ARCHIVES
Vol.
OF BIOCHEMISTRY
285, No. 1, February
AND
BIOPHYSICS
15, pp. 116-119,199l
Phosphatidylserine Vesicles Increase Ca*+ Uptake by Rat Brain Synaptosomes M. Floreani, Department
Received
P. Debetto, of Pharmacology,
September
and F. Carpenedol University
of Padova, Large E. Meneghetti
2, 35131 Padova, Italy
7, 1990
Phosphatidylserine (PS) vesicles incorporated into rat brain synaptosomes increased total Ca2+ uptake. Total Caz+ uptake was resolved in three components: K+ depolarization-induced Ca2+ uptake, Na+/Ca2+ exchange, and passive Ca2’ entry, which were differently affected by PS depending on the amount of incorporated phospholipid. K+ depolarization-induced Ca2’ uptake was stimulated by 0.06-O. 10 pmol PS/mg protein while 0. lo0.30 Fmol PS/mg protein increased Na+/Ca2+ exchange activity and passive Ca2’ entry but not K+ depolarizationinduced Ca2+ uptake. High amounts of incorporated PS also increased passive Rb’ uptake. o 1991 Academic press, IIIC.
The influx of Ca2+ into synaptosomes is regulated mainly by the voltage-dependent Ca2+channels (1,2) and Na+/C!a2+ exchange (2-5). Synaptosomes, retaining a membrane potential, can be depolarized by external K+, thus causing the opening of Ca2+ channels with consequent influx of Ca2+ (6-8). Moreover, in the presence of an outwardly directed Na+ gradient, synaptosomes exchange Na+ for Ca2+ (2-5). The dependence of Na+/C!a2+ exchange activity on acidic phospholipids, such as phosphatidylserine (PS),’ is well defined only in cardiac tissue (g-12), whereas the regulation of Ca2+ channels by membrane phospholipids, as studied in skeletal muscle (13, 14), is still debated. In cerebral tissue, the regulation of intracellular free Ca2+levels is fundamental for the coupling of depolarization to neurotransmitter release (15) and PS vesicles have been reported to influence norepinephrine turnover in hypothalamus (16) and acetylcholine output from cerebral cortex (17). It seems, therefore, particularly relevant to elucidate the modulation of membrane Ca2+fluxes by PS in brain. i To whom correspondence should be addressed. FAX: 049-831878. * Abbreviations used: PS, phosphatidylserine; Mops, 4-morpholinepropanesulfonic acid.
In the present study, we investigated the influence of PS on Ca2+ uptake in rat brain synaptosomes, showing that PS, upon incorporation into synaptosomal membrane, increased total Ca2+ uptake mediated by voltagedependent Ca2+ channels, Na+/Ca2+ exchange, and passive permeability. However, the three Ca2+uptake mechanisms were affected differently depending on the amount of incorporated PS. MATERIALS
AND
METHODS
Preparation and treatment of synaptosomes. Synaptosomes were prepared from rat brain according to Whittaker and Barker (18). Preparation of PS vesicles by sonication in 0.3 M sucrose in 25 mM TrisHCl (pH 7.4) and incorporation of PS into synaptosomal membranes were performed as described previously (19). Either control or PS-treated synaptosomes were resuspended in 160 mM NaCl-20 mM Mops/T& (pH 7.4) at a final protein concentration of about 4 mg/ml and incubated overnight at 4°C to allow Na+ to equilibrate across the synaptosomal membrane. For experiments designed to measure mRbf uptake, the sedimented synaptosomes were suspended in 0.32 M sucrose. Protein content was assayed by the method of Lowry et al. (20) using bovine serum albumin as standard, and the phospholipid content, assayed as total phosphorus, was measured by the method of Bartlett (21). Integrity of control and PS-treated synaptosomes was tested by assaying spectrophotometrically the lactate dehydrogenase activity as reported previously (22). Measurements of Gas+ uptake. Ca2+ uptake in synaptosomes was studied under experimental conditions allowing the influx of Cal+ through voltage-dependent Ca2+ channels to be distinguished from that coming via Na+/Ca’+ exchange and passive permeability. Aliquots of 3 pl (lo-12 pg of protein) of Na+-loaded synaptosomes were diluted into uptake media containing 160 mM KCl, choline chloride, or NaCl in 20 mM Mops/Tris (pH 7.4), containing 40 pM GCaC12 in a total volume of 200 pl, and incubated at 37°C. The Ca’+-uptake reactions were stopped after 30 s by rapid filtration (Millipore, 0.45 pm) of 70-pl aliquots. The filters were washed with 2 X 2.5 ml of ice-cold 160 mM KCl-20 mM Mops/Tris (pH 7.4) containing 1 mM LaCls, dried, and counted by liquid scintillation spectroscopy. Passive Ca*+ influx was the Cax+ uptake rate of Na+-loaded synaptosomes suspended in NaCl medium; Na+/Cax+ exchange was the Cazf uptake rate of Na+-loaded synaptosomes suspended in choline chloride medium minus the passive Ca2+ influx; Ca2+ influx through voltagedependent channels was the Ca2+ uptake rate of Na+-loaded synaptosomes suspended in KC1 medium minus both Na+/Ca2+ exchange and passive Ca’+ influx.
