Heteroexchange of amino acids in incubated slices of brain

ARCHIVES

OF

BIOCHEMISTRY

AND

Heteroexchange LEONTINO New

York

State

BIOPHYSICS

161,

of Amino BATTISTIN,2 Research

Acids

for

New January

(1972)

in Incubated

FEDERICO

Institute

Received

102-111

PICCOLP

Neurochemistyy York, New York

and 10095

10, 1972; accepted

Drvg

April

Slices AND

ABEL

Addiction,

of Brain’ LAJTHA Ward’s

Island,

8, 1972

Exchange diffusion (flow induced by counterflow, or countertransport) of amino acids has been demonstrated in brain slices by the increase in influx of a single amino acid in the medium after the tissue was preloaded with the same or a related amino acid. The observable increase in influx was smaller if the preloading amino acid was present not only in the tissue but also in the medium, with competitive inhibition of uptake from the outside counteracting the activation of influx from the inside. The increased influx of one amino acid was not always accompanied by an increased efflux of the exchanging amino acid; the fact that increases in influx and elllux often were not equal indicates that not all exchange occurs in a 1:l manner. The patterns of exchange of neutral, basic, and acidic amino acids indicated substrate specificity of exchange similar to that shown in studies of transport classes in concentrative uptake. In exchange, basic amino acids were the most active. No heteroexchange could be observed in the presence of cyanide; the absence of Na+, which strongly inhibits influx, lowered exchange less. The differences between cerebral amino acid uptake and exchange indicate that the same transport classes participate in the two processes, but to a different degree.

Transport of amino acids into and out of brain cells occurs continuously under physiological conditions (l-3); such movements occur mostly through mediated processes that require metabolic energy (4-8). Transport constitutes an important feature of the brain barrier system and can determine metabolite levels and rates of metabolism in the nervous system (9, 10). A question that has not been clearly answered in brain or in other systems is whether the various aspects of metabolite passage-processes of uptake, exit, and exchange-are mediated through the same transport carriers. In a number of systems evidence has been found for a common site

for unidirectional and exchange fluxes (ll), but Christensen and co-workers (12, 13) have shown the existence of a transport system in Ehrlich cells that seems to mediate predominantly the exchange of neutral amino acids; this exchange system, labeled “L,” could be distinguished from the “A” system, the major mediator of the active accumulation of neutral amino acids. Further evidence that different carriers mediate exchange and active uptake has been found (14, 15). Exchange diffusion is usually distinguished from active transport by assuming that it does not require energy (17, 18) ; however, the possibility of an energy requirement has also been found (3, 19, 20). llfany features of amino acid uptake and exit in nervous tissues were studied in other laboratories and in ours, both in vivo and in vitro (2, 21) ; moreover, evidence t’hat homoexchange of amino acids takes place in the brain has been presented (3, 22, 23). In the

1 This investigation was supported in part by Public Health Service Research Grant No. 04360 from the National Institute of Neurological Diseases and Stroke. * Present address: Clinics Malattie Nervose e Mentali, University of Padua, Padua, Italy. a Present, address: Clinica Malattie Nervose e Mentali, University of Palermo, Palermo, Italy. 102 Copyright All rights

@ 1972 by Academic Press, of reproduction in any form

Inc. reserved.

EXCHANGE

OF

CEREBRAL

present paper we investigated whether heteroexchange of amino acids occurs in the brain, and whether these processes are influenced by the available energy. The investigation was carried out on brain slices with four amino acids, glutamic acid, glycine, leucine, and lysine, representative of four transport classes, namely, acidic, small neutral, large neutral, and basic amino acids. EXPERIMENTAL

AMINO

37%. At the end of the incubation the tissue was filtered, frozen in dry ice, weighed, and homogenized in 2 ml of 3y0 w/v perchloric acid. After centrifugation, 0.5 ml of the supernatant was mixed with 16 ml of modified Prockop-Ebert scintillation fluid (24), and radioactivity was measured in a Nuclear Chicago Mark I Liquid Scintillation Counter. In the first set of experiments (Tables I and II), 2 mMW amino acid was present in standard mt dium (10 mM glucose, 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl,, 1.2 mM MgSOI, 10 rnM Naz, HPO;, and 100 mM Tris at pH 7.4) during the measurement of exchange flux. To measure the steady-state level of concentrative uptake, incubation with l4C amino acid was carried out for 65 min, at the end of which time slices were rapidly filtered and frozen. To measure exchange, the exchanging 3H amino acid was pipetted into the flask after 60 min of incubation in the presence of the first ‘4C amino acid, and the incubation was continued for 5 min; slices were then collected and frozen. Control 5min flux was measured after incubating the slices

