J o u r n d of Nrurochenwfry. 1977. Vol. 29, pp. 873-881. Pergarnun Press. Printed in Great Britain.

KINETIC STUDY OF GLUTAMATE TRANSPORT IN RAT BRAIN MITOCHONDRIA A. MI"'

and J. GAYET

Laboratoire de Physiologie Generale, UniversitC de Nancy 1, 54 037 Nancy, France (Received 31 January 1977. Revised 21 April 1977. Accepted 2 M a y 1977)

Abstract-The experiments reported here confirm that glutamate can penetrate the inner membrane of isolated rat brain non-synaptosomal mitochondria, either on a glutamate-hydroxyl antiporter or on a glutamate-aspartate antiporter. An inhibition of respiratory activity of mitochondria with glutamate as substrate was obtained in the presence of avenaciolide or N-ethylmaleimide. Swelling of the mitochondria in iso-osmotic N H ~ - L glutamate was inhibited in the presence of avenaciolide and N-ethylmaleimide, but mersalyl, kainic acid, glisoxepide and amino-oxyacetic acid had no effect on the glutamate-hydroxyl exchange. Glutamate induced the reduction of intramitochondrial NAD(P), as estimated by double-beam spectrophotometry, and this reduction was inhibited on the one hand by N-ethylmaleimide, avenaciolide or fuscine, on the other hand by aminooxyacetic acid. A direct estimation of the penetration of ~-['~C]glutamate into brain mitochondria was performed by using the centrifugation-stop procedure. This penetration followed saturation kinetics, with a mean apparent K , of 1 . 5 6 m ~at pH 7.4 and at 20°C the value of V,,, was 4.34 nmol per min per mg protein in the same conditions. N-Ethylmaleimide slowed down the initial rate of glutamate penetration, and this inhibition appeared to be non-competitive with a K iof 0.7 mMat pH 7.4 and at 20°C. The entry of glutamate was pH-dependent and it increased 2-fold in the pH range of 7.4 to 6.4. A temperature-dependence of glutamate transport was also shown between 2 and 25°C; the Arrhenius plot was a straight line, with a calculated EA of 12.8 kCal per mol of glutamate and a Q l o of 2.16. The activity of y-glutamyl transpeptidase was practically absent in these rat brain mitochondria. Oxidation of extramitochondrial NADH by the 'malate-aspartate shuttle' reconstituted in vitro was followed in rat brain non-synaptosomal mitochondria. In the absence of extramitochondrial malate or glutamate the 'shuttle' did not function, and in the absence of extramitochondrial aspartate the rate of NADH oxidation was low. Glutamine or y-aminobutyrate did not replace glutamate efficiently. A high inhibition of the 'malate-aspartate shuttle' occurred in the presence of avenaciolide or mersalyl, and a moderate one in the presence of N-ethylmaleimide, glisoxepide or n-butylmalonate. Glutaminase activity in intact brain mitochondria was inhibited in the presence of extramitochondrial glutamate

GLUTAMATE translocation across the inner membrane chondrial glutamate for intramitochondrial aspartate of isolated rat liver mitochondria has been extensively (the glutamate-aspartate antiport system) (LA NOUE studied. Glutamate may enter these mitochondria et al., 1974); when the inner mitochondria1 membrane according to two separate mechanisms (AZZI et al., is energized, this exchange is unidirectional (MEIJER 1967). The first one involves the penetration of gluta- & VANDAM,1974; TISCHLER et al., 1976). Only glismate together with a proton (the glutamate-proton oxepide, a blood glucose lowering sulphonylurea, was symport system) or by exchange for a hydroxyl (the shown to be an inhibitor of the glutamate-aspartate glutamate-hydroxyl antiport system) (MEIJERet al., antiport system (SLING & SECK,1975). 1972). This mechanism may be inhibited either by It was postulated that the heterogeneity of brain some molecular analogues of glutamate such as mitochondria is related to the metabolic compart3-hydroxy-~-glutamateand erythro-hydroxyaspartate mentation of glutamate in the organ (VANDENBERG (AZZI et al., 1967), or by non-polar and liposoluble et at., 1975). Glutamate is considered to be distributed thiol reagents such as N-eth ylmaleimide, avenaciolide into at least two major metabolic compartments in and fuscin (MEYER & VIGNAIS,1973), and also by tan- the brain represented by neuronal and glial mitochonnic acid (KING& DIWAN,1972). The second mechan- dria. Indirect evidence has indicated that the so-called ism involves an electrogenic exchange of extramito- 'large' compartment may consist of particles present in various neuronal ,structures, whereas the 'small' ' Attache de Recherche au Centre National de la one, which is associated with high glutamine content, has been thought to consist of glial mitochondria Recherche Scientifique. 1975). Moreover, recent data suggest that Abbreviations used: MES, (n-morpho1ino)ethane sul- (QUASTEL, phonic acid; FCCP, carbonylcyanide p-fluoromethoxy- the 'small' glutamate compartment consists of two subpopulations of mitochondria with differing prophen ylh ydrazone. N.C. 2 9 / 5 4

