Energetics of amino acid transport into brain slices: Effects of glucose depletion and substitution of krebs' cycle intermediates

Brain Research, 131 (1977) 321-334 © Elsevier/North-Holland Biomedical Press

321

E N E R G E T I C S OF A M I N O A C I D T R A N S P O R T I N T O B R A I N SLICES: E F F E C T S OF G L U C O S E D E P L E T I O N A N D S U B S T I T U T I O N OF K R E B S ' CYCLE INTERMEDIATES

D. N. TELLER*, M. BANAY-SCHWARTZ, T. DEGUZMAN and A. LAJTHA New York State Research Institute for Neurochemistry and Drug Addiction, Ward's Island, New York, N.Y. 10035 (U.S.A.)

(Accepted December 3rd, 1976)

SUMMARY Amino acid uptake by slices of brain is greatly diminished by incubation of the slices in glucose-free media. Uptake can be restored by the addition of a number of substrates, e.g., lactate, citrate, or oxaloacetate. The mixture of succinate, malate, and pyruvate (SMP, 20, 5, and 5 m M ) restored amino acid uptake better than glucose after brain slices were incubated in glucose-free media to deplete endogenous energy stores. The degree of restoration of uptake was different with the various amino acid transport classes and was independent of the restoration of ATP levels in the tissue. After restoration of uptake with SMP the amino acid uptake was resistant to NaF, but was markedly more sensitive to arsenite and oligomycin. The results indicate the coupling of mitochondrial energy transducing systems to transport.

INTRODUCTION Active transport of amino acids into brain slices requires a metabolic energy sourcea, 9. Inhibitors of glycosis and of mitochondrial enzymes are also potent inhibitors of amino acid transport into brain slices 2,a,5. However, most media used for studies of amino acid transport in brain slices, cells, or subcellular particles use glucose as an energy source. We were interested in finding a substitute for glucose because mitochondrial enzyme inhibitors seemed more potent than inhibitors of glycolytic enzymes in blocking amino acid transporta,5; in other tissues, for instance, in the kidney, glucose is mildly inhibitory towards amino acid 17 or morphine transport 30,

* To whom correspondence should be addressed at: Department of Psychiatry and Behavioral Sciences, University of Louisville Medical School, P.O. Box 1055 MDR 517, Louisville, Ky. 40201, U.S.A.

322 and is ineffective for support of alpha-aminoisobutyric acid (AIB) uptake in liver slices 31. Precedents for using Krebs' cycle intermediates to supply energy for transport can easily be traced through studies on amino acid and sugar transport into isolated bacterial vesiclesa,21, 32,33. Such investigations usually employed only one or two non-glycolytic energy sources, e.g., lactate, or phenazine methosutfate (PMS) plus ascorbate. Earlier investigations had shown strong inhibition of active transport by blockade of mitochondrial electron transport with rotenone, antimycin-A, or 2-nheptyl-4-hydroxyquinoline-N-oxide, whereas atractylate, atractyloside, phlorizin, phloretin, and other specific inhibitors of nucleoside or sugar transport were not inhibitory 3,5,29,30. Thus, one of the control points for energy transduction to transport appeared to lie at the flavin-ubiquinone-cytochrome b level, in a mitochondrial, rather than glycolytic, enzyme complex 19,2°. Moreover, inhibition of transport could be reversed by succinate, a further indication of energy coupling at this levepS,2~, 30. Underlying the experimental approach were the assumptions that (a) if the energy stores of the tissue are removed without causing irreversible damage, such stores might be replaced or substituted; (b) if the removal process (which is termed 'depletion' in this report) causes some irreversible damage, the uptake process will not be completely restored; and (c) if the tissue does not have to perform much work while undergoing depletion, damage will be minimized. D-Glutamate and alphaaminoisobutyric acid (D-GIu and a-AIB) were chosen as 'unmetabolized' amino acids because of an initial assumption that alteration of the energy source in the media might give false results by stimulating metabolism of amino acids, with subsequent retention of isotope. However, analyses of the amino acid composition of the media and tissues indicated that this problem does not occur to a significant degree. In addition, the uptake of non-metabolized amino acid is a good index of cell viability ~6. Here we demonstrate that glucose and glycolysis are not required for low affinity active transport of D-GIu, AIB, glycine, valine, histidine, ~,-aminobutyrate (GABA) or lysine. In addition, the degree of inhibition of D-GIu or AIB uptake by various metabolic inhibitors depends upon the substrate utilized after glucose is depleted. MATERIALS AND METHODS Brain (cortex) slices were prepared from adult male or female mice bred from a Swiss-Webster strain as previously reported z,a,29. Left and right cerebral cortices were sliced transversely at 0.42 mm with a McIlwain-Mickle mechanical chopper. The cerebral hemispheres were kept in iced media prior to slicing. Hepes-2 medium was prepared as described previously 2,a,29 : (a) Hepes (--) G ~ Hepes-2 without glucose; (b) SMP = Hepes (--) G with addition of 20 m M succinate, 5 mM malate, and 5 m M pyruvate (as sodium salts). Sources of drugs, inhibitors, and substrates have been listed previously3, 3°. Measurements of dry weight, swelling, and inulin space were performed by methods described 1. Radioactive amino acids were obtained from New England Nuclear, AmershamSearle or Calatomic, as in previous studies 2,a,~9. The specific activities were reduced

