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Veratridine-induced release of endogenous glutamate from rat brain cortex slices: a reappraisal of the role of calcium
Brain Research, 461 (1988) 377-380
377
Elsevier BRE 23126
Veratridine-induced release of endogenous glutamate from rat brain cortex slices: a reappraisal of the role of calcium S. Villanueva, Patricia Frenz, Y. Dragnic and F. Orrego Department of Physiology and Biophysics, Faculty of Medicine, Universityof Chile, Santiago (Chile) (Accepted 21 June 1988)
Key words: Endogenous glutamate; Brain cortex; Calcium; Veratridine
The efflux of endogenous glutamate from thin slices of rat brain cortex superfused in vitro with artificial cerebrospinal fluid (ACSF) was studied. Initially, glutamate efflux was very high (2.5 nmol/mg protein/min), possibly because of the cutting procedure, but declined sharply, and at 30 min of superfusion was 25 pmol/mg protein/min. In ACSF without added calcium, spontaneous glutamate efflux was always higher than that in calcium-containing medium, e.g. at 30 min it was 75 pmol/mg protein/min. Addition of 10 ktM veratridine for 2 min, between 30 and 32 min of superfusion, led, in ACSF with calcium, to an increase in glutamate efflux of 288%, when the maximum efflux following veratridine is compared to the glutamate efflux that immediately preceded the application of this drug (from 25 to 97 pmol/mg protein/min), while in ACSF without added calcium, veratridine induced an increase of only 117% (from 75 to 163 pmol/mg protein/min). These results are interpreted as due to the dual effect of veratridine. In calcium-containing ACSF, veratridine increases sodium influx which depolarizes the neurons and opens voltage-sensitive calcium channels. The increased intraneuronal calcium induces glutamate release from synaptic vesicles, while increased intracellular sodium enhances the release of soluble cytoplasmic glutamate by the reverse operation of the plasma membrane, sodium-dependent glutamate carrier. In ACSF without calcium, the release of vesicular glutamate is suppressed, while the sodium-dependent mechanism remains. This appears as if veratridineinduced glutamate efflux were only partially calcium-dependent.
The role of glutamate as a classical transmitter at n u m e r o u s excitatory synapses in the m a m m a l i a n CNS has gained considerable support from the detailed characterization of receptors for excitatory amino acids 4'16 and, especially, from the measurement of a high concentration of glutamate inside synaptic vesicles 12'13, for which an active u p t a k e mechanism has been found 9. In a p p a r e n t discrepancy with a classical transmitter role for glutamate, are the various reports that veratridine or drugs that act by a similar mechanism, induce a large release of g l u t a m a t e that is i n d e p e n d e n t of extracellular calcium, (Ca)o 1"6'10. Recently, a detailed analysis has been m a d e of the effects of veratridine and (Ca)o on the release of the classical transmitter noradrenaline, and also on that of the nontransmitter amino acid a - a m i n o i s o b u t y r a t e 11. These studies have led us to re-examine the question of
whether calcium-dependent glutamate release induced by veratridine can be d e m o n s t r a t e d . A positive answer to this would solve the discrepancies noted above, and further support a transmitter role for glutamate. We first tested w h e t h e r calcium ions had any effect on the sensitive and specific r a d i o r e c e p t o r assay used for glutamate 12'13 (Fig. 1). A l t h o u g h , as is well known 4, the cations present in artificial cerebrospinal fluid ( A C S F ) r e d u c e d the specific (3H)kainic acid ( K A ) binding to its receptors, no further change in specific or non-specific ( 3 H ) K A binding due to calcium ions could be seen (Fig. 1A). The sensitivity of the r a d i o r e c e p t o r assay for glutamate, was also unaffected by calcium (Fig. 1B). The ' s p o n t a n e o u s ' release of e n d o g e n o u s glutamate from superfused thin brain cortex slices was at first very high (Fig. 2), possibly due to the cutting
Correspondence: F. Orrego, Department of Physiology and Biophysics, Faculty of Medicine, Universidad de Chile, Casilla 70055, Santiago, Chile. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
378 A
lease between both tissue preparations were highly significant (Fig. 3). Regarding these results, two questions arise. First, if glutamate is a classical brain cortex transmitter, why is its veratridine-induced release not entirely suppressed in the absence of calcium, as is the case for (3H)noradrenaline under identical conditionsU? A likely interpretation for this is that, while noradrenaline is almost exclusively located in synaptic vesicles, glutamate, aside from a vesicular location,
