stimuli such as thrombin, ADP, both in platelets of SHR and of hypertensive patients or adrenalin, collagen Distal to receptor (l-4,21,37,38) tends to rule out such an hypothesis. various an increased phospholipase C activity has been reported in occupancy, SHR including erythrocytes (391, platelets (20,211 arterial Wall tissues of the Indeed, cultured vascular smooth muscle cells and (41). (40) hypersensitivity of thrombin-mediated phospholipase C activity in platelets of amounts of the two second messengers would produce greater SHR, which account for the could likely inositol trisphosphate, diacylglycerol and that we report in this work. An agonist-induced phosphorylation increased alternative explanation might reside in alterations at the level of GTP-binding believed to couple receptors to phospholipase C (42). proteins, which are Although we have experimental evidence by immunoblotting and PertUSSiS-toxin mediated ADP-ribosylation experiments that the levels of the probed G Proteins unpublished of SHR and WKY (Koutouzov et al, were similar in platelets the involvement of pertussis-toxin insensitive G proteins in the results),
PROTEIN PHOSPHORYLATION
484
altered excluded
receptor-mediated (431.
transmembrane
signalling
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can nevertheless
not
be
In conclusion, this study provides evidence to suggest that enhanced thrombin-induced protein phosphorylation displayed by platelets of SHR is unlikely to directly involve aberrations in protein kinase C and/or myosin light chain kinase, per se. Alternatively, this feature, which is closely linked to the platelet hyperresponsiveness of SHR may reflect an abnormal receptor-mediated transmembrane signalling. Further studies are undoubtedly necessary to determine the molecular level at which this occurs in various cells of hypertensive subjects.
Acknowledgments: nx87037
This
work was supported
by a grant
from
Bayer-Pharma-INSERM
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signals.
Anna1 -*
RECEPTOR-DEPENDENT AND -INDEPENDENT PROTEIN PHOSPHORYLATIONIN PLATELETS OF SPONTANEOUSLYHYPERTENSIVE RATS.
Sophie Koutouzov, Isabelle INSERM U’I/CNRS UA 318, Department 161 rue de Sevres,
(Received
1.2.1990;
accepted
Limon, and Pierre Marche of Pharmacology, Hospital 75015 Paris, France
in revised form 15.5.1990
Necker,
by Editor M.C. Boffa)
ABSTRACT This study investigates the role of protein kinase C and of myosin light chain kinase in mediating platelet hyperresponsiveness spontaneously hypertensive rats (SHR). For this purpose, 32P-labeltdn washed platelets of both SHR and normotensive controls Wistar-Kyoto were challenged either with a receptor-mediated agonist (WKY) (thrombin) or with direct activators of myosin light chain kinase and protein kinase C. Such enzymatic activities were assessed by measuring changes in 32P-labeling of their respective target proteins, namely myosin light chain (20 KDa) and the 47 KDa protein. In resting platelets, the patterns of protein phosphorylation were similar between SHR and WKY, suggesting that the two cell types were in a comparable quiescent status. By contrast, in both dose-response and time-course studies, thrombin promoted a significantly greater phosphorylation of the 20- and 47 KDa proteins in platelets of SHR compared with that for WKY. Sensitivity of myosin light chain kinase to the calcium ionophore A23187 and of protein kinase C to both phorbol ester and dioctanoylglycerol was apparently not different between The data indicate that the exaggerated the two cell types. thrombin-induced protein phosphorylation observed for platelets of SHR is not linked to alterations in protein kinase C and/or myosin light chain kinase per se. These results therefore suggest that platelet hyperresponsiveness in SHR is likely to be related, at to abnormalities in receptor-mediated transmembrane least in part, signalling. INTRODUCTION Blood platelets genetic hypertension when abnormalities the features of
with of patients with essential hypertension and of rats SHR) display multiple (spontaneously hypertensive rats, The major controls. compared with cells of normotensive rate of are an enhanced modified platelet profile
Key words: Hypertension, platelet, protein kinase C, phospholipase
protein C 475
phosphorylation,
PROTEIN PHOSPHORYLATION
476
adhesion/aggregation stimuli (l-4). atherosclerosis coronary artery
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and an exaggerated release reaction in response to many These phenomena seem to be associated with progressive and a tendancy to thrombosis, which are involved in obstructive disease (5,6).