873

8 74

A. Mi"

and J. GAYET

portions of y-aminobutyrate transaminase and glutamate dehydrogenase (REIJNIERSErt al., 1975). Whatever the population o r subpopulation of mitochondria may be, glutamate may have to cross the mitochondrial membranes in order to be metabolizcd. BRAND& CHAPPELL(1974) had concluded that isolated brain mitochondria did not possess a glutamate-hydroxyl antiport system, but did possess only a glutamate-aspartatc antiport system. However, the swelling of brain mitochondria in iso-osmotic NH;L-glutamate and its inhibition by N-ethylmaleimide and tannic acid did confirm unequivocally the existence of a glutamate-hydroxyl antiport system (MI" & GAYET,1973; 1974; MINN tit al., 197.5; GAYET& MINN, 1975). Recently, DENNIS et a/. (1976) had confirmed these results and the functioning of both a glutamate-hydroxyl antiporter and a glutamateaspartate antiportcr in isolated rat brain mitochondria. In this paper, we report the kinetic properties of the glutamate -hydroxyl antiport system in isolated non-synaptosomal rat brain mitochondria, and also the effects of some inhibitors on the functioning of this antiport system. The glutamatc-aspartate antiport system is also studied in the reconstructed 'malate-aspartate shuttle' (BORST,1963), which allows the transfer of reducing equivalents between the cytosol and the mitochondrial matrix. Finally, the activity of brain mitochondria glutaminase is investigated, in order to prove that glutamate does enter the particles by the glutamate-hydroxyl antiporter. Preliminary reports of these investigations were published previously (GAYET & MI", 1975; MINN & GAYET,1976).

were isolated by the method of CLARK& NICKLAS ( I 970). Measurement of the swelliny of' mitochondria. The swelling of isolated brain mitochondria was monitored at 25°C from the variation of optical density, at 520 nm, in a Beckman Acta 111 recording spectrophotometer, as prcviously described (MI" et al., 1975). Estimation of the respiratory rate of' mitochondria. The respiratory rate was estimated in freshly isolated mitochondria, oxygen uptake being recordcd polarographically using a Clark type electrode (Yellow Springs Instruments, Yellow Springs, OH) in a Gilson oxygraph KM. The incubation medium was the 5mM-K+ medium of CLARK& NICKLAS(1970). Determination of chanyes in thc redox state of intramitochondriul nicotinumide nucleotides. NAD(P)H formation was followed by double-beam spectrophotometry at 340-373 nm in an Aminco-Chance spectrophotometer (AZZI et al., 1967). Mitochondria (2mg of protein) were suspended in 3 ml of a medium containing 100 mM-KCI, 10 mM-Tris ~HCI,10 m ~ - 2(n-morpho1ino)ethane sulphonic acid (MES) pH 7.5, 2.5 pwcarbonylcyanide fl-fluoromethoxyphenylhydrazone (FCCP) (an uncoupler), at 21 "C.After 2 min, 8 fiM-rotenone were added. The changes in optical density was followed for 5 to 7 min after the addition of 0.2-2 mM-L-glutamate or aspartate, as potassium salt. With aspartate as substrate, the previous reduction of NAD(P)+ was induced by addition of 1 mM a-ketoglutarate in the presence of inorganic phosphate and malonate. Measurement of glutamute transport. Penetration of thc mitochondrial matrix by glutamate was measured by the centrifugation-stop procedure (MEYER& VIGNAIS, 1973). Mitochondria (1.2-1.5 mg of protein) were preincubated for 30 s in a medium containing 50 mM-Tris-HC1, 15 mM-KCI, 5 mM-MgCI,, 2 mM-K+-EDTA, pH 7.4, 4 pg antimycin A, I .S pCi/ml 3HZ0. Incubation was started by the rapid acid (0.5 pCi), with addition of 1 m~-~-[U-'~C]glutamic the help of a push button adjustable Hamilton syringe CR 7W200 model automatically controlled by a Jaquet electromagnetic chronoscope 310c model (accuracy of 0.1 s), and it was stopped by a rapid separation of the mitochonMATERIALS AND METHODS dria by centrifugation in an Eppendorf 3200 microcentriAnimals. Wistar rats (25&300 g), 3-month-old and bred fuge. The supernatant fluid was discarded and acidified in the laboratory were used. with 0.70~-HClO, to a 0.35 M-final concentration; the Chemicals. Disodium-ADP, NADH, L-y-glutamyl-p- tightly packed pellet was quickly rinsed with chilled nitranilide, glutamate-oxaloacetate transaminase (EC 0.25 M-SUCrOSe, then it was extracted in lOOpl 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37)were from 0.70 M-HCIO,. The radioactivity C3H] and ['"C] of a SO pl Boehringer Mannheim France (Paris). Kainic acid, N-cth- sample of the deproteinized supernatant fluid and of the ylmaleimide (NEM) and rotenone were from Sigma (Saint 100pI perchloric acid extract of the pellet was cstirnated Louis, MO). Antimycin A was from Calbiochem (Los in a LS 150 Beckman liquid-scintillation spectrometer, Angeles, CA). Ficoll was from Pharmacia (Uppsala) and with Instagel (Packard) as a scintillator. Assays were run was dialysed twice for I2 h against deionized water at 0°C. in parallel in which [14C] sucrose was added instead of Aminooxyacetic acid was from K and K Laboratories ['4C]glutamate, and the radioactivity of the pellet was (Plainview, NY). Mersalyl (Salyrgan), avenaciolidc and glis- measured as described for ['4C]glutamate. It is assumed oxepide were gifts respectively from Hoechst (Frankfurt), that the matrix space represents the difference between the from Dr. W. B. TURNER. Imperial Chemical Industries total water space (permeable to 3 H 2 0 ) and the extra (Macclesfield)and from Dr. H. PLUMPE,Bayer A.G. (Wup- matrix space (permeable to ['4C]sucrose). According to pertal). Fuscin extracted from Uidiodendrumfuscum Robak the experimental conditions the matrix space ranged from (Imperial College 0.5 to 0.7 p1 per mg protein, and it was kept constant durwas a gift of Professor D. H. R. BARTON ing the short times of incubation applied, whereas the of Science and Technology, London). L-[U-'~C]Glutamic acid (specific radioactivity 200 mCi/ sucrose space varied from 2 to 3 pl per mg protein. Reconstruction of the malate-aspartate shuttle. As shown mmol) and 3 H 2 0 (specific radioactivity 0.5 mCi/ml) were by CHAPPELL (1969) in isolated rat liver mitochondria and from the Centre dEtudes Nucleaires (Saclay). [U-'4C]Sucrose (specific radioactivity 15 mCi/mmol) was from the by BRAND& CtiAPPnLL (1974) and GAYET& MINN(1975) in isolated rat brain mitochondria, the malate-aspartate Radiochemical Centre (Amersham). Isolation of mitochondria. Rat cerebral hemispheres were shuttle which causes the transfer of reducing power across obtained immediately after decapitation, and mitochondria the mitochondrial membrane may be reconstructed in uitro