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Fig. 1. Effect of calcium on the radioreceptor assay for glutamate. Glutamate was measured using a sensitive and specific receptor assay 12J3, in which binding of (3H)kainic acid (KA) (60 Ci/mmol, New England Nuclear) to rat brain synaptic membranes prepared following Enna and SnydeP is performed. Incubation was for 30 min at 4 °C in a volume of 2 ml. Final concentrations were: (3H)KA 2 nM; Tris-citrate, pH 7.0, 120 mM; synaptic membranes, 0.5 mg/ml. Non-specific binding, shown as hatched bars, was obtained by adding 200MM unlabeled KA. ACSF indicates that the binding assay contained 0.8 ml of artificial cerebrospinal fluid (concentration (mM): NaCI 124; KCI 5; KH2PO 4 1.24; MgSO 4 1.3; CaCI 2 1; NaHCO 3 26; glucose 10). Following the incubation, the tube contents were filtered on Whatman GF/B glass fiber filters, and washed 3 times with 4 ml of ice-cold 20 mM, pH 7.0, Tris-citrate buffer. In A each bar is the means of quadruplicate measurements. Bars represent total binding, and the non-hatched area is the specific binding. One S.E.M. is indicated. In B, Hill plots of the specific binding of L-glutamate (unlabeled) to the KA receptors is shown. The left plot was done in the presence of ACSF as indicated above, and the right plot also with ACSF but with no calcium added to it. Each point is the mean value of triplicate measurements.
procedures, and then decreased progressively. At all time points spontaneous release was higher in the medium without added calcium. Addition to control slices of 10#M veratridine for a 2-min period, led to a very large and prolonged increase in glutamate release. While in tissue superfused without added calcium, the release induced by veratridine was much smaller and of lesser duration. The differences in re-
500
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Fig. 2. Efflux of endogenous glutamate from brain cortex thin slices. Slices, about 0.35 mm thick, were cut with blade and blade-guide, and placed on Mcllwain quick-transfer electrodess, which were immersed in 3.5 ml of ACSF in a waterjacketed vessel at 36.5 °C. The fluid was continuously bubbled with 5% CO 2 in 0 2. It was aspirated and replenished with fresh fluid every 2 min. The first 2-min fraction was discarded. At the end of the experiment, endogenous glutamate was measured in duplicate in each aspirated fraction. The slices were homogenized and their protein content measured 7. Each curve represents 4 independent experiments. S.E.M. and presence or absence of calcium are indicated. Asterisks indicate P < 0.02, or less, when comparing by Student's t-test, spontaneous glutamate efflux between corresponding points in the two curves. Veratridine (10/~M) was present between minute 30 and 32 of superfusion. Four control, no veratridine, experiments in which superfusion was with ACSF throughout, were also done. The efflux curve, not shown, coincided entirely with the ACSF one in the figure up to 30 min.
379
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1 -I00 Fig. 3. Comparison of calcium effects on veratridine-induced efflux of glutamate. In the middle and right columns, the mean efflux of the 3 fractions following the application of veratridine (30 min of superfusion) are compared to the 3 fractions immediately preceding the drug. In the control, left hand column, the 3 fractions that follow min 30 of superfusion are compared to the 3 that precede it. One S.E.M. is indicated. *P < 0.001 relative to control efflux; **P < 0.01 relative to control; ***P < 0.02 of veratridine-induced release in the absence of calcium, relative to that in the presence of calcium.
is also present, as all amino acids, in the soluble cytoplasm, both in glutamatergic and in o t h e r transmitter-secreting neurons. This soluble amino acid increases its efflux from neurons when intracellular sodium, (Na)i, increases, by the action of veratridine, because of the reversibility of plasma m e m b r a n e , sod i u m - d e p e n d e n t , glutamate transporters 5. In fact, as in low (Ca)o the increase in (Na)i induced by veratri-
1 Benjamin, A.M. and Quastel, J.H., Locations of amino acids in brain slices from the rat, Biochem. J., 128 (1972) 631-646. 2 Caterall, W.A., Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes, Annu. Rev. Pharmacol. Toxicol., 20 (1980) 15-43. 3 Enna, S.J. and Snyder, S.H., Properties of GABA receptor binding in rat brain synaptic membranes, Brain Research, 100 (1975) 81-97. 4 Foster, A.C. and Fagg, G.E., Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103-164.