As with many other cell types, platelets respond to Ca2+-mobilizing agonists (e.g. thrombin, collagen, thromboxane) with a rapid phospholipase Cmediated hydrolysis of membrane inositol-containing phospholipids (7,8). This reaction generates a transient accumulation of two intracellular messengers, namely diacylglycerol (DG) and inositol trisphosphate (IPa) (7,9). These products have specific functions: DG activates protein kinase C (PKC) and IPs mobilizes Ca2+ from internal stores (10) leading to the activation of Ca2+ /calmodulin-dependent kinases such as myosin light chain kinase (MLCK) (11,12). Stimulation of this signal transduction pathway evokes phosphorylation of specific intracellular platelet proteins, - in particular of myosin light chain (20 KDa) and of the 47 KDa protein -, by myosin light chain kinase and protein kinase C, respectively (13-15). Many studies indicate that these phosphorylation processes are closely related to platelet functional responses: MLCK-dependent phosphorylation of the 20 KDa myosin light chain may trigger shape change whereas PKC-induced phosphorylation of the 47 KDa protein may regulate aggregation and release reactions (16-19). Recently, studies on the metabolism of phosphoinositides in platelets of SHR suggested that enhanced thrombin-induced functional responses may be linked to a receptor-mediated hypersensitivity of phospholipase C (20,211. On the other hand, the recent finding of an increased protein kinase C activity in platelets from SHR (22) has led to the suggestion that this enzyme may be involved in hypertension. In order to assess the relative contributions of protein kinase C and of myosin light chain kinase in mediating enhanced functional responses in between the hypertensive and compared, platelets of SHR, we have analysed the patterns of protein strain and Wistar-Kyoto (WKY) normotensive controls, phosphorylation obtained either with a receptor-mediated agonist (thrombin) or For the latter, a phorbol ester with direct activators of the protein kinases. (or a diacylglycerol analogue) and a Ca2+ionophore, which by-pass receptor have been used to probe the PKC-dependent pathway and the Ca2+ activation, pathway of platelet activation, respectively (14,23,24).
MATERIALS AND METHODS Animals Male SHR of the Okamoto strain and normotensive WKY were obtained from C.E.R.J. (St Berthevin, France) and were studied at 12-16 weeks of age. measured by the tail-cuff method was 202 + 10 Systolic arterial blood pressure, (n=6; mean + SEMI. and 132 i: 7 mm Hg for SHR and WKY, respectively Preparation
of
P-labeled
platelets.
Blood was withdrawn from the abdominal aorta of rats under light ether anaesthesia into 10% (v/v) of ACD-C (170 mM trisodium citrate, 130 mM citric acid and 4% dextrose) as anticoagulant. Platelet-rich plasma (PRP) was obtained by centrifugation at 230 g for 15 min at room temperature and was further centrifuged at 120 g for 8 min to sediment contaminating red and white cells. Platelets in PRP were labeled at 10’ cells/ml with 3 pCi 32P-orthophosphate/10s
PROTEIN PHOSPHORYLATION
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477
cells for 2 hours at 37OC ilmCi/ml, Amersham, Buckinghamshire, UK). Thereafter, all subsequent steps were performed at room temperature. Labeled platelets were isolated by centrifugation at 400 g for 15 min and resuspended in a Ca*+-f ree Tyrode’s buffer (NaCl, 137 mM; KCl, 2.6 mM; NaHCOs, 12 mM; MgS04, 0.9 mM; glucose, 5.5 mM; gelatin, 0.25%; pH 6.5). Platelets were washed once in this buffer and were finally resuspended in a Ca*+-free Tyrode’s / HEPES buffer (NaCl, 137 mM; KCl, 2.6 mM; MgSO,, 0.9 mM; glucose, 5.5 mM; HEPES, 5 mM; gelatin, 0.25%; pH 7.4). Platelet suspensions from SHR and WKY were adjusted to equivalent concentrations using a Coulter Counter (3-5 lo8 cells /ml) and were maintained for 30-40 min at 37OC before experimentation. Measurement
a@
separation
of
P-labeled
proteins.
