Age-dependent changes in glutamate oxidation by non-synaptic and synaptic mitochondria from rat brain

Mechanisms o f Ageing and Development, 13 (1980) 75 -81

75

© Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

A G E - D E P E N D E N T C H A N G E S IN G L U T A M A T E O X I D A T I O N BY NONS Y N A P T I C AND S Y N A P T I C M I T O C H O N D R I A F R O M R A T B R A I N

D. R. DESHMUKH and M. S. PATEL* Departments of Biochemistry and Nutrition, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 (U.S.A.)

(ReceivedOctober 8, 1979;in revised form January 30, 1980)

SUMMARY The oxidation of glutamate by non-synaptic and synaptic mitochondria from brains of 3-, 12- and 24-month-old rats was studied. With glutamate plus malate as substrates, non-synaptic mitochondria showed higher respiration rates than synaptic mitochondria in all the three age groups studied. The rate of oxidation of L-[1-14C] glutamate and the activities of NAD-glutamate dehydrogenase and aspartate aminotransferase were also higher in non-synaptic mitochondria compared with synaptic mitochondria in three age groups. With glutamate plus malate as substrates, a significant reduction in state 3 respiration was observed in both rnitochondrial populations from 12- and 24-month-old rats compared with 3-month-old animals. Although an age-dependent decrease in the oxidation of L-[1-14C] glutamate was observed in both non-synaptic and synaptic mitochondria from aging rats, the oxidation of [1-14C]-2-oxoglutarate was unaltered in non-synaptic and synaptic mitochondria from senescent rats. The activity of NAD-glutamate dehydrogenase was decreased with age in both mitochondrial populations, whereas aspartate aminotransferase was not altered with age. The results indicate that the oxidation rate of glutamate in rat brain mitochondria is decreased during aging.

INTRODUCTION Glutamate, a putative neurotransmitter, is present in high concentrations and metabolized rapidly in the brain [1-3]. Many investigators [4-8] have proposed that in the brain at least two distinct tricarboxylic acid cycles 'are localized in separable mitochondria and that these two tricarboxylic acid cycles are associated with large and small pools of glutamate [9, 10]. Until recently, isolation of metabolically active and relatively pure mitochondria of synaptic origin was a major problem for studying compartmentation of glutamate in the brain [10]. *To whom all correspondence should be addressed: Department or' Biochemistry, School of Medicine, Case Western ReserveUniversity,Cleveland,Ohio 44106, U.S.A.

76 It is well-documented that oxidative metabolism is reduced significantly in aging animals (for review, see ref. 11). Hhnwich [12] reported that the concentrations of glutamic acid and glutamine were decreased in the brains of aging rats. The activities of glutamate dehydrogenase [ 13] and glutamic acid decarboxylase [14] were also shown to alter in brain with development and aging. From these studies it appears that the metabolism of glutamate in the brain may be altered during aging. Although there are several reports to show that the metabolism of glutamate is altered in the mitochondria from non-neuronal tissues like heart and skeletal nmscle [15] and in rat liver [16] during aging, very little is known about the changes in glutanrate metabolism in non-synaptic and synaptic mitochondria from brains of aging rats. The present studies were undertaken to investigate possible changes in glutamate oxidation in non-synaptic and synaptic mitochondria from rat brain during aging. METHODS AND MATERIALS