PROCEDURES

The preparation of the brain slices has been described in deta,il previously (7, 24). Briefly, adult Swiss mice were decapitated, the brain was quickly removed, and slices O.&mm thick were prepared from each hemisphere using a McIlwain tissue chopper; the Islices from each hemisphere were then placed in IZ 25-ml Erlenmeyer flask containing the indicated incubation medium. After the flasks were gassed wi.th 0% for 2 min and stoppered, incubations were carried out in a shaker bath at TABLE EFFECT

OF PRELO~DING

ON THE UPTaKE

Preloading

Compound

None Glycine Alanine AIB Valine Leucine GABA Aspartic acid Glutamic acid DAB Lysine Arginine Histidine

amino

acid

Uptake pmoles/ml

I

OF GLYCINE

AND GLUTAMIC

ACID

IN MOUSE

Glycine

Uptake of preloading amino acid percent

103

ACIDS

Glutamic

Uptake wMoles/ml 5.96

f

0.23

BRAIN

Percent

Uptake of preloading amino acid percent

100

42.8 21.2 25.4 8.7 4.1 60.8 92.3

i f zt i i f f

0.7 0.3 0.7 0.2 0.1 1.7 1.5

106 95 99 94 116 100

3.76 4.61 4.10 4.30 3.81 3.08

f. 0.25 f 0.36 f 0.31 f 0.35 f 0.22 f 0.31

63 77 69 72 64 52

60.3

f

3.5

91

1.95

f

0.42

33

25.4 6.4 8.9 40.4

f f f f

0.6 0.1 0.2 0.8

100 104 96 98

4.35 4.35 4.49 3.12

f f f f

0.27 0.51 0.34 0.59

73 73 75 52

SLICESO acid

Uptake rMoles/ml

Percent

110 111

11.91 8.82 11.15

i f f

0.72 0.82 0.48

100 74 94

98 97 104 91

11.30 10.36 8.65 0.53

f i f f

0.18 0.10 0.20 0.22

95 87 73 5

98 85 108

11.74 9.89 10.01

i f f

0.92 0.16 0.99

99 83 84

0 The following abbreviations are used in the tables: AIB = 01 aminoisobutyric acid; GABA = 7 aminobutyric acid; DAB = 2-4 diaminobutyric acid. Slices of mouse brain were incubated in 4.5 ml of standard medium with 2 mM preloading amino acid at 37°C. To obtain the uptake of the preloading amino acid incubation was carried out for 65 min. To measure exchange at 60 min of incubation 0.5 ml of medium was added containing the exchanging amino acid (glycine or glutamate) (final concentration 2 mM) Results are expressed as concentrative uptake bmoles/ml intracellular water-pmoles/ml medium). In experiments with glutamic acid and aspartic acid the W-labeled compounds were used because of loss of label when 3H-labeled compounds were used. Averages of 12-24 experiments f SEM are given.

104

BATTISTIN,

PICCOLI,

AND

TABLE EFFECT

OF PRELOADING

ON

THE

UPT.AK~

Preloading amino acid

None Glycine Alanine AIB Valine Leucine GABA Aspartie Glutamic DAB Lysine Arginine Histidine Ornithine