dine is enhanced 2'14, it seems also possible that sodiu m - d e p e n d e n t glutamate efflux induced by veratridine, is also enhanced in low (Ca)o. A second question relates to why this calcium dependency of veratridine-induced release has usually not been seen in previous work t,6A°. O n e reason may be that endogenous, and not r a d i o l a b e l e d glutamate was m e a s u r e d in the present work, as significant differences in cellular and subcellular localization exist between them ~5. Also, detection was of glutamate itself and not of a fluorescent or other chemical derivative, since derivatization of acidic amino acids frequently is not quantitative. Still a n o t h e r reason may be that a relatively low concentration of veratridine was applied for a short time, conditions shown to favour a high but reversible induction of transmitter release 11. In conclusion, the results p r e s e n t e d suggest that veratridine induces glutamate release from glutamatergic synaptic vesicles by a mechanism that has an absolute r e q u i r e m e n t on (Ca)o, together with a (Na)i-dependent efflux of soluble cytoplasmic, nontransmitter glutamate, that m a y be increased in low (Ca)o. The coexistence of both mechanisms appears, under the present e x p e r i m e n t a l conditions, as if veratridine induced a glutamate release that is only partially d e p e n d e n t on (Ca)o. U n d e r other experimental conditions, it seems possible that (Na)i-dependent glutamate efflux m a y entirely conceal the calciumd e p e n d e n t release of vesicular glutamate. W e are grateful to P. Cancino for technical assistance, and to Y o l a n d a Montoille for secretarial help. S u p p o r t e d by projects of F O N D E C Y T and of Dep a r t a m e n t o T6cnico de Investigaci6n, Universidad de Chile.
5 Kanner, B.I. and Sharon, I., Active transport of L-glutamate by membrane vesicles isolated from rat brain, Biochemistry, 17 (1978) 3949-3953. 6 Levi, G., Gallo, V. and Raiteri, M., A reevaluation of veratridine as a tool for studying the depolarization-induced release of neurotransmitters from nerve endings, Neurochem. Res., 5 (1980) 281-295. 7 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 8 Mcllwain, H. and Rodnight, R., Practical Neurochemistry, J.A. Churchill, London, 1962. 9 Naito, S. and Ueda, T., Characterization of glutamate up-
380 take into synaptic vesicles, J. Neurochem., 44 (1985) 99-109. 10 Norris, P.J., Dhaliwal, D.K., Druce, D.P. and Bradford, H.F., The suppression of stimulus-evoked release of amino acid neurotransmitters from synaptosomes by verapamil, J. Neurochem., 40 (1983) 514-521. 11 Pizarro, M., Valdivieso, M.P. and Orrego, F., Differential effects of veratridine and calcium on the release of (3H)noradrenaline and (lac)a-aminoisobutyrate from rat brain cortex slices, Neurochem. Int., 8 (1986) 207-212. 12 Riveros, N., Fiedler, J., Lagos, N., Mufioz, C. and Orrego, F., Glutamate in rat brain cortex synaptic vesicles: influence of the vesicle isolation procedure, Brain Research, 386
(1986) 405-408. 13 Riveros, N. and Orrego, F., A search in rat brain cortex synaptic vesicles for endogenous ligands for kainic acid receptors, Brain Research, 236 (1982) 492-496. 14 Straub, R., Die Wirkungen von Veratridine und Ionen auf das Ruhepotential markhaltiger Nervenfasern Frosches, Helv. Physiol. Acta, 14 (1956) 1-28. 15 Van den Berg, C.J., Krzali6, L., Mela, P. and Waelsch, H., Compartmentation of glutamate metabolism in brain, Biochem. J., 113 (1969) 281-290. 16 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21 (1981) 165-204.
