Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
August 14, 1992
Pages 1582-1587
I N H I B I T I O N O F P H O S P H A T I D Y L S E R I N E S Y N T H E S I S BY G L U T A M A T E , A C E T Y L C H O L I N E , T H A P S I G A R G I N A N D I O N O P H O R E A23187 I N G L I O M A C6 C E L L S
M.Czarny ~, P.Sabala ~, A.Ucieklak:, L.Kaezmarek 3 and J.Barafiska ~° 1Dept. o f Cellular Biochemistry, 2Dept. of Cell Biology, 3Cell Culture Unit, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland Received June
15, 1992
Summary: Phosphatidylserine synthesis was studied in glioma C6 cells with [14C]serine and in the presence or absence of agents which increase the level of [Ca2+]~. It was found that glutamate and acetylcholine inhibited this synthesis by up to 40%, whereas thapsigargin and the ionophore A23187 inhibited by up to 70%. The inhibitory effect of thapsigargin and the A23187 was observed in Ca2+-free medium. The data show that the inhibition of this synthesis is caused by the Ca2+-depletion from endoplasmic reticulum, suggesting that the synthesis of phosphatidylserine occurs on the luminal side of these structures and can be regulated by transmembrane signaling systems. ® 1992 Academic P . . . . . Inc.
In mammalian cells phosphatidylserine (PS) is believed to be synthesized solely by the base exchange reaction that occurs mainly in the endoplasmic reticulum [1,2]. This reaction takes place in the absence of metabolic energy and is characterized by a requirement for Ca2÷. Therefore, any changes in Ca2÷ level in the cell should influence on the biosynthesis of this phospholipid. In order to investigate this, we used a number of agents which are known to alter the intracellular Ca 2÷, as glutamate, acetylcholine, thapsigargin and the calcium ionophore A23187, and examined their effect on PS synthesis in glioma C6 cells. Glutamate and acetylcholine are neurotransmitters which, by transmembrane signaling via cell surface receptors, lead to a rapid and sustained increase in the cytosolic Ca:+ concentration [3-5]. This is due to both the release of Ca2÷ from a non-mitochondrial intracellular stores caused by inositol(1,4,5)trisphosphate (InsP3), and the influx of extracellular Ca"-+ into the cell. Thapsigargin, a plant-derived sesquiterpene lactone, has been shown to be an effective and selective inhibitor of endoplasmic reticulum Ca:+-ATPase [6-8]. Thapsigargin mimics agonists' action by a rapid and marked increase in the concentration of cytosolic free Ca2+. This occurs also v/a a direct discharge of intracellular Ca"`+ stores
" To whom correspondence should be addressed. Abbreviations: [Ca2+]i - intracellular free calcium concentration; EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; PBS - phosphate buffer saline; PS - phosphatidylserine. 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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and Ca :+ influx, but without hydrolysis of inositolphospholipids [6-8]. The third type of agent used, the ionophore A23187 makes cell membranes permeabilized to Ca :+ and is consequently a convenient tool for the modulation of intracellular Ca:+ level. The present study demonstrates that all the agents used inhibit PS synthesis in glioma C6 cells. The inhibitory effect of ionophore and tapsigargin was observed in calcium-free medium. These data suggest that the PS synthesis occurs on the luminal side of the endoplasmic reticulum and can be regulated by intracellular events responsible for calcium fluxes. Materials and Methods Materials: Dulbecco's modified Eagle Medium, calf serum, trypsin solution and PBS were from Gibco BRL, UK. Glutamic acid, acetylcholine, eserine, the ionophore A23187 and EGTA were purchased from Sigma Chemical Co.,St.Louis,USA, and thapsigargin was from LC Services Corporation, Woburn, USA. Fura-2/AM was obtain from Molecular Probes, Inc.. L-[U-14C]serine was purchased from Praque, Czechoslovakia. TLC aluminium sheets silica gel 60 (without fluorescent indicator) were from Merck. Cell culture: Glioma C6 cells were cultured in Dulbecco's modified