acid acid

Uptake of preloading amino acid percent

II

OF LEUCINE

Leucine

AND

-~ Uptake pMoles/ml

LAJTHA

Percent

100 103 95 94

1.45 0.75 1.25 0.76 1.27

f f f & f

0.06 0.17 0.19 0.22 0.10

100 52 86 52 88

106 93 94 109 97 98 112

0.17 0.29 1.88 2.30 2.33 2.74

0* i f f f f f

0.12 0.15 0.41 0.15 0.11 0.24

0 12 20 130 159 161 189

LYSINE

IN

~.~ ~__ Uptake of preloading amino acid percent

MOUSE

BRAIX

Lysine Uptake .____-___ pMoles/ml

Percent

105 109

1.39 1.09 0.73

& 0.049 f 0.17 f 0.20

100 78 53

100 98 96 100 114 99

1.44 0.96 1.96 0.99 0.40 1.73

f f f i i f

0.06 0.12 0.15 0.21 0.11 0.30

104 69 141 71 29 124

103 93 109

1.97 2.32 1.38

f i f

0.10 0.06 0.17

142 167 99

* For experimental details see legend to Table I. Control concentrative uptake of ornithine 0.6 pmoles/ml intracellular wat.er. Averages of 12-24 experiments i SEM are given. *With GABA intracellular leucine concentration was below that in the medium. for 60 min in an amino acid-free medium followed by a 5.min incubation in the presence of the 3Hlabeled amino acid. In the experiment in which the exit of an amino acid was measured, after incubation of slices for 60 min in standard medium cont,aining 2 mrvr i4C amino acid, the medium was removed by gentle filtration through a Hirsch funnel, and the slices were transferred into a fresh amino acid-free standard medium, incubated for 5 min, collected, and frozen. When the effect of a second amino acid on the exit of the first was measured, the exit medium contained the second amino acid at a concentration of 2 mM. A similar procedure was followed when the exchange was measured with the preloading amino acid absent from the medium (Tables IV and V). After the slices were incubated with one amino acid for 66 min, they were transferred into a medium containing only the second labeled amino acid for a 5-min exchange experiment. In order to determine exchange when transport is inhibited (KCN or Na+-free medium) it was first necessary to establish the time of incubation and the proper concentration of the preloading amino acids in the medium, to achieve in the tissue an intracellular concentration of the compounds comparable to that obtained in the absence of inhibitors. All amino acids were n-isomers, except aspartic and glutamie acid, whose n-isomers were used. All

SLICES”

was 9.1 Z!Z

the unlabeled amino acids were purchased California Biochemical Co.; the radioactive pounds were purchased from New England clear Co.

from comNu-

CALCULATIONS Counts per gram of tissue final wet weight (CPG) were computed from the measured counts per minute above background (CPM) of the 0.5 ml perchloric acid extract by the relation CPG = CPM X (2 + 0.8 X Mi)/O.5 X W, where If’ is tissue final wet weight; this corrects for the contribution of tissue water of the sample to the volume of the perchloric acid extract. When two amino acids with different label were present at the same time in the tissue the respective CPMs were calculated with a double-label computer program in a Mathatron 4280. MMoles of amino acid/ml of intracellular tissue water (Intracellular Concentration) were computed according to the formula: Intracellular Concentration = (1.25 X CPG) - (M X E)/(I E), where h4 is concentration of the amino acid in the medium at the end of the incubation, and E is extracellular space (ml inulin space/ml tissue water). Here multiplication by 1.25 assumes an 80% water content in the tissue samples, and that all the amino acids are in the tissue water and the medium level is in equilibrium with the level in the extracellular water in the sample.

EXCHANGE

OF

CEREBRAL

Concentrative uptake was calculated as follows: Concentra,tive Uptake = Intracellular Concentration - M; this gives pmoles of amino acid/ ml of intracellular tissue water above the medium, therefore the amount that cannot be taken up by the tissue by passive diffusion alone. In the case of exit into amino acid-free medium, the amino acid level of the medium was assumed to be zero. RESULTS

Exchange when Both Amino Acids are Present in the Incubation Medium In the first’ set of experiments (Tables I and II) the influx of four amino acids was compared in slices that had been previously incubated in amino acid-free medium with slices that had been preloaded with various amino acids. The level of the “preloading” amino acid and its possible alteration during the exchange period was also measured. In the experiments described in Tables I and II, the preloading amino acid was present, in both the tissue and the incubation medium, to represent a possible situation in the living organism in which an amino acid is elevated in the blood and in the brain. Under such clonditions, the preloading amino acid may have several, sometimes opposite, effects. Its presence in the medium may competitively inhibit influx from the medium (22) and its presence in the tissue may, by heteroexchange, increase influx from the medium (3, 14, 16). The preloading amino acid in addition to its effects on influx may also have the effect of increasing or decreasing the exit of the amino acid measured. The final result, the summation of all these effects, will depend on the affinities of the respective amino acids to the carriers and their concentration in the extra- and intracellular spacles. The influx. of glycine was inhibited by the amino acids tested (Table I); the strongest effect was by the acidic, the weakest by the basic, amino acids. The stronger inhibition of glycine flux by leucine in comparison to ar-amino-isobutyric acid (AIB) is of interest, In experiments in which initial glycine influx or efflux was measured, the inhibition of AIB was greater than that of leucine (22,25), again demonstrating that when an amino acid is present on both sides of the membrane