Glutamate transport in rat brain mitochondria by adding to the incubation medium the substrates and the enzymes involved in the functioning of this shuttle. Mitochondria (0.4-0.6 mg of protein) were suspended in 2.7ml of the 5mM-K+ medium of CLARK& NICKLAS (1970) containing 0.15 mM-NADH. Oxidation of NADH was followed in a Beckman Acta 111 recording spectrophotometer, at 340nm and at 25"C, with a constant electromagnetic stirring. 50 pg of glutamate-oxaloacetate transaminase, 25 pg of malate dehydrogenase, 2 mM-K+-Lglutamate, 2.5 mM-K+-L-malate and 2 mM-K+-L-aspartate were successively added to the mitochondrial suspension. In experiments made in the presence of an inhibitor of the malate-aspartate shuttle, the inhibitor together with the enzymes and the substrates were added to the incubation medium before the addition of mitochondria, in order to estimate the initial rate of NADH oxidation in this experimental condition. As pointed out by CHAPPELL (1969) due to the very small amount of mitochondria used in these experiments the redox-changes in the intramitochondrial NAD(P) were only a small part of the total optical density changes. Estimation of the y-glutamyltranspeptidase activity. The method of ORLOWSKI& MEISTER(1963) modified by MEYER(1975) was used. The incubation medium had the following composition : to 20 ml of 100 mM-Tris-HC1 buffer, pH 8.2, were added 45mg of MgCl,, 132mg of glycine and 22.9 mg of y-glutamyl-p-nitroanilide. Two ml of this freshly prepared medium were poured into a spectrophotometer cell, and the reaction was initiated by adding mitochondria. The variations of optical density was followed at 405 nm and at 3 7 T , for 10 min with constant electromagnetic stirring. Estimation of the glutarninase activity. Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) activity was estimated at 25°C by measuring ammonia formation with an NH:-sensitive glass electrode (Select-ion electrode Beckman No. 39 047) with a calomel reference electrode in a Beckman research pH-meter connected to a Gilson polygraph. To 4.6 ml of I50 mM-choline hydrochloride and 10 mM-Tris-HC1 buffer, pH 7.4, were added 5 pg of antimycin A, after 5 min mitochondria ( 3 4 mg of protein) were added and the incubation was performed with constant electromagnetic stirring up to a steady potential, then

1

0 01 pAt 0 2 min

-

a 'a/

2 min

FIG.2. Effect of the thiol group reagents, avenaciolide and mersalyl, on the swelling rate of isolated rat brain mitochondria. A. Effect of avenaciolide on the swelling rate of mitochondria incubated in iso-osmotic NHf -L-glutamate. Mitochondria (1-1.2 mg protein) were incubated in 2 ml of 100 mM-NHa-L-glutamate, 5 mM-Tris-HC1, 0.1 mMEGTA, at pH 7.4 and at 25"C, in the presence of 4 pg-antimycin A and 0 to 120 pM-avenaciolide. The irreversibility of the effect of avenaciolide was checked by the addition of 1 mwcysteine, as indicated by the arrow. B. Effect of mersalyl on the swelling rate of brain mitochondria incubated in iso-osmotic NHi-phosphate. The experimental conditions were the same as in Fig. 2 A, except that 120 mM-NHf-phosphate was used instead of 100 mMNHf-L-glutamate. See Methods for details. The arrow accompanying the scale indicates the direction of swelling. 5 mM-L-glutamine and 20 mM-Tris-phosphate were added to the suspension. The NHa-sensitive glass electrode was calibrated with NH,C1 solution as a standard. Protein determination. Protein was estimated by the method of LOWRYet al. (1951) with bovine serum albumin (Fraction V) as a standard.

RESULTS

Oxidation of glutamate

Mitochondria Glutamate

I-

875

27 p M Avenmiolie

\ 38

FIG. 1. Effect of avenaciolide on the oxygen uptake of brain mitochondria oxidizing L-glutamate. Mitochondria (3 mg protein) were incubated in the polarographic medium containing 5 mM-K+, at pH 7.4 and at 28°C; the final volume was 1.8 ml. Oxygen uptake was recorded during oxidation of 2 mM-L-glutamate (state 4), in the presence of 0 to 38 pM-avenaciolide.

It was previously shown that brain mitochondria oxidized L-glutamate, this oxidation being stimulated & NICKLAS,1970; in the presence of L-malate (CLARK MI" et al., 1975). In the presence of avenaciolide added to the polarographic medium, the inhibition of oxygen uptake occurred only for the first minutes of incubation and then a strong increase of respiratory activity took place (Fig. I.). This increase probably originated from the change of the permeability of the inner mitochondria1 membrane resulting from the detergent effect of avenaciolide (GODINOT, 1974). A clear inhibition of glutamate oxidation was obtained when mitochondria were pre-incubated in the presence of avenaciolide for 15 s to 2 min, a n d subsequently when incubation was stopped by inactivating avenaciolide with added 1 mwcysteine. Similar results were obtained in these experimental conditions with N-ethylmaleimide, but this was less efficient than avenaciolide (Fig. 3 A).