Samples of 32P-labeled platelets (0.4 ml) were placed in aggregometer tubes and preincubated while stirring (1000 rpml for 1 min at 37’C in the presence of 1.3 mM CaC12. Then thrombin, phorbol 12-myristate 13-acetate (PMA), ionophore dioctanoylglycerol or calcium A23187 (all dissolved in dimethylsulfoxide except for thrombin) were added at concentrations and for periods indicated in the specific experiments. Incubations were terminated by addition of 0.2 ml of SDS-stop buffer (20% glycerol, 10% 2-mercaptoethanol and 0.004% bromophenol blue) and immediately thereafter, samples were placed in a boiling water bath for 3 min. Samples (40 pl aliquots, 25-35 ug protein) were electrophoresed on SDS-polyacrylamide (11%) gels (26) and after staining, destaining and drying procedures, the gels were autoradiographed using MP films integrator-linked (Amersham, UK), The autoradiographs were scanned with an 32P-incorporation into densitometer (Ultroscan XL-2200, LKB, Sweden) to assess specific proteins. Statistical
analysis
described Individual animals were used in the experiments Data are expressed as the measurements were performed in duplicate. and tests of significance were performed using the unpaired Student’s
above and means f SEM t test.
RESULTS
were
In unstimulated platelets, comparable between cells
of
the patterns SHR and WKY.
of basal
protein
phosphorylation
TABLE 1 Levels
of Protein
Phosphorylation 32P-20
in Unstimulated
Platelets 32P-47
KDa
of SHR and WKY. KDa
SHR (n=19)
0.32
f 0.03
(1.5
+ 0.1)
0.48
f 0.04
(2.1
? 0.11
WKY (n=20)
0.31
+ 0.04
(1.4
* 0.1)
0.47
f 0.06
(2.2
f 0.11
?-labeled platelet proteins were separated on 11% polyacrylamide gels. Labeled Values represent the means + SEM polypeptides were located by autoradiography. the densitometric measurements of the autoradiographic bands and are given of Values in brackets represent the percentage of in arbitrary units (OD x area). P-20 KDa and -47 KDa proteins versus the radioactivity incorporated in all protein bands of the platelet sample.
PROTEIN PHOSPHORYLATION
478
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there was no significant difference between the two rat strains In particular, with respect to the radioactivity associated with the 20- and 47 KDa proteins of the total radioactivity which represented 1.5 and 2.1%, respectively, incorporated into proteins (Table 1 & Fig lA, lanes 1 & 7). When platelets were challenged with thrombin (0.1 U/ml), the two proteins of molecular weight 20- and 47 KDa were rapidly phosphorylated within the first 20-30 set, followed by a plateau thereafter (Fig 1A). A comparative study of the kinetics of protein phosphorylation in response to thrombin indicated that 32P-incorporation into the 20 KDa protein was from 10 set and up to 90 set, in platelets from SHR than from WKY (Fig lA,lB). significantly greater of SHR exhibited a significantly Likewise, platelets during this period, than extent in the radioactivity associated into the 47 KDa protein greater cells of WKY (Fig lA,lC) .