AMINO

105

ACIDS

its influence is a composite of its effect on influx and efflux. Glutamate influx was not markedly affected by most of the amino acids tried, except aspartic acid, which was strongly inhibitory (Table I). When the influx of leucine and lysine was measured in the presence of the same ‘preloading amino acids, somewhat different effects were found. The acidic amino acids again showed a strongly inhibitory effect and neutral amino acids were also inhibitory, but the basic amino acids significantly increased leucine and lysine flux. y-Aminobutyric acid strongly inhibited leucine flux but increased lysine flux in these experiments (Table II). During the 5-min influx experiments, there was relatively little change in the intracellular concentration of the preloading amino acid in each case. Changes in e$ux of the intracellular amino acid. To minimize the inhibition of influx of the amino acid to be measured by the preloading amino acid, in a new set of experiments, after preloading with one amino acid the slices were transferred to a medium conTABLE CHANGES

IN EXIT

-

DURING

-

Percent

6O-min L Con*j-min C entra. . ’ Exit tive nIptakf _ Glycine Alanine Valine Aspartic acid DAB Arginine Histidine

III THE EXCHANGH

--

-

change in exit in presence of:

-

-

GILI1tamic acid

Glytine .-

42.8 21.2 8.7 92.3

5.41 2.50 2.92 1.65

87 47 28

25.4 8.9 40.4

2.63 1.19 1.94

0 47 200

-

PERIODS

0 134

0 0

:

Leutine

LYsine

0 164 25 0

0 66 18 382

26 35 116

0 0 ‘28

_-

D Mouse brain slices were incubated in 5 ml of a 2 mM amino acid in standard medium at 37’ C for 60 min. To measure control 5-min exit, slices were transferred (see Methods) to amino acid-free medium and incubated 5 min. To measure exit in the presence of another amino acid, the second medium contained 2 mM (exchanging) amino acid; incubation was also 5 min. Control exit data areexpressed as difference in ~moles/ml of intracellular water between 60 min uptake and after 5 min incubation in amino acid-free medium.

106

BATTISTIN,

PICCOLI, TABLE

HETEROEXCHANGE

AND LAJTHA IV

OF GLYCINE

AND

GLUTAMIC

Glycine Preloading amino acid

Glycine Alanine Valine Aspartic acid DAB Arginine Histidine

Glutamic acid

pMoles/ml intracellular water Extra exit of preload- Exchange of ing amino glycine acid 2.18 1.38 0.45 0 0.56 3.89

0.02 1.64 (-1.61) 0.25 0.56 2.71

ACIDS

pMoles/ml intracellular water Percent exchange

0.3 27.5

Extra exit of preload- Exchange of ing amino glutamic acid acid 0 3.35 0

(-Q.95)

C-27) 4.4 9.4 45.5

1.52 1.59

0 0

1.90 2.55

Percent exchange

12.8 13.4 (-5:? 16.0 21.4

0 Bracketed figures indicate inhibition of exchange. Mouse brain slices were incubated in 5 ml of 2 mM preloading amino acid in standard medium for 60 min and transferred into 5 ml of a medium containing 2 mM glycine or glutamic acid but free of the preloading amino acid for 5 min of further incubation. Both the preloading and the exchanging amino acid were labeled CH and W, respectively). Data are presented a8 rmoles amino acid/ml intracellular water; exchange as the difference between the intracellular concentration in the absence and in the presence of the preloading amino acid. Percent exchange = percent change in concentrative uptake. The 5 min uptake in the absence of preloading amino acid was: glycine 7.86, glutamio acid 13.8 pmoles/ml intracellular water. Averages of six experiments are given.

taining only the exchanging amino acid (the influx of which was to be measured). Under such conditions, a significant efflux of the preloading amino acid occurred during the 5-min exchange; therefore the exit of the preloading amino acid was measured, and also the effect on this exit of the presenceof the exchanging amino acid in the medium during the 5-min exchange period. The uptake in 60 min, and the subsequent exit during the following 5 min, of the preloading amino acids are presented in Table III. The 5-min exit of glycine did not change upon the addition of an amino acid to the medium, but in a number of casesexit of the other amino acids was increased; e.g., there was more than fourfold increase in the exit of aspartate when lysine was present in the medium. In Tables IV and V this increase in exit is compared with the increasein influx of the amino acid from the medium. Exchange When Only One Amino Acid is Present in the Incubation Medium Glycine heteroexchange could be shown by the increase of glyeine influx in slicespre-