116 All
Copyright 0 1991 rights of reproduction
0003.9861/91 $3.00 by Academic Press, Inc. in any form reserved.
PHOSPHATIDYLSERINE
AND
CA’+
UPTAKE
IN
RAT
BRAIN
117
SYNAPTOSOMES
Effect of Incorporated PS on Ca2+ Uptake
0.504
I 0
0.25
0.50 pmol added
0.75 PS/mg
1.00
125
prot
FIG. 1. Increase in phospholipid content of rat brain synaptosomes treated with increasing amounts of PS vesicles. Phospholipid content was assayed as total phosphorus content as reported under Materials and Methods. Inset, incorporation of PS as a function of PS added exogenously to synaptosomes. Incorporation of PS was calculated as the difference between phospholipid contents of PS-treated and control synaptosomes. Data are means f SE from nine duplicate experiments.
Assay of passive %Rb+ uptake. About 0.3 mg of synaptosomal proteins suspended in 0.32 M sucrose was diluted into 0.5 ml of 160 mM choline chloride in 50 mM Tris/HCl (pH 7.4) containing 0.2 mM ouabain and 0.65 mM ffiRbC1. After different periods of time at 37”C, Rb+ uptake was stopped by rapid Millipore filtration of loo-p1 aliquots. The filters were washed with 2 X 2.5 ml of ice-cold 160 mM choline chloride in 20 mM Mops/Tris (pH 7.4), dried, and counted by liquid scintillation spectroscopy. Chemicals. Bovine brain PS was kindly provided by Fidia S.P.A. (Abano Terme, Italy). Before use, PS was acid-washed and then salified by the addition of 0.034% (w/v) MgC&. Tris, Mops, and ouabain were from Sigma Chemical Co. (St. Louis, MO). Calcium ionophore A23187 was from Calbiochem Corp. (San Diego, CA). “5CaC12 was obtained from New England Nuclear (Florence, Italy), %RbCl was from Amersham (Amersham, England). All other reagents were pure-grade.
RESULTS Incorporation
Ca2+ uptake in synaptosomes was markedly increased by incorporation of increasing amounts of PS (Fig. 2). PS increased the total Ca2+uptake when the uptake reactions were measured either at the initial rate (10 s) or the steady-state (4 min) (data not shown). The effect of PS was evident only upon incorporation of the phospholipid into synaptosomal membrane. PS vesicles, added directly to synaptosomes in the Ca2+-uptake media in the same amounts of incorporated PS, did not affect Ca2+uptake (data not shown). The total Ca2+ uptake by synaptosomes could be resolved in three components by changing the composition of the Ca2+-uptake medium (see Materials and Methods for details). Passive Ca2+ influx, K+ depolarization-induced Ca2+ uptake, and Na+/Ca2+ exchange accounted for 10,44, and 46% of the total Ca2+uptake, respectively. The three components were differently affected depending on the amounts of incorporated PS (Fig. 3). K+ depolarization-induced Ca2+ uptake was stimulated by low amounts (lower than 0.1 pmol PS/mg protein) of incorporated PS, falling to control levels when higher amounts of incorporated PS were used. At concentrations of PS higher than 0.1 pmol/mg protein, an increase in either Na+/Ca2+ exchange activity or passive Ca2+ influx was evident. To exclude the possibility that the effects of incorporated PS on the three components of Ca2+ uptake into synaptosomes could be due to different bindings of 45Ca2+ to PS vesicles in the three assay media, control experiments were performed by adding PS vesicles directly to the three different Ca2+-uptake media in the same amounts of the incorporated phospholipid and in the absence of synaptosomes. The cpm measured under these experimental conditions represented about 510% of the
of PS into Synaptosomes
Rat brain synaptosomes incubated with increasing amounts of PS vesicles increased their phospholipid content in a concentration-dependent way (Fig. 1). Saturation was achieved when about 0.3 pmol of PS were incorporated per milligram of protein. A linear relationship between incorporated and exogenously added PS was evident at the lowest concentrations used (0.04-0.32 pmol PS/mg protein) (Fig. 1, inset). The incorporation of PS into synaptosomes was time-dependent, reaching its maximum within 30 min of incubation (data not shown). Under these experimental conditions, all incorporated PS was unmodified at the membrane level as already reported (23). When synaptosomes were incubated for 30 min with 0.64 pmol PS/mg protein, the percentage amount of PS in respect to the total phospholipid content of synaptosomal membranes was 11 +- 1.5 (SE, n = 15) in the controls and 32 f 2.3 (SE, n = 15) in PS-treated synaptosomes (data not shown).