Eagle medium (DMEM) suplemented with 10% (v/v) calf serum, penicilin (50 IU/ml), streptomycin (50 #g/ml) and 2 mM L-glutamine under humidified atmosphere of 95% 02/5% CO,, at 37°C. The cells were passaged when confluent by using trypsin (0.25%), and the medium was changed twice a week. Cells for experiment were washed once with PBS. Phosphatidylserine synthesis in cultured cells: Cells were plated into 60 mm dishes at a concentration of 0.5 x l& cells/dish. Before measurements were made the medium was replaced with the following mixture (buffer A): 137 mM NaCI, 2.7 mM KC1, 1 mM Na:HPO4, 25 mM glucose, 20 mM Hepes (pH 7.4), 1 mM MgCI: and 1% (v/v) bovine serum albumin with the addition of L-[U-14C]serine (4.5 #M, 0.5 #Ci/ml). The cells were then incubated at 37°C in the buffer A for various periods of time and at various calcium concentration in the presence or absence of glutamate, acetylcholine, thapsigargin, A23187 and EGTA as indicated in Figures. After incubation, the medium was removed and the cells were washed with ice-cold PBS, scrapped off with cell lifter, collected in test tubes, spun down and resuspended in PBS. Lipids were extracted using chloroform/methanol (1 : 2; v/v) according to Bligh and Dyer [9]. Phospholipids were analysed by two dimensional TLC. The solvent in the first dimension was: chloroform/methanol/ammonium hydroxide/water (55:33:4:2 v/v); and in the second: chloroform/methanol/acetic acid/water (30:15:6:1 v/v). The spots of phospholipids were visualised with iodine and ninhidrin and scrapped off for radioactivity counting. Measurements of intracellular calcium: Cells were attached to glass coverslips, washed twice with PBS and once with buffer A and incubated in this buffer with 1 #M fura-2 (fura-2/acetoxymetyl ester) for 40 min at 37°C. Thereafter they were washed three times with buffer A. The third wash was left for 20 min at 37°C. Coverslips were mounted in a chamber over an Nikon Diaphot inverted-stage microscope equipped with a x20 fluorescence objective lens. All substances were added as a buffer A solutions. Digital fluorescent microscopy was used to determine the spatial distribution of [Ca:÷]i. Cells were alternately illuminated with 340 nm and 380 nm light from a xenon lamp. Emitted light was passed through a 500 nm barrier filter into an image-intensified camera (CCD). Experiments were carried out on a Joyce-Loebl Ltd. computerizing image-processing system Magiscan for calcium analyzis using Tardis V6.2. Determination of protein was preformed according to Lowry et al.[10] with the modification of Nalgcz et al [11]. Results Fig. I. shows that in treated glioma C6 cells PS synthesis is significantly altered compared to the basal condition. Approximately 30%-40% of this process was inhibited by neurotransmitters, whereas 1583
V o l . 186, N o . 3, 1992
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Fig. 1. Concentration-dependent effect of glutamate, acetylcholine and thapsigargin on PS synthesis in glioma C6 cells. Cells were incubated for 15 min with 2 mM CaCI 2 and [U-14C]serine as described in Materials and Methods, in the absence or presence of indicated concentration of glutamate (A), acetylcholine 03) and thapsigargin (C). Values are expressed as percentage of control which avaraged 9.0+0.8 pmoles [14C]phosphatidylserine formed per mg protein. The data are means + standard errors from two individual experiments. ~7~, Controls; F"--I, additions.
thapsigargin resulted in a marked (up to 70%) inhibition of the serine incorporation into PS. In contrast, the total uptake of serine by the cells was not affected by any of these compounds (not shown), demonstrating that the decrease in serine incorporation is not caused by changes in the uptake of this arninoacid. Glutamate was shown to induce an ihibition of PS synthesis in range of 10-6-10-3 M, whereas as low as 10 4 M acetylcholine was able to produce similar effect. Thapsigargin was effective in the concentration 0,5 x 10-S-l.5 x 10 -7 M (Fig.l). Similar results were already detectable 10 min after of incubation of the cells.