loaded with histidine, valine, or arginine (Table IV). The increase in glycine influx was approximately equal to the increase in histidine, valine, or arginine efflux in these experiments: a true one-to-one relationship of influx of one and efllux of another amino acid seemsto be the mechanism involved in this process. Glycine in the medium stimulated the exit of intracellular alanine, but intracellular alanine did not stimulate the influx of glycine from the medium-precluding an equimolar exchange in this case. A one-to-one exchange was not found in the case of glutamic acid. Glutamate influx was increased in most cases, showing that glutamate exchanged with most of the amino acids tested, but not with valine (Table IV). The increase in glutamate influx was not paralleled by an increase in the efflux of the preloading amino acid; there was no increase in the exit of arginine, histidine, or glycine, although they stimulated glutamate influx, while the increase in alanine efflux was more than twice the increase in glutamate influx caused by alanine. As in the case of all four amino acids tested for exchange (glycine,

EXCHANGE

OF CEREBRAL

AMINO

ACIDS

107

TABLE V HETEROEXCHANGE OF LEUCINE AND LYSINE~ Leucine

Lysine

pMoles/ml intracellular water Preloading amino acid

Extra exit of preloading amino acid

Exchange of leucine

0 4.10 0.73 0 0.69 0.42 2.25

0 0.74 0.20 (-0.51) 0 0.46 2.96

Glycine Alanine Valine Aspartic acid DAB Arginine Histidine

rMoles/ml intracellular water Percent exchange

Extra exit of preloading amino acid

Exchange of lysine

0 51.0 13.8 (-35.2) 0 31.7 204.1

0 1.65 0.52 6.30 0 0 0.55

0.25 (-0.32) 0.50 (-0.81) 1.88 2.03 1.55

Percent exchange

(2:) 135 146 112

(1Experimental details are as in Table IV except leucine or lysine was added instead of glycine or glutamic acid as an exchange amino acid. The 5-min uptake in the absence of preloading amino acid was: leucine 3.3!3, lysine 1.39 pmoles per ml intracellular water. Averages of six experiments are given. Figures in brackets indicate inhibition of exchange. TABLE VI HETEROEXCH~NGE IN PRESENCE OF KCNQ

-

Compound

Glycine Valine Arginine Histidine

Preloading amino acid pMoles/ml water

Glutamic acid exchange

Glycine exchange

Lysine exchange

In medium

Intracellular

20 20 10 20

33.5 24.1 13.7 20.5

pMoles/ml

Percent

(-0.58)

(-28.2)

(-0.57)

(-27.7)

uMoles/ml

Percent

0

-

(-0.07) 0.04

-

(-12.3) 7.0

~Moles/ml

Percent

(-0.5)

(-50.5)

0.13 (-0.18)

13.1 (-18.0)

5 The incubation time with the preloading amino acid was 30 min with 1 mM KCN in the standard medium. For exchange 5-min uptake in the presence of 2 mM exchanging amino acid and 1 mM KCN (but in the absence of preloading amino acid in the medium) was measured. The 5-min uptake in the absence of preloading amino acid but in the presence of cyanide was: glycine 2.06, glutamic acid 0.57, lysine 0.99 pmoles/ml intracellular water. Figures in brackets indicate inhibition of exchange. Averages of six experiments are given.

l,ysine, and leucine), preloading with aspartic acid strongly inhibited influx. This inhibition was found only with aspartic acid, and it may have been due to some damage to the tissue caused by the high levels of aspartate reached during the preloading. Swelling and structural damage were reported when brain slicestook up high levels of acidic amino acids (26).

glutamate,

Leucine exchange was found with large neutral (alanine, valine) and large basic (arginine, histidine) but not with small neutral (glycine) and small basic (diaminobutyrate) amino acids. With lysine, heteroexchange was especially high with the basic amino3 acids; some exchange occurred also w&h~ glycine and valine. Aspartate, and in this casealanine, were inhibitory (Table V) . Indi-

108

BATTISTIN,

PICCOLI, TABLE

HETEROEXCHANGE

Compound

Preloading amino acid, pMoles/ml water

Glycine exchange

rMole/ml

Percent

AND

LAJTHA

VII

IN I Na+-FREE

MEDIUMS

Glutamic acid exchange

pMole/ml

Percent

0.06

60.0

0.08 (-0.03)

80.0 (-39)

Leucine exchange

pMole/ ml

Percent

Lysine exchange

PM;)/

Percent

~~ Glycine DAB Arginine Histidine

(-0.11)

(-9.6)