PZO
-
---II
WKY
TIME (seconds)
._I
SHR
TIME (secondsI
P-labeled Time-course for thrombin-induced protein phosphorylation. Fisure 1. platelets from SHR (01 and WKY (01, were stimulated with thrombin (0.1 U/ml) for the designated times. Platelet samples were electrophoresed and protein (a): a phosphorylation was quantified as described in Materials and Methods. typical autoradiogram: the Mr values (x 10s3) determined from coelectrophoresed standards are indicated by the arrows. results are presented as Lower panel: the means _+ SEM (n=6-8) and express the percentage of 32P in 20 KDa (b) and 47 KDa (cl proteins in stimulated samples related to that in unstimulated samples (arbitrary taken as 100%). Asterisks indicate significant differences between SHR and WKY. *, p ( 0.05; +, p ( 0.02; **, p ( 0.01; ***, p ( 0.001.
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PROTEIN PHOSPHORYLATION
479
The thrombin-induced phosphorylation of both 20- and 47 KDa proteins also occured in a dose-dependent and saturable manner (Fig 2) with maximal increases 32P-incorporation being 3 to 3.5 fold above control values at high doses of iie agonist (0.3 U/ml) . From such dose-profiles, it was evident that, even at very low doses of thrombin (0.025-0.05 U/ml) and up to 0.1-0.2 U/ml, platelets from SHR displayed higher levels of 32P-incorporation (-, 1.3 fold greater) into both the 20- (Fig 2A) and 47 KDa (Fig 2B) proteins than platelets from WKY. However, the difference between the two rat strains was no longer significant for doses of thrombin ranging between 0.2 and 0.3 U/ml.
T
0
0.05
0.1
0.P
THROMBIN(U/ml)
0.8
0
0.1
0.3
0.2
THROMBIN
B
W/ml)
Figure 2. Dose-response for thrombin-induced protein phosphorylation. Platelets from SHR (01 and WKY (01 were prelabeled with 32P and challenged with the doses of thrombin for 20 sec. Proteins were separated by indicated and phosphorylation was assesed.as described in Materials and electrophoresis Data are presented as the means f SEM (n=5-11, except for O.lU/ml Methods. and express the percentage of 32P in 20 KDa (A) and 47 KDa (B) where n=25) relative to that in unstimulated controls stimulated samples proteins in Asterisks indicate significant differences between (arbitrary taken as 100%). t, p ( 0.02; **, p ( 0.01; ***, p ( 0.001; N.S., not SHR and WKY. *, p ( 0.05; significant.
platelets were challenged with In a second series of experiments, activators of either protein kinase C or of myosin light chain kinase. In all the agents were dissolved in dimethylsulfoxide (DMSO) (0.2%, studies, concentration did not affect the level of concentration) ; such a solvent proteins in unstimulated controls. Activation of protein kinase C was first studied with a concentration sufficient to induce protein phosphorylation to phorbol (PMA, lo-*M)
direct these final 32Pof levels
400
PROTEIN PHOSPHORYLATION
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comparable to those observed with 0.1 U/ml thrombin (see above). Incorporation of 32P into the 47 KDa protein was time-dependent and after 2 min reached = 170% of control values (Fig 3A). Under such conditions, no significant increase in the extent of 32P-incorporation into the 20 KDa protein could be observed. This figure additionally shows that the time-dependent phosphorylation of the 47 KDa protein was comparable between SHR and WKY. Exposure of platelets to increasing concentrations of PMA (10-s - 5.10D7 M) for 20 set resulted in a dose-dependent, albeit differential, phosphorylation of both the 47- and 20 KDa proteins (Fig 3B). The maximum increase (4-5 fold above resting levels) of 32Pincorporation into the 47 KDa protein occured at concentration of PMA between 10’7 - 5.10-7M. The increase in the radioactivity associated to the 20 KDa protein was barely detectable at low concentrations of PMA but reached =: 150% values at maximum doses of this agonist. of, control These figures clearly demonstrate PMA-induced protein phosphorylation of each 20- and 47 KDa proteins to be similar for platelets of SHR and WKY.