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Fig: 2. Effect of glutamate (A), acetylcholine 03) and thapsigargin (C) on intracellular Ca2+ in fura-2 loaded glioma C6 cells. A. Glutamate added (I00 ~M); B. acetylcholine added (0.1 ~M); C. thapsigargin added (0.1 ~M). Stimulation of cells were performed in the presence of external 2 mM Ca2÷. The addition of glutamate, acetylcholine and thapsigargin is indicated by arrows. Loading of cells with fura-2 is described in Materials and Methods. The results are from a single experiment typical of two others. Data points were taken every 1.1 s. 1584
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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Fig. 3. Effect of calcium ionophore A23187 and thapsigarginon phosphatidylserineformation at varying Ca~* concentration. Cells were incubated for 15 min with [14C]serinein the absence (control) or presence of 5 #M A23187 (O) or 0.1 #M thapsigargin (A) at varying CaCI: concentration as indicated. The values obtained when using untreated cells are taken as 100% (basal line, control). Incubation of cells with Ca:+-free medium and with 200/zM EGTA diminished the control level by about 50%, whereas in the presence of CaCIz it was similar. Data are means 4- standard error from five experiments. Fig. 4. Time dependent effect of A23187 and thapsigargin on the [14C]phosphatidylserineformation. Glioma C6 cells were incubated with [14C]serine in the absence (controls, O) and presence of 5 #M A23187 (O) or 0.1 #M thapsigargin (A). The experiment shown is representative of two identical experiments.
To examine if glutamate, acetylcholine and thapsigargin indeed induce the increase in [Ca2+]i in glioma C6 cells, the effect of these agents was measured with fura-2 (Fig.2). Although the rise in [Ca2+]i mediated by 104 M glutamate was slower than the rapid initial spike induced by 10-7M acetylcholine, both neurotransmitters led to a sustained increase in the cytosolic calcium, higher than the basal level (about 100 nmoles) of resting cells. Treatment of fura-2 labeled cells by thapsigargin caused a slow increase in cytosolic calcium, and after a lag time period of 250 s, a sustained [Ca2+]~ rise. It is notable that using an image-processing Magiscan system one can observe a considerable heterogeneity in individual cell responses [12]. Nevertheless, the most of the cells responded in the manner shown in Fig.2. Fig.3 demonstrates the effect of calcium ionophore A23187 and thapsigargin on PS formation at varying Ca2÷ concentration. Thapsigargin inhibited this process in both the presence or absence of external calcium, and only at 10 mM CaC12 was this effect not so visible. The inhibition of PS synthesis by the ionophore A23187 occured in Ca2+-free medium, and continued at a low calcium concentration (100 #M). On the other hand, at 2mM CaC12, the ionophore was without effect, and at 10 mM CaCI:, it distinctly stimulated PS synthesis. 1585
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These results point to the direct regulation of PS synthesis by the intracellular calcium. However, when the cells are incubated at low CaCI2 concentration (100/~M), the addition of A23187 increases the level of calcium in the cytosol to the same value, which is a thousand fold higher than in the resting cells. Thus, the inhibition of PS synthesis observed in these conditions, can be explained only by the release of Ca2+ from the intracellular stores, i.e. from the endoplasmic reticulum, where a 103M concentration; of Ca2+ can be achieved [15]. The same phenomenon takes place in Ca2+-free medium. The data presented in Fig.1 and Fig.3 proceeded for 15 minutes intervals. Fig.4 shows a time-dependent effect of thapsigargin and A23187 on PS formation. As observed here, the inhibitory effect of thapsigargin is maintained for at least 4 h. However, A23187 expressed a lack of effect for the first 15 rain of incubation (see also Fig.3) but at longer times (1-4 h) strongly inhibited PS synthesis (up to 70%).
