Comparison of the Release of Exogenous and Endogenous Excitatory Amino k i d s f b m Rat Cerebral Cortex A. M. C. T. REITER," AND M. BOISCHELLER" Dcparhncna of "Pyhiat?y and Phamaa&y University ofPimbutgh Medkal Center Pimbutgh, Pnrnsylpania 15213 The utility of the efflux of preaccumulated ~[3H]aspar&ate(a nonmetabolizable acidic amino acid) as an index of the release of endogenous Gaspartate and Gglutamate has been questioned, largely because of a study by Ferkany and Coyle using hippocampal slices.' This showed that kainic acid (KA), a naturally occurring excitotoxin, induced a Ca2+-dependent increase in the efflux of endogenous excitatory amino acids (EAAs), but had no e&ct on the efflux of preaccumulated ~ [ ~ H ] a s p a r t aand t e glutamate am ate. A major difficulty of such studies is to distinguish release from uptake. This is particularly relevant fbr KA because the reported effects of this toxin on EAA release were apparent at concentrations known to inhibit h@ affinity EAA uptake, 0.1-1 mM.3 Although Ferkany and Coyle supefised slices (0.25 mllmin), the extent to which u p take processes confbunded their measures is not dear.Our study therefore reexamines the effect of KA on the efflux of preaccumulated ~ [ ~ H l a s p a r t aand t e endogenous aspa art ate and tglutamate using a superfusion rate over six times greater than that employed by Ferkany and Coyle.
MATERIALS AND METHODS Slices of rat cerebral cortex (cut in two planes separated by 4 5') were added to 10 ml of ice-cold buffer solution (pH 7.4) which had the following composition (mM): NaCll28, KCI 5, MgW4 1.2, Na2PO4 1.0, CaCl2 2.6, glucose 10, N-2-hydroxyethylpipcrazine-K-2-ethanesulfbnic acid (Hepes) 20, and was then oxygenated (95% 0 2 5% C02). The minislices were dispersed, allowed to settle, oxygenated and incubated in a water bath (37OC, 5 min). ~[JHIaspartatewas then added to give a final concentration of 200 nM and the incubation continued fbr an additional 25 minutes. After washing four times with 6 ml of either normal media or media with CaC12 replaced by equimolar MgC12, the suspension (4 ml) was mixed over a magnetic stirrer and 200 PL aliquots transkrred to each chamber (300 PL in volume) of the Supefision 20 system (Brandel, Gaithersberg, MD). Following a 30-minute period of supefision (35OC, 1.6 mL/min), 12 successive 0.5 min fractions were collected. KA (0.01-1 mM) 361
362
ANNALS NEW YORK ACADEMY OF SCIENCES
TABLE 1. Comparison of the K+evoked Release of Exogenous and Endogenous Excitatory Amino Acid# ~[~Hlaspartate
waspartate
L-glutamate
50 mM K+ 100 100 100 5 mM K+ 2.4 f 0.2c 10.5 f 3.1) 1.5 f 0.4c 50 mM K+,0 mM Ca2+ 10.7 f 3.lC 31.1 f 20.1 4.7f 3.2) 50 mM K+, 1 pM TTX 77.0 f 12.4 81.7 f 6.7 83.0f 12.3 a Data are expressed as a percent of control values (50mM K+) f SEM for three separate experiments. Mean control values (* SEM) for D[JH]aspartate, taspartate, and Gglutamate were 34,066 f 5,470 dpmlmg protein, 327 f 93 pmollmg protein, and 858 f 147 pmollmg protein, respectively. l T X : teuodotoxin. b p < 0.05,c p < 0.01(analysis of variance followed by paired t-tests). Statistical analyses were performed on original data.
-
was added at t 15-36 min. In order to examine the Ca2+-dependencyof release, CaC12 was replaced by equimolar MgC12 in some experiments. Superfusate fractions were collected into vials and 100 C(L aliquots h z e n at -8OOC until HPLC analysis of amino acids4 was carried out and the radioactivity of the remaining 700 C(L was determined by liquid scintillation spectrometry.
