Pergamon Press
Life Sciences, Vol. 47, pp. 1419-1425 Printed in the U.S.A.
PROTEIN KINASE C TRANSLOCATION
IN HUMAN
BLOOD PLATELETS
Hoau-Yan Wang and Eitan Friedman Departments of Psychiatry and Pharmacology Medical College of Pennsylvania 3200 Henry Avenue Philadelphia, PA 19129 (Received in final form August 10, 1990)
Summarv Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 PM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 ,vM) and the specific 5-HT, receptor agonist, l-(2,5-dimethoxy-4-iodophenyI)-2-aminopropane (DOI) (IO-100 PM) but not the 5-HT,, or 5-HT,, agonists, ( f I (8-OH-DPAT) or 5-methoxy-3-3B-hydroxy-dipropylamino-tetralin (1,2,3,6-tetrahydro-4-pyridin) 1 H-indole succinate (RU 249691 induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT, receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT, receptors. Protein phosphorylation is an important step in the transduction of extracellular signals into meaningful cellular responses. Protein kinase C, a phosphorylating enzyme which is distributed in a wide variety of cells is found in high amounts in the blood platelet (1). This enzyme can be activated either by diacylglycerol (DAG) which is formed by receptor-mediated phospholipid hydrolysis (2) or by the DAG analogues, phorbol esters (3). In response to DAG or to phorbol esters, the enzyme is redistributed from the cytosolic to membrane fractions of the cell (4,5). Platelets are known to respond to various agents such as 5-HT or thrombin as part of their physiological role in clotting. In response to these and other external stimuli, phosphoinositide turnover is rapidly increased and DAG as well as IP, are formed (6,7). In platelets the synthetic DAG, diolein, increases the phosphorylation of a 40 KDa protein which is associated with the release of stored platelet 5-HT (8), suggesting a role for PKC in mediating biological responses in platelets. In the present communication, we report (1) that platelet PKC translocation occurs in response to direct stimulation of PKC and indirectly probably via cell surface receptors for thrombin or 5-HT and (2) that the serotonin-activated platelet PKC translocation is mediated by a 5-HT, receptor subtype. 0024-3205/90 $3.00 + .oo Copyright (c) 1990 Pergamon Press plc
1420
Platelet PKC Translocation
Vol. 47, No. 16, 1990
Methods Venous blood (30 ml) was collected from consenting healthy volunteers in EDTAcontaining Monovettes (Sarstedt, Princeton, N.J.). Blood was centrifuged at 120 X g for 10 min at 4°C. The supernatant was centrifuged at 2500 X g for 10 min at 4°C and the platelet pellet was suspended in 4 ml of oxygenated 0.32 M sucrose/5 mM HEPES buffer (pH 7.5). Aliquots (0.8 ml) taken from the platelet suspension were incubated with various agents in a total volume of 1 ml for 10 minutes at 37°C. Following incubation, reaction was stopped by adding 1 ml of 0.32 M sucrose buffered with 20 mM Tris HCI (pH 7.5) and containing 2 mM EDTA, 1 .O mM EGTA, 50 fig/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride and 0.1% 2mercaptoethanol. The reaction mixture was sonicated for 1 min and centrifuged at 25,000 x g for 15 min. The supernatant (2 ml) was applied onto washed diethylaminomethyl cellulose anion exchange columns (Whatman, DE52) and eluted from the column as previously described (9) prior to measuring PKC activity (cytosol). The pellet was sonicated in 200 ~1 of the above buffer containing 1% Nonidet P-40 (Sigma), allowed to stand on ice for 1 hr and diluted with 1.8 ml of buffer. The tube was centrifuged at 25,000 x g for 15 minutes and the supernatant applied onto DE52 columns and PKC activity determined in column eluates (membrane-bound). Columns were washed with 4 ml of buffer followed by 0.5 ml of buffer/O.1 M NaCl and enzyme was eluted with 1.5 ml of buffer/O.1 M NaCl and used immediately for PKC activity determination. The final assay mixture (volume of 250~1) consisted of: 5 mM NaCI, 0.025 mM EGTA, 0.2 mM EDTA, 24 mM Tris HCI, 0.015% 2-mercaptoethanol, 30pg/ml leupeptin, 0.01 mM phenylmethylsulfonyl fluoride, 0.25 mg/ml histone type III (lysine-rich, Sigma), 1.2 mM CaCI,, 2Oflg phosphotidylserine, 8.1 nM PMA, 10 mM magnesium acetate and 30,~M i3’Pl y-ATP (50-75 cpm/pmole, New England Nuclear). Assay mixtures were preincubated at 30°C. The protein phosphorylation reaction was initiated by addition of 25 /II of column eluates and incubated for 1 minute at 30°C. Reactions were terminated by transferring 25 /II aliquots of the incubate onto a 1 x 2 cm phosphocellulose strip (Whatman ~81) and immersed in 75 mM phosphoric acid (10 ml per sample). Following washing and drying in air, the radioactivity in the strips was determined by liquid scintillation counting (LKB 1214 RACKBETA). Protein was determined by the method of Lowry et al. (10). Statistical significance was evaluated by the two-tailed Student’s t test or by ANOVA followed by Newman-Keul’s Multiple comparison.
