- Email: [email protected]
Calcium mobilization evoked by amyloid β-protein involves inositol 1,4,5-trisphosphate production in human platelets
UC sciences, VOI. 62, No. It, pp. 705-713,1998 CQpyrigtlt0 1998 E?kbier science Inc. Printed in the USA. All rights resewed om-32mpJ3 519.00 + .oo
PII !?4024-3205(97)01169-7
CALCIUM MOBlLlZATlON
WOKED
INOSITOL 1,4,5-TRISPHOSPHATE
BY AMYLOID PRODUCTlON
f3-PROTEIN INVOLVES IN HUMAN PLATELETS
Hiroki Ishikawa, Hiroki Ozawa, Toshikazu Saito’,Naohiko Takahata and Haruo Takemura* Departments of Neuropsychiatry and *Pharmacology, School of Medicine, ‘Department of Occupa tr‘0nal Therapy, School of Health Science, Sapporo Medical University, South-l, West-l 6, Chuo-ku, Sapporo 060, Japan (Received in fmal form December 2,1997)
Summary
We examined the effects of amyloid p-protein (A@ on Ca*+ mobilization in human platelets. The addition of Af3 fragments 25-35 (Af325-35) gradually After the increased the cytoplasmic free Ca*+ concentration @*‘Ii). maximum response, [Ca*ri decreased and then reached a sustained, higher level of [Ca*+li . Similar effects were also observed with A6 l-40, whereas l-28 , 12-28 and 31-35 did not affect the Ca*+ response. In the absence of extracellular Ca2+, Af3 2535 caused a transient increase in [Ca*qi, which U73122, a phospholipase C inhibitor, returned to the resting level. completely abolished Ca*+ mobilization induced by thrombin and Aj3 25-35. Furthermore, A6 enhanced the production of inositol 1,4,5Msphosphate (IPJ in platelets. These findings suggest that Ca*+ mobilization induced by A6 2535 is due to phospholipase C activation and IP, production. key Wordr:amyloid p-protein, Ca2’ mobilization, phospholipase C, platelet, Alzheimer ’ s disease, human Amyloid B-protein (AfI), which contains 39-43 amino acid residues, is a major Component of senile plaques and is implicated in the pathogenesis of Alzheimer disease (AD). It has been shown to have neurotoxic effects (l-3), and these effects of A6 or its fragment A6 25-35, which is the active fragment (1,4), have been suggested to result from the ability of these peptides to affect cellular calcium homeostasis. Recently, several studies have described changes in calcium homeostasis caused by A6 in neuronal and peripheral cells (5-10). These reports suggest that effects of Ag on Ca*’ mobilization are different among various types of cells. Platelets have often been considered to serve as a peripheral model for the exploration of neuronal metabolism in a number of neuropsychiatric disorders (11-14). Corresponding author: Hiroki Ozawa, MD., Ph.D., Fax: 81-l l-6443041. E-Mail: [email protected]
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In platelets, the basal level of [Ca2+]i has been reported to increase in aging and AD (15) but there is no information about whether Ap mobilizes cytoplasmic calcium. Therefore, we investigated the effect and mechanism of AfI on calcium mobilization in human platelets. Materials and Methods Materials Af325-35 was obtained from Peptide Institute (Osaka, Japan) and other Ap fragments were obtained from Sigma. Fura- acetoxy methyl ester (fura2/AM) was obtained from Molecular Probes. U73122 was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). The inositol 1,4,5trisphosphate assay system was obtained from Amersham Corp. Other materials and chemicals were obtained from commercial sources. Cytop/asmic Ca2+ flCa2+]i) measurement Human blood, drawn from healthy volunteers, was collected into one-tenth volume of acid citrate dextrose anticoagulant (4.0 g of citric acid, 11.O g of sodium citrate and 12.25 g of dextrose in 500 ml of water, pH 4.5) and then centrifuged at 360x9 for 10 min at room temperature. Measurement of [Ca2+‘ji was performed according to a modification of the method used for Jurkat T lymphocytes (16). Briefly, platelet-rich plasma (PRP) was obtained and incubated with 3 PM fura-2/AM for 15 min at 37°C. After incubation, PRP was centrifuged at 700xg for 10 min at room temperature, and the platelet pellet was resuspended in Krebs-Ringer-HEPES medium (KRH), composed of 145 mM NaCI, 5 mM KCI, 1 mM MgCl2, 0.5 mM Na2HP04, 0.2 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4. The platelets were adjusted to about 2x10’ cells/ml. Fluorescence of the fura-2-loaded platelet suspension was monitored with a Hitachi F-2006 (Hitachi Corp., Tokyo, Japan). Samples were prewarmed in a cuvette at 37°C for 1 min to measure the resting level and then Aj3 or thrombin was added to the incubation medium. After dilution with 10% dimethyl sulfoxide buffer A@was used within 30 min because aggregation of Afi does not seem to occur in this period (17). The intracellular calcium concentration, [Ca2+ Ii, was estimated as described by Grynkiewicz et al. (16). For the experiments in the absence of extracellular Ca2+, 1 mM EGTA was added to the platelet suspension 40 set before drug stimulation to chelate extracellular Ca2’. When U73122, a phospholipase C (PLC) inhibitor (19) was used, it was preincubated for 300 set before addition of A6 25-35 or thrombin. All determinations of the platelet intracellular free calcium level were completed within three hours of obtaining blood samples.