RESULTS Compared with basal values, 50 mM K+ produced an increase in efflux of D-[%] aspartate (42-tbld), L-aspartate (10-tbld), and L-glutamate (68-bld) that was largely Ca2+-dependentand tetrodotoxin insensitive WLE 1). In the presence of 5 mM K+, KA caused a dose-dependent increase in the efflux of ~-[~H]aspartate, L-aspartate, and Lglutamate in the presence of Ca2+;in the absence of this cation KA showed a trend to increase efflux but was only significant tbr L-glutamate in the presence of 1 mM KA. Under stimulated conditions (50 mM K+),KA caused a dosedependent inhibition of the release of ~-[jH]aspartatein both the presence and absence of Ca2+; release of L-aspartate and L-glutamate were not significantly affected.
DISCUSSION Elevated concentrations of K+ evoked a large Ca2+dependent, tetrodotoxininsensitive increase in the efflux of both exogenous and endogenous EAA, which contrasts with the relatively modest, tetrodotoxin-sensitive release reported by Ferkany and Coyle.2 Under basal conditions, KA stimulated the efflux of D-[jH]aspartateand endogenous EL4A.s to a similar extent. This again contrasts with the study of Ferkany and Coylql which indicated that KA increased efflux of endogenous, but not exogenous EAAs. These discrepancies are probably related to the k t e r rate of perfusion (and thus reduced influence of uptake processes) and shorter collection time in our study. It is therefore concluded that, at least under the conditions employed here, the release of preaccumulated ~-[3H]aspartatedoes indeed provide a reliable marker of the release of endogenous Gaspartate and cglutamate.
0.01 0.03 0.1 0.3 1
0
100 92 f 102 f 87 f 77 f 70 f
13 (6) 5 (6) 12 (6) 10 (5) 10 (6)b
2.6 mM Caz+ 100 96 f 6 (6) 119 f 6 (6)b 128 f 13 (6) 160 f 13 (5)c 197 f 23 (6)c 100 86 f 84 f 80 f 50 f 42 f 12 (3) 3 (3) 25 (3) 5 (3) 3 (3)b
0 mM Caz+ 100 110 f 6 (3) 130 f 0 (3) 193 f 38 (3) 173 f 9 (3) 203 f 3 (3)
~[3H]aspartate
100 93 f 12 (6) 101 f 4 (6) 90 f 12 (6) 89 f 7 (5) 87 f 13 (6)
2.6 mM Caz+ 100 79 f 14 (6) 115 f 18 (6) 132 f 22 (6) 131 f 23 (5) 220 f 36 (6)d 100 70 f 83 f 100 f 79 f 108 f
15 (3) 14 (3) 26 (3) 13 (3) 22 (3)
0 mM Caz+ 100 80 f 5 (3) 103 f 8 (3) 151 f 39 (3) 167 f 12 (3) 190 f 10 (3)
L-aspartate
100 92 f 101 f 94 f 99 f 124 f
16 (6) 5 (6) 16 (6) 10 (5) 22 (6)
2.6 mM Caz+ 100 86 f 10 (6) 143 f 21 (6) 148 f 25 (6) 206 f 31 (5)d 325 f 56 (6)&
b p < 0.05,c p < 0.01,
100 89 f 16 (3) 91 f 4 (3) 119 f 36 (3) 95 f 5 (3) 130 f 10 (3)
0 mM Caz+ 100 94 f 13 (3) 130 f 21 (3) 220 f 58 (3) 230 f 40 (3) 300 f 36 (3)b
Gglutamate
Data are presented as percent of control (with number of experiments in parentheses). < 0.001 (analysis of variance followed by paired &tests). Data analysis was performed on original data.