Discussion
In a mixed rat brain microsomal population, serine base-exchange enzyme is believed to be located on the luminal surface of the microsomes [14-15]. We have previously suggested a similar localization of the enzyme in the liver microsomes [16]. The results presented here provide further evidence that PS synthesis proceeds on the luminal leaflet of the endoplasmic reticulum, and can be regulated not only by Ca2+-ATPase [16], but by most other signals which control cellular Ca2+ fluctuations. The specific inhibition of PS synthesis during activation of Jurkat T lymphocytes was first reported by Pelassy et al. [17,18]. The authors suggested that this process is regulated similarly as increasing inositolphospholipids turnover, i.e. by transmembrane signaling, but the direct mechanism of this inhibition was obscure. The present study clarifies this problem by demonstrating that the inhibition of PS synthesis is induced by those events which lead to the depletion of Ca:+ from the endoplasmic reticulum. This was shown clearly by the effect of neurotransmitters which via InsP3 release Ca2+ from the intracellutar stores and decrease PS synthesis, and by the action of A23187 at low calcium concentrations, and in Ca2+-free medium. It was also indicated by the effect of thapsigargin, which being a specific blocker of the endoplasmic reticulum Ca2+ pump, prevents reuptake of calcium into these structures and so markedly inhibits PS synthesis. It has been reported that the thapsigargin-releasable Ca2+ pool within the cell is larger than InsP3 sensitive Ca:+-pool [7,8]. The evidence presented by Gill and coworkers indicates three different calcium pools in smooth muscle cell line [7]. Two of them are sensitive to thapsigargin, and only one releases Ca:+ uppon addition of InsP3, In neurons, the endoplasmic reticulum contains also at least two Ca:÷-stores [19]. The various inhibitory effect of neurotransmitters and thapsigargin on PS synthesis might suggest that also this process could occur in different subcompartments (pools) of the endoplasmic reticulum, one sensitive to InsP3 (smaller), and the other (larger) sensitive to thapsigargin. The results reported above show that the Ca2+ ionophore A23187 decreased of [14C]serine incorporation into PS after a long incubation time of cells (Fig. 4). Since it occured in the presence of 2 mM CaC12 it can not be explained by the inhibition of the synthesis of this phospholipid. This most probably reflects 1586
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phospholipasas activation and their action on already formed PS, and will be a subject of our further studies. Acknowledgment This work was supported by Committee for Scientific Research grant nr 406109101. References 1. Kanfer, J.N. (1980) Can. J. Biochem. 58, 1370-1380. 2. Baratiska, J. (1983) Adv. Lipid Res. 19, 163-184. 3. Murphy, S.N,. and Miller, R.J. (1988) Proc. Natl. Acad. Sci. USA, 87, 2466-2470. 4. Jensen, A.M., and Chiu, S.Y. (1991)J. Neurosci. 11, 1674-1684. 5. Murphy, N.P., Vaughan, P.F.T., Ball, S.G., and McCornack, G.J. (1991) J. Neurochem. 57, 21162123. 6. Thastrup, O., Cullen, P.J., Drobak, B., Hanley, M.R., and Dawson, A.P. (1991) Proc. Natl. Acad. Sci. USA, 87, 2466-2470. 7. Bian, J., Ghosh, T.K., Wang, J.-G., and Gill, D.L. (1991) J. Biol. Chem., 266, 8801-8806. 8. Ely, J.A., Ambroz, C., Baukal, A.J., Christensen, S.B., Balla, T., and Chem. 266, 186635-18640.
Catt, K.J. (1991) J. Biol.
9. Bligh, M.J., and Dyer, W.J., (1959) Can. J. Biochem. 37, 911-917. 10. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265275. 11. Nalgcz, M.J., Na/~cz, K.A., and Azzi, A. (1986)In Membrane Proteins (Azzi,A., Masotti, L., and Vecli, A., Eds.) pp. 67-75, Springer-Verlag, Berlin, Heidelberg, New York. 12. Hanley,M.R., Jackson, T.R., Cheung, W.T., Dreher, M., Gatti, A., Hawkins, P., Patterson, S.I., Vallejo, M., Dawson, A.P.. and Thastrup. O (1988) Cold Spring Harbor Symp. on Quatitative Biology 53, 435-445. 13. Brattin, W.J.,Jr., Waller, R.L., and Recknagel, R.O. (1982) J. Biol. Chem. 257, 10044-10051. 14. Bauchanan,A.G., and Kanfer, J.N. (1980) J. Neurochem. 34, 720-725. 15. Corazzi, L., Zborowski, J., Roberti, R., Binaglia, L., and Arienti, G. (1987) Bull. Mol. Med., 12, 19-31. 16. Baradska, J. (1989) FEBS Lett. 256, 33-37. 17. Pelassy, C., Aussell, C., and Fehlmann, M. (1989) Cell Signaling 1, 99-105. 18. Pelassy, C., Dallanegra, A., Aussell, C., and Fehlmann, M. (1989) Moll. Immun. 26, 1801-1806. 19. Henzi, V., and MacDermott, A.B. (1992) Neuroscience 46, 251-273.