0.81

56.3

0.06 1.07 0.40 0.77

9.1 162.1 60.6 116.6

0 Slices were incubated for 30 min with the preloading amino acid at indicated concentrations in a Na+-free medium and then transferred into 2 mM solution of exchanging amino acid in a Na+-free medium for 5 min of incubation. The composition of the Na+-free medium was: 10 rnM glucose, 128 mM choline chloride, 5 mM KCI, 2.7 mM CaC% 1.2 mM MgSOd, 5 mu KtHPOl and 50 mM Tris at pH 7.4. The 5-min uptake in the absence of preloading amino acid in the Na+-free medium was: glycine 1.14, glutamic acid 0.10, leucine 1.44, lysine 0.66 rmoles/ml intracellular water. Averages of three experiments are given. Figures in brackets indicate inhibition of exchange.

cations for a one-to-one exchange were found tine with histidine could be shown, although for the following pairs: lysine-valine, leu- this was less than the exchange in the presence of high Na+ in the medium (compare tine-histidine, and leucine-arginine. The effects of cyanide and la& of Na+ on Table VII with Table V). ez&ange. The presently available evidence doea not, allow a definite decision on whether TABLE VIII exchange diffusion is distinguishable from acHETEROEXCHANGE OF AMINO ACIDS AT Low tive transport by assuming that unlike acCONCENTRATIONS” - ___.tive transport it is not inhibited by the lack Glycine Lysine of energy. To contribute observations to this Glutamic acitiL exchange exchange problem, experiments were performed either Pr+oadi?g in the presence of a strong metabolic inhibitor, cyanide, or in a Na+-free medium. No ammoacid heteroexchange could be observed in the presence of cyanide (Table VI). Since cyanide strongly inhibits uptake, the amino acid 12 Glycine concentration in the preloading medium had Valine 0.05 4.5 to be increased from 2 mM to lo-20 m&I in Arginine 0.18a 12 160 order to reach intracellular levels similar to 0.60b 21 those in the absence of cyanide. AS shown H&i&e 0.38 34.2 68 I I previously (2, 6, 7) cyanide inhibited the in0 a = 0.1, b = 0.2 mM glutamic acid in the exflux of the three amino acids tried (Glu, Gly, Lys) and this influx was not increased when changing medium. Incubation period with 2 mM the cells were preloaded with amino acids preloading amino acid was 60 min followed by (except a small increase in lysine flux by 5-min incubation with varying concentra.tions of exchanging amino acid without the preloading arginine) . amino acid. The 5-min uptake in the absence of Lowering Na+ in the medium also lowered preloading amino acid was: glycine (0.1 mM) 1.11, influx, but it did not abolish heteroexchange glutamic acid (0.1 mM) 1.53, (0.2 mM) 2.81, lysine in each case (Table VII). The exchange of (1.0 mM) 1.26 Fmoles/ml intracellular water. lysine with basic amino acids and that of leu- Averages of four experiments are given.

EXCHANGE

OF

CEREBRAL

The lowering or absence of exchange in the presence of cyanide or low Na cannot be completely explained by the generally lowered level of influx. Exchange occurred at low medium amino acid levels when the amount taken up by the slices and influx was much lower than in the previous experiment,s (Table VIII). Although heteroexchange seemed to be lower when the influx of the exchanging amino acid was lower, exchange could be shown in each case tested, and in one case (lysine-arginine) the exchange at low and high amino acid levels was equal. The intracellular concentration of the amino acids tested :at low level (Table VIII) was comparable to that reached in the presence of cyanide (Table VI). DISCUSSION

Although t#he cerebral level of each amino acid is constant under most conditions in the living organism and the composition of the amino acid pool is characteristic for t’he brain, experiments with tracer doses administered indicate a rapid flux of most amino acids between plasma and brain (1, 10, 27). Upon constant intravenous infusion with tracer doses (Seta and Lajtha, in preparation), the specific activity of some amino acids rapidly became equal in plasma and blood, showing rapid and complete exchange; with a few amino acids the existence of a very slowly exchangeable pool was also revealed. There is also a measureable bidirectional flux of those amino acids (glutamate, aspartate, glycine) that show no significant net increase in the brain when plasma levels are elevated. Exchange of amino acids between medium and tissue has also been observed upon incubation of sl.ices from brain (3). Although brain slices retain most of their endogenous pool of amino acids upon incubation (28,29), this pool is in a dynamic state, and the levels in the slice are determined by the influx of amino acids (21). Most of the flux of cerebral amino acids occurs through mediated transport (10). Detailed ‘knowledge of the mechanism of exchange and its relationship to the mechanism of net transport is lacking. A number of differences were found between the properties of exch.ange and net transport; an example is the finding of Christensen and co-