Results Effects of 5-HT. PMA and Thrombin in Inducing Protein Kinase C Translocation. Protein kinase C activity and its translocation in response to 5-HT, PMA and thrombin were assessed in human platelets. The results summarized in Table I and Figure 1 indicate that (a) the distribution of PKC between cytosolic and particulate fractions was about 60:40, (b) PMA and thrombin elicited a marked redistribution of the enzyme from cytosol to membrane, and (cl 5-HT induced PKC translocation in a dose-dependent fashion. The catecholamines, norepinephrine and epinephrine at 10 PM concentrations did not elicit redistribution of platelet PKC (Figure 2).
vol. 47, No. 16, 1990
Platelet PKC Translocation
1421
TABLE I Effect of PMA and Thrombin on PKC Translocation
CYTOSOL
MEMBRANE-BOUND
PKC Activity (pmollminlyg protein) CONTROL THROMBIN (0.5 U/ml), 1 min PMA (50 nM1, IO min
2.41 f 0.10 1.74 f 0.06’ 1.54 zk 0.05b
1.81 f 0.07 2.47 f 0.05’ 2.69 f 0.05b
Human blood platelets were incubated for 10 min with HEPES buffer (control), 9 min buffer/l min with thrombin or 10 min with PMA. The tissues were collected, sonicated and cytosolic and membrane-bound PKC activities were determined following partial purification on DE52 columns. Each value represents the mean f SEM of 20 individual experiments performed in duplicate. “p < 0.01; bpCO.005
compared with respective control group.
0 CONTROL EZJ 5-HT 3.00
2.50
2.00
1.50
1.00
0.50
0.00
0.5
1.0 kHT1
10
100
(@f)
FIG 1 Human blood platelets were incubated for 10 minutes with various concentrations of 5-HT. Cytosolic and membrane-bound PKC activities were measured. Each bar indicates the mean f SEM of 5-20 determinations. Significant 5-HT dose-dependent effect was obtained (p CO.01, one-factor ANOVA).
1422
Vol. 47, No. 16, 1990
Platelet PKC Translocation
Characterization of the 5HT Receptor Subtype Responsible for 5-HT-Induced Protein Kinase C Translocation. Experiments aimed at characterizing the 5HT receptor subtype which is responsible for activation of platelet PKC translocation employed various receptor agonists and antagonists (Table II, Figures 2 and 3). While the selective 5-HT, agonist, DOI induced a concentration-dependent redistribution of the enzyme (Figure 21, the specific 5-HT,, agonist, 8-OH-DPAT and the 5HT,, agonist, RU24969 were inactive (Figure 2). Platelet PKC translocation elicited by 5-HT was inhibited by increasing doses of the selective 5-HT, antagonist, ketanserin (Table II). The effect of 5-HT was also inhibited by 10 PM spiroperidol and by the 5-HT, and 5-HT,, antagonist, mianserin (1OpM) but not by the 1OpM of 5-HT, antagonist, 3-tropanyl-indole-3-carboxylate (ICS205930) (Figure 3). In addition, neither the a,-adrenergic antagonist, prazosin nor the D, antagonist, I-sulpiride were found to block 5-HT-induced PKC redistribution. These results suggest that 5-HT-induced platelet PKC translocation is mediated through 5-HT,-type 5-HT serotonin receptors.