377
Elsevier BRE 23126
Veratridine-induced release of endogenous glutamate from rat brain cortex slices: a reappraisal of the role of calcium S. Villanueva, Patricia Frenz, Y. Dragnic and F. Orrego Department of Physiology and Biophysics, Faculty of Medicine, Universityof Chile, Santiago (Chile) (Accepted 21 June 1988)
Key words: Endogenous glutamate; Brain cortex; Calcium; Veratridine
The efflux of endogenous glutamate from thin slices of rat brain cortex superfused in vitro with artificial cerebrospinal fluid (ACSF) was studied. Initially, glutamate efflux was very high (2.5 nmol/mg protein/min), possibly because of the cutting procedure, but declined sharply, and at 30 min of superfusion was 25 pmol/mg protein/min. In ACSF without added calcium, spontaneous glutamate efflux was always higher than that in calcium-containing medium, e.g. at 30 min it was 75 pmol/mg protein/min. Addition of 10 ktM veratridine for 2 min, between 30 and 32 min of superfusion, led, in ACSF with calcium, to an increase in glutamate efflux of 288%, when the maximum efflux following veratridine is compared to the glutamate efflux that immediately preceded the application of this drug (from 25 to 97 pmol/mg protein/min), while in ACSF without added calcium, veratridine induced an increase of only 117% (from 75 to 163 pmol/mg protein/min). These results are interpreted as due to the dual effect of veratridine. In calcium-containing ACSF, veratridine increases sodium influx which depolarizes the neurons and opens voltage-sensitive calcium channels. The increased intraneuronal calcium induces glutamate release from synaptic vesicles, while increased intracellular sodium enhances the release of soluble cytoplasmic glutamate by the reverse operation of the plasma membrane, sodium-dependent glutamate carrier. In ACSF without calcium, the release of vesicular glutamate is suppressed, while the sodium-dependent mechanism remains. This appears as if veratridineinduced glutamate efflux were only partially calcium-dependent.
The role of glutamate as a classical transmitter at n u m e r o u s excitatory synapses in the m a m m a l i a n CNS has gained considerable support from the detailed characterization of receptors for excitatory amino acids 4'16 and, especially, from the measurement of a high concentration of glutamate inside synaptic vesicles 12'13, for which an active u p t a k e mechanism has been found 9. In a p p a r e n t discrepancy with a classical transmitter role for glutamate, are the various reports that veratridine or drugs that act by a similar mechanism, induce a large release of g l u t a m a t e that is i n d e p e n d e n t of extracellular calcium, (Ca)o 1"6'10. Recently, a detailed analysis has been m a d e of the effects of veratridine and (Ca)o on the release of the classical transmitter noradrenaline, and also on that of the nontransmitter amino acid a - a m i n o i s o b u t y r a t e 11. These studies have led us to re-examine the question of
whether calcium-dependent glutamate release induced by veratridine can be d e m o n s t r a t e d . A positive answer to this would solve the discrepancies noted above, and further support a transmitter role for glutamate. We first tested w h e t h e r calcium ions had any effect on the sensitive and specific r a d i o r e c e p t o r assay used for glutamate 12'13 (Fig. 1). A l t h o u g h , as is well known 4, the cations present in artificial cerebrospinal fluid ( A C S F ) r e d u c e d the specific (3H)kainic acid ( K A ) binding to its receptors, no further change in specific or non-specific ( 3 H ) K A binding due to calcium ions could be seen (Fig. 1A). The sensitivity of the r a d i o r e c e p t o r assay for glutamate, was also unaffected by calcium (Fig. 1B). The ' s p o n t a n e o u s ' release of e n d o g e n o u s glutamate from superfused thin brain cortex slices was at first very high (Fig. 2), possibly due to the cutting
Correspondence: F. Orrego, Department of Physiology and Biophysics, Faculty of Medicine, Universidad de Chile, Casilla 70055, Santiago, Chile. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
378 A
lease between both tissue preparations were highly significant (Fig. 3). Regarding these results, two questions arise. First, if glutamate is a classical brain cortex transmitter, why is its veratridine-induced release not entirely suppressed in the absence of calcium, as is the case for (3H)noradrenaline under identical conditionsU? A likely interpretation for this is that, while noradrenaline is almost exclusively located in synaptic vesicles, glutamate, aside from a vesicular location,