Vol. 62, No. 8, 1998
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/no&to/ 1,4,5Wsphosphate (rPg assay Measurement of IP, was performed as described previously (16). Platelets (2.4~10~ cells/l20 ~1)were incubated at 37°C for 5 min and then stimulated with Ab 2535 or thrombin for 30 sec. Then the reactions were terminated by the addition of 50 ml of ice-cold 10% perchloric acid (PCA) followed by a 30 min incubation in an ice bath. After centrifugation at 2000xg for 10 min at 4°C the supernatant (150 ~1) was recovered and its pH was adjusted to 6.5-7.5 with a 1.73 M KOH solution containing 75 mM HEPES. KC104 was precipitated for 30 min at 4°C and sedimented at 2000xg for 10 min at 4°C. The amount of IP, in the resulting supernatant (100 PI) was determined using the IP, assay system. Statistical comparison was performed by ANOVA.
Results The addition of A6 25-35 (10 $vl) gradually increased [Ca2’ji with a peak latency (the time from the stimulation to the peak of the change) of about 40 set, whereas thrombin (0.5 U/ml) more rapidly increased [Ca*‘ji with a peak latency of about 1 sec. After the maximum response, [Ca2’ji elevated by A6 25-35 decreased and then reached a sustained, higher level (Fig. 1). These effects of Ca2+ mobilization occurred in a dose-dependent manner (2-25 PM) (Fig. 1). Similar effects were also observed with A6 l-40, whereas 31-35, l-26 and 12-26 did not affect the Ca2+ response. The response of Aj3 lo-20 was very weak (Fig. 2). In the absence of extracellular Ca2’, A6 25-35 caused a transient increase in [Ca2’ji that thereafter returned to the resting level (Fig. 3) suggesting that the elevation of [Ca2*]i induced by A6 2535 was due to both Ca2+ entry from extracellular medium and release from intracellular Ca2+ stores. The response induced by thrombin after stimulation with A6 was smaller than that induced by thrombin alone. On the other hand, after stimulation with thrombin, Af3 caused no response. These results suggested that A6 mobilized Ca 2t from the intracellular Ca2+ store and that A6 and thrombin shared the same Ca2+ store. We examined whether Ca2+ mobilization induced by A6 involves PLC activation and IP, accumulation. U73122, a PLC inhibitor, completely abolished thrombin- and Ap 2535- induced Ca2+ mobilization, suggesting that A6 as well as thrombin caused Ca2+ mobilization due to PLC activation (Fig. 4). In addition, thrombin and A6 25-35 significantly increased the cellular IP, level to the same degree (Fig. 5).