Effect of Ketanserin on 5-HT-Induced
PKC Translocation in Platelets
CYTOSOL
MFMBRANE-BCUNQ
PKC Activity (pmol/mln/~g protein)
CONTROL KETANSERIN, 10 PM 5-HT, 10 yM 5-HT + Ketanserin, 0.1 PM 5-HT + Ketanserin, 1 .O PM 5-HT + Ketanserin, 10 PM
2.89 2.85 2.15 2.49 2.76 2.87
f f f f f f
0.04 0.02 0.04’ 0.05b 0.04” 0.10”
2.17 2.26 2.97 2.76 2.39 2.15
f f f f f f
0.04 0.03 0.05’ 0.06b 0.03” 0.03”
Human blood platelets were incubated with various concentrations of ketanserin for 10 minutes. Tissues were subsequently exposed to 5-HT (10 Platelet PKC activity in cytosolic and membranous NM) for 10 minutes. fractions were measured. Values indicate the mean f SEM of 4 individual experiments. Significance (p < 0.01) by one factor ANOVA was found for the ketanserin doses. *p < 0.005 for 5-HT response vs. control by two-tailed Student’s ‘t’ test bp < 0.05; “p < 0.01 Comparison of 5-HT + Ketanserin vs. 5-HT by NewmanKeuls multiple comparison
Vol. 47, No. 16, 1990
Platelet PKC Tranelocation
0 a m LXI 6XY BE8 m
CONTROL 5-HT 6-OH-DPAT RU24969 DOI NOREPINEPHRINE EPINEPHRINE
**
\
4 i
1.oo
I g
0.50
d ;
0.00
10
I
10
10
10 100
10
10
[AGONIST] (phi)
FIG. 2 Human blood platelets were exposed (IO min) to various agents. Cytosolic and membrane-bound PKC activities were determined. Each bar represents the mean f SEM of 8 individual experiments. Statistical differences were evaluated using the two-tailed Student’s ‘t’ test. “p
Life Sciences, Vol. 47, pp. 1419-1425 Printed in the U.S.A.
PROTEIN KINASE C TRANSLOCATION
IN HUMAN
BLOOD PLATELETS
Hoau-Yan Wang and Eitan Friedman Departments of Psychiatry and Pharmacology Medical College of Pennsylvania 3200 Henry Avenue Philadelphia, PA 19129 (Received in final form August 10, 1990)
Summarv Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 PM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 ,vM) and the specific 5-HT, receptor agonist, l-(2,5-dimethoxy-4-iodophenyI)-2-aminopropane (DOI) (IO-100 PM) but not the 5-HT,, or 5-HT,, agonists, ( f I (8-OH-DPAT) or 5-methoxy-3-3B-hydroxy-dipropylamino-tetralin (1,2,3,6-tetrahydro-4-pyridin) 1 H-indole succinate (RU 249691 induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT, receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT, receptors. Protein phosphorylation is an important step in the transduction of extracellular signals into meaningful cellular responses. Protein kinase C, a phosphorylating enzyme which is distributed in a wide variety of cells is found in high amounts in the blood platelet (1). This enzyme can be activated either by diacylglycerol (DAG) which is formed by receptor-mediated phospholipid hydrolysis (2) or by the DAG analogues, phorbol esters (3). In response to DAG or to phorbol esters, the enzyme is redistributed from the cytosolic to membrane fractions of the cell (4,5). Platelets are known to respond to various agents such as 5-HT or thrombin as part of their physiological role in clotting. In response to these and other external stimuli, phosphoinositide turnover is rapidly increased and DAG as well as IP, are formed (6,7). In platelets the synthetic DAG, diolein, increases the phosphorylation of a 40 KDa protein which is associated with the release of stored platelet 5-HT (8), suggesting a role for PKC in mediating biological responses in platelets. In the present communication, we report (1) that platelet PKC translocation occurs in response to direct stimulation of PKC and indirectly probably via cell surface receptors for thrombin or 5-HT and (2) that the serotonin-activated platelet PKC translocation is mediated by a 5-HT, receptor subtype. 0024-3205/90 $3.00 + .oo Copyright (c) 1990 Pergamon Press plc
1420
Platelet PKC Translocation
Vol. 47, No. 16, 1990