0 vmol
FIG. 2. Effect Ca2+ uptake in KC1 medium as rf- SE from nine
0.1 Incorporated
0.2 PS/mg
I
0.3 prot
of increasing amounts of incorporated PS on the total synaptosomes. Total Ca2+ uptake was assayed in the reported under Materials and Methods. Data are means duplicate experiments.
118
FLOREANI,
DEBETTO,
7.5-
AND
CARPENEDO 41
0
O-
0
9
0.1 pm01 incorporated
0.2 PS/mg
0.3 prot
FIG. 3. Effect of increasing amounts of incorporated PS on Cazf uptake in synaptosomes mediated by K+ depolarization (O), Na+/Ca’+ exchange (0), and passive Ca2+ influx (A). Ca*+ uptake through the three components was assayed as reported under Materials and Methods. Data are means + SE from nine duplicate experiments.
cpm counted in the presence of synaptosomes (data not shown). This low binding of 45Ca2f to PS could not account for the effects caused by different concentrations of PS on Ca2+ uptake. Effect of Incorporated PS on Synaptosomal Membrane Permeability Since high amounts of incorporated PS increased both the Naf/Ca2+ exchange activity and the passive Ca2+ influx, we investigated whether Ca2+uptake could be partly increased by an alteration of membrane permeability. The release of Ca2+ taken up, in the absence of a Na+ gradient, by synaptosomes which incorporated different amounts of PS (0.1 and 0.3 pmol PS/mg protein) was measured. Figure 4 shows that Ca2+ taken up by synaptosomes with 0.3 pmol incorporated PS/mg protein was increased by 108% with respect to that of control synaptosomes. Under the same experimental conditions, passive Ca2+ uptake increased by 39% in synaptosomes with 0.1 pmol PS incorporated/mg protein. When either control or PS-treated synaptosomes were treated with the Ca2+ ionophore A23187, a reduction in Ca2+ uptake was observed (Fig. 4), suggesting that most of the Ca2+ associated to synaptosomes entered and was not externally bound to membrane negatively charged phospholipids. Moreover, 0.3 pmol incorporated PS/mg protein but not 0.1 pmol incorporated PS/mg protein significantly increased passive %Rb+ uptake into synaptosomes (Fig. 5). DISCUSSION
Present results show that PS, upon incorporation into rat brain synaptosomal membranes, increases total Ca2+
I
2 3 time (mkn)
4
5
I 6
FIG. 4. Passive %a*+ uptake in control and PS-treated synaptosomes. Control synaptosomes (0) and synaptosomes with 0.1 (A) and 0.3 (0) rmol incorporated PS/mg protein, after a 3-min incubation at 37’C in the NaCl medium were diluted (4) 1:50 into a Ca*+-free medium containing 160 mM NaCl-20 mM Mops/Tris (pH 7.4), and 50 pM A23187. Data are means + SE from four duplicate experiments.
uptake mediated by passive permeability, K’ depolarization-induced Ca2+ uptake, and Na+/Ca2+ exchange. The three components contribute to the increase in total Ca2+ uptake to different extents, being differently affected by the phospholipid depending on the amounts of incorporated PS. K+ depolarization-induced Ca2+ uptake is stimulated by low amounts of incorporated PS and accounts for the 75~30% of the increase in total Ca2+uptake, the remaining 25-20% being sustained by passive entry. Since it is generally accepted that the K+ depolarization-induced Ca2+ uptake is mediated by the opening of the voltage-depen-
time (mm) FIG. 5. Passive =Rb+ uptake in control and PS-treated synaptosomes. %Rb+ uptake was evaluated in control synaptosomes (0) and in synaptosomes with 0.1 (A) and 0.3 (0) pmol incorporated PS/mg protein as described under Materials and Methods. Data are means f SE from four duplicate experiments.