A/3 and Ca2’ Mobilization in Platelet
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loo0 (l&f)
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1 min
Vol. 62, No. 8, 1998
lcmo(nM)
1 min
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liuombin
600-
m-
E
1
‘wo-
: ml-
2Ot-
loo -i 0
i
lb
li
26
A
3h
Fig. 1 Upper : The effects of 0.5 U/ml thrombin and 10 PM Afl 25-35 on [Ca2+li in human platelets. Each trace is a representative response from at least 3 separate experiments. Lower : Dose-response study of Ap25-35 on [Ca2+]i in human platelets. Values are means&.E. (n=3-12)
Vol. 62, No. 8, 1998
A@ and Ca2+ Mobiiition
in Platelet
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GG
- 300 $_ -0
f A$ l-40 25 pM
-0
-7-Afi 31-35 2.5 w
AfJ lo-20 25 pM
;=
- 300
$ c
-0
-i---
f
A/3 l-28 10 pM
z::” 1 min
Fig. 2 The effects of Afl25-35, Ap l-40, Ab 10-20, Afi 31-35, Afi l-28 and Ab 12-28 on [Ca2+]i in human platelets. Each tracs is a representative
response from at least 3 separate experiments.
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A/3 and Ca*’ Mobilization in Platelet
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Vol. 62, No. 8, 1998
(W 500 (nM)
5oo (nM) -
1 1 min
0
-
A$25-35 25W
lluombin OSU/ml
Fig. 3 The effects of 25 @I Ab 25-35 and 0.5 U/ml thrombin on [Ca2+)i in the absence of extracellular Ca2+, 1 mM EGTA was added to the platelet suspension 40 set before drug stimulation to chelate extracellular Ca2’. Each trace is a representative response from at least 3 separate experiments. (A) Thrombin was added 100 seconds after stimulation with Af3 25-35. (B) AfT 25-35 was added 100 seconds after stimulation wtth thrombin. (4 1000 (nM)
loo0 (nM) 1
I
1
t
Ilvombin OSU/ml
Thornbin O.SU/ml
(W
Ap 25-35 25PM
1 min
The effects of 0.5 U/ml thrombin (A) and 25 @t Af325-35 (B) on [Ca2p after addition of 5 ufvl U73122 (preincubation time = 300 set) in human platelets. Each trace is a representative response from at least 3 separate experiments. (A) Left: thrombin alone, Right: U73122+thrombin(B) Left: Ab 25-35 alone, Right: U73122+A@ 25-35
Vol. 62, No. 8, 1998
Ap and Ca2’ Mobilizationin Platelet
Basal
Aft 25-35
711
Thrombin
Fig. 5 The effects of 25 pM A$ 25-35 and 1.0 U/ml thrombin on IP, formation at 30 set after stimulatiin. Values are mean2S.E. (n=S-a). Significant differences from control are indicated by lp
Discussion Several studies of neuronal or peripheral cells have shown that Af3 causes direct Ca2+ influx from the extracellular medium into cells (9, 10, 20) or potentiates an increase in [Ca2$ evoked by various stimuli (5-7, 21). Furthermore, AB mobilizes Ca2+ in the absence of extracellular Ca2+ in human leukemic HL-80 cells (8). In the above studies, the mechanisms of Ca2+ mobilization and potentiation of elevation of [Ca2+J induced by Ap were not clarified. The present study clearly showed that Afi increased [Ca2+# due to Ca2’ release from intracellular stores as well as Ca2+ entry from extracellular medium in human platelets. In addition, Afi induced the release of Ca2+ from thrombin-sensitive intracellular stores (Fig. 3) and increased IP, accumulation (Fig. 5). Thus, Ca2+ release caused by Af3 is likely to be due to an elevation of IP, mobilized from IP,-sensitive intracellular Ca2+ stores. However, the mechanism of the activation of Ca2+ entry in non-excitable cells, including platelets, is less understood than that of Ca2+ release. A popular hypothesis for the activation of Ca2+ entry is that it occurs via the capacitative Ca2’ entry pathway by which depletion of Ca2+ in the intracellular store activates Ca2+ entry. We reported that thapsigargin, a microsomal Ca2+-ATPase inhibitor, caused both the release of Ca2+ from IP@ensitiie intracellular stores and the entry of Ca2+ from extracellular medium via an IP,independent mechanism (22). Capacitative Ca2+ entry is widely