323 by addition of unlabelled carrier amino acids to 1-5 × 104 disint./min//~mole. The stock, concentrated amino acid (10-100 m M ) was prepared in Hepes-G. The overall incubation period was usually divided into three phases: depletion, restoration and uptake. The depletion phase was usually 30 min at 37 °C, shaking at 1.6 -2 Hz in media (Hepes-2 or Hepes-G) that had been oxygenated for 2-4 min at 25 °C. The depletion phase control was the 'preincubation', or more correctly, the standard 30 min temperature equilibration period3, 3° before incubation with radioactive amino acids. The restoration phase began after 30 min of depletion by addition of 0.5 ml of SMP (10 times concentrated) or another energy source to 4.5 ml Hepes-G, or by transfer of tissue to fresh SMP. Slices were filtered from media using a Hirsch funnel. Uptake incubation started 20-30 min after restoration by the addition of 14C-amino acid to the appropriate medium. At the end of the uptake incubation the slices were filtered2,3, za from the medium and were frozen in pulverized CO2. The frozen tissue masses were weighed and homogenized in 2 ml of ice-cold 3 % (w/v) perchloric acid (PCA). The homogenates were quickly centrifuged at high speed (approx. 12-15,000 ref × gav., for 4 min at 4-8 °C) to prepare supernatants for ATP analysis. I f only Na + and K + were to be determined, the homogenization and centrifugation were performed more leisurely. If only radioactivity was to be measured, the frozen tissues were digested in scintillation vials with 1 ml 1 N N a O H for 30 rain at 60 °C; then 1.5 ml 1 N HC1, 2.4 ml H 2 0 and 8.8 ml of tT76 (toluene, Triton) 2a scintillator were added, and shaken to produce a thixotropic gel at l0 °C. The radioactivity of PCA supernatants was determined using 0.5 ml/16 ml of modified Prockop-Ebert scintillation fluid 29. Scintillation counting procedures, glassware calibration, and quench correction techniques have been described previously ~a. Na + and K + contents of diluted PCA supernatants were measured with an IL-343 flame photometer; ATP was determined with a duPont Luminescence Biometer a. Calculation procedures have been detailed elsewhere 2,a for measurement of concentrative uptake (CU = / ~ m o l e s of amino acid in 1 ml ofintracellular fluid, above the final medium concentration at the end of incubation.) Amino acid analysis of tissue incubated in Hepes-2 and SMP media were performed by the methods of Neidle et al. 22 to determine the extent of changes in the pattern of amino acids in the tissue. Media samples examined by the same methods (of which a more detailed account is in preparation) indicated that, with the exception of glutamate, there was no significant metabolism that would bias the uptake data by more than 5 % vs. the radioactivity (direct) measurement (see also ref. 26). Uptake from 1 m M D- or L-Glu indicated that 30 % of the total radioactivity from [14C]Glu after 30 min at 37 °C (with Hepes-2 or SMP) was associated with glutamine in the medium. Therefore, the C U data from incubations with D-[laC] - or L-[14C]Glu do underestimate the t o t a l molecules of Glu taken into the slice, because a significant portion appears to preferentially exit from the mouse brain slices into the medium as glutamine. However, for comparative purposes, since the [14C]glutamine did not accumulate in the slices, nor did it vary with 1 or 2 m M external D- or L-Glu (based on the specific activity at the start of incubation and the radioactivity in the slices at the end) it does not appear to be affected in a manner that invalidates the general observations which we report in this study. For example,