PHOSPHATIDYLSERINE
AND
CA*+
UPTAKE
dent Ca2+ channels (6-E!), the stimulating effect of low amounts of incorporated PS under depolarizing conditions may suggest a regulation of Ca2+ channels by PS. However, it remains to be evaluated whether PS stimulates directly the Ca2+ channel activity or increases the capacity of the system because of an alteration of membrane lipid environment. The possibility that PS modulates the functioning of Ca 2+ channels is supported by data of Coronado (13) who found that Ca2+ channels from skeletal muscle reconstituted into PS planar bilayers have decreased open-channel lifetimes. In our synaptosomal preparation, the decrease in Kf depolarization-induced Ca2+ uptake caused by high amounts of incorporated PS may be due either to a direct effect of high PS amounts on Ca2+ channels or to an inhibitory effect of internal Ca2+, whose concentration is increased as a consequence of an increase in both passive permeability and Na+/Ca2+ exchange activity. In fact, high [Ca2+]i inhibit voltage-dependent Ca2+ channels in several cell systems [for a review see (24)] and in synaptosomes (personal observation). Both Ca2+ membrane permeability and Na+/Ca’+ exchange activity are stimulated by the highest amounts of incorporated PS and account for the increase in total Ca2+ uptake by 40 and 60%, respectively. The increase in membrane permeability involves not only Ca2+ but also monovalent ions, as suggested by the increase in “Rb+ uptake. An altered ion membrane permeability after incorporation or fusion of PS vesicles with cells has been already reported by Papahadjopoulos et al. (25) and Poste (26). Moreover, an increase in passive membrane permeability has been hypothesized to explain the stimulation of Na+/K+ ATPase activity caused by the high amount of incorporated PS both in intact rat brain synaptosomes (19) and in BHK 21 cells (22). The concomitant increase in ion membrane permeability and Na+/Ca2+ exchange activity caused by high amounts of incorporated PS makes it difficult to establish the mechanisms of PS effects on Na+/Ca2+ exchange. An increase in [Ca2+]i consequent to the increase in passive Ca2+ entry may stimulate Na+/Ca2+ exchange activity, as reported in cardiac sarcolemmal vesicles (27,28). On the other hand, as reported in reconstituted cardiac sarcolemma1 vesicles (10-12) a creation of domains of altered fluidity that facilitates the functioning of the exchanger and/or a direct activation of the protein(s) by PS cannot be excluded. In conclusion, we demonstrated that PS modifies Ca2+ uptake upon incorporation into the membranes of rat brain synaptosomes. Although the molecular mechanisms underlying this effect are not fully elucidated, our results are particularly relevant to the understanding of Ca2+dependent, pharmacological effects of PS in brain.
IN
RAT
BRAIN
119
SYNAPTOSOMES
ACKNOWLEDGMENTS We thank Prof. R. M. Gaion for critical revision of this manuscript. This research was supported by a grant from M.P.I. (60%).
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K. D. (1985)
10. Luciani,
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11. Vemuri, R., and 937,258-268. 12. Debetto, Biophys. 13. Coronado,
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247, 617-655. U., and Silver,
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14. Flockerzi, V., Oeken, H. J., Hofmann, F., Pelzer, D., Cavalib, A., and Trautwein, W. (1986) Nature (London) 323,66-68. 15. Harvey, A. M., and Macintosh, F. C. (1940) J. Physiol. 97, 408416. G., Leon, A., Mazzari, S., Savoini, G., Teolato, S., and 16. Toffano, Orlando, P. (1978) Life Sci. 23, 1093-1102. 17. Casamenti, F., Mantovani, J. Neurochem. 32,529-533. 18. Whittaker, l-52. 19. Floreani, Biophys. 20. Lowry, (1951) 21. Bartlett, 22. Floreani, Commun.
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V. P., and Barker, M., Bonetti, Res. Commun.