Isolation of non-synaptic and synaptic mitochondria Male Sprague-Dawley rats, 3, 12 and 24 months of age, maintained on Purina Chow and water ad libiturn were used in all the experiments. Non-synaptic and synaptic mitochondria were isolated from brain using the procedure of Lai and Clark [10] with minor changes as described previously [17]. Briefly, two brains were pooled, chopped and washed in isolation medium [0.32 M sucrose, 1 mM EDTA (potassium salt), 10 mM Tris'HC1, pH 7.4], and homogenized (20%, w/v) in isolation medium with a Teflon pestle and glass homogenizer (clearance 0.1 ram), and fractionated as follows. The crude mitochondrial pellet was suspended in 3 ml of isolation medium and layered on a gradient of 7 ml of 7.5% Ficoll-sucrose on 7 ml of 13% Ficoll-sucrose solutions. The separation of the crude mitochondrial fraction on this gradient was achieved at 99 000 g for 30 min in the Beckman L2-65B ultracentrifuge with a 6 × 17 ml SW-27 swing-out rotor. The non-synaptic (free) mitochondrial pellet was resuspended in 10 ml of isolation medium containing fatty acid free bovine albumin (0.5 mg/ml), centrifuged at 12000 g for 10 min in a Sorvall Superspeed RC2-B and resuspended in about I ml of isolation medium. The synaptosomal fraction was further processed as described by Lai and Clark [10] with minor volume adjustments to isolate synaptic mitochondria which were washed with isolation medium containing fatty acid free bovine albumin (0.5 mg/ml). The pellet was resuspended in 0.5 ml of isolation medium. Mitochondrial respiration Using a Clark-type microelectrode, oxygen consumption was measured polarographically in a thermostatically controlled chamber of 1.0 ml volume at 25 °C. The medium (pH 7.4) consisted of 225 mM mannitol, 75 mM sucrose, 5 mM KC1, 5 mM phosphate-Tris (pH 7.4), 10 mM Tris-HC1 (pH 7.4) and 0.05 mM EDTA (potassium salt). State 3 condition was initiated by the addition of 0.25 mM ADP in the presence of substrates as shown in Table I. About 1 mg of mitochondrial protein was used in each experiment.

77

Oxidation of labelled substrates by mitochondria Mitochondria were incubated in a medium containing 75 mM mannitol, 57 mM sucrose, 100 mM KC1, 5 mM potassium phosphate-Tris (pH 7.4), 10 mM Tris-HC1 (pH 7.4), 50 /aM EDTA (potassium salt), 1 mM ADP and 2.5 mM L-malate with either 5 mM L-[1-14C]glutamate or 5 mM [1-14C]-2-oxoglutarate (New England Nuclear, Boston, MA, U.S.A.), as indicated in Table II. Reaction was started by the addition of about 1 mg of mitochondrial protein. The flasks were gassed with O2 + CO2 (95:5, v/v), sealed with a rubber serum stopper equipped with a hanging polyethylene center well, and incubated for 15 min in a shaking water-bath at 37 °(2. At the end of the incubation period, ~4CO2 was trapped in hyamine 10-X injected into the well and radioactivity was determined. Enzyme assays NAD-glutamate dehydrogenase (EC 1.4.1.2) [10, 18] and aspartate aminotransferase (EC 2.6.1.1) [19] were assayed spectrophotometrically at 37 °C. Initial rates were proportional to the amount of enzyme added (at least at two protein concentrations). An aliquot of mitochondrial preparation was treated with Triton X-100 (t'mal concentration 0.05%, w/v). A unit of activity is defined as 1 /amole of substrate utilized or product formed per minute at 37 °C. Protein was measured by Lowry's method [20] with bovine serum albumin as a standard. Statistical analyses were performed using Student's t-test.

RESULTS AND DISCUSSION Although several investigators [15, 16, 21] have reported age-dependant changes in enzyme activities and metabolism of mitochondria from different non-neuronal tissues, studies on possible alterations in non-synaptic and synaptic mitochondria from brains of senescentanimals have been limited. The major obstacle has been the lack of a suitable procedure for the isolation of metabolically active and relatively pure mitochondria from brain. Non-synaptic and synaptic mitochondria isolated from brains of 3-, 12- and 24month-old rats by the method of Lai and Clark [10] were metabolically active and well coupled, as seen from high respiratory control ratios (values above 5 for non-synaptic mitochondria and above 4 for synaptic mitochondria metabolizing glutamate plus malate, Table I). High respiratory control ratios were also obtained in both mitochondrial populations when pyruvate plus malate were used as substrates [17]. It was also observed that non-synaptic and synaptic mitochondria obtained by this method were almost devoid of contamination by subcellular components as seen from very low activities of lactate dehydrogenase (mean -+S.E.M. of the total of 18 observations for three groups were 133 -+ 11 and 40 + 3 munits/mg of protein for non-synaptic and synaptic mitochondria, respectively) and acetylcholinesterase (mean +S.E.M. of 18 observations were 11 + 1 and 9 + 1 munits/mg of protein for non-synaptic and synaptic mitochondria, respectively) [17]. With glutamate plus malate as substrates, non-synaptic mitochondria showed higher state 3 respiration rates (63%) than synaptic mitochondria in all three age groups studied.