324 we did n o t observe radioactivity in aspartate after i n c u b a t i o n of slices in D-Glu ( - - ) glucose, as might be expected with L-Glu (see Table I of Benjamin a n d Quastell0). RESULTS Table I shows the effects of the i n c u b a t i o n conditions on the b r a i n slice components a n d o n the uptake of D-Glu, A1B, or L-Lys. ATP, K +, and concentrative uptake of the a m i n o acids decrease when the slices are i n c u b a t e d in the absence of glucose, while N a + a n d swelling increase. These changes are partially reversible if glucose is added after a period of depletion. However, the uptake of a m i n o acid and the reduction of swelling are restored to a m u c h better degree t h a n are the levels of ATP, N a ÷, or K ÷. A m i n o acid uptake can be partially restored if, instead of glucose, other metabolizable substrates are added to the medium. The s o d i u m salts of lactate, o f p y r u v a t e , or the mixture of succinate, malate, a n d pyruvate, ( S M P ; 20, 5, and 5 m M , respectively) added after glucose depletion instead of glucose, also restore the tissue levels of TABLE I Tissue components and amino acid uptake in the presence or absence o f glucose Tissue treatment Fresh, unincubated slices

Complete medium, 30 rain*

Tissue components (/tmoles/ml tissue H20) ATP 0.40 1.12 60 106 Na + K÷ 140 7O H20§§ 3.54 5.27 Per cent swelling§§§ 0 49 Concentrative uptaket o-Glu (1 mM) A1B (2 mM) L-Lys (2 raM)

14.4 9.7 2.3

Depletion, 30 min**

Depletion, 60 rain***

Depletion, 30 min; Restoration, 30 rain§

0.09 148 20 5.58 58

0.06 146 22 6.47 83

0.45 137 49 5.42 53

8.7 5.8 1.0

6.7 4.8 0.8

11.8 8.3 1.4

* Thirty minute temperature equilibration in Hepes-2 at 37 ~C before ~4C-amino acid addition. ** Thirty minute temperature equilibration in Hepes-2 (--) glucose (Hepes-G) at 37 'C ('depletion') before amino acid addition. *** Sixty minute depletion before amino acid addition. § Thirty minute depletion, plus 30 rain of 'restoration' (10 mM glucose added) before amino acid addition. §§ Tissue water, ml/g frozen fresh tissue. §§§ Per cent swelling, (H20 in slices after incubation (ml/g) frozen weight/H~O in unincubated slices) × 100 (--) 100. Swelling data is only for incubations with D-Glu; in AIB and Lys incubations it is somewhat lower. t Concentrative uptake of 14C-amino acid, for 10 rain in presence of 10 mM glucose after the respective tissue treatment. Data are averages from 3-12 separate incubations with estimated standard error (e.s.e.) < 6 %.