Stimulated (50mM K+)
Basal (5mM K+)
KainiC Acid (mM) 0 0.01 0.03 0.1 0.3 1
TABLE 2. The Influence of Kainic Acid on the Efflux of Exogenous and Endogenous Excitatory Amino Acids fiom Rat Cerebral Cortex"
a
51 0
E
m
k
I
R
a
EiP
P
364
ANNALS NEW YORK ACADEMY OF SCIENCES REFERENCES
1 . FERKANY,J. W.& J. T. COYLE, 1983. Evoked release of aspartate and glumate: Disparities between prelabeling and dkct measurement. Brain Rs.278: 279-282. 2. FERKANY,J. W.& J. T. C ~ Y L E1983. . Kainic add selectively stimulates the releasc of endogenous excitatory acidic amino acids. J. Pharmacol. Exp. Ther. 225: 399406. 3. JOHNSON, G.A. R, S. M. E. KENNEDY & B. TWITCHIN. 1979. Action of the neurotoxin kainic acid on high affinity uptake of Gglumic acid in rat brain slices. J. Neurochem. 32: 121-127. 4. REYNOLDS, I. J. & A. M. PALMER.1991. Regional variations in [3H]MK801 binding to rat brain N-methyl-maspartatereceptors. J. Neurochem. 56: 1731-1740.
MATERIALS AND METHODS Slices of rat cerebral cortex (cut in two planes separated by 4 5') were added to 10 ml of ice-cold buffer solution (pH 7.4) which had the following composition (mM): NaCll28, KCI 5, MgW4 1.2, Na2PO4 1.0, CaCl2 2.6, glucose 10, N-2-hydroxyethylpipcrazine-K-2-ethanesulfbnic acid (Hepes) 20, and was then oxygenated (95% 0 2 5% C02). The minislices were dispersed, allowed to settle, oxygenated and incubated in a water bath (37OC, 5 min). ~[JHIaspartatewas then added to give a final concentration of 200 nM and the incubation continued fbr an additional 25 minutes. After washing four times with 6 ml of either normal media or media with CaC12 replaced by equimolar MgC12, the suspension (4 ml) was mixed over a magnetic stirrer and 200 PL aliquots transkrred to each chamber (300 PL in volume) of the Supefision 20 system (Brandel, Gaithersberg, MD). Following a 30-minute period of supefision (35OC, 1.6 mL/min), 12 successive 0.5 min fractions were collected. KA (0.01-1 mM) 361
362
ANNALS NEW YORK ACADEMY OF SCIENCES
TABLE 1. Comparison of the K+evoked Release of Exogenous and Endogenous Excitatory Amino Acid# ~[~Hlaspartate
waspartate
L-glutamate
50 mM K+ 100 100 100 5 mM K+ 2.4 f 0.2c 10.5 f 3.1) 1.5 f 0.4c 50 mM K+,0 mM Ca2+ 10.7 f 3.lC 31.1 f 20.1 4.7f 3.2) 50 mM K+, 1 pM TTX 77.0 f 12.4 81.7 f 6.7 83.0f 12.3 a Data are expressed as a percent of control values (50mM K+) f SEM for three separate experiments. Mean control values (* SEM) for D[JH]aspartate, taspartate, and Gglutamate were 34,066 f 5,470 dpmlmg protein, 327 f 93 pmollmg protein, and 858 f 147 pmollmg protein, respectively. l T X : teuodotoxin. b p < 0.05,c p < 0.01(analysis of variance followed by paired t-tests). Statistical analyses were performed on original data.
-
was added at t 15-36 min. In order to examine the Ca2+-dependencyof release, CaC12 was replaced by equimolar MgC12 in some experiments. Superfusate fractions were collected into vials and 100 C(L aliquots h z e n at -8OOC until HPLC analysis of amino acids4 was carried out and the radioactivity of the remaining 700 C(L was determined by liquid scintillation spectrometry.
RESULTS Compared with basal values, 50 mM K+ produced an increase in efflux of D-[%] aspartate (42-tbld), L-aspartate (10-tbld), and L-glutamate (68-bld) that was largely Ca2+-dependentand tetrodotoxin insensitive WLE 1). In the presence of 5 mM K+, KA caused a dose-dependent increase in the efflux of ~-[~H]aspartate, L-aspartate, and Lglutamate in the presence of Ca2+;in the absence of this cation KA showed a trend to increase efflux but was only significant tbr L-glutamate in the presence of 1 mM KA. Under stimulated conditions (50 mM K+),KA caused a dosedependent inhibition of the release of ~-[jH]aspartatein both the presence and absence of Ca2+; release of L-aspartate and L-glutamate were not significantly affected.