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AB and Ca2’ Mobilizationin Platelet
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confirmed in platelets (23-25). It is, thus, likely that Ca2+ entry caused by Af3is due to capacitative Ca2+ entry in human platelets. A study on rat cortical membranes has demonstrated that A6 enhances the binding of IP, (26), however, whether Af3alone increased IP, accumulation was not examined. In the present study, A$ increased IP, accumulation (Fig. 5) and the elevation of [Ca2ri induced by A& was abolished by pretreatment with U73122, a PLC inhibitor (Fig. 4). These results show for the first time that IP, increases accumulation by itself, presumably due to the activation of PLC. Although receptors for thrombin link G-proteins that modulate PLC activity, the mechanisms of Af3-induced IP, production at receptor and G-protein levels are unknown. However, the hydrophobic C terminal portion of Af3 is buried in the membrane and the flexible N terminal exposed to the membrane environment (27). In the present study, the increase of IP, accumulation induced by Af3 occurred via a different mechanism from that induced by thrombin because the peak latency to mobilize Ca2+ and the shape of changes in [Ca2’]i induced by the addition of Af3 and thrombin were different (Fig. 1). Therefore one might consider that this intercalation of Ap in membranes may modulate the coupling of receptors and effecters with G proteins independent of agonist-like action. Indeed, recent studies on neuronal cell culture suggest that A6 acts on the coupling of receptors and G proteins via oxidative stress (26). It has recently been reported that Af3 damages endothelial cells of blood vessels to produce an excess of superoxide radicals and that this damage upregulates Af3 production (29). Furthermore, it has been shown that platelets are the primary source of Ap in human blood, indicating that these cells may be a potential source for the amyloid deposits in the vessels and brain (39). Thus, further study is necessary to examine whether the response of A6 to platelets is essential for the pathogenesis of AD and whether it can serve as a peripheral marker for AD. Acknowledgments The authors wish to thank Prof. H. Ohshika for his helpful suggestions and encouragement. This research was supported by the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan (H. Ozawa, T. Saito), and the Ministry of Heatth and Welfare of Japan (H. Ozawa, T. Saito) References 1. 2. 3.
B.A.YANKNER, L.K.DUFFY and D.A.KIRSCHNER, Science. 250 279-262 (1999). C.J.PIKE, A.J.WALENCEWICS, C. G.GlABE and C. G.COTMAN, Brain Res. 663 311-314 (1991) KUEDA, Y.FUJII and HKAGEYAMA, Brain Res. 639 240-244 (1994)
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4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19. 29. 21. 22. 23. 24.
25. 26. 27. 28.
29. 30.
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C.J.PIKE,, A.J.WALENCEWICS-WASSERMAN,J.KOSMOSKI, D.H.CRIBBS, C. G.GLABE and C. G.COTMAN, J. Neurochem. 64 253-265 (1995) H.HARTMANN, A.ECKERT and W.E.MULLER, Biochem. Biophys. Res. Commun. 194 12j6-1220 (1993) A.ECKERT, H.FORSTL and H.HARTMANN, Neuroreport. 6 1199-1202 (1995) M. P.MAlTSON, B.CHEUNG, D. DAVIS, K.BRYANT, I.LIEBERBURG and R.E. RYDEL, J. Neurosci. 12 376-389 (1992) T.TAKENOUCHI and E.MUNEKATA, Peptides. 16 1019-I 024 (1995) R.JOSEPH and E.HAN, Biochem. Biophys. Res. Commun. 164 1441-1447 (1992) R.FUKUYAMA, K. C.WADHWANI, Z.GALDICKI, S. IRAPOPORT and G.EHRENSTEIN , Brain Res. 667 269-272 (1994) M.MIKUNI, AKAGAYA, K.TAKAHASHI, and H. Y. MELTZER Psychopharmacol. 166 31 I-31 4 (1992) A.ECKERT, HGANN, D.RIEMANN, J.ALDENHOFF and W.E.MULLER, Biol. Psychiatry. 