325 A T P , N a + o r K ÷. I n all cases, the A T P remains low ( a b o u t 40 ~o o f control); the N a ÷ is elevated (25-50 ~o), a n d the K ÷ is decreased ( a b o u t 60 ~o o f control). Nevertheless, the u p t a k e o f D-GIu, AIB, L-Lys, L-Val, L-His, a n d L-GIu is p a r t i a l l y r e s t o r e d after glucose d e p l e t i o n by the a d d i t i o n o f either lactate, pyruvate, or glucose, or is c o m pletely r e s t o r e d using S M P (Table II). There are some difference3; S M P restores glut a m a t e u p t a k e better, b u t lysine u p t a k e less well, t h a n glucose. Lactate, alone, is able to p a r t i a l l y restore u p t a k e o f D-GIu, A I B , a n d Val, whereas pyruvate, alone, is inhibitory to A I B , Lys, a n d G l y uptake. A l p h a - g l y c e r o p h o s p h a t e d i d n o t restore u p t a k e o f the a m i n o acids a n d was n o t tested further. T h e a d d i t i o n o f succinate to m a l a t e or p y r u v a t e increased the u p t a k e o f a m i n o acids over that achieved with m a l a t e or p y r u v a t e alone (Table III). F u r t h e r m o r e , m a l a t e e n h a n c e d the recovery o f the slice u p t a k e o f lysine, even t h o u g h the A T P , N a ÷, a n d K + levels were identical to those o f tissues that h a d no energy source at all ( ' n o n e ' ) . Lactate, pyruvate, succinate, a n d citrate were less effective t h a n glucose, b u t 5 m M oxaloacetic acid s u p p o r t e d g l u t a m a t e u p t a k e better t h a n d i d glucose, a n d could s u p p o r t some o f this u p t a k e after extended p e r i o d s o f glucose d e p l e t i o n (Table IV). Oxaloacetate, alone, was n o t as effeztive in restoring u p t a k e o f D-GIu after depletion,

TABLE II Restoration o f amino acid uptake with different substrotes

Brain slices were incubated for 30 min in a medium lacking glucose (depletion), then for 30 min in the presence of a substrate (restoration), before the addition of the labeled amino acid used for uptake measurement. Substrate levels: D, L-lactate, Na-pyruvate, and glucose were 10 mM; SMP was Na-succinate 20 mM, Na-malate 5 mM, and Na-pyruvate 5 mM; a-glycerophosphate was 20 mM. In control experiments the depletion incubation was omitted. Averages of 9 experiments are given, with an e.s.e, within 5 ~ of the mean. Uptake Control Concentrative antino acid uptake (l~moles/ml intracellular water (rain) glucose above medium) None

D-GIu (1 mM) a-AIB (2 raM) e-Lys (2 mM) e-Val (1 mM) L-His (1 mM) Gly (1 mM)

10 20 30 10 20 30 10 20 30 20 20 20

13.5 22.9 28.6 9.9 18.1 22.1 2.03 3.63 5.40 8.8 11.9 19.2

1.96 2.94 3.26 2.47 5.27 7.24 1.35 1.88 2.1 2.4 6.4

Glucose

11.6 19.6 25.4 7.57 15.5 18.9 1.20 3.00 3.81 7.09 10.2 13.4

Lactate Pyruvate

8.25 16.3 22.8 5.65 13.0 16.1 0.85 2.36 2.98 5.1 5.1 8.9

7.34 13.6 16.0 2.68 4.30 4.94 0.71 1.11 2.69 2.1 4.2 4.5

a-Gly-P S M P

2.58 4.82 5.77 2.39 5.90 5.50 0.88 2.37 3.36 3.1 3.0 4.8

16.1 24.4 30.5 8.76 17.9 22.6 0.90 2.63 3.55 6.8 10.8 16.3

Slice contents, I~moles/ml tissue water

ATP Na + K÷

I0 10 10

1.12 106 70

0.07 165 24

0.47 136 49

0.36 140 43

0.40 154 45

0.36 177 44

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327 TABLE IV Restoration o f glutamate uptake by various substrates

Brain slices were incubated with 1 mM [14C]D-glutamate for 10 min. Averages of 5 experiments are given with an e.s.e, within 6 ~ of the mean. Substrate