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Pages 1582-1587
I N H I B I T I O N O F P H O S P H A T I D Y L S E R I N E S Y N T H E S I S BY G L U T A M A T E , A C E T Y L C H O L I N E , T H A P S I G A R G I N A N D I O N O P H O R E A23187 I N G L I O M A C6 C E L L S
M.Czarny ~, P.Sabala ~, A.Ucieklak:, L.Kaezmarek 3 and J.Barafiska ~° 1Dept. o f Cellular Biochemistry, 2Dept. of Cell Biology, 3Cell Culture Unit, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland Received June
15, 1992
Summary: Phosphatidylserine synthesis was studied in glioma C6 cells with [14C]serine and in the presence or absence of agents which increase the level of [Ca2+]~. It was found that glutamate and acetylcholine inhibited this synthesis by up to 40%, whereas thapsigargin and the ionophore A23187 inhibited by up to 70%. The inhibitory effect of thapsigargin and the A23187 was observed in Ca2+-free medium. The data show that the inhibition of this synthesis is caused by the Ca2+-depletion from endoplasmic reticulum, suggesting that the synthesis of phosphatidylserine occurs on the luminal side of these structures and can be regulated by transmembrane signaling systems. ® 1992 Academic P . . . . . Inc.
In mammalian cells phosphatidylserine (PS) is believed to be synthesized solely by the base exchange reaction that occurs mainly in the endoplasmic reticulum [1,2]. This reaction takes place in the absence of metabolic energy and is characterized by a requirement for Ca2÷. Therefore, any changes in Ca2÷ level in the cell should influence on the biosynthesis of this phospholipid. In order to investigate this, we used a number of agents which are known to alter the intracellular Ca 2÷, as glutamate, acetylcholine, thapsigargin and the calcium ionophore A23187, and examined their effect on PS synthesis in glioma C6 cells. Glutamate and acetylcholine are neurotransmitters which, by transmembrane signaling via cell surface receptors, lead to a rapid and sustained increase in the cytosolic Ca:+ concentration [3-5]. This is due to both the release of Ca2÷ from a non-mitochondrial intracellular stores caused by inositol(1,4,5)trisphosphate (InsP3), and the influx of extracellular Ca"-+ into the cell. Thapsigargin, a plant-derived sesquiterpene lactone, has been shown to be an effective and selective inhibitor of endoplasmic reticulum Ca:+-ATPase [6-8]. Thapsigargin mimics agonists' action by a rapid and marked increase in the concentration of cytosolic free Ca2+. This occurs also v/a a direct discharge of intracellular Ca"`+ stores
" To whom correspondence should be addressed. Abbreviations: [Ca2+]i - intracellular free calcium concentration; EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; PBS - phosphate buffer saline; PS - phosphatidylserine. 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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and Ca :+ influx, but without hydrolysis of inositolphospholipids [6-8]. The third type of agent used, the ionophore A23187 makes cell membranes permeabilized to Ca :+ and is consequently a convenient tool for the modulation of intracellular Ca:+ level. The present study demonstrates that all the agents used inhibit PS synthesis in glioma C6 cells. The inhibitory effect of ionophore and tapsigargin was observed in calcium-free medium. These data suggest that the PS synthesis occurs on the luminal side of the endoplasmic reticulum and can be regulated by intracellular events responsible for calcium fluxes. Materials and Methods Materials: Dulbecco's modified Eagle Medium, calf serum, trypsin solution and PBS were from Gibco BRL, UK. Glutamic acid, acetylcholine, eserine, the ionophore A23187 and EGTA were purchased from Sigma Chemical Co.,St.Louis,USA, and thapsigargin was from LC Services