A. MINNand J. GAYET

swelling was insensitive to added 5 p ~ - F C C P ,in the presence of antimycin A. N-Ethylmaleimide and avenaciolide inhibit swelling in iso-osmotic N H ~ - L * ’, glutamate, but this inhibition was not reversible and f--’Phosphot‘ the presence of an excess of added cystein prevented -_-O it. The inhibition by avenaciolide was efficient at a P -/ Avenociolide concentration of 15 to 70 p~ (Fig. 2 A), but at higher concentrations rapid swelling occurred due to a detergent-like effect of avenaciolide. In comparative assays, when brain mitochondria were incubated in isoosmotic sucrose, swelling occurred after the addition / L of 120 pM-avenaciolide, which confirmed the deterI 2 min gent-like effect of this reagent. The presence of avenaciolide did not affect the swelling of brain mitochonFIG.3. Effect of preincubating rat brain mitochondria in the presence of non-ionic thiol group reagents. Mitochon- dria in iso-osmotic NHf-phosphate, but it was inhidria (3 mg protein) were preincubated for 0-2 min in the bited strongly in the presence of 50 pwmersalyl, presence of avenaciolide or N-ethylmaleimide as described which is an ionic thiol reagent, the inhibition due in Methods, and then they were immediately used for the to this latter being suppressed by added cystein (Fig. , had assays. A. Percentage of inhibition of oxygen uptake during 2 B). Used at a concentration of 5 0 p ~ mersalyl oxidation of 2 rnw-L-glutamate (state 4). Mitochondria no effect on the intramitochondrial penetration of were preincubated in 66 pM-avenaciolide(O), or in 400 pw- glutamate. When mitochondria were previously preinN-ethylmaleimide (NEM) (0).B. Percentage of inhibition cubated for 15 s-2 min in the presence of 400 ,uM-Nof the swelling rate of mitochondria incubated in iso-osmo- ethylmaleimide and then supplied with cystein, they tic NHa-phosphate (A)and in iso-osmotic NHa-L-glutashowed an inhibition of swelling when subsequently mate ( 0 ) ;mitochondria were preincubated in 400 ~ M - N incubated in iso-osmotic NHf +glutamate, this inhiet hylmaleimide. bition being stronger with a longer time of incubation. Phosphate permeation seemed to be more sensitive to the inhibitory effect of N-ethylmaleimide (Fig. Rate of swelling of mitochondria 3 B). On the other hand, mitochondria submitted to Rat brain mitochondria swelled rapidly when they cystein prior to a preincubation in the presence of were incubated in iso-osmotic NHi-L-glutamate or N-ethylmaleimide or avenaciolide did not exhibit the K +-L-glutamate in the presence of valinomycin inhibitory effect of these two thiol reagents, confirm(MI” et al., 1975). In the present experiments, this ing that the inhibitory effect on glutamate penetration E

A

,/

i

0

/J

15 pM Fuscin 22

1

‘0‘

Glu

f,

:1 pM NEM

FIG.4. Effect of glutamate and aspartate on the redox state of intramitochondrial NAD(P). Rat brain mitochondria (2 mg protein) were incubated in 3 ml of the medium containing 100 mM-KC1, 10 mM-TrisHC1, 10 m ~ - 2(n-morpho1ino)ethane sulphonic acid, pH 7.5, in the presence of 6 ~ M - F C C P . After 2-3 min, 8 pw-rotenone (Rot) were added, then the oxidizable substrate was added. Reduction of intramitochondrial NADP was recorded in double-beam spectrophotometry at 340 and 373 nm and at 21°C. A. Effect of the presence of N-ethylmaleimide (NEM) or fuscin on the reduction rate of intramitochondrial NADP by added 0.67 mM-L-glutamate (Glu). B. Reoxidation of intramitochondrial NAD(P)H by added 0.67 mw-aspartate (Asp) and enhancing of this reoxidation by 0.67 mw-glutamate added subsequently; added 0.3 mw-glisoxepide slowed down the reoxidation. Intramitochondrial NAD(P) were previously reduced by successively added 1 mw-u-ketoglutarate (u-KG), 1 mM-tnalOnate (Malon) and 1 mM-phosphate (P).

Glutamate transport in rat brain mitochondria is related to the binding of the reagents on the thiol groups of the translocator in the inner mitochondria1 membrane. Kainic acid, which is a cyclic analogue of glutamate (OLNEY et al., 1974), glisoxepide, which is an inhibitor of the exchange of glutamate and aspartate in liver mitochondria (SOLING& SECK, 1975) and amino-oxyacetic acid, which is an inhibitor of transaminases (WEBB,1966), had no effect on the rate of swelling of brain mitochondria incubated in iso-osmotic NHl-L-glutamate.

877

0

a

E"

1.5

\

2

0

E X 0

0 + P

0.5

Rate ofchanges in the redox state of intramitochondrial nicotinarnide nucleotides The estimation of the redox state of intramitochondrial NAD(P) in double-beam spectrophotometry showed that addition of glutamate to the mitochondrial suspension induced a reduction of intramitochondrial NAD(P), the initial rate of which increased with increasing concentrations of added glutamate. The initial rate of reduction of intramitochondrial NAD(P) induced by glutamate was inhibited by thiol reagents, such as N-ethylmaleimide and fuscine at a concentration of 24 PM which were previously shown acting on the transport of glutamate in liver mitochondria (MEIJERet al., 1972; MEYER& VIGNAIS, 1973) (Fig. 4 A). Moreover, 1 mM-amino-oxyacetic acid did not prevent reduction of intramitochondrial NAD(P). Reduction of intramitochondrial nicotinamide nucleotides was rapidly induced by cc-ketoglutarate plus malonate in the presence of inorganic phosphate, and aspartate subsequently added induced a reoxidation of intramitochondrial NAD(P), this reoxidation being enhanced by adding glutamate (Fig. 4B). In the same sort of experiment in which glutamate was subsequently added instead of ppartate, the reoxidation of intramitochondrial NAD(P)H was negligible. In the presence of 0.3 mM-glisoxepide a decrease in the reoxidation of intramitochondrial NAD(P)H induced by added aspartate occurred. The determination of the changes in the initial rate of reduction of intramitochondrial NAD(P) in relation with the concentration of added glutamate allows us to estimate indirectly the kinetic constants of the penetration of glutamate. The mean apparent K , is 0.75 mM at 21°C and pH 7.5, and the V,,, is 2.8 nmol NAD(P) per min per mg protein (Fig. 6A).