Concentrative uptake o f glutamate, I~moles/ml intracellular water above medium No depletion*

Substrate o, L-lactate, 5 mM Na-pyruvate, 5 mM Na-succinate, 5 mM Na-citrate, 2 mM Na-oxaloacetate, 5 mM Glucose, 10 mM None

8.5 6.9 3.4 3.5 15.9 13.0 2.2

No restoration* * 30 min

60 min

6.0 6.1 2.1 1.5 5.6 8.7 2.2

3.9 3.5 1.8 1.4 8.6 6.7 1.6

Depletion and restoration* * *

7.2 7.7 4.0 4.3 9.4 11.8 1.9

* The tissue was incubated for 30 min with substrate, then with glutamate for 10 min. The tissue was incubated for 30 or 60 min without substrate, then with substrate and glutamate for 10 min. *** The tissue was incubated for 30 rain without substrate, for 30 rain with substrate, then with glutamate for 10 min. **

as was the SMP combination. In separate experiments (not shown in Table IV) fumarate and citrate (1-5 mM) did not restore uptake of Lys, Glu, or AIB. We compared the ability to restore uptake of the Tris- and potassium-neutralized tricarboxylic acids with that of the sodium-neutralized ones. Generally, the utilization of Tris-neutralized succinate and malate, with potassium pyruvate, increased D-GIu uptake markedly in comparison with the results from incubations with the corresponding sodium salts, whether the substrates were tested singly, in pairs, or as SMP. For example, the 20 min concentrative uptake of D-Glu increased from 6.1 with sodiumsuccinate (20 mM) plus malate (5 mM) to 15.3 when Tris replaced Na +. With pyruvate, alone, the increase was from 12.7 (sodium salt) to 17.8 (potassium salt). This effect of Na + on glutamate uptake was not observed with other amino acids. With AIB, Val, His, and Lys we did not observe significant differences between restoration of uptake with the Tris or sodium salts of the Krebs cycle intermediates. These results are summarized in Table V. Not all compounds show similar restoration of uptake with added substrates. For example, G A B A uptake was restored best; Leu and Trp were also restored; while the uptake of ornithine (Orn), tryptamine, choline, and decanoic acid was not restored (Table VI). The lack of inhibition of tryptamine and decanoic acid uptake by glucose depletion indicates that the uptake of these compounds is not energy dependent under these conditions.

328 TABLE V Effect of Na + and Tris salts of substrates on the restoration of amino acid uptake Slices were incubated for 30 rain without glucose (depletion), then 30 min with the substrate (restoration), and then 20 min with substrate and amino acid (uptake). The concentrations were: D-glutamic acid, 1 mM; a-aminoisobutyric acid, 2 mM; glucose, 10 mM; succinate, 20 mM; malate, 5 mM; pyruvate, 5 mM. Uptake is given as concentrative uptake (level in intracellular water above medium) ATP and K + as level in tissue water. Averages of 6 experiments are given, with an e.s.e, within 5 % of the mean. Substrate

Glucose Na-SMP Tris-SMP

D-glutamic acid (#moles/ml water)

a-aminoisobutyric acid (t~moles/ml water)

Uptake

.4 TP

K+

Uptake

A TP

K+

19 22 29

0.49 0.31 0.35

44 44 58

14 17 17

0.79 0.46 0.49

38 43 45

Indications o f specificity o f energy sources f o r transport o f different amino acids The particular substrate utilized for energy alters the uptake o f a m i n o acids to various degrees, not only when substituted for glucose, but also when energy m e t a b o l i s m is decreased by some m e t ab o l i c inhibitors. Substitutions for glucose altered inhibition o f A I B or G l u uptake by various ' m e t a b o l i c inhibitors' (Table VII). Inhibition by arsenite, oligomycin and o u ab ai n was greatest in the presence o f pyruvate, whereas the inhibition o f a m i n o acid uptake by other c o m p o u n d s , such as fluoroacetate, 2 , 4 - D N P , or rotenone, was greatest in the presence o f lactate. M a r k e d differences could be observed using other substrate and inhibitor c o m b i n a t i o n s : inhibition by TABLE VI Restoration of uptake of additional test compounds in brain slices Values given are the average of 4 experiments with an e.s.e, within 7 % of the mean. Compound