*so -
I
0
10
20
30
00
TIME
(seconds)
120
I
10-a
1
I
I
10-7 [PMA(M)]
1
5.10-'
1
Figure 3. Effects of PMA on protein phosphorylation. Platelets from SHR (01 and WKY (01 were prelabeled with 32P and exposed to PMA (lo-sM) for the indicated times (Al or with the designated doses of PMA for 20 set (B). Data are given as the means ? SEM (n=3-51 and express the percentage phosphorylation of the 47 KDa protein (A) or of both 20- and 47 KDa proteins (B) in stimulated samples relative to that in 0.2% DMSO-containing unstimulated controls (arbitrary taken as 100%).
Vol59, No. 3
PROTEIN PHOSPHORYLATION
481
Stimulation of protein kinase C by 100 pM of dioctanoylglycerol (a diacylglycerol analogue), evoked a rapid 32P-phosphorylation of the 47 KDa target protein, which peaked after 15-20 set at a level 4-5 fold above control values (Fig 4). Our data indicate, at all times studied, a comparable level of radioactivity into the 47 KDa protein, in both platelets of SHR and WKY.
TIME (seconds)
Ficrure 4. Kinetics of dioctanoylglycerol-induced protein phosphorylation. Platelets from SHR (0) and UKY (0) were prelabeled with 32P and challenged with dioctanoylglycerol (100 uM in DMSO, 0.2% final concentration) for the indicated times. Data are expressed as the means f SEM (n=3-5) and express the percentage phosphorylation of the 47 KDa protein in stimulated samples relative to that in DMSO-containing control samples (taken as 100%).
Activation of myosin light chain kinase through the exposure of 32Plabeled platelets to calcium ionophore A23187 (0.25-5 pM) for 30 set, promoted phosphorylation of the 20 KDa protein in a dose-dependent manner (Fig 5). The into the 20 KDa protein was obtained for 2 maximum extent of 32P- incorporation A23187 also evoked phosphorylation of the 47 M A23187 (160-170% of controls). KDa protein which was barely detectable at 0.25-0.5 uM and reached maximum levels (280% of control values) at 3 uM of the agent (not shown). Figure 5
PROTEIN PHOSPHORYLATION
482
indicates that and WKY, as it
the labeling was the case
of myosin light chain for the 47 KDa protein
I
0
1
Vol59, No. 3
was not different (not shown).
I
I
I
I
2
3
4
5
A23187
between
SHR
I
(phi)
Figure 5. Dose-response for A23187-induced protein phosphorylation. 32 P-labeled platelets from SHR (01 and WKY (01 were exposed to various concentrations of A23187 (dissolved in 0.2% DMSO, final concentration) for 30 sec. Results are the means + SD (n=3) and express the percentage of 32P associated to the 20 KDa protein in stimulated samples relative to DMSO-containing unstimulated controls (taken as 100%).
DISCUSSION This study was designated to investigate the biochemical pathways involved in the thrombin-induced hyperresponsiveness displayed by platelets from the hypertensive strain SHR. In this respect, we have prepared and isolated 32Plabeled washed platelets under conditions previously described (21) in which enhanced thrombin-induced aggregation and release of serotonin were observed in the hypertensive-derived cells. Our results indicate that, the extent in 32P-labeling of myosin light chain (20 KDal and of the 47 KDa protein was found to be similar in platelets at rest from SHR and WKY, suggesting that before stimulation, the two cell types were in a comparable quiescent status. Our data also show, both in time-course and dose-response under stimulation of platelets with thrombin, phosphorylation chain (20 KDal and of the 47 KDa protein was markedly enhanced to WKY (Fig l-2).
studies, that of myosin light in SHR compared
Vol59, No. 3
PROTEIN PHOSPHORYLATION
Our results confirm and extend those reported by Huzoor-Hakbar and Anwer since we provide in this work evidence for a leftward shift, at concentrations of thrombin, of the dose-response for protein phosphorylation stimulated platelets of SHR.