A 80
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60
E
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40
20
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ACSF
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CONTROL
lO000
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06
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-7
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-5
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I
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I
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1000
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Fig. 1. Effect of calcium on the radioreceptor assay for glutamate. Glutamate was measured using a sensitive and specific receptor assay 12J3, in which binding of (3H)kainic acid (KA) (60 Ci/mmol, New England Nuclear) to rat brain synaptic membranes prepared following Enna and SnydeP is performed. Incubation was for 30 min at 4 °C in a volume of 2 ml. Final concentrations were: (3H)KA 2 nM; Tris-citrate, pH 7.0, 120 mM; synaptic membranes, 0.5 mg/ml. Non-specific binding, shown as hatched bars, was obtained by adding 200MM unlabeled KA. ACSF indicates that the binding assay contained 0.8 ml of artificial cerebrospinal fluid (concentration (mM): NaCI 124; KCI 5; KH2PO 4 1.24; MgSO 4 1.3; CaCI 2 1; NaHCO 3 26; glucose 10). Following the incubation, the tube contents were filtered on Whatman GF/B glass fiber filters, and washed 3 times with 4 ml of ice-cold 20 mM, pH 7.0, Tris-citrate buffer. In A each bar is the means of quadruplicate measurements. Bars represent total binding, and the non-hatched area is the specific binding. One S.E.M. is indicated. In B, Hill plots of the specific binding of L-glutamate (unlabeled) to the KA receptors is shown. The left plot was done in the presence of ACSF as indicated above, and the right plot also with ACSF but with no calcium added to it. Each point is the mean value of triplicate measurements.
procedures, and then decreased progressively. At all time points spontaneous release was higher in the medium without added calcium. Addition to control slices of 10#M veratridine for a 2-min period, led to a very large and prolonged increase in glutamate release. While in tissue superfused without added calcium, the release induced by veratridine was much smaller and of lesser duration. The differences in re-
500
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tL
TTI
r
i,--
50
VTD
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20
30
40
50
TIME
( min )
Fig. 2. Efflux of endogenous glutamate from brain cortex thin slices. Slices, about 0.35 mm thick, were cut with blade and blade-guide, and placed on Mcllwain quick-transfer electrodess, which were immersed in 3.5 ml of ACSF in a waterjacketed vessel at 36.5 °C. The fluid was continuously bubbled with 5% CO 2 in 0 2. It was aspirated and replenished with fresh fluid every 2 min. The first 2-min fraction was discarded. At the end of the experiment, endogenous glutamate was measured in duplicate in each aspirated fraction. The slices were homogenized and their protein content measured 7. Each curve represents 4 independent experiments. S.E.M. and presence or absence of calcium are indicated. Asterisks indicate P < 0.02, or less, when comparing by Student's t-test, spontaneous glutamate efflux between corresponding points in the two curves. Veratridine (10/~M) was present between minute 30 and 32 of superfusion. Four control, no veratridine, experiments in which superfusion was with ACSF throughout, were also done. The efflux curve, not shown, coincided entirely with the ACSF one in the figure up to 30 min.
379
300
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200
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~
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1 -I00 Fig. 3. Comparison of calcium effects on veratridine-induced efflux of glutamate. In the middle and right columns, the mean efflux of the 3 fractions following the application of veratridine (30 min of superfusion) are compared to the 3 fractions immediately preceding the drug. In the control, left hand column, the 3 fractions that follow min 30 of superfusion are compared to the 3 that precede it. One S.E.M. is indicated. *P < 0.001 relative to control efflux; **P < 0.01 relative to control; ***P < 0.02 of veratridine-induced release in the absence of calcium, relative to that in the presence of calcium.