Time course of mitochondria

['4C]glutamate

penetration in brain

The time course of the penetration of ['4C]glutamate in rat brain mitochondria is represented in Fig. 5. At 20"C, the curve is linear for about the first 20 s of incubation and an equilibrium is reached after 40 to 50s of incubation. The actual time of incubation is obtained by extrapolating the initial linear part of the curve towards the time axis, because glutamate penetration goes on for the first seconds of the rapid centrifugation used for stopping the reaction. We had verified that the value for the time necessary to stop the intramitochondrial penetration of labelled gluta-

FIG.5. Time course of ~-['~C]glutamate penetration into rat brain mitochondria. Mitochondria (1.4 mg protein) were added to an incubation medium containing 15 mM-KC1, 50 mM-Tris-HC1, 5 mM-MgCI,, 2 mM-K+EDTA, 3 H 2 0 1.4 pCi, at pH 7.4 and at 20T, in the presence of 2 pg rotenone and 10 pg antimycin A. After 30 sec, 1 m~-~-['~C]glutamate (0.4pCi) is added and rapidly mixed with the suspension. Freshly isolated brain mitochondria (0);brain mitochondria previously preincubated in 400p~-N-ethylmaleimidefor 2 min (A).See Methods for experimental details.

mate by rapid centrifugation did not change for concentrations of glutamate ranging from 0.2 to 4 m . The initial rate of the penetration of ['4C]glutamate into mitochondria decreased when the particles were previously preincubated for 1-2 min in the presence of 400 pi-N-ethylmaleimide and then submitted to cystein (Fig. 5). In Dixon plot, the inhibition of ['4C]glutamate penetration by N-ethylmaleimide appears to be non-competitive, with a K i of 0 . 7 m ~ at 20°C and pH 7.4. As shown in Fig, 6 B the penetration of ['4C]glutamate in rat brain mitochondria follows a saturation kinetics. A mean apparent K , of 1 . 5 6 m (values ranging from 0-75 to 1 . 6 m ~ at ) 20°C and pH 7.4 was estimated from eight experiments with concentration of glutamate ranging from 0.2 to 5 m ~ and , a V,, of 4.34nmol per min per mg protein at 20°C and pH 7.4. This value of K , is in good correlation with that estimated indirectly in measuring the redox state of intramitochondrial NAD(P) (Fig. 6 A). In lowering the H + concentration in the incubation medium from pH 7.4 to pH 6.4, the initial rate of transport of [''C]glutamate into mitochondria increased more than 2-fold (Fig. 7). The temperature dependence of the transport of ['4C]glutamate into brain mitochondria, when labelled glutamate was used at a concentration of 1 mM, is illustrated in Fig. 8. Between 2°C and 25°C the Arrhenius plot is a straight line, and the activation energy calculated from the slope is 12.8 kcal per mol of glutamate; from this last value we obtain a Q l 0 of 2.16. Our measurements of the penetration of ['4cjglutamate into mitochondria were performed in the pres-

A. MINNand J. GAYET

878

0 3.4

FIG.6. Lineweaver Burk plot of reduction of intramitochondrial NAD(P) (A) and of glutamate penetration into rat brain mitochondria (B). A. Mitochondria were incubated in the same conditions as those in Figure'4 except that no inhibitor or anion transport was added. B. Mitochondria were incubated in the same conditions as those in Figure 5. ence of antimycin A, thus in the absence of available energy from electron transfer. In subsequent experiments made in the presence of available energy, as added ATP or succinate in the presence of rotenone instead of antirnycin A it appeared that the initial rate of penetration of labelled glutamate greatly increased. MEIJERr t ul. (1972) had emphasized the structural analogy between N-ethylmaleimide and pyrrolidone-2-carboxylic acid (pyroglutamic acid) which is considered as an intermediate in the mechanism of transport of amino acids by the y-glutamyl cycle

," i m

E

60

6.5

70

7.5

8.0

PH

FIG.7. Effect of pH on the initial rate of [*4C]glutamate penetration into rat brain mitochondria. The experimental conditions were similar to those in Figure 6 B, the concentration of added ['4C]glutamate being 1 mM. The pH of the incubation medium varied from 6.2 to 7.6. For the estimation with pH values lower than 7.0, 10 m~-2(nmorpho1ino)ethane sulphonic acid were used instead of 10 mM-Tris-HC1.

+

3.5

3.6

x 10'

FIG. 8. Effect of temperature on the initial rate of [14C]glutamate penetration into rat brain mitochondria (Arrhenius plot). The experimental conditions wcrc thc same as those in Fig. 6 B, the concentration of added [I4C]glutamate being 1 mM. The temperature of the incubation medium varied from 2°C to 25°C.