GABA L-Leu Trp L-Orn Trp-NH2 Choline Decanoic acid

hfftial medium concentration (raM)

Concentrative uptake (l~moles per ml intracellular water above medium) No depletion*

No substrate**

+ SMP***

1 1 0.1 1 1 0.001 0.01

24.1 3.3 1.4 4.4 6.4 1.2 0.3

9.3 1.0 0.4 2.8 6.2 0.5 0.2

20.0 2.4 1.0 2.8 6.2 0.6 0.2

* Thirty minutes temperature equilibration in presence of 10 mM glucose prior to incubation for 20 rain uptake. ** Thirty minutes depletion, no substrate added. *** Thirty minutes depletion, 30 min restoration with Na-succinate (20 mM), Na-malate (5 mM), and Na-pyruvate (5 mM) (SMP) prior to incubation.

329 TABLE VII Dependence o f the inhibition o f amino acid uptake upon the substrate supplying energy

Depletion and restoration of slices was for 30/30 min. Amino acid uptake was for 10 min with 1 mM D-glutamate or 2 mM aminoisobutyrate. Glucose, lactate, and pyruvate were 10 mM; SMP, as before (20, 5, 5 raM). Averages of 7 experiments with an e.s.e, within 6 ~ of the mean are given. Inhibitor concentrations shown are only 1-3, of up to 9 examined, within the range listed under '(mM)' or '(/~M)'. Uptake as per cent o f control (undepleted) slices

Inhibitor

Glucose

Lactate

Pyruvate

SMP

Glu

AIB

Glu

AIB

Glu

A1B

Glu

AIB

86

86

71

71

59

24

106

99

(mM) 0.05 0.2 2.0 10.0 2.0 20.0 0.3 0.1 0.4 0.02 0.04

93 88 77 25 99 99 22 87 69 77 54

72 62 58 12 85 55 56 63 48 89 68

75 21 55 2 56 27 57 47 34 25 2

33 8 43 15 34 14 43 44 17 43 8

16 4 52 43 79 76 56 49 35 51 18

16 9 22 33 19 18 32 22 9 19 15

53 8 124 103 114 85 99 106 11 79 5

34 8 93 75 87 49 46 81 22 88 19

(#M) 0.02 0.10 0.1 0.5 0.2 0.9 11.5 0.45 1.8 1.0 10.0

80 50 68 40 91 71 68 66 56 55 35

62 35 68 40 99 87 60 52 27 49 17

32 5 39 13 57 33 9 30 8 70 32

38 11 49 22 44 25 10 25 8 56 15

43 7 36 7 33 14 9 8 2 47 27

23 10 17 8 23 17 8 13 6 20 12

82 3 68 15 57 33 4 23 5 68 42

63 15 66 28 67 45 15 29 13 30 --

None Arsenite NaF Fluoroacetate lodoacetate Amytal 2,4-DNP

Rotenone Antimycin A Oligomycin

Valinomycin Ouabain

N a F was greater than that o f o l i g o m y c i n with glucose, but m u c h less than o l i g o m y c i n with SMP. Responses o f neutral a m i n o acid t r a n s p o r t to inhibitors after substrate substitution were generally similar to those shown for A I B in Tab l e V I I : 62-65 ~ o f the uninhibited, glucose-restored u p t a k e was b l o ck ed by 10 m M N a F in glucosec o n t a i n i n g media, while u p t a k e in S M P was n o t blocked (Leu) or was inhibited only 5-9 ~ (Val, Gly). Generally, substitution o f S M P for glucose further reduced u p t a k e after t r e a t m e n t with inhibitors o f m i t o c h o n d r i a l electron t r a n s p o r t or p h o s p h o r y l a tion, while lactate a n d S M P decreased inhibition caused by ouabain. Differential r e s t o r a t i o n o f t r a n s p o r t o f a m i n o acids within an d between carrier classes was observed. Typical data are sh o w n in Tab l e I I ; whereas lactate restored two-thirds to three-quarters o f the u p t a k e o f valine or A I B , it restored less t h a n h a l f