is also present, as all amino acids, in the soluble cytoplasm, both in glutamatergic and in o t h e r transmitter-secreting neurons. This soluble amino acid increases its efflux from neurons when intracellular sodium, (Na)i, increases, by the action of veratridine, because of the reversibility of plasma m e m b r a n e , sod i u m - d e p e n d e n t , glutamate transporters 5. In fact, as in low (Ca)o the increase in (Na)i induced by veratri-
1 Benjamin, A.M. and Quastel, J.H., Locations of amino acids in brain slices from the rat, Biochem. J., 128 (1972) 631-646. 2 Caterall, W.A., Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes, Annu. Rev. Pharmacol. Toxicol., 20 (1980) 15-43. 3 Enna, S.J. and Snyder, S.H., Properties of GABA receptor binding in rat brain synaptic membranes, Brain Research, 100 (1975) 81-97. 4 Foster, A.C. and Fagg, G.E., Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103-164.
dine is enhanced 2'14, it seems also possible that sodiu m - d e p e n d e n t glutamate efflux induced by veratridine, is also enhanced in low (Ca)o. A second question relates to why this calcium dependency of veratridine-induced release has usually not been seen in previous work t,6A°. O n e reason may be that endogenous, and not r a d i o l a b e l e d glutamate was m e a s u r e d in the present work, as significant differences in cellular and subcellular localization exist between them ~5. Also, detection was of glutamate itself and not of a fluorescent or other chemical derivative, since derivatization of acidic amino acids frequently is not quantitative. Still a n o t h e r reason may be that a relatively low concentration of veratridine was applied for a short time, conditions shown to favour a high but reversible induction of transmitter release 11. In conclusion, the results p r e s e n t e d suggest that veratridine induces glutamate release from glutamatergic synaptic vesicles by a mechanism that has an absolute r e q u i r e m e n t on (Ca)o, together with a (Na)i-dependent efflux of soluble cytoplasmic, nontransmitter glutamate, that m a y be increased in low (Ca)o. The coexistence of both mechanisms appears, under the present e x p e r i m e n t a l conditions, as if veratridine induced a glutamate release that is only partially d e p e n d e n t on (Ca)o. U n d e r other experimental conditions, it seems possible that (Na)i-dependent glutamate efflux m a y entirely conceal the calciumd e p e n d e n t release of vesicular glutamate. W e are grateful to P. Cancino for technical assistance, and to Y o l a n d a Montoille for secretarial help. S u p p o r t e d by projects of F O N D E C Y T and of Dep a r t a m e n t o T6cnico de Investigaci6n, Universidad de Chile.
5 Kanner, B.I. and Sharon, I., Active transport of L-glutamate by membrane vesicles isolated from rat brain, Biochemistry, 17 (1978) 3949-3953. 6 Levi, G., Gallo, V. and Raiteri, M., A reevaluation of veratridine as a tool for studying the depolarization-induced release of neurotransmitters from nerve endings, Neurochem. Res., 5 (1980) 281-295. 7 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 8 Mcllwain, H. and Rodnight, R., Practical Neurochemistry, J.A. Churchill, London, 1962. 9 Naito, S. and Ueda, T., Characterization of glutamate up-
380 take into synaptic vesicles, J. Neurochem., 44 (1985) 99-109. 10 Norris, P.J., Dhaliwal, D.K., Druce, D.P. and Bradford, H.F., The suppression of stimulus-evoked release of amino acid neurotransmitters from synaptosomes by verapamil, J. Neurochem., 40 (1983) 514-521. 11 Pizarro, M., Valdivieso, M.P. and Orrego, F., Differential effects of veratridine and calcium on the release of (3H)noradrenaline and (lac)a-aminoisobutyrate from rat brain cortex slices, Neurochem. Int., 8 (1986) 207-212. 12 Riveros, N., Fiedler, J., Lagos, N., Mufioz, C. and Orrego, F., Glutamate in rat brain cortex synaptic vesicles: influence of the vesicle isolation procedure, Brain Research, 386
(1986) 405-408. 13 Riveros, N. and Orrego, F., A search in rat brain cortex synaptic vesicles for endogenous ligands for kainic acid receptors, Brain Research, 236 (1982) 492-496. 14 Straub, R., Die Wirkungen von Veratridine und Ionen auf das Ruhepotential markhaltiger Nervenfasern Frosches, Helv. Physiol. Acta, 14 (1956) 1-28. 15 Van den Berg, C.J., Krzali6, L., Mela, P. and Waelsch, H., Compartmentation of glutamate metabolism in brain, Biochem. J., 113 (1969) 281-290. 16 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21 (1981) 165-204.