(MEISTER, 1974). On the other hand, the presence of glutamyl-glutamate, which is an intermediate in the transport of glutamate in the cycle, was demonstrated in the monkey brain (REICHELT,1970). The activity of y-glutamyl transpeptidase was very low in rat cerebral hemisphere homogenate compared to that estimated in kidney homogenate, and it was practically absent in rat brain mitochondria. Glutamatt-aspartate exchange

As previously shown by BRAND& CHAPPELL (1974), the transfer of reducing equivalents between the incubation medium and the mitochondrial matrix may be initiated in uitro if malate, malate dehydrogenase, aspartate, aspartate aminotransferase and glutamate are present in the extramitochondrial medium. Table I shows that in the absence of malate or glutamate the 'malateaspartate shuttle' for the oxidation of extramitochondrial NADH did not operate, which confirms that malate is the intramitochondrial precursor of oxaloacetate which is necessary for the transamination of glutamate. In the absence of extramitochondrial aspartate, the rate of NADH oxidation was 25% lower than the maximal rate recorded in the presence of glutamate, malate and aspartate. The presence of glutamine or y-aminobutyrate instead of glutamate in the extramitochondrial medium allowed only a reduced activity of the 'shuttle', and in the absence of extramitochondrial aspartate, added or-ketoglutarate did not greatly alter the maximal rate of NADH oxidation. The functioning of the malate-aspartate shuttle involves three processes of anion exchange at the level of the inner mitochondrial membrane, namely the glutamate-aspartate, the glutamate-hydroxyl and the malate-a-ketoglutarate exchanges. In Table 2 the values of the rate of oxidation of extramitochondrial NADH in the presence of some inhibitory reagents of these three anion exchanges are collected. A high inhibition of the shuttle occurred with added avena-

Glutamate transport in rat brain mitochondria

879

TABLE1. PART PLAYED

BY EXTRAMITOCHONDRIAL SUBSTRATE-ANIONS IN THE FUNCTIONING OF THE ‘MALATEASPARTATE SHUTTLE’ I N ISOLATED GRAIN MITOCHONDRIA

Rate of NADH oxidation

Added substrates

+ L-aspartate + L-malate 100 (Arbitrary unit) L-Glutamate + L-aspartate 0 to 10 L-Aspartate + L-malate 0 to 10 L-Glutamate + L-malate 77 L-Malate + L-aspartate + L-glutamine 36 L-Malate + L-aspartate + y-aminobutyrate 18 L-Malate + L-glutamate + a-ketoglutarate 65 L-Glutamate

/ Glu

t

Glm

PiGlrn

~

Mitochondria (0.4 rng protein) were incubated in the 5 mM-KCI medium of CLARK& NICKLAS (1970) at 25”C, in the presence of 25 pg-malate dehydrogenase, 50 pg-glutamate-oxaloacetate transaminase and 0.15 mM-NADH. Oxidation of NADH was recorded spectrophotometrically at 340nm, after an addition of the following substrates used as K+-salts (pH 7.4): 2.5 mM-L-malate, 2 mM-L-glutamate, or L-aspartate, or L-glutamine, or y-aminobutyrate or sc-ketoglutarate. The values of the rate of oxidation of NADH given in the Table were corrected from the rate of oxidation of NADH estimated in the presence of the two added enzymes, before the addition of the substrates. The maximal rate obtained corresponded to an oxidation rate of 79 nmol NADH per min per mg protein.

FIG.9. Glutaminase activity in rat brain mitochondria. Mitochondria (3.54.0 mg protein) were incubated in a K + free medium containing 150 mu-choline hydrochloride, 10 mM-Tris-HC1, at pH 7.4 and at 2 5 T , the final volume being 5 ml. After 5 min glutamine (Glm), glutamate (Glu), succinate (SUC)were added as Tris salts at a concentration of 5 mM, Tris-phosphate (P) was added at a concentration of 20 mM, 4 FM-FCCP and 25 pM-N-ethylmaleimide (NEM) were also added as indicated by the arrows. Glutaminase activity was determined from the amount of NHf liberated in the suspension and estimated by means of an N H f sensitive glass electrode under constant electromagnetic stirring. See Methods for experimental details. Control experiments are indicated in dotted line.

ciolide or mersalyl, and a moderate one with added N-ethylmaleimide, glisoxepide or n-butylmalonate. No inhibition occurred in the presence of phenylpyruvate or 1, 2, 3-benzene-tricarboxylate.

that glutamate penetrated into these mitochondria (Fig. 9.). FCCP was an efficient inhibitor of glutaminase activity, as N-ethylmaleimide added at a concentration which was previously shown to inhibit the swelling of brain mitochondria incubated in isoosmotic NHi-L-glutamate (MI” et al., 1975). Mitochondrial glutaminase activity was enhanced in the presence of extramitochondrial 5 mwsuccinate or citrate, but no effect was observed in the presence of 5 mM-N-acetylaspartate, although this derivative

Glutaminase activity A high rate of glutaminase activity in brain mitochondria was initiated by adding phosphate to the incubation medium, and extramitochondrial glutamate acted as an inhibitor of this activity proving TABLE2. EFFECTOF

THE PRESENCE OF SOME INHIBITORS OF ANION TRANSLOCATORS ON

THE ACTIVITY OF THE ‘MALATE-ASPARTATE SHUTTLE’ IN ISOLATED BRAIN MITOCHONDRIA

Translocator inhibited

Inhibitor None Mersalyl 40 p~ Mersalyl I60 p ~ } n-Butylmalonate 2.6 mM Phenylpyruvate 1 mM 1, 2, 3-Benzene tricarboxylate 1 mM N-Ethylmaleimide 66 PM N-Ethylmaleimide 165 p~ N-Ethylmaleimide 330 PM Avenaciolide 6 p~ Avenaciolide 13 p~ Avenaciolide 16 p~ Glisoxepide 0.3 mM Glisoxepide 0.6 mM}

I

I

Pi/OH Malate/Pi Pyruvate/OHCitrate/malate Pi/OH Glutamate/OH Glutamate/OHGlutamate/aspartate

Rate of NADH oxidation 100 (Arbitrary unit) 41 37 75 100 100 81 63 52 78 28 16 82 61

Experimental conditions were the same as those in Table 1, but malate dehydrogenase, glutamate-oxaloacetate transaminase and the substrate-anions, L-glutamate, L-aspartate and L-malate as well as the inhibitory reagents were added in the cuvette of the spectrophotometer before the addition of mitochondria.