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0. H., Rosebrough, N. J., Farr, J. Biol. C&m. 193,265-275. G. R. (1959)
L., and Pepeu,
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Carpenedo,
Chem.
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Biochem.
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23. Floreani, M., and Carpenedo, F. (1984) Biochem. Commun. 119,821-827. 24. Pelzer, D., Pelzer, S., and McDonald, T. F. (1990) Biochem. Pharmacol. 114,107-207. 25. Papahadjopoulos, D., Poste, in Membrane Biology (Kern, New York.
Reu. Physiol.
G., and Wail, W. J. (1979) in Methods E. D., Ed.), Vol. 10, pp. I-110, Plenum,
26. Poste, G. (1980) in Liposomes in Biological Systems (Gregoriadis, G., and Allison, A. C., Eds.), pp. 101-151, Wiley, New York. 27. Reeves, C23.
J. P., and Poronnik,
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28. Debetto, P., Luciani, S., Tessari, M., Floreani, F. (1989) Biochem. Pharmacol. 38, 1137-1145.
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252,
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M., and Carpenedo,
Vol.
OF BIOCHEMISTRY
285, No. 1, February
AND
BIOPHYSICS
15, pp. 116-119,199l
Phosphatidylserine Vesicles Increase Ca*+ Uptake by Rat Brain Synaptosomes M. Floreani, Department
Received
P. Debetto, of Pharmacology,
September
and F. Carpenedol University
of Padova, Large E. Meneghetti
2, 35131 Padova, Italy
7, 1990
Phosphatidylserine (PS) vesicles incorporated into rat brain synaptosomes increased total Ca2+ uptake. Total Caz+ uptake was resolved in three components: K+ depolarization-induced Ca2+ uptake, Na+/Ca2+ exchange, and passive Ca2’ entry, which were differently affected by PS depending on the amount of incorporated phospholipid. K+ depolarization-induced Ca2’ uptake was stimulated by 0.06-O. 10 pmol PS/mg protein while 0. lo0.30 Fmol PS/mg protein increased Na+/Ca2+ exchange activity and passive Ca2’ entry but not K+ depolarizationinduced Ca2+ uptake. High amounts of incorporated PS also increased passive Rb’ uptake. o 1991 Academic press, IIIC.
The influx of Ca2+ into synaptosomes is regulated mainly by the voltage-dependent Ca2+channels (1,2) and Na+/C!a2+ exchange (2-5). Synaptosomes, retaining a membrane potential, can be depolarized by external K+, thus causing the opening of Ca2+ channels with consequent influx of Ca2+ (6-8). Moreover, in the presence of an outwardly directed Na+ gradient, synaptosomes exchange Na+ for Ca2+ (2-5). The dependence of Na+/C!a2+ exchange activity on acidic phospholipids, such as phosphatidylserine (PS),’ is well defined only in cardiac tissue (g-12), whereas the regulation of Ca2+ channels by membrane phospholipids, as studied in skeletal muscle (13, 14), is still debated. In cerebral tissue, the regulation of intracellular free Ca2+levels is fundamental for the coupling of depolarization to neurotransmitter release (15) and PS vesicles have been reported to influence norepinephrine turnover in hypothalamus (16) and acetylcholine output from cerebral cortex (17). It seems, therefore, particularly relevant to elucidate the modulation of membrane Ca2+fluxes by PS in brain. i To whom correspondence should be addressed. FAX: 049-831878. * Abbreviations used: PS, phosphatidylserine; Mops, 4-morpholinepropanesulfonic acid.