34 565-568 (1993) S. L.DUBOVSKY, J.CHRISTIANO, L. C. DANIEL, Ft. D. FRANKS, J. MURPHY, L. ALDER, , N. BAKER and A.HARRIS Arch. Gen. Psychiatry. 46 632-637 (1989) Y. OKAYMOTO, A. KAGAYA, H. SHINNO, N. MOTOHASHI and S YAMAWAKI. Life Sci. 56 327-332 (1995) K. H.LE QUAN SANG, E. MIGNOT, J. C.GIBERT, R.HUGUET, J. P.AQUINO, 0. REGNIER and M. A.DEVYNCK, Biol. Psychiatry. 33 391-393 (1993) S. SAKANO, H.TAKEMURA, K.YAMADA, K.IMOTO, MKANEKO and H.OHSHIKA, J. Biol. Chem. 271 11148-l 1155 (1996) H.MORI, I K.ISHI, T.TOMIYAMA, Y.FURIYA, NSAHARA, S.ASANO, N.ENDO, T.SHIRASAKA, and K. TAKIO Tohoku J. Exp. Med. 174 251-262 (1994) G.GRYNKIEWICS, M.POENIE and R. T. TSIEN, J. Biol. Chem. 266 3440-3450 (1985) Y. OKAMOTO, H.NINOMIYA, S.MIWA and T.MASAKI Biochem. Biophys. Res. Commun. 212 96-96 (1995) I.ENGSTROM, G.RONQUIST, L.PElTERSSON and A.WALDENSTRCM, Eur. J. Clin. Invest. 25 471-476 (1995) A.ECKERT, H.HARTMANN, H.FCRSTL and W.E.MULLER, Life Sci. 65 20192029 (1994) H.TAKEMURA, A. R.HUGHES, O.,TAHSTRUP and J. W. Jr. PUTNEY, J. Biol. Chem. 264 12266-12271 (1989) D. S.DESHMUKH, S.KUISON and H.BROCKENHOFF, Cell calcium 12 645654 (1991) S. O.SAGE, P.SARGEANT, J. W. M.HEEMSKERK and M. P.MAHAUT-SMITH, Mechanisms of Platelet Activation and Control. , K. S. Authi (Eds.) 69-82, Plenum Press, New York (1993) . J. G.VOSTAL and B.SCHAFER, J. Biol. Chem. 271” 19524-19529 (1996) R. F.COWBURN, B. WIEHAGER and E.SUNDSTROM, Neurosci. Lett. 191 31-34 (1995) T. KOHNO, K. KOBAYASHI, T.MAEDA, K.SATO and A. TAKASHIMA, Biochemistry 35 16094-l 6194 (1996) J. F. KELLY, K. FURUKAWA, S. W. BARGER, M. R. RENGEN, R. J. MARK , E. M. BLANC, G. S. ROTH and M. P. MAlTSON,Proc. Natl. Acad. Sci. USA 93 6753-6758 (1996) T.THOMAS, G.THOMAS, C.MCLEDON, T.SUlTON and M.MULtAN. Nature. 369 168-171 (1996) M.CHEN, N. C.INESTRISA, G. S.ROSS and H. L. FERNANDEZ, Biochem. Biophys. Res. Commun. 213 96-103 (1995)
PII !?4024-3205(97)01169-7
CALCIUM MOBlLlZATlON
WOKED
INOSITOL 1,4,5-TRISPHOSPHATE
BY AMYLOID PRODUCTlON
f3-PROTEIN INVOLVES IN HUMAN PLATELETS
Hiroki Ishikawa, Hiroki Ozawa, Toshikazu Saito’,Naohiko Takahata and Haruo Takemura* Departments of Neuropsychiatry and *Pharmacology, School of Medicine, ‘Department of Occupa tr‘0nal Therapy, School of Health Science, Sapporo Medical University, South-l, West-l 6, Chuo-ku, Sapporo 060, Japan (Received in fmal form December 2,1997)
Summary
We examined the effects of amyloid p-protein (A@ on Ca*+ mobilization in human platelets. The addition of Af3 fragments 25-35 (Af325-35) gradually After the increased the cytoplasmic free Ca*+ concentration @*‘Ii). maximum response, [Ca*ri decreased and then reached a sustained, higher level of [Ca*+li . Similar effects were also observed with A6 l-40, whereas l-28 , 12-28 and 31-35 did not affect the Ca*+ response. In the absence of extracellular Ca2+, Af3 2535 caused a transient increase in [Ca*qi, which U73122, a phospholipase C inhibitor, returned to the resting level. completely abolished Ca*+ mobilization induced by thrombin and Aj3 25-35. Furthermore, A6 enhanced the production of inositol 1,4,5Msphosphate (IPJ in platelets. These findings suggest that Ca*+ mobilization induced by A6 2535 is due to phospholipase C activation and IP, production. key Wordr:amyloid p-protein, Ca2’ mobilization, phospholipase C, platelet, Alzheimer ’ s disease, human Amyloid B-protein (AfI), which contains 39-43 amino acid residues, is a major Component of senile plaques and is implicated in the pathogenesis of Alzheimer disease (AD). It has been shown to have neurotoxic effects (l-3), and these effects of A6 or its fragment A6 25-35, which is the active fragment (1,4), have been suggested to result from the ability of these peptides to affect cellular calcium homeostasis. Recently, several studies have described changes in calcium homeostasis caused by A6 in neuronal and peripheral cells (5-10). These reports suggest that effects of Ag on Ca*’ mobilization are different among various types of cells. Platelets have often been considered to serve as a peripheral model for the exploration of neuronal metabolism in a number of neuropsychiatric disorders (11-14). Corresponding author: Hiroki Ozawa, MD., Ph.D., Fax: 81-l l-6443041. E-Mail: [email protected]