Methods Venous blood (30 ml) was collected from consenting healthy volunteers in EDTAcontaining Monovettes (Sarstedt, Princeton, N.J.). Blood was centrifuged at 120 X g for 10 min at 4°C. The supernatant was centrifuged at 2500 X g for 10 min at 4°C and the platelet pellet was suspended in 4 ml of oxygenated 0.32 M sucrose/5 mM HEPES buffer (pH 7.5). Aliquots (0.8 ml) taken from the platelet suspension were incubated with various agents in a total volume of 1 ml for 10 minutes at 37°C. Following incubation, reaction was stopped by adding 1 ml of 0.32 M sucrose buffered with 20 mM Tris HCI (pH 7.5) and containing 2 mM EDTA, 1 .O mM EGTA, 50 fig/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride and 0.1% 2mercaptoethanol. The reaction mixture was sonicated for 1 min and centrifuged at 25,000 x g for 15 min. The supernatant (2 ml) was applied onto washed diethylaminomethyl cellulose anion exchange columns (Whatman, DE52) and eluted from the column as previously described (9) prior to measuring PKC activity (cytosol). The pellet was sonicated in 200 ~1 of the above buffer containing 1% Nonidet P-40 (Sigma), allowed to stand on ice for 1 hr and diluted with 1.8 ml of buffer. The tube was centrifuged at 25,000 x g for 15 minutes and the supernatant applied onto DE52 columns and PKC activity determined in column eluates (membrane-bound). Columns were washed with 4 ml of buffer followed by 0.5 ml of buffer/O.1 M NaCl and enzyme was eluted with 1.5 ml of buffer/O.1 M NaCl and used immediately for PKC activity determination. The final assay mixture (volume of 250~1) consisted of: 5 mM NaCI, 0.025 mM EGTA, 0.2 mM EDTA, 24 mM Tris HCI, 0.015% 2-mercaptoethanol, 30pg/ml leupeptin, 0.01 mM phenylmethylsulfonyl fluoride, 0.25 mg/ml histone type III (lysine-rich, Sigma), 1.2 mM CaCI,, 2Oflg phosphotidylserine, 8.1 nM PMA, 10 mM magnesium acetate and 30,~M i3’Pl y-ATP (50-75 cpm/pmole, New England Nuclear). Assay mixtures were preincubated at 30°C. The protein phosphorylation reaction was initiated by addition of 25 /II of column eluates and incubated for 1 minute at 30°C. Reactions were terminated by transferring 25 /II aliquots of the incubate onto a 1 x 2 cm phosphocellulose strip (Whatman ~81) and immersed in 75 mM phosphoric acid (10 ml per sample). Following washing and drying in air, the radioactivity in the strips was determined by liquid scintillation counting (LKB 1214 RACKBETA). Protein was determined by the method of Lowry et al. (10). Statistical significance was evaluated by the two-tailed Student’s t test or by ANOVA followed by Newman-Keul’s Multiple comparison.
Results Effects of 5-HT. PMA and Thrombin in Inducing Protein Kinase C Translocation. Protein kinase C activity and its translocation in response to 5-HT, PMA and thrombin were assessed in human platelets. The results summarized in Table I and Figure 1 indicate that (a) the distribution of PKC between cytosolic and particulate fractions was about 60:40, (b) PMA and thrombin elicited a marked redistribution of the enzyme from cytosol to membrane, and (cl 5-HT induced PKC translocation in a dose-dependent fashion. The catecholamines, norepinephrine and epinephrine at 10 PM concentrations did not elicit redistribution of platelet PKC (Figure 2).
vol. 47, No. 16, 1990
Platelet PKC Translocation
1421
TABLE I Effect of PMA and Thrombin on PKC Translocation
CYTOSOL
MEMBRANE-BOUND
PKC Activity (pmollminlyg protein) CONTROL THROMBIN (0.5 U/ml), 1 min PMA (50 nM1, IO min
2.41 f 0.10 1.74 f 0.06’ 1.54 zk 0.05b
1.81 f 0.07 2.47 f 0.05’ 2.69 f 0.05b
Human blood platelets were incubated for 10 min with HEPES buffer (control), 9 min buffer/l min with thrombin or 10 min with PMA. The tissues were collected, sonicated and cytosolic and membrane-bound PKC activities were determined following partial purification on DE52 columns. Each value represents the mean f SEM of 20 individual experiments performed in duplicate. “p < 0.01; bpCO.005
compared with respective control group.