AMINO

ACIDS

109

workers (12, 30) on the L and A systems mediating neutral amino acid flux, that the L (leucine-preferring) can mediate exchange well whereas A (alanine-preferring) mediates net uptake well and exchange poorly. Exchange in a number of studies showed lower dependence than influx on Na+ (12, 31, 32). Some difference in brain slices was found between the heat inactivation of exchange and uphill transport (33). It was pointed out that such observations could be explained equally by the existence of two independent systems -separate ones for uphill transport and for exchange-or of systems that take part in both uphill transport and exchange (11, 19, 34). In brain slices, as in other systems, further difficulties arise from the heterogeneity of the tissue and of the amino acid distribution; quantitative differences were found in amino acid uptake between neurons and glia (35). In our experiments some interference with influx by the preloading amino acid can be assumed, even when during the measurement of exchange there was no added preloading amino acid in the medium, because some of the amino acids exit rapidly from the slice during the experiment and are present also in the extracellular matter. This inhibition of influx by the extracellular component would diminish the effect of increasing influx via exchange by the intracellular component. Such inhibition of influx may be responsible for occasionally fmding a larger increase in efflux than in influx but would not explain greater increases in influx. The difference in the pattern of exchange between neutral and basic amino acids supports the existence of transport classes in exchange similar to those shown in studies OE uptake competition (21, 23, 24, 36) and indicates that at least in brain more than one carrier is involved in exchange. The finding that K, of uptake and exchange are the same in some systems (37) but different in others (34) supports the idea that the various carriers participate in uphill transport and in exchange to a different degree (36). The participation of an acidic amino acid carrier in exchange is more difficult to assess since high levels of these compounds result in swelling, ion changes, and structural damage in brain tissue (26).

110

BATTISTIN,

PICCOLI,

Although in principle there is no requirement for energy in exchange, homoexchange (3) and heteroexchange (Table VI) in brain slices are decreased by metabolic inhibitors. In some studies inhibitors of uptake in some systems had no effect on exchange (17,lS); in others exchange was also inhibited (19, 20). Such inhibition may be indirect, since maintenance of membranes or of ion balances may be affected by the lack of available energy. Differences between the requirements of exchange and of concentrative uptake shown by the effects of metabolic inhibitors are also emphasized by the lower dependence of exchange on Na+. In brain slices uptake of amino acids requires Naf (6, 38) ; homoexchange (3) and heteroexchange (Table VII) are affected less, corresponding to lower Na+ dependence of exchange in other systems (12, 31, 36). Inhibition of exchange by tribromethanol without affecting net uptake (19) further supports differences in properties, indicating that the quantitative participation of the carrier sites is different for uptake and exchange. Alterations of amino acid levels in vivo and in vitro affect amino acid transport, metabolism, and other metabolism, such as protein synthesis (10, 39). Under physiological conditions amino acids are present in brain and in plasma; therefore exchange must occur in the living brain. Heteroexchange and transport inhibition may alter the cerebral levels of a number of compounds when the level of one is elevated in the blood, as in aminoacidurias; the changes in brain composition could in turn be responsible for the mental changes reported in a number of these diseases. REFERENCES 1. LAJTHA, A.: AND MELA, P. (1961) J. Neurothem. 7, 210-217. 2. NEAME, K. D. (1968) in Applied Neurochemistry (Davidson, A. N., and Dobbing, J., eds.), p. 119, Blackwell, Oxford. 3. BLASBERG, R., LEVI, G., AND LAJTHA, A. (1970) Biochim. Biophys. Acta 203, 464483. 4. ABADOM, P. N., AND SCHOLEFIELD, P. G. (1962) Can. J. Biochem. Physiol. 40, X031618. 5. LAJTHA, A. (1967) in Problems of Brain Biochemistry (Galoyan, A. A., ed.), Vol. 3, p. 31. Acad. Sci. Armenian SSR, Yerevan.