L-Glutamate (2.5 mM) plus L-malate (2.5 mM)

Substrate added

126 -+ 3 111 -+ 2 (<0.005) 80 -+ 6 (<0.001)

12

24

16 +- 1 (<0.001)

27 +- 1 (ns)

26 -+ 1

5.1 -+ 0.3

4.1 +- 0.1

5.0 +- 0.3

43 ± 3 (<0.001)

45 -+ 1 (<0.001)

77 +- 6

11 -+ 0.6 (<0.005)

14 ± 0.4 (<0.005)

19 +- 1

State 4 ( - ADP)

State 3 (+ADP)

RCR

State 3 (+ADP)

State 4 (-ADP)

Synaptic mitochondria

Non-synaptic mitochondria

Respiration rate (natoms of 0 per min per mg of protein)

3

Age (months)

4.0 -+ 0.3

3.2 +- 0.1

4.0 -+ 0.2

RCR

The results are expressed as mean ± S.E.M. of six expernnents, p values are shown in parentheses; ns indicates not significant (p > 0.05). Respiratory control ratio (RCR) = State 3 respiration/State 4 respiration.

RATES OF RESPIRATION AND THE RESPIRATORY CONTROL RATIOS OF NON-SYNAPTIC AND SYNAPTIC MITOCHONDRIA FROM BRAINS OF 3-, 12-AND 24-MONTH-OLD RATS

TABLE I

79 TABLE II THE OXIDATION OF LABELLED SUBSTRATES TO 14CO2 BY NON-SYNAPTIC AND SYNAPTIC MITOCHONDRIA FROM BRAINS OF 3-, 12- AND 24-MONTH-OLD RATS Results are the means ± S.E.M. for six experiments, p values are shown in parentheses; ns indicates not significant (p > 0.05).

Labelled substmte

Age [months)

nmoles of labelled substrate oxidized per mg of protein per 15 min Non-synaptic mitochondria

L-[ 1-14C] Glutamate (5 mM) plus L-malate (2.5 mM)

• [ 1-14C]-2-Oxoglutarate (5 mM) plus L-malate (2.5 mM)

Synaptic mitochondria

3

1375 ± 41

645 ± 35

12

827 ± 18 (<0.001)

448 +-17 (<0.001)

24

816 ± 21 (<0.001)

473 ± 20 (<0.005)

3

1939 ± 56

1041 ± 44

24

2055 £ 46

(ns)

981 ± 44

(ns)