In the present study, we investigated the influence of PS on Ca2+ uptake in rat brain synaptosomes, showing that PS, upon incorporation into synaptosomal membrane, increased total Ca2+ uptake mediated by voltagedependent Ca2+ channels, Na+/Ca2+ exchange, and passive permeability. However, the three Ca2+uptake mechanisms were affected differently depending on the amount of incorporated PS. MATERIALS
AND
METHODS
Preparation and treatment of synaptosomes. Synaptosomes were prepared from rat brain according to Whittaker and Barker (18). Preparation of PS vesicles by sonication in 0.3 M sucrose in 25 mM TrisHCl (pH 7.4) and incorporation of PS into synaptosomal membranes were performed as described previously (19). Either control or PS-treated synaptosomes were resuspended in 160 mM NaCl-20 mM Mops/T& (pH 7.4) at a final protein concentration of about 4 mg/ml and incubated overnight at 4°C to allow Na+ to equilibrate across the synaptosomal membrane. For experiments designed to measure mRbf uptake, the sedimented synaptosomes were suspended in 0.32 M sucrose. Protein content was assayed by the method of Lowry et al. (20) using bovine serum albumin as standard, and the phospholipid content, assayed as total phosphorus, was measured by the method of Bartlett (21). Integrity of control and PS-treated synaptosomes was tested by assaying spectrophotometrically the lactate dehydrogenase activity as reported previously (22). Measurements of Gas+ uptake. Ca2+ uptake in synaptosomes was studied under experimental conditions allowing the influx of Cal+ through voltage-dependent Ca2+ channels to be distinguished from that coming via Na+/Ca’+ exchange and passive permeability. Aliquots of 3 pl (lo-12 pg of protein) of Na+-loaded synaptosomes were diluted into uptake media containing 160 mM KCl, choline chloride, or NaCl in 20 mM Mops/Tris (pH 7.4), containing 40 pM GCaC12 in a total volume of 200 pl, and incubated at 37°C. The Ca’+-uptake reactions were stopped after 30 s by rapid filtration (Millipore, 0.45 pm) of 70-pl aliquots. The filters were washed with 2 X 2.5 ml of ice-cold 160 mM KCl-20 mM Mops/Tris (pH 7.4) containing 1 mM LaCls, dried, and counted by liquid scintillation spectroscopy. Passive Ca*+ influx was the Cax+ uptake rate of Na+-loaded synaptosomes suspended in NaCl medium; Na+/Cax+ exchange was the Cazf uptake rate of Na+-loaded synaptosomes suspended in choline chloride medium minus the passive Ca2+ influx; Ca2+ influx through voltagedependent channels was the Ca2+ uptake rate of Na+-loaded synaptosomes suspended in KC1 medium minus both Na+/Ca2+ exchange and passive Ca’+ influx.
116 All
Copyright 0 1991 rights of reproduction
0003.9861/91 $3.00 by Academic Press, Inc. in any form reserved.
PHOSPHATIDYLSERINE
AND
CA’+
UPTAKE
IN
RAT
BRAIN
117
SYNAPTOSOMES
Effect of Incorporated PS on Ca2+ Uptake
0.504
I 0
0.25
0.50 pmol added
0.75 PS/mg
1.00
125
prot
FIG. 1. Increase in phospholipid content of rat brain synaptosomes treated with increasing amounts of PS vesicles. Phospholipid content was assayed as total phosphorus content as reported under Materials and Methods. Inset, incorporation of PS as a function of PS added exogenously to synaptosomes. Incorporation of PS was calculated as the difference between phospholipid contents of PS-treated and control synaptosomes. Data are means f SE from nine duplicate experiments.
Assay of passive %Rb+ uptake. About 0.3 mg of synaptosomal proteins suspended in 0.32 M sucrose was diluted into 0.5 ml of 160 mM choline chloride in 50 mM Tris/HCl (pH 7.4) containing 0.2 mM ouabain and 0.65 mM ffiRbC1. After different periods of time at 37”C, Rb+ uptake was stopped by rapid Millipore filtration of loo-p1 aliquots. The filters were washed with 2 X 2.5 ml of ice-cold 160 mM choline chloride in 20 mM Mops/Tris (pH 7.4), dried, and counted by liquid scintillation spectroscopy. Chemicals. Bovine brain PS was kindly provided by Fidia S.P.A. (Abano Terme, Italy). Before use, PS was acid-washed and then salified by the addition of 0.034% (w/v) MgC&. Tris, Mops, and ouabain were from Sigma Chemical Co. (St. Louis, MO). Calcium ionophore A23187 was from Calbiochem Corp. (San Diego, CA). “5CaC12 was obtained from New England Nuclear (Florence, Italy), %RbCl was from Amersham (Amersham, England). All other reagents were pure-grade.