330 of the glycine uptake. It can also be seen that pyruvate, alone, was mildly inhibitory to neutral amino acid uptake, although it permitted some restoration of transport of D-Glu, an amino acid of another transport class. Thus, among amino acids with high capacity, low-affinity transport in slices (i.e. CU typically greater than 5 with 1-2 m M initial external medium levels), uptake of a basic amino acid seems to be restored by malate (and by a-glycerophosphate) and is inhibited by pyruvate; the converse holds for the acidic amino acid, whereas neither pyruvate nor malate (alone) restores uptake of AIB. No evidence was found for energy coupling via flavins directly from electron transport. D N P inhibition was not reversed by amytai (Table VIII), while 10 # M CPZ and 2.5/~M PMS did not block uptake when pyruvate, succinate, lactate, or glucose were substrates for uptake of D-Glu. Despite the lack of effect on amino acid uptake, the addition of CPZ or PMS lowered the slice ATP levels, which indicated that these compounds did produce the expected uncoupling of the tissue's high-energy phosphate metabolism. Slice uptake is unrelated to the A T P level

In these experiments with glucose-depleted slices, as in previous experiments with undepleted slices3, 7, the changes in ATP levels were not parallel to changes in uptake. ATP content was higher with glucose than with SMP; in contrast, amino acid uptake was higher with SMP than with glucose (Table V). Previously, it was shown that when substances containing high-energy phosphate (5-10 m M phosphoenol pyruvate, 10 m M ADP, 0.4 m M pyridoxal phosphate, 5 mM cyclic AMP) were added to the media, the ATP level was increased 25-94 ~ in slices TABLE VIII Amytal does not reverse 2, 4-dinitrophenol ( D NP) inhibition of amino acid uptake in glucose-depleted slices after substrate substitution

Depletion and restoration (30/30 rain) were as described before. Twenty minute amino acid uptake was measured with D-glutamate and histidine, at 1 mM; a-aminoisobutyrate, 2 raM. Substrates were glucose and lactate: 10 mM; SMP, (succinate, malate, pyruvate), 20:5:5 raM. Averages of four experiments are given, with a standard error of 8 ~ of the mean. Substrate

Inhibitor

Per cent of control Concentrative uptake

Glucose SMP L-lactate

DNP (50/~M) Amytal (20/~M) DNP ÷ Amytal DNP (20/~M) Amytal (10/~M) DNP -t- Amytal DNP (20/~M) Amytal (10 #M) DNP ÷ Amytal