706
A/3 and Ca2’ Mobilization in Platelet
Vol. 62, No. 8, 1998
In platelets, the basal level of [Ca2+]i has been reported to increase in aging and AD (15) but there is no information about whether Ap mobilizes cytoplasmic calcium. Therefore, we investigated the effect and mechanism of AfI on calcium mobilization in human platelets. Materials and Methods Materials Af325-35 was obtained from Peptide Institute (Osaka, Japan) and other Ap fragments were obtained from Sigma. Fura- acetoxy methyl ester (fura2/AM) was obtained from Molecular Probes. U73122 was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). The inositol 1,4,5trisphosphate assay system was obtained from Amersham Corp. Other materials and chemicals were obtained from commercial sources. Cytop/asmic Ca2+ flCa2+]i) measurement Human blood, drawn from healthy volunteers, was collected into one-tenth volume of acid citrate dextrose anticoagulant (4.0 g of citric acid, 11.O g of sodium citrate and 12.25 g of dextrose in 500 ml of water, pH 4.5) and then centrifuged at 360x9 for 10 min at room temperature. Measurement of [Ca2+‘ji was performed according to a modification of the method used for Jurkat T lymphocytes (16). Briefly, platelet-rich plasma (PRP) was obtained and incubated with 3 PM fura-2/AM for 15 min at 37°C. After incubation, PRP was centrifuged at 700xg for 10 min at room temperature, and the platelet pellet was resuspended in Krebs-Ringer-HEPES medium (KRH), composed of 145 mM NaCI, 5 mM KCI, 1 mM MgCl2, 0.5 mM Na2HP04, 0.2 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4. The platelets were adjusted to about 2x10’ cells/ml. Fluorescence of the fura-2-loaded platelet suspension was monitored with a Hitachi F-2006 (Hitachi Corp., Tokyo, Japan). Samples were prewarmed in a cuvette at 37°C for 1 min to measure the resting level and then Aj3 or thrombin was added to the incubation medium. After dilution with 10% dimethyl sulfoxide buffer A@was used within 30 min because aggregation of Afi does not seem to occur in this period (17). The intracellular calcium concentration, [Ca2+ Ii, was estimated as described by Grynkiewicz et al. (16). For the experiments in the absence of extracellular Ca2+, 1 mM EGTA was added to the platelet suspension 40 set before drug stimulation to chelate extracellular Ca2’. When U73122, a phospholipase C (PLC) inhibitor (19) was used, it was preincubated for 300 set before addition of A6 25-35 or thrombin. All determinations of the platelet intracellular free calcium level were completed within three hours of obtaining blood samples.
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/no&to/ 1,4,5Wsphosphate (rPg assay Measurement of IP, was performed as described previously (16). Platelets (2.4~10~ cells/l20 ~1)were incubated at 37°C for 5 min and then stimulated with Ab 2535 or thrombin for 30 sec. Then the reactions were terminated by the addition of 50 ml of ice-cold 10% perchloric acid (PCA) followed by a 30 min incubation in an ice bath. After centrifugation at 2000xg for 10 min at 4°C the supernatant (150 ~1) was recovered and its pH was adjusted to 6.5-7.5 with a 1.73 M KOH solution containing 75 mM HEPES. KC104 was precipitated for 30 min at 4°C and sedimented at 2000xg for 10 min at 4°C. The amount of IP, in the resulting supernatant (100 PI) was determined using the IP, assay system. Statistical comparison was performed by ANOVA.