880

A. MINNand J. GAYET

maleimide. Our present data showing the inhibitory effect of avenaciolide on the respiratory activity, the activatory effect of glutamate on the reoxidation of intramitochondrial NAD(P)H by aspartate and its inhibitory effect on glutaminase activity, as well as the DlSCUSSION inhibitory effect of both N-ethylmaleimide and The results of the experiments reported in this avenaciolide on the extrusion of glutamate from mitopaper prove the existence of two carrier systems chondrial matrix thus slowing down glutaminase acallowing glutamate to cross the membranes of iso- tivity, give an additional evidence for the functioning lated rat brain non-synaptosomal mitochondria, of a glutamate-hydroxyl translocator in rat brain namely a glutamate-hydroxyl antiporter and a gluta- mitochondria. DENNISet ul. (1976) found in their oxygen uptake mate-aspartate antiporter. The sensitivity of the glutamate-hydroxyl anti- experiments that when brain mitochondria oxidized porter towards uncharged liposoluble thiol group re- 10 mwglutamate plus 2.5 mwmalate, 70% of extramiagents (N-ethylmaleimide. avenaciolide and fuscin), tochondrial glutamate entered mitochondrial matrix its dependency upon the pH gradient between the by exchange with aspartate. Our present results show mitochondrial matrix and the extramitochondrial that the rate of respiratory activity in brain mitochonmedium, and its specificity for L-glutamate are the dria oxidizing 2 mM-glutamate, withqut added malate, same as those previously shown for isolated liver is greatly slowed down when the particles have been mitochondria (BRADFORD & MCGIVAN,1973; MEYER preincubated in the presence of N-ethylmaleimide or & VIGNAIS,1973; MEYER1975). These properties avenaciolide, these reagents having no effect on the together with the kinetic features of the penetration glutamate-aspartate translocator. When glutamate of ['4C]glutamate into mitochondria and its depen- plus malate are used a s oxidizable substrate for brain dency towards temperature prove that the glutamate- mitochondria, malate enters mitochondrial matrix hydroxyl antiporter in brain mitochondria possesses and becomes the precursor of intramitochondrial enzyme-like characteristics. The sensitivity of the glu- oxaloacetate for transamination: it was previously tamate-hydroxyl antiporter towards uncharged lipo- shown that addition of malate to crude brain mitosoluble thiol group reagents, as well as the irreversibi- chondria oxidizing glutamate greatly increased asparlity of the inhibition, differentiate the glutamate car- tate synthesis (GAYETet al., 1970). The increase of rier from the phosphate carrier which is sensitive to intramitochondrial aspartate induces a stimulation of charged hydrophilic thiol group reagents (such as the penetration of glutamate into the mitochondrial mersalyl). The phosphate-hydroxyl antiporter in matrix by exchange with aspartate, followed correlatibrain mitochondria is probably located in an outer vely by an increased rate of oxygen uptake (CLARK and hydrophilic part of the membrane, and the gluta- & NICKLAS,1970). matehydroxyl antiporter is located in a hydrophobic The second carrier system present in brain mitopart of the membrane, as equally postulated pre- chondria is the glutamate-aspartate antiporter, as viously for liver mitochondria (MEYER& VIGNAIS, previously shown by BRAND& CHAPPELL (1974). Our 1973). The K , and V,,, values of the glutamate-hyd- present data confirms the presence of this glutamate roxyl carrier in brain mitochondria as calculated from translocator, since the reoxidation of intramitochonour data are in close range with those reported for drial NAD(P)H in uncoupled mitochondria in the liver mitochondria (BRADFORD & MCGIVAN,1973; presence of FCCP is obtained by added aspartate. O n MEYER& VIGNAIS,1973). These results allow us to the other hand, in the reconstituted 'malate-aspartate consider that the glutamate-hydroxyl translocator in shuttle' the rate of oxidation of extramitochondrial brain mitochondria is quite similar to that extensively NADH estimated in the absence of added aspartate studied in liver mitochondria. The low rate of swelling is also consistent with the functioning of the shuttle. of isolated brain particles incubated in iso-osmotic However, the maximal rate of oxidation of extramitoNH:-L-glutamate would be physically caused by the chondrial NADH is obtained after the addition of intricated ordering of the intramitochondrial cristae, extramitochondrial aspartate (Table l), this being as revealed in situ in electron-microscopy (PALAY & probably due to a greater activity of the extramitoCHAN-PALAY, 1973). chondrial transaminase than that of the intramitoBRAND& CHAPPELL (1974) concluded the non-exis- chondrial one. It was recently shown that external tence of a glutamate-hydroxyl antiport system in aspartate inhibited glutamate uptake via the glutabrain mitochondria from the absence of swelling of mateaspartate exchange in rat liver mitochondria et al., 1976). The in uiuo cytosolic level of the particles incubated in iso-osmotic NHZ-L-gluta- (TISCHLER mate, as well as the absence of an inhibition of the aspartate may thus control directly the functioning glutaminase activity in the presence of extramitochon- of the malate-aspartate shuttle. In these conditions, drial glutamate. However, recently DENNISet al. and in brain mitochondria, the entry of glutamate (1976) have shown that brain mitochondria exhibited would proceed via the glutamate-hydroxyl exchange, glutamate uptake and swelling in iso-osmotic N H ~ - L - which explains the high inhibitory effect of N-ethylglutamate, both of which were inhibited by N-ethyl- maleimide or avenaciolide and the restricted inhibi-

was previously shown to exert an efficient activation of the enzyme in frozen and thawed brain mitochondria (WEIL-MALHERBE, 1969).