0 CONTROL EZJ 5-HT 3.00
2.50
2.00
1.50
1.00
0.50
0.00
0.5
1.0 kHT1
10
100
(@f)
FIG 1 Human blood platelets were incubated for 10 minutes with various concentrations of 5-HT. Cytosolic and membrane-bound PKC activities were measured. Each bar indicates the mean f SEM of 5-20 determinations. Significant 5-HT dose-dependent effect was obtained (p CO.01, one-factor ANOVA).
1422
Vol. 47, No. 16, 1990
Platelet PKC Translocation
Characterization of the 5HT Receptor Subtype Responsible for 5-HT-Induced Protein Kinase C Translocation. Experiments aimed at characterizing the 5HT receptor subtype which is responsible for activation of platelet PKC translocation employed various receptor agonists and antagonists (Table II, Figures 2 and 3). While the selective 5-HT, agonist, DOI induced a concentration-dependent redistribution of the enzyme (Figure 21, the specific 5-HT,, agonist, 8-OH-DPAT and the 5HT,, agonist, RU24969 were inactive (Figure 2). Platelet PKC translocation elicited by 5-HT was inhibited by increasing doses of the selective 5-HT, antagonist, ketanserin (Table II). The effect of 5-HT was also inhibited by 10 PM spiroperidol and by the 5-HT, and 5-HT,, antagonist, mianserin (1OpM) but not by the 1OpM of 5-HT, antagonist, 3-tropanyl-indole-3-carboxylate (ICS205930) (Figure 3). In addition, neither the a,-adrenergic antagonist, prazosin nor the D, antagonist, I-sulpiride were found to block 5-HT-induced PKC redistribution. These results suggest that 5-HT-induced platelet PKC translocation is mediated through 5-HT,-type 5-HT serotonin receptors.
Effect of Ketanserin on 5-HT-Induced
PKC Translocation in Platelets
CYTOSOL
MFMBRANE-BCUNQ
PKC Activity (pmol/mln/~g protein)
CONTROL KETANSERIN, 10 PM 5-HT, 10 yM 5-HT + Ketanserin, 0.1 PM 5-HT + Ketanserin, 1 .O PM 5-HT + Ketanserin, 10 PM
2.89 2.85 2.15 2.49 2.76 2.87
f f f f f f
0.04 0.02 0.04’ 0.05b 0.04” 0.10”
2.17 2.26 2.97 2.76 2.39 2.15
f f f f f f
0.04 0.03 0.05’ 0.06b 0.03” 0.03”
Human blood platelets were incubated with various concentrations of ketanserin for 10 minutes. Tissues were subsequently exposed to 5-HT (10 Platelet PKC activity in cytosolic and membranous NM) for 10 minutes. fractions were measured. Values indicate the mean f SEM of 4 individual experiments. Significance (p < 0.01) by one factor ANOVA was found for the ketanserin doses. *p < 0.005 for 5-HT response vs. control by two-tailed Student’s ‘t’ test bp < 0.05; “p < 0.01 Comparison of 5-HT + Ketanserin vs. 5-HT by NewmanKeuls multiple comparison
Vol. 47, No. 16, 1990
Platelet PKC Tranelocation
0 a m LXI 6XY BE8 m
CONTROL 5-HT 6-OH-DPAT RU24969 DOI NOREPINEPHRINE EPINEPHRINE
**
\
4 i
1.oo
I g
0.50
d ;
0.00
10
I
10
10
10 100
10
10
[AGONIST] (phi)
FIG. 2 Human blood platelets were exposed (IO min) to various agents. Cytosolic and membrane-bound PKC activities were determined. Each bar represents the mean f SEM of 8 individual experiments. Statistical differences were evaluated using the two-tailed Student’s ‘t’ test. “p