The oxidation of L-[1-14C] glutamate and [1-14C]-2-oxoglutarate was also higher in nonsynaptic compared to synaptic mitochondria (Table II). Specific activities of NAD-glutamate dehydrogenase and of aspartate aminotransferase were also higher in non.synaptic compared with synaptic mitochondria. Our findings agree with that of Van den Berg [9] who reported that NAD-glutamate dehydrogenase activity was higher in non-synaptic than synaptic mitochondria. However, Lai and Clark [10] reported that synaptic mitochondria had higher activity of NAD-glutamate dehydrogenase than non-synaptic mitochondria. Although a reason for this discrepancy is not apparent, Clark and his colleagues also observed higher rates of respiration in the presence of glutamate plus malate [10] and higher rates of glutamate synthesis from ~ketoglutarate and ammonium acetate [22] by non-synaptic than synaptic mitochondria. These observations, although not consistent with their findings on the activity of NAD-glutamate dehydrogenase in two mitochondrial populations, are in agreement with our data (Tables I and II). With glutamate plus malate as substrates, an age-dependent decrease in state 3 respiration was found in non-synaptic mitochondria in 12- and 24-month-old rats (13% and 37%, respectively) compared to 3-month-old animals. Synaptic mitochondria also showed a decrease in state 3 respiration, with glutamate plus malate as substrates in 12- and 24month-old rats compared with 3-month-old animals. Similar age-dependent reduction in the oxidation of L-[1-14C]glutamate was observed in non-synaptic (40%)and synaptic (30%) mitochondria from brains of senescent animals, whereas the oxidation of [1-14C] 2-oxoglutarate by non-synaptic and synaptic mitochondria was not altered in 24-month-

80 TABLE III SPECIFIC ACTIVITIES OF NAD-GLUTAMATE DEHYDROGENASE AND ASPARTATE AMINOTRANSFERASE IN NON-SYNAPTIC AND SYNAPTIC MITOCHONDRIA FROM BRAINS OF 3-, 12- AND 24-MONTH-OLD RATS The results are the means ± S.E.M. for six experiments, p values are shown in parentheses; ns indicates not significant (p > 0.05).

Enzyme

NAD-glutamate dehydrogenase

Aspartate aminotransferase

Age (months)

munits/mg of protein Non-synaptic mitochondria

Synaptic mitochondria

3

1548 ± 96

805 ± 20

12

11605 100 (< 0.02)

437 -+46 (<0.001)

24

1227 ± 53 (<0.05)

462 _+26 (<0.001)

3

3996 ± 138

2866 _+132

12

4149 ± 85 (ns)

3330 ± 128 (ns)

24

4449 ± 250 (ns)

2792 _+74 (ns)

old rats. The activity o f NAD-glutamate dehydrogenase was found to decrease with age in both non-synaptic (25%) and synaptic (46%) mitochondria. However, the activity o f aspartate aminotransferase was not altered with age in both mitochondrial populations. It has been shown recently that non-synaptic mitochondria from rat brain possess b o t h g l u t a m a t e - a s p a r t a t e and g l u t a m a t e - O H - translocases [23, 2 4 ] . The transamination reaction is the major pathway for the oxidation o f glutamate, accounting for about 90% o f the glutamate uptake in rat brain mitochondria [25]. The results reported here show that, although the activity o f aspartate aminotransferase (Table III) and the oxidation o f 2-oxoglutarate (Table II) were unaltered in mitochondria from brains of all three age groups studied, the oxidation o f glutamate in the presence of malate was significantly decreased in both mitochondrial populations from aging rats (Tables I and II). These findings suggest that glutamate transport across the inner mitochondrial membrane may be altered during aging.

ACKNOWLEDGEMENTS This work was supported by the National Institutes of Health Grants AG-01345, AG-00146 and NS-11088. We thank Cindy Raefsky and Carolyn Brown for technical assistance.