AND

LAJTHA

6. BATTISTIN, L., GRYXB.\UM, A., AND L~JTH.~, A. (1969) Bruin Res. 16, 187-197. 7. B.INAY-SCHU’ARTZ, M., PIRO, L., AND LJTEI.~, A. (1971) Arch. Biochem. Biophys. 146, 199210. 8. GUROFF, G.: KING, W., AND UDENFRIEND, S. (1961) J. Biol. Chem. 236, 1773-1777. 9. LEVI, G., KANDER.4, J., AND LAJTH~, A. (1967) Arch. Biochem. Biophys. 119, 303-311. 10. L.~JTH~, A. (1968) in Progress in Brain Research (Lajtha, A. and Ford, D. H., eds.), Vol. 29, Brain Barrier Systems, p. 201, Elsevier, Amsterdam. 11. JOHNSTONE, R. M., AND SCHOLEFIELD, P. G. (1961) J. Biol. Chem. 236, 1419-1424. 12. OXENDER, D. L. AND CHRISTENSEN, H. N. (1963) J. BioE. Chem. 238, 3686-3699. 13. INUI, Y., AND CHRISTENSEN, H. N. (1966) J. Gen. Physiol. 60, 203-224. 14. JACQUEZ, J. A. (1967) Biochim. Biophys. Acta 135, 751-755. 15. BELKHODE, M. L. AND SCHOLEFIELD, P. G. (1969) Biochim. Biophys. Acta 173, 290-301. 16. USSING, H. II. (1947) Nature (London) 160, 262-263. 17. JEQUIER, J.-CL., ROBINSON, J. W. L., AND FELBER, J.-P. (1965) Biochem. Biophys. Res. Commun. 18, 507-511. 18. JXQUEZ, J. A., AND SHERMAN, J. H. (1965) Biochim. Bigphys. Acta 109, 128-141. 19. OXENDER, n. L., AND WHITMORE, B. (1966) Fed. Proc. 26, 592. 20. SCHWARTZMAN, L., BLAIR, A., AND SEGAL, S. (1967) Biochim. Biophys. Acta 136, 136-145. 21. COHEN, S. R., AND LAJTHA, A. (1971) in Handbook of Neurochemistry (Lajtha, A, ed.), Vol. 7, p. 545, Plenum Press, New York. 22. BLASBERG, R., AND LAJTH.~, A. (1966) Brain Res. 1, 86-104. 23. LAJTHA, A., LEVI, G., AND BLASBERG, R. (1967) in Brain Edema (Klatzo, I., and Seitelberger, F., eds.), p. 367, Springer Co., New York. 24. BLASBERG, R., AND LAJTHA, A., (1965) Arch. Biochem. Biophys. 112, 361-377. 25. LEVI, G., BLBSBERG, R., AND LAJTHA, A., Arch. Biochem. Biophys. 114.339-351. 26. TSUK~DA, Y., NAGATA, Y., HIRANO, S., AND M.~TsUT~NI, T. (1963) J. Neurochem. 10, 241-256. 27. OLDENDORF, W. H. (1971) Amer. J. Physiol. 221, 214 -217. 28. NEIDLE, A., KANDERA, J., AND CHEDEKEL, M. (1970) Fed. Proc. 29, 911. 29. JONES, D. A., END MCILWAIN, H. (1971) J. Neurobiol. 2, 311-326. 30. CHRISTENSEN, H. N., AND HANDLOGTEN, M. E. (1968) J. Biol. Chem. 243, 5428-5438.

EXCHANGE

OF CEREBRAL

31. JOHNSTONE, R. M., AND SCHOLIWIELD, P. G. (1965) Biochim. Biophys. Acta 94, 130-135. 32. BELKHODE, M. L.? AND SCHOLEFIELD, P. G. (1969) Bio’chim. Biophys. Acta 173, 290-301. 33. YAMAGUCHI, T., YAMAGUCHI, M., .~ND LAJTHA, A. (1972) Biochim. Biophys. Acta 266, 422435. 34. J~CQTJEZ,

J. A., SHERMAN, J. H., AND TERRIS, J. (1970) Biochim. Biophys. Acta 203, 150166. 35. HAMBERGER, A. (1971) Brain Res. 31,169-178.

AMINO

ACIDS

36. CHRISTENSEN, H. N., AND LIANG, J. Biol. Chem. 240, 3601-3608. 37. SCHOLEB’IELD, P. G., AND CLAYMAN,

111 M. (1965)

S. (1968) in Progress in Brain Research (Lajtha, A., and Ford, D. H., eds.), Vol. 29, Brain Barrier Systems, p. 173, Elsevier, Amsterdam. 38. LAHIRI, S., SND LAJTHA, A. (1964) J. Neurocham. 11, 77-86. 39. ROBERTS, S. (1968) in Progress in Brain Research (Lajtha A., and Ford, D., eds.), Vol. 29, Brain Barrier Systems, p. 235, Elsevier, Amsterdam.