RESULTS Incorporation
Ca2+ uptake in synaptosomes was markedly increased by incorporation of increasing amounts of PS (Fig. 2). PS increased the total Ca2+uptake when the uptake reactions were measured either at the initial rate (10 s) or the steady-state (4 min) (data not shown). The effect of PS was evident only upon incorporation of the phospholipid into synaptosomal membrane. PS vesicles, added directly to synaptosomes in the Ca2+-uptake media in the same amounts of incorporated PS, did not affect Ca2+uptake (data not shown). The total Ca2+ uptake by synaptosomes could be resolved in three components by changing the composition of the Ca2+-uptake medium (see Materials and Methods for details). Passive Ca2+ influx, K+ depolarization-induced Ca2+ uptake, and Na+/Ca2+ exchange accounted for 10,44, and 46% of the total Ca2+uptake, respectively. The three components were differently affected depending on the amounts of incorporated PS (Fig. 3). K+ depolarization-induced Ca2+ uptake was stimulated by low amounts (lower than 0.1 pmol PS/mg protein) of incorporated PS, falling to control levels when higher amounts of incorporated PS were used. At concentrations of PS higher than 0.1 pmol/mg protein, an increase in either Na+/Ca2+ exchange activity or passive Ca2+ influx was evident. To exclude the possibility that the effects of incorporated PS on the three components of Ca2+ uptake into synaptosomes could be due to different bindings of 45Ca2+ to PS vesicles in the three assay media, control experiments were performed by adding PS vesicles directly to the three different Ca2+-uptake media in the same amounts of the incorporated phospholipid and in the absence of synaptosomes. The cpm measured under these experimental conditions represented about 510% of the
of PS into Synaptosomes
Rat brain synaptosomes incubated with increasing amounts of PS vesicles increased their phospholipid content in a concentration-dependent way (Fig. 1). Saturation was achieved when about 0.3 pmol of PS were incorporated per milligram of protein. A linear relationship between incorporated and exogenously added PS was evident at the lowest concentrations used (0.04-0.32 pmol PS/mg protein) (Fig. 1, inset). The incorporation of PS into synaptosomes was time-dependent, reaching its maximum within 30 min of incubation (data not shown). Under these experimental conditions, all incorporated PS was unmodified at the membrane level as already reported (23). When synaptosomes were incubated for 30 min with 0.64 pmol PS/mg protein, the percentage amount of PS in respect to the total phospholipid content of synaptosomal membranes was 11 +- 1.5 (SE, n = 15) in the controls and 32 f 2.3 (SE, n = 15) in PS-treated synaptosomes (data not shown).
0 vmol
FIG. 2. Effect Ca2+ uptake in KC1 medium as rf- SE from nine
0.1 Incorporated
0.2 PS/mg
I
0.3 prot
of increasing amounts of incorporated PS on the total synaptosomes. Total Ca2+ uptake was assayed in the reported under Materials and Methods. Data are means duplicate experiments.
118
FLOREANI,
DEBETTO,
7.5-
AND
CARPENEDO 41
0
O-
0
9
0.1 pm01 incorporated
0.2 PS/mg
0.3 prot
FIG. 3. Effect of increasing amounts of incorporated PS on Cazf uptake in synaptosomes mediated by K+ depolarization (O), Na+/Ca’+ exchange (0), and passive Ca2+ influx (A). Ca*+ uptake through the three components was assayed as reported under Materials and Methods. Data are means + SE from nine duplicate experiments.
cpm counted in the presence of synaptosomes (data not shown). This low binding of 45Ca2f to PS could not account for the effects caused by different concentrations of PS on Ca2+ uptake. Effect of Incorporated PS on Synaptosomal Membrane Permeability Since high amounts of incorporated PS increased both the Naf/Ca2+ exchange activity and the passive Ca2+ influx, we investigated whether Ca2+uptake could be partly increased by an alteration of membrane permeability. The release of Ca2+ taken up, in the absence of a Na+ gradient, by synaptosomes which incorporated different amounts of PS (0.1 and 0.3 pmol PS/mg protein) was measured. Figure 4 shows that Ca2+ taken up by synaptosomes with 0.3 pmol incorporated PS/mg protein was increased by 108% with respect to that of control synaptosomes. Under the same experimental conditions, passive Ca2+ uptake increased by 39% in synaptosomes with 0.1 pmol PS incorporated/mg protein. When either control or PS-treated synaptosomes were treated with the Ca2+ ionophore A23187, a reduction in Ca2+ uptake was observed (Fig. 4), suggesting that most of the Ca2+ associated to synaptosomes entered and was not externally bound to membrane negatively charged phospholipids. Moreover, 0.3 pmol incorporated PS/mg protein but not 0.1 pmol incorporated PS/mg protein significantly increased passive %Rb+ uptake into synaptosomes (Fig. 5). DISCUSSION
Present results show that PS, upon incorporation into rat brain synaptosomal membranes, increases total Ca2+
I
2 3 time (mkn)
4
5
I 6
FIG. 4. Passive %a*+ uptake in control and PS-treated synaptosomes. Control synaptosomes (0) and synaptosomes with 0.1 (A) and 0.3 (0) rmol incorporated PS/mg protein, after a 3-min incubation at 37’C in the NaCl medium were diluted (4) 1:50 into a Ca*+-free medium containing 160 mM NaCl-20 mM Mops/Tris (pH 7.4), and 50 pM A23187. Data are means + SE from four duplicate experiments.