Corporation, Woburn, USA. Fura-2/AM was obtain from Molecular Probes, Inc.. L-[U-14C]serine was purchased from Praque, Czechoslovakia. TLC aluminium sheets silica gel 60 (without fluorescent indicator) were from Merck. Cell culture: Glioma C6 cells were cultured in Dulbecco's modified Eagle medium (DMEM) suplemented with 10% (v/v) calf serum, penicilin (50 IU/ml), streptomycin (50 #g/ml) and 2 mM L-glutamine under humidified atmosphere of 95% 02/5% CO,, at 37°C. The cells were passaged when confluent by using trypsin (0.25%), and the medium was changed twice a week. Cells for experiment were washed once with PBS. Phosphatidylserine synthesis in cultured cells: Cells were plated into 60 mm dishes at a concentration of 0.5 x l& cells/dish. Before measurements were made the medium was replaced with the following mixture (buffer A): 137 mM NaCI, 2.7 mM KC1, 1 mM Na:HPO4, 25 mM glucose, 20 mM Hepes (pH 7.4), 1 mM MgCI: and 1% (v/v) bovine serum albumin with the addition of L-[U-14C]serine (4.5 #M, 0.5 #Ci/ml). The cells were then incubated at 37°C in the buffer A for various periods of time and at various calcium concentration in the presence or absence of glutamate, acetylcholine, thapsigargin, A23187 and EGTA as indicated in Figures. After incubation, the medium was removed and the cells were washed with ice-cold PBS, scrapped off with cell lifter, collected in test tubes, spun down and resuspended in PBS. Lipids were extracted using chloroform/methanol (1 : 2; v/v) according to Bligh and Dyer [9]. Phospholipids were analysed by two dimensional TLC. The solvent in the first dimension was: chloroform/methanol/ammonium hydroxide/water (55:33:4:2 v/v); and in the second: chloroform/methanol/acetic acid/water (30:15:6:1 v/v). The spots of phospholipids were visualised with iodine and ninhidrin and scrapped off for radioactivity counting. Measurements of intracellular calcium: Cells were attached to glass coverslips, washed twice with PBS and once with buffer A and incubated in this buffer with 1 #M fura-2 (fura-2/acetoxymetyl ester) for 40 min at 37°C. Thereafter they were washed three times with buffer A. The third wash was left for 20 min at 37°C. Coverslips were mounted in a chamber over an Nikon Diaphot inverted-stage microscope equipped with a x20 fluorescence objective lens. All substances were added as a buffer A solutions. Digital fluorescent microscopy was used to determine the spatial distribution of [Ca:÷]i. Cells were alternately illuminated with 340 nm and 380 nm light from a xenon lamp. Emitted light was passed through a 500 nm barrier filter into an image-intensified camera (CCD). Experiments were carried out on a Joyce-Loebl Ltd. computerizing image-processing system Magiscan for calcium analyzis using Tardis V6.2. Determination of protein was preformed according to Lowry et al.[10] with the modification of Nalgcz et al [11]. Results Fig. I. shows that in treated glioma C6 cells PS synthesis is significantly altered compared to the basal condition. Approximately 30%-40% of this process was inhibited by neurotransmitters, whereas 1583
V o l . 186, N o . 3, 1992
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thapsigargin resulted in a marked (up to 70%) inhibition of the serine incorporation into PS. In contrast, the total uptake of serine by the cells was not affected by any of these compounds (not shown), demonstrating that the decrease in serine incorporation is not caused by changes in the uptake of this arninoacid. Glutamate was shown to induce an ihibition of PS synthesis in range of 10-6-10-3 M, whereas as low as 10 4 M acetylcholine was able to produce similar effect. Thapsigargin was effective in the concentration 0,5 x 10-S-l.5 x 10 -7 M (Fig.l). Similar results were already detectable 10 min after of incubation of the cells.