DISCUSSION Elevated concentrations of K+ evoked a large Ca2+dependent, tetrodotoxininsensitive increase in the efflux of both exogenous and endogenous EAA, which contrasts with the relatively modest, tetrodotoxin-sensitive release reported by Ferkany and Coyle.2 Under basal conditions, KA stimulated the efflux of D-[jH]aspartateand endogenous EL4A.s to a similar extent. This again contrasts with the study of Ferkany and Coylql which indicated that KA increased efflux of endogenous, but not exogenous EAAs. These discrepancies are probably related to the k t e r rate of perfusion (and thus reduced influence of uptake processes) and shorter collection time in our study. It is therefore concluded that, at least under the conditions employed here, the release of preaccumulated ~-[3H]aspartatedoes indeed provide a reliable marker of the release of endogenous Gaspartate and cglutamate.
0.01 0.03 0.1 0.3 1
0
100 92 f 102 f 87 f 77 f 70 f
13 (6) 5 (6) 12 (6) 10 (5) 10 (6)b
2.6 mM Caz+ 100 96 f 6 (6) 119 f 6 (6)b 128 f 13 (6) 160 f 13 (5)c 197 f 23 (6)c 100 86 f 84 f 80 f 50 f 42 f 12 (3) 3 (3) 25 (3) 5 (3) 3 (3)b
0 mM Caz+ 100 110 f 6 (3) 130 f 0 (3) 193 f 38 (3) 173 f 9 (3) 203 f 3 (3)
~[3H]aspartate
100 93 f 12 (6) 101 f 4 (6) 90 f 12 (6) 89 f 7 (5) 87 f 13 (6)
2.6 mM Caz+ 100 79 f 14 (6) 115 f 18 (6) 132 f 22 (6) 131 f 23 (5) 220 f 36 (6)d 100 70 f 83 f 100 f 79 f 108 f
15 (3) 14 (3) 26 (3) 13 (3) 22 (3)
0 mM Caz+ 100 80 f 5 (3) 103 f 8 (3) 151 f 39 (3) 167 f 12 (3) 190 f 10 (3)
L-aspartate
100 92 f 101 f 94 f 99 f 124 f
16 (6) 5 (6) 16 (6) 10 (5) 22 (6)
2.6 mM Caz+ 100 86 f 10 (6) 143 f 21 (6) 148 f 25 (6) 206 f 31 (5)d 325 f 56 (6)&
b p < 0.05,c p < 0.01,
100 89 f 16 (3) 91 f 4 (3) 119 f 36 (3) 95 f 5 (3) 130 f 10 (3)
0 mM Caz+ 100 94 f 13 (3) 130 f 21 (3) 220 f 58 (3) 230 f 40 (3) 300 f 36 (3)b
Gglutamate
Data are presented as percent of control (with number of experiments in parentheses). < 0.001 (analysis of variance followed by paired &tests). Data analysis was performed on original data.
Stimulated (50mM K+)
Basal (5mM K+)
KainiC Acid (mM) 0 0.01 0.03 0.1 0.3 1
TABLE 2. The Influence of Kainic Acid on the Efflux of Exogenous and Endogenous Excitatory Amino Acids fiom Rat Cerebral Cortex"
a
51 0
E
m
k
I
R
a
EiP
P
364
ANNALS NEW YORK ACADEMY OF SCIENCES REFERENCES
1 . FERKANY,J. W.& J. T. COYLE, 1983. Evoked release of aspartate and glumate: Disparities between prelabeling and dkct measurement. Brain Rs.278: 279-282. 2. FERKANY,J. W.& J. T. C ~ Y L E1983. . Kainic add selectively stimulates the releasc of endogenous excitatory acidic amino acids. J. Pharmacol. Exp. Ther. 225: 399406. 3. JOHNSON, G.A. R, S. M. E. KENNEDY & B. TWITCHIN. 1979. Action of the neurotoxin kainic acid on high affinity uptake of Gglumic acid in rat brain slices. J. Neurochem. 32: 121-127. 4. REYNOLDS, I. J. & A. M. PALMER.1991. Regional variations in [3H]MK801 binding to rat brain N-methyl-maspartatereceptors. J. Neurochem. 56: 1731-1740.