uptake mediated by passive permeability, K’ depolarization-induced Ca2+ uptake, and Na+/Ca2+ exchange. The three components contribute to the increase in total Ca2+ uptake to different extents, being differently affected by the phospholipid depending on the amounts of incorporated PS. K+ depolarization-induced Ca2+ uptake is stimulated by low amounts of incorporated PS and accounts for the 75~30% of the increase in total Ca2+uptake, the remaining 25-20% being sustained by passive entry. Since it is generally accepted that the K+ depolarization-induced Ca2+ uptake is mediated by the opening of the voltage-depen-
time (mm) FIG. 5. Passive =Rb+ uptake in control and PS-treated synaptosomes. %Rb+ uptake was evaluated in control synaptosomes (0) and in synaptosomes with 0.1 (A) and 0.3 (0) pmol incorporated PS/mg protein as described under Materials and Methods. Data are means f SE from four duplicate experiments.
PHOSPHATIDYLSERINE
AND
CA*+
UPTAKE
dent Ca2+ channels (6-E!), the stimulating effect of low amounts of incorporated PS under depolarizing conditions may suggest a regulation of Ca2+ channels by PS. However, it remains to be evaluated whether PS stimulates directly the Ca2+ channel activity or increases the capacity of the system because of an alteration of membrane lipid environment. The possibility that PS modulates the functioning of Ca 2+ channels is supported by data of Coronado (13) who found that Ca2+ channels from skeletal muscle reconstituted into PS planar bilayers have decreased open-channel lifetimes. In our synaptosomal preparation, the decrease in Kf depolarization-induced Ca2+ uptake caused by high amounts of incorporated PS may be due either to a direct effect of high PS amounts on Ca2+ channels or to an inhibitory effect of internal Ca2+, whose concentration is increased as a consequence of an increase in both passive permeability and Na+/Ca2+ exchange activity. In fact, high [Ca2+]i inhibit voltage-dependent Ca2+ channels in several cell systems [for a review see (24)] and in synaptosomes (personal observation). Both Ca2+ membrane permeability and Na+/Ca’+ exchange activity are stimulated by the highest amounts of incorporated PS and account for the increase in total Ca2+ uptake by 40 and 60%, respectively. The increase in membrane permeability involves not only Ca2+ but also monovalent ions, as suggested by the increase in “Rb+ uptake. An altered ion membrane permeability after incorporation or fusion of PS vesicles with cells has been already reported by Papahadjopoulos et al. (25) and Poste (26). Moreover, an increase in passive membrane permeability has been hypothesized to explain the stimulation of Na+/K+ ATPase activity caused by the high amount of incorporated PS both in intact rat brain synaptosomes (19) and in BHK 21 cells (22). The concomitant increase in ion membrane permeability and Na+/Ca2+ exchange activity caused by high amounts of incorporated PS makes it difficult to establish the mechanisms of PS effects on Na+/Ca2+ exchange. An increase in [Ca2+]i consequent to the increase in passive Ca2+ entry may stimulate Na+/Ca2+ exchange activity, as reported in cardiac sarcolemmal vesicles (27,28). On the other hand, as reported in reconstituted cardiac sarcolemma1 vesicles (10-12) a creation of domains of altered fluidity that facilitates the functioning of the exchanger and/or a direct activation of the protein(s) by PS cannot be excluded. In conclusion, we demonstrated that PS modifies Ca2+ uptake upon incorporation into the membranes of rat brain synaptosomes. Although the molecular mechanisms underlying this effect are not fully elucidated, our results are particularly relevant to the understanding of Ca2+dependent, pharmacological effects of PS in brain.
IN
RAT
BRAIN
119
SYNAPTOSOMES
ACKNOWLEDGMENTS We thank Prof. R. M. Gaion for critical revision of this manuscript. This research was supported by a grant from M.P.I. (60%).
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