Results The addition of A6 25-35 (10 $vl) gradually increased [Ca2’ji with a peak latency (the time from the stimulation to the peak of the change) of about 40 set, whereas thrombin (0.5 U/ml) more rapidly increased [Ca*‘ji with a peak latency of about 1 sec. After the maximum response, [Ca2’ji elevated by A6 25-35 decreased and then reached a sustained, higher level (Fig. 1). These effects of Ca2+ mobilization occurred in a dose-dependent manner (2-25 PM) (Fig. 1). Similar effects were also observed with A6 l-40, whereas 31-35, l-26 and 12-26 did not affect the Ca2+ response. The response of Aj3 lo-20 was very weak (Fig. 2). In the absence of extracellular Ca2’, A6 25-35 caused a transient increase in [Ca2’ji that thereafter returned to the resting level (Fig. 3) suggesting that the elevation of [Ca2*]i induced by A6 2535 was due to both Ca2+ entry from extracellular medium and release from intracellular Ca2+ stores. The response induced by thrombin after stimulation with A6 was smaller than that induced by thrombin alone. On the other hand, after stimulation with thrombin, Af3 caused no response. These results suggested that A6 mobilized Ca 2t from the intracellular Ca2+ store and that A6 and thrombin shared the same Ca2+ store. We examined whether Ca2+ mobilization induced by A6 involves PLC activation and IP, accumulation. U73122, a PLC inhibitor, completely abolished thrombin- and Ap 2535- induced Ca2+ mobilization, suggesting that A6 as well as thrombin caused Ca2+ mobilization due to PLC activation (Fig. 4). In addition, thrombin and A6 25-35 significantly increased the cellular IP, level to the same degree (Fig. 5).
A/3 and Ca2’ Mobilization in Platelet
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loo0 (l&f)
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1 min
Vol. 62, No. 8, 1998
lcmo(nM)
1 min
I A$2s-35
liuombin
600-
m-
E
1
‘wo-
: ml-
2Ot-
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i
lb
li
26
A
3h
Fig. 1 Upper : The effects of 0.5 U/ml thrombin and 10 PM Afl 25-35 on [Ca2+li in human platelets. Each trace is a representative response from at least 3 separate experiments. Lower : Dose-response study of Ap25-35 on [Ca2+]i in human platelets. Values are means&.E. (n=3-12)
Vol. 62, No. 8, 1998
A@ and Ca2+ Mobiiition
in Platelet
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GG
- 300 $_ -0
f A$ l-40 25 pM
-0
-7-Afi 31-35 2.5 w
AfJ lo-20 25 pM
;=
- 300
$ c
-0
-i---
f
A/3 l-28 10 pM
z::” 1 min
Fig. 2 The effects of Afl25-35, Ap l-40, Ab 10-20, Afi 31-35, Afi l-28 and Ab 12-28 on [Ca2+]i in human platelets. Each tracs is a representative
response from at least 3 separate experiments.