Glutamate -transport in rat brain mitochondria

881

A. I., LIEBERC. S., BEATTIED. S. & RUBIN tory effect of glisoxepide on the reconstructed malate- CEDERBAUM E. (1973) Archs Biochem. Biophys. 158, 763-781. aspartate shuttle (Table 2). Recent data have shown J. B. (1969) in Inhibitors-Tools in Cell Research that the inhibitory effect of glisoxepide on the gluta- C~APPELL (BUCHERTH.& SIESH., eds) pp. 335-350. Springer, Berlin. mate-aspartate exchange in rat liver mitochondria W. J. (1970) J . biol. Chem. 245, CLARKJ. B. & NICKLAS appears to be not strictly specific (MEYER,J. & CUBER, 47244731. J. C., personal communication). On the other hand, DENNISS. C., LANDJ. M. & CLARKJ. B. (1976) Biochem. the inhibitory effect of mersalyl on the glutamateJ . 156, 323-331. aspartate exchange (Table 2 ) must not be ascribed GAYET J. & MINNA. (1975) 10th Meet. FEBS, Abstract to the blockade of the phosphate translocator, the No. 1166, Paris, 20-25 July. stoechiometric malate-a-ketoglutarate exchange GAYETJ., MINN A. & LEHRP. (1970) Brain Res. 18, 368-37 1. allowing the functioning of the malate-aspartate shutC. (1974) FEBS Lett. 46, 138-140. tle, but rather to the direct effect of the reagent on GODINOT the external glutamate-oxaloacetate transaminase as KINGM. J. & DIWANJ. J. (1972) A r c h Biochem. Biophys. 152, 670476. previously shown in liver mitochondria (CEDERBAUM M. E. (1976) in Mitochondria LA NOUEK. F. & TISCHLER et al., 1973). (PACKER L. & GOMEZ-PUYOU A., eds) pp. 61-78. AcaAn uncertainty remains concerning the carrier demic Press, New York. mechanism involved in the entry of [14C]glutamate LA NOUEK. F., MEIJERA. J. & BROUWERA. (1974) A r c h in brain mitochondria. However, the relatively close Biochem. Biophys. 161, 544-550. values of the apparent K,, calculated both from the LOWRY0. H., ROSEBROUGH N. J. FARRA. L. & RANDALL R. J. (1951) J . bid. Chem. 193, 265-275. estimation of the initial rate of reduction of intramitochondrial NAD(P) and the measurement of the net MEIJERA. J. & VAN DAMK. (1974) Biochim. biophys. Acta 346, 213-244. penetration of [“C]gIutamate, point out a unique MEIJER A. J., BROUWER A., REIJNGOUDD. J., HOEKJ. B. mechanism for glutamate translocation. The sensi& TAGERJ. M. (1972) Biochim. biophys. Acta 283, tivity of the glutamate carrier system in the two ex421429. perimental methods towards N-ethylmaleimide, MEISTER A. (1974) in Brain Dysfunction in Metabolic Diswhich was previously shown to be without effect on orders (PLUMF., ed.) pp. 273-291. Raven Press, New the glutamate-aspartate exchange in liver mitochonYork. dria (LA NOUE et al., 1974), and the increase of the MEYERJ. (1975) ThPse Doctorat &-Sciences Naturelles, rate of [“Clglutamate penetration when the pH graUniversite de Grenoble, 20 dtc. dient between the mitochondria1 matrix (alkaline) and MEYERJ. & VIGNAISP. M. (1973) Biochim. biophys. Acta 325, 375-384. the incubation medium rises, confirm the transport of glutamate together with a proton or in exchange MINNA. & GAYETJ. (1973) J . Physiol., Paris 67, 352 A. with a hydroxyl. Moreover, this carrier system is the MINNA. & GAYETJ. (1974) C.r. hebd. Sianc. Acad Sci, Paris 279 D, 1019-1022. only one possible when the external p H value is lower MINNA. & GAYETJ. (1976) 1st. Meet. Eur. SOC. Neurothan 7.0, since in these experimental conditions the chem., Bath, U.K., 19-23 Sept., abstract No. 31 P. glutamate-aspartate exchange is greatly slowed down MINNA,, GAYET J. & DELQRME P. (1975) J . Neurochem. (LA NOUE& TISCHLER,1976). 24, 149-156. OLNEY J. W., RHEEV. & HO 0. L. (1974) Brain Res. 77, Acknowledgements-The estimation of the redox state of 507-5 12. intramitochondrial NAD(P) was made in the Laboratoire M. & MEISTER A. (1963) Biochim. biophys. Acta de Biochimie, Departement de Recherche Fondamentale, ORLQWSKI 73, 679481. Centre #Etudes Nucleaires, Grenoble (Head: Prof. P. V. V. (1973) in Metabolic ComVIGNAIS); we thank the members of this department for PALAYS. L. & CHAN-PALAY partmentation in the Brain (BALAZS R. & CREMER J. E., their help. We wish to thank Prof. P. V. VIGNAIS and Dr. eds) pp. 187-207. Macmillan, London. P. M. VIGNAIS for useful comments during preparation of QUASTEL J. H. (1975) in Metabolic Compartmentation and the manuscript, and also Prof. C. LIEBECQ and Dr. F. E. Neurotransmission (BERLS., CLARKE D. D. & SCHNEIDER SLUSE for suggesting technical improvements in our methoD., eds) pp. 337-361. Plenum Press, New York. dology. This work was supported by grants from the Centre National de la Recherche Scientifique (E.R.A. 331) REICHELTK. L. (1970) J . Neurochem. 17, 19-25. and from the Fondation pour la Recherche Medicale REIJNIERSE G. L. A., VELDSTRAH., VAN DEN BERGc. J. (1975) Biochem. J . 152, 469475. Franqaise. SLING H. D. & SECKA. (1975) FEBS Lett. 51, 52-59. TISCHLER M. E., PACHENCE J., WILLIAMSON J. R. & LA REFERENCES NOUEK. F. (1976)A r c h Biochem. Biophys. 173, 448-462. D. F., RONDAG., REIIAzzi A,, CHAPPELLJ. 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