81 REFERENCES 1 A. M. Benjamin and J. H. Quastel, Metabolism of amino acids and ammonia in rat brain cortex slices in vivo. Z Neurochem., 25 (1975) 197~206. 2 C. J. Van den Berg, in A. Lajtha (ed.), Handbook ofNeurochemistry, Vol. 3, Plenum Press, New York, 1970, pp. 355-379. 3 G. Guroff, in R. W. Albert, G. J. Siegel, R. Katzman and B. W. Agranoff (eds.), Transport and Metabolism of Amino Acids, Churchill Livingstone, 1972, pp. 191-206. 4 A. Neidle, C. J. Van den Berg and A. Grynbaum, The heterogeneity of rat brain mitochondria isolated on continuous sucrose gradients. Z Neurochem., 16 (1969) 225-234. 5 C. J. Van den Berg, L. J. Krazalic, P. Mela and H. Waelsch, Compartmentation of glutamate metabolism in brain. Biochem. Z, 113 (1969) 281-290. 6 C. J. Van den Berg, D. F. Matheson, G. Ronda, A. L. A. Reijnierse, A. A. D. Blockhuis, M. C. Kroon, D. D. Clarke and D. Garfinkel, in S. Berl, D. D. Clarke and D. Schneider (eds.), Metabolic Compartmentation andNeurotransmission, Plenum Press, New York, 1975, pp. 515-543. 7 R. Balazs and J. E. Cremer, in R. Balazs and J. E. Cremer (eds.), Metabolic Compartmentation in the Brain, Macmillan, London, 1973, pp. 167-184. 8 S. Bed, D. D. Clarke and D. Schneider (eds.), Metabolic Compartmentation and Neurotransmission, Plenum Press, New York, 1975. 9 C. J. Van den Berg, Model of compartmentation in mouse brain based on glucose and acetate metabolism. In R. Balazs and J. E. Cremer (eds.), Metabolic Compartmentation in the Brain, Macmillan, London, 1973, pp. 137-166. 10 J. C. K. Lai and J. B. Clark, Preparation and properties of mitochondria derived from synaptosomes. Biochem. Z, 154 (1976) 423-432. 11 D. R. Sanadi, Metabolic changes and their significance in aging. In C. E. Finch and L. Hayflick (eds.), Handbook o f the Biology of Aging, Van Nostrand Reinhold, New York, 1977, pp. 73-98. 12 W. A. Himwich, Neurochemical patterns in the developing and aging brain. In M. Rockstein (ed.), Development and Aging in the Nervous System, Academic Press, New York, 1973, pp. 151-170. 13 G. Kaur and M. S. Kanungo, Alterations in glutamate dehydrogenase of the brain of rats of various ages. Can. J. Biochem., 48 (1970) 203-206. 14 M. H. Epstein and C. H. Barrows, Jr., The effects of age on the activity ofglutamic acid decarboxylase in various regions of the brains of rats. J. Gerontol., 24 (1969) 136-139. 15 C. J. Chen, J. B. Warsaw and D. R. Sanadi, Regulation of mitochondrial respiration in senescence. Z Cell. Physiol., 80 (1972) 141-148. 16 E. C. Weinbach and J. Garbus, Oxidative phosphorylation in mitochondria from aged rats. J. Biol. Chem., 234 (1959) 412-417. 17 D. R. Deshmukh, O. E. Owen and M. S. Patel, Effect of aging on the metabolism of pyruvate and 3-hydroxybutyrate in non-synaptic and synaptic mitochondria from rat brain. J. Neurochem., 34 (1980) 1219-1224. 18 E. Schmidt, in H. U. Bergmeyer (ed.), Methods in Enzymatic Analysis, Academic Press, New York, 1963, pp. 752-756. 19 R. Balazs, D. Dahl and J. R. Harwood, Subcellular distribution of enzymes of glutamate metabolism in rat brain. J. Neurochem., 13 (1966) 897-905. 20 O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with the folin phenol reagent. J. Biol. Chem., 193 (1951) 265-275. 21 B. Bulos, S. Shukla and B. Sacktor, Bioenergetic properties of mitochondria from flight muscle of aging blowflies. Arch. Biochem. Biophys., 149 (1972) 461-469. 22 S. G. C. Dennis and J. B. Clark, The synthesis of glutamate by rat brain mitoehondria. J. Neurochem., 31 (1978) 673--680. 23 S. C. Dennis, J. M. Land and J. B. Clark, Glutamate metabolism and transport in rat brain mitochondria. Biochem. J., 156 (1976) 3 2 3 - 3 3 1 . 24 A. Minn and J. Gayet, Kinetic study of glutamate transport in rat brain mitochondria. J. Neurochem., 29 (1977) 873-881. 25 R. Balazs, Control of glutamate oxidation in brain and liver mitochondrial systems. Biochem. Z, 95 (1965) 497-508.