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A/3 and Ca*’ Mobilization in Platelet
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Vol. 62, No. 8, 1998
(W 500 (nM)
5oo (nM) -
1 1 min
0
-
A$25-35 25W
lluombin OSU/ml
Fig. 3 The effects of 25 @I Ab 25-35 and 0.5 U/ml thrombin on [Ca2+)i in the absence of extracellular Ca2+, 1 mM EGTA was added to the platelet suspension 40 set before drug stimulation to chelate extracellular Ca2’. Each trace is a representative response from at least 3 separate experiments. (A) Thrombin was added 100 seconds after stimulation with Af3 25-35. (B) AfT 25-35 was added 100 seconds after stimulation wtth thrombin. (4 1000 (nM)
loo0 (nM) 1
I
1
t
Ilvombin OSU/ml
Thornbin O.SU/ml
(W
Ap 25-35 25PM
1 min
The effects of 0.5 U/ml thrombin (A) and 25 @t Af325-35 (B) on [Ca2p after addition of 5 ufvl U73122 (preincubation time = 300 set) in human platelets. Each trace is a representative response from at least 3 separate experiments. (A) Left: thrombin alone, Right: U73122+thrombin(B) Left: Ab 25-35 alone, Right: U73122+A@ 25-35
Vol. 62, No. 8, 1998
Ap and Ca2’ Mobilizationin Platelet
Basal
Aft 25-35
711
Thrombin
Fig. 5 The effects of 25 pM A$ 25-35 and 1.0 U/ml thrombin on IP, formation at 30 set after stimulatiin. Values are mean2S.E. (n=S-a). Significant differences from control are indicated by lp
Discussion Several studies of neuronal or peripheral cells have shown that Af3 causes direct Ca2+ influx from the extracellular medium into cells (9, 10, 20) or potentiates an increase in [Ca2$ evoked by various stimuli (5-7, 21). Furthermore, AB mobilizes Ca2+ in the absence of extracellular Ca2+ in human leukemic HL-80 cells (8). In the above studies, the mechanisms of Ca2+ mobilization and potentiation of elevation of [Ca2+J induced by Ap were not clarified. The present study clearly showed that Afi increased [Ca2+# due to Ca2’ release from intracellular stores as well as Ca2+ entry from extracellular medium in human platelets. In addition, Afi induced the release of Ca2+ from thrombin-sensitive intracellular stores (Fig. 3) and increased IP, accumulation (Fig. 5). Thus, Ca2+ release caused by Af3 is likely to be due to an elevation of IP, mobilized from IP,-sensitive intracellular Ca2+ stores. However, the mechanism of the activation of Ca2+ entry in non-excitable cells, including platelets, is less understood than that of Ca2+ release. A popular hypothesis for the activation of Ca2+ entry is that it occurs via the capacitative Ca2’ entry pathway by which depletion of Ca2+ in the intracellular store activates Ca2+ entry. We reported that thapsigargin, a microsomal Ca2+-ATPase inhibitor, caused both the release of Ca2+ from IP@ensitiie intracellular stores and the entry of Ca2+ from extracellular medium via an IP,independent mechanism (22). Capacitative Ca2+ entry is widely
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confirmed in platelets (23-25). It is, thus, likely that Ca2+ entry caused by Af3is due to capacitative Ca2+ entry in human platelets. A study on rat cortical membranes has demonstrated that A6 enhances the binding of IP, (26), however, whether Af3alone increased IP, accumulation was not examined. In the present study, A$ increased IP, accumulation (Fig. 5) and the elevation of [Ca2ri induced by A& was abolished by pretreatment with U73122, a PLC inhibitor (Fig. 4). These results show for the first time that IP, increases accumulation by itself, presumably due to the activation of PLC. Although receptors for thrombin link G-proteins that modulate PLC activity, the mechanisms of Af3-induced IP, production at receptor and G-protein levels are unknown. However, the hydrophobic C terminal portion of Af3 is buried in the membrane and the flexible N terminal exposed to the membrane environment (27). In the present study, the increase of IP, accumulation induced by Af3 occurred via a different mechanism from that induced by thrombin because the peak latency to mobilize Ca2+ and the shape of changes in [Ca2’]i induced by the addition of Af3 and thrombin were different (Fig. 1). Therefore one might consider that this intercalation of Ap in membranes may modulate the coupling of receptors and effecters with G proteins independent of agonist-like action. Indeed, recent studies on neuronal cell culture suggest that A6 acts on the coupling of receptors and G proteins via oxidative stress (26). It has recently been reported that Af3 damages endothelial cells of blood vessels to produce an excess of superoxide radicals and that this damage upregulates Af3 production (29). Furthermore, it has been shown that platelets are the primary source of Ap in human blood, indicating that these cells may be a potential source for the amyloid deposits in the vessels and brain (39). Thus, further study is necessary to examine whether the response of A6 to platelets is essential for the pathogenesis of AD and whether it can serve as a peripheral marker for AD. Acknowledgments The authors wish to thank Prof. H. Ohshika for his helpful suggestions and encouragement. This research was supported by the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan (H. Ozawa, T. Saito), and the Ministry of Heatth and Welfare of Japan (H. Ozawa, T. Saito) References 1. 2. 3.
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