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Fig: 2. Effect of glutamate (A), acetylcholine 03) and thapsigargin (C) on intracellular Ca2+ in fura-2 loaded glioma C6 cells. A. Glutamate added (I00 ~M); B. acetylcholine added (0.1 ~M); C. thapsigargin added (0.1 ~M). Stimulation of cells were performed in the presence of external 2 mM Ca2÷. The addition of glutamate, acetylcholine and thapsigargin is indicated by arrows. Loading of cells with fura-2 is described in Materials and Methods. The results are from a single experiment typical of two others. Data points were taken every 1.1 s. 1584
Vol. 186, No. 3, 1992
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(min)
Fig. 3. Effect of calcium ionophore A23187 and thapsigarginon phosphatidylserineformation at varying Ca~* concentration. Cells were incubated for 15 min with [14C]serinein the absence (control) or presence of 5 #M A23187 (O) or 0.1 #M thapsigargin (A) at varying CaCI: concentration as indicated. The values obtained when using untreated cells are taken as 100% (basal line, control). Incubation of cells with Ca:+-free medium and with 200/zM EGTA diminished the control level by about 50%, whereas in the presence of CaCIz it was similar. Data are means 4- standard error from five experiments. Fig. 4. Time dependent effect of A23187 and thapsigargin on the [14C]phosphatidylserineformation. Glioma C6 cells were incubated with [14C]serine in the absence (controls, O) and presence of 5 #M A23187 (O) or 0.1 #M thapsigargin (A). The experiment shown is representative of two identical experiments.
To examine if glutamate, acetylcholine and thapsigargin indeed induce the increase in [Ca2+]i in glioma C6 cells, the effect of these agents was measured with fura-2 (Fig.2). Although the rise in [Ca2+]i mediated by 104 M glutamate was slower than the rapid initial spike induced by 10-7M acetylcholine, both neurotransmitters led to a sustained increase in the cytosolic calcium, higher than the basal level (about 100 nmoles) of resting cells. Treatment of fura-2 labeled cells by thapsigargin caused a slow increase in cytosolic calcium, and after a lag time period of 250 s, a sustained [Ca2+]~ rise. It is notable that using an image-processing Magiscan system one can observe a considerable heterogeneity in individual cell responses [12]. Nevertheless, the most of the cells responded in the manner shown in Fig.2. Fig.3 demonstrates the effect of calcium ionophore A23187 and thapsigargin on PS formation at varying Ca2÷ concentration. Thapsigargin inhibited this process in both the presence or absence of external calcium, and only at 10 mM CaC12 was this effect not so visible. The inhibition of PS synthesis by the ionophore A23187 occured in Ca2+-free medium, and continued at a low calcium concentration (100 #M). On the other hand, at 2mM CaC12, the ionophore was without effect, and at 10 mM CaCI:, it distinctly stimulated PS synthesis. 1585
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These results point to the direct regulation of PS synthesis by the intracellular calcium. However, when the cells are incubated at low CaCI2 concentration (100/~M), the addition of A23187 increases the level of calcium in the cytosol to the same value, which is a thousand fold higher than in the resting cells. Thus, the inhibition of PS synthesis observed in these conditions, can be explained only by the release of Ca2+ from the intracellular stores, i.e. from the endoplasmic reticulum, where a 103M concentration; of Ca2+ can be achieved [15]. The same phenomenon takes place in Ca2+-free medium. The data presented in Fig.1 and Fig.3 proceeded for 15 minutes intervals. Fig.4 shows a time-dependent effect of thapsigargin and A23187 on PS formation. As observed here, the inhibitory effect of thapsigargin is maintained for at least 4 h. However, A23187 expressed a lack of effect for the first 15 rain of incubation (see also Fig.3) but at longer times (1-4 h) strongly inhibited PS synthesis (up to 70%).