Tissue level

D-Glu

a-A1B

L-His

ATP

73 97 71 81 95 67 36 83 38

67 96 68 54 94 49 41 101 41

77 96 75 71 90 54 53 90 43

---53 66 55 35 71 39

331 that were not depleted of glucose, while uptake was decreased from 5-70 ~ (ref. 2). In the present study, combination of acetyl phosphate (1-2 mM) and CoA (1-2 mg/ml) increased ATP levels up to 45 ~ without affecting uptake; acetylphosphate increased the slice K + level as well, but by itself, had slight inhibitory effects on the uptake of L-His or o-Glu. In addition, when 10-7 M rotenone was added with the D-[14C]GIu after restoration with SMP, uptake was reduced 50 ~ . However, when the rotenone was added to the depleted slices with the SMP, the subsequent uptake of D-Glu was blocked by 85 ~ ; then, although addition of 10 m M phosphocreatine increased the slice ATP level to the same point as when 50 ~ of the o-Glu uptake remained, the transport was still blocked. These results suggest that the transport of o-Glu cannot be maintained by internally generated ATP in the presence of an inhibitor that blocks the reduction of ubiquinone. DISCUSSION These results appear to indicate that active amino acid transport in brain slices does not require glycolysis, but that glucose indirectly maintains mitochondrial energy transducing systems that are coupled to transport. In all cases, mitochondrial electron transport inhibitors are the most potent blocking agents of low-affinity uptake and are active at 10-7-10 -6 M regardless of the energy source used. In contrast, l0 -4 M or higher concentrations of non-specific electron transport inhibitors (i.e., CN-), mercurials and SH-oxidants, and glycolytic inhibitors (i.e., phlorizin, atractyloside, fluoride) are required to block active transport a,a°. When the energy source in the medium allows the slice to bypass the sites of action of glycolytic inhibitors, much higher concentrations of glycolytic inhibitors are inactive (e.g. NaF). The corollary of such an assumption also was tested, and the results support the assumption: agents that might increase the effectiveness of mitochondrial inhibitors, while innocuous themselves, would further block transport. One example is that fluoroacetate (FAA) inhibition is increased by SMP or lactate, while with glucose or pyruvate its inhibition is reduced. Certain substrates are expected to increase the conversion of FAA to fluorocitrate, the active inhibitor of aconitate hydratase12, 25. The results indicate that internally generated ATP may not be used directly for fueling transport. (1) Levels of NaF that block ATPases (5-10 raM) did not block transport under certain conditions (i.e., with SMP). (2) While the addition of high energy phosphate compounds increased the tissue ATP levels, they did not reverse rotenone blockade of uptake. (3) Addition of electron transport traps (i.e., uncouplers of oxidative phosphorylation, CPZ or PMS) affected the tissue ATP levels when various substrates were used, but had no effect on transport. (4) Amytal did not reverse blockade of uptake by 2,4-DNP. For these reasons it does not appear likely that amino acid transport into slices can be fueled directly by electron transport systems (as it can in bacteria and vesicles), nor was evidence obtained for unknown phosphorylative mechanisms. Indeed, the low concentrations of oligomycin (Table VII) that can block transport indicate that one of the key steps in the conversion of substrate energy for the pumping of amino acids may be the PI-H20 exchange mechanism aS.

332 One reason for amytal sensitivity in SMP, pyruvate, or lactate fueled medium might be decreased p H level within' the mitochondria by acidification of the mitochondria utilizing these acids, optimal inhibition being observed at a p H below the p K a 15,27. In agreement with this concept is the fact that inhibition by 20 # M 2 , 4 - D N P was greater with lactate, which tends to lower the mitochondrial p H the most. These data indicate that mitochondrial enzymes at the level o f ubiquinone reduction and coupling to cytochrome b may be those most closely involved with providing energy for amino acid transport into brain slices. There is no reason why other ketogenic substances might not be tested for their ability to support transport functions in slices. That such materials readily penetrate into CSF 34 suggests the p3ssibility o f fueling transport in vivo under conditions that interfere with glycolysis or when glucose is not well utilized, since S M P as a glucose substitute works for the A system (glycine, AIB), the L system (Leu, Val, His), and the acidic system (D-glutamate) 13. Considerable further work remains. For instance, there is the possibility that the A T P level in the tissue is a residue from processes that c o m p e t e for energy with transport mechanisms. In incubated slices o f infant rat cerebral cortex, glycolysis is less active than in the adult, yet A T P levels are higher 28, while as the cortex matures, transport capabilities for m a n y amino acids increase, but A T P levels decrease. Furthermore, several saturable transport systems may operate in parallel, e.g. glial and neuronal, but m a y be fueled independently 24. This would obfuscate a clear demonstration of the specificity o f energy sources for transport. However, a similar problem has been partially overcome in studies o f intestinal transport 11, and amino acid transport mechanisms o f brain slices may only apparently be more complex. ACKNOWLEDGEMENTS This investigation was supported in part by National Institute of Health Grants NB 03226 and RR5707.

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