483
(20) low in
The enhanced thrombin-induced phosphorylation of the 20- and 41 KDa protein that we observe in the hypertensive-derived cells could possibly result from alterations in intrinsic activities of myosin light chain kinase and/or of protein kinase C. Nevertheless, our data indicate that A23187, which activate platelets from intracellular stores (14,18,24) through Ca2+ influx and Ca2+ mobilization evoked phosphorylation of myosin light chain (20 KDa) to a similar extent in platelets of SHR and WKY. Furthermore, the Ca2+-mediated phosphorylation of the 47 KDa protein (27) was also found to be similar between the two cell types. These results therefore suggest that sensitivity of both myosin light chain kinase and protein kinase C to Ca2+ ions seems to be unaltered in platelets of the hypertensive strain. intrinsic protein kinase C activity was probed On the other hand, by agents which by-pass the receptor-mediated activation of the enzyme and act in the capacity of diacylglycerol mimetics (23,281. Since many studies suggest that tumor-promoting phorbol diesters and DG analogues may not be fully equivalent activators of protein kinase C, partly owing to the rapid conversion of DG into the phosphatidic acid derivative and to the differential effects of both types of compounds upon intracellular Ca2+ mobilization (29-321, we have used both PMA and dioctanoylglycerol to assess and compare the intrinsic ability of PKC to be activated in platelets of SHR and WKY. Our data (Fig 3,4) indicate that regardless of the agonist used, activity of the endogenous protein kinase C was similar between platelets of both substrains. These are at variance with those of Takaori et al (22) who used an in vitro results assay for the enzymatic measurement of platelet protein kinase C activity in this activity was determined using SHR and WKY. However, high-speed supernatants, in which Ca2+- and phospholipid independent kinases, phosphatases and perhaps inhibitors may be present (33,34) in differing amounts in SHR and WKY. Our findings nevertheless do concur with recent studies which reported comparable protein kinase C activities in cultured smooth muscle cells from SHR and WKY (35,361. Taken together, our results strongly suggest that the exaggerated thrombin-induced phosphorylation of platelet proteins in SHR may be ascribed to an impaired receptor-mediated activation of both myosin light chain kinase and could reflect altered thrombin receptors protein kinase C. This abnormality However, the fact that enhanced cell functional properties in SHR platelets. responses have been observed with different stimuli such as thrombin, ADP, both in platelets of SHR and of hypertensive patients or adrenalin, collagen Distal to receptor (l-4,21,37,38) tends to rule out such an hypothesis. various an increased phospholipase C activity has been reported in occupancy, SHR including erythrocytes (391, platelets (20,211 arterial Wall tissues of the Indeed, cultured vascular smooth muscle cells and (41). (40) hypersensitivity of thrombin-mediated phospholipase C activity in platelets of amounts of the two second messengers would produce greater SHR, which account for the could likely inositol trisphosphate, diacylglycerol and that we report in this work. An agonist-induced phosphorylation increased alternative explanation might reside in alterations at the level of GTP-binding believed to couple receptors to phospholipase C (42). proteins, which are Although we have experimental evidence by immunoblotting and PertUSSiS-toxin mediated ADP-ribosylation experiments that the levels of the probed G Proteins unpublished of SHR and WKY (Koutouzov et al, were similar in platelets the involvement of pertussis-toxin insensitive G proteins in the results),
PROTEIN PHOSPHORYLATION
484
altered excluded
receptor-mediated (431.
transmembrane
signalling
Vol59, No. 3
can nevertheless
not
be
In conclusion, this study provides evidence to suggest that enhanced thrombin-induced protein phosphorylation displayed by platelets of SHR is unlikely to directly involve aberrations in protein kinase C and/or myosin light chain kinase, per se. Alternatively, this feature, which is closely linked to the platelet hyperresponsiveness of SHR may reflect an abnormal receptor-mediated transmembrane signalling. Further studies are undoubtedly necessary to determine the molecular level at which this occurs in various cells of hypertensive subjects.
Acknowledgments: nx87037
This
work was supported
by a grant
from
Bayer-Pharma-INSERM
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