Discussion
In a mixed rat brain microsomal population, serine base-exchange enzyme is believed to be located on the luminal surface of the microsomes [14-15]. We have previously suggested a similar localization of the enzyme in the liver microsomes [16]. The results presented here provide further evidence that PS synthesis proceeds on the luminal leaflet of the endoplasmic reticulum, and can be regulated not only by Ca2+-ATPase [16], but by most other signals which control cellular Ca2+ fluctuations. The specific inhibition of PS synthesis during activation of Jurkat T lymphocytes was first reported by Pelassy et al. [17,18]. The authors suggested that this process is regulated similarly as increasing inositolphospholipids turnover, i.e. by transmembrane signaling, but the direct mechanism of this inhibition was obscure. The present study clarifies this problem by demonstrating that the inhibition of PS synthesis is induced by those events which lead to the depletion of Ca:+ from the endoplasmic reticulum. This was shown clearly by the effect of neurotransmitters which via InsP3 release Ca2+ from the intracellutar stores and decrease PS synthesis, and by the action of A23187 at low calcium concentrations, and in Ca2+-free medium. It was also indicated by the effect of thapsigargin, which being a specific blocker of the endoplasmic reticulum Ca2+ pump, prevents reuptake of calcium into these structures and so markedly inhibits PS synthesis. It has been reported that the thapsigargin-releasable Ca2+ pool within the cell is larger than InsP3 sensitive Ca:+-pool [7,8]. The evidence presented by Gill and coworkers indicates three different calcium pools in smooth muscle cell line [7]. Two of them are sensitive to thapsigargin, and only one releases Ca:+ uppon addition of InsP3, In neurons, the endoplasmic reticulum contains also at least two Ca:÷-stores [19]. The various inhibitory effect of neurotransmitters and thapsigargin on PS synthesis might suggest that also this process could occur in different subcompartments (pools) of the endoplasmic reticulum, one sensitive to InsP3 (smaller), and the other (larger) sensitive to thapsigargin. The results reported above show that the Ca2+ ionophore A23187 decreased of [14C]serine incorporation into PS after a long incubation time of cells (Fig. 4). Since it occured in the presence of 2 mM CaC12 it can not be explained by the inhibition of the synthesis of this phospholipid. This most probably reflects 1586
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phospholipasas activation and their action on already formed PS, and will be a subject of our further studies. Acknowledgment This work was supported by Committee for Scientific Research grant nr 406109101. References 1. Kanfer, J.N. (1980) Can. J. Biochem. 58, 1370-1380. 2. Baratiska, J. (1983) Adv. Lipid Res. 19, 163-184. 3. Murphy, S.N,. and Miller, R.J. (1988) Proc. Natl. Acad. Sci. USA, 87, 2466-2470. 4. Jensen, A.M., and Chiu, S.Y. (1991)J. Neurosci. 11, 1674-1684. 5. Murphy, N.P., Vaughan, P.F.T., Ball, S.G., and McCornack, G.J. (1991) J. Neurochem. 57, 21162123. 6. Thastrup, O., Cullen, P.J., Drobak, B., Hanley, M.R., and Dawson, A.P. (1991) Proc. Natl. Acad. Sci. USA, 87, 2466-2470. 7. Bian, J., Ghosh, T.K., Wang, J.-G., and Gill, D.L. (1991) J. Biol. Chem., 266, 8801-8806. 8. Ely, J.A., Ambroz, C., Baukal, A.J., Christensen, S.B., Balla, T., and Chem. 266, 186635-18640.
Catt, K.J. (1991) J. Biol.
9. Bligh, M.J., and Dyer, W.J., (1959) Can. J. Biochem. 37, 911-917. 10. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265275. 11. Nalgcz, M.J., Na/~cz, K.A., and Azzi, A. (1986)In Membrane Proteins (Azzi,A., Masotti, L., and Vecli, A., Eds.) pp. 67-75, Springer-Verlag, Berlin, Heidelberg, New York. 12. Hanley,M.R., Jackson, T.R., Cheung, W.T., Dreher, M., Gatti, A., Hawkins, P., Patterson, S.I., Vallejo, M., Dawson, A.P.. and Thastrup. O (1988) Cold Spring Harbor Symp. on Quatitative Biology 53, 435-445. 13. Brattin, W.J.,Jr., Waller, R.L., and Recknagel, R.O. (1982) J. Biol. Chem. 257, 10044-10051. 14. Bauchanan,A.G., and Kanfer, J.N. (1980) J. Neurochem. 34, 720-725. 15. Corazzi, L., Zborowski, J., Roberti, R., Binaglia, L., and Arienti, G. (1987) Bull. Mol. Med., 12, 19-31. 16. Baradska, J. (1989) FEBS Lett. 256, 33-37. 17. Pelassy, C., Aussell, C., and Fehlmann, M. (1989) Cell Signaling 1, 99-105. 18. Pelassy, C., Dallanegra, A., Aussell, C., and Fehlmann, M. (1989) Moll. Immun. 26, 1801-1806. 19. Henzi, V., and MacDermott, A.B. (1992) Neuroscience 46, 251-273.
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