Regional Differences in Cerebral Amino Acid Transport

219

Regional Differences in Cerebral Amino Acid Transport G I U L I O LEV1 Istiticto Superiore d i Sanita, Viale Regina Elena 299, Rome (Italy)

INTRODUCTION

The anatomical, physiological and biochemical heterogeneity of various areas of the brain has been object of a number of studies (for a recent symposium see: Kety and Elkes, 1961). In particular, the physiological level of most free amino acids was found to be different in various regions of the central nervous system (CNS), the regional distribution pattern varying from one compound to the other (Berl and Waelsch, 1958; Okumura et al., 1959; Lajtha and Mela, 1961; Bart et al., 1962; Singh and Malhotra, 1964; Shaw and Heine, 1965; Ramirez de Guglielmone and Gomez, 1966). It has been suggested that the heterogeneous distribution of cerebral amino acids may be determined by regional differences in the transport of these compounds (Lajtha and Mela, 1961) but very few workers investigated this problem (Lajtha, 1961 ; Nakamura and Nagayama, 1966). The work to be presented here summarizes experiments in vitro and in vivo on regional differences in cerebral amino acid transport. It is known that amino acid transport in brain slices occurs through an active, carrier-mediated process (Stern et al., 1949; Guroff et a/., 1962; Neame, 1962; Abadom and Scholefield, 1962; Tsukada et al., 1963; Lahiri and Lajtha, 1964; Blasberg and Lajtha, 1965 and 1966; Levi et al., 1966); we have shown that amino acid transport processes of brain slices are in many ways similar to those operating in the living brain, except for the marked restriction of the passage of amino acids from the blood to the CNS in vivo (Lajtha et al., 1966), which is generally attributed to the presence of a complex “brain barrier system”. Brain slices appear to be a useful tool to study one aspect of this complex system: the permeability properties of nervous tissue cells, in particular their unrestricted transport capacity. For this reason the problem of regional amino acid transport was approached both in brain slices and in the living brain, and an attempt was made to compare and correlate the results obtained. The aim of these studies was not only to try to clarify some of the mechanisms by which amino acids are transported into the nervous tissue, but also to further investigate the role that transport processes may have in the determination and in the maintenance of the physiological amino acid levels in different areas of the CNS. References pp. 227-228

220

G. L E V 1 RESULTS

Table I shows the degree of steady state accumulation of five amino acids in slices from various areas of the CNS. The amino acids chosen belong to different classes and have different affinities for the membrane carriers (Blasberg and Lajtha, 1966). More areas than those presented were analysed, but only the most representative are shown in this and in the following tables. TABLE I S T E A D Y S T A T E A C C U M U L A T I O N O F A M I N O A C I D BY S L I C E S FROM D I F F E R E N T A R E A S O F T H E RAT

CNS

Tissue to medium ratio

Area

a-Aminoisobutyrate*

Cortex White matter Midbrain Cerebellum Pons-medulla

8.8 4.4

11

1.7 9.1

Leucine

Lysine *

2.1 1.7 2.4 2.8 2.2

4.0 2.6 3.3 2.8 2.5

Taurine 20 18 22 15 12

9.6 4.1 27 3.6 12

Slices were incubated for 70 min in a Krebs-Ringer medium containing initially 2 mM 14C amino acid. pmoles/ml tissue water after incubation. pmoles/ml medium From Levi and Lajtha, 1965.

Tissue to medium ratio

*

=

It can be seen that the degree of accumulation varies with each of the amino acids tested. Among them D-glutamate is concentrated to the highest degree, leucine to the lowest. The regional pattern of accumulation also varies with each amino acid. None of the areas studied has in every case the highest accumulation, and none has in every case the lowest. Some area may have the highest accumulation with one amino acid, and the lowest with an other, such as cerebellum with leucine and taurine respectively. The degree of regional variation of accumulation also varies with the amino acid tested, and is relatively small with leucine and great with taurine. The difference between lowest and highest accumulation is of the order of two-fold with a-aminoisobutyric acid (AIB), leucine, lysine and D-glutamate, but is almost eight-fold with taurine. In previous experiments (Levi et al., 1966) we have shown that the degree of amino acid accumulation at steady state is determined by the relative extent of the following processes: ( I ) Accumulation rate (influx); (2) Exit rate (efflux); (3) Homoexchange into and out of thecells. With brain slices it is often sufficient to know the extent of influx and efflux in order to be able to predict with some approximation the degree of accumulation at steady

REGIONAL DIFFERENCES I N CEREBRAL AMINO A C I D T R A N S P O R T

221

state. In order to investigate whether the regional differences of amino acid steady state accumulation could be explained on the basis of regional differences of influx or efflux, these fluxes were studied separately, with three amino acids (AIB, D-ghtamate, and lysine). With AIB it was found that regional differences of influx were very similar to those of steady state accumulation (Levi and Lajtha, 1965), making it likely that regional differences in A1B exit would not be great. Table I 1 shows that indeed the exit of AIB is very similar in all the areas except cerebellum, where it is slightly higher. This finding confirms that with AIB the heterogeneity of steady state accumulation is mostly due to regional differences of the accumulation rates and not to differences of the exit rates. T A B L E 11 E X I T O F A M I N O A C I D S FROM S L I C E S F R O M D I F F E R E N T A R E A S O F T H E R A T

CNS*

Per ceirt ititracellular loss Area

Cortex White matter Midbrain Cerebel I urn Pons-medulla

u-Anrinoisobittyrate

D-Gliitamale

Lysitie

41 18 28 25 28

57 49 61 66 64

31 33 30 39 29

Slices were preincubated for 30 min in amino acid containing medium, then transferred to amino acid free medium for 30 min. Average intracellular level after preincubation: AIB I3 ,umoles/ml

*

D-GIu 33 pnoles/rnl Lys 5.5 pmoles/ml

From Levi, Cherayil and Lajtha, 1965.

T A B L E 111 C O M P A R l S O N OF L-LYSINE F L U X E S IN D I F F E R E N T AREAS O F T H E R A T

Area Cortex

White matter Midbrain Cerebellum Pons-medulla

Relative initial accittnulat ion*

Relative steady state accumulation*

Relative exit * *

101

127 87 106 91 83

94

83 104 I02 98

Relative values are calculated taking the average of total areas as 100. * Recalculated from Lcvi and Lajtha, 1965. +* Recalculated from Levi, Cherayil and Lajtha, 1965. Rtfiwnces pp. 227-2211

CNS

80 100 108 106

222

G . LEV1

With D-glutamate the regional heterogeneity of influx did not parallel exactly that of steady state (Levi and Lajtha, 1965), making it likely that in this case regional differences in both influx and efflux play a role in determining the degree of accumulation at steady state. The second column of Table I1 shows in fact that D-glutamate exit varied from area to area, being highest in cortex, about average in midbrain, cerebellum, and pons-medulla, and lowest in white matter. Also with lysine the regional pattern of influx did not parallel that of steady state accumulation; in agreement with this lysine exit was not homogeneous in the various areas, as shown in the third column of Table 11. Table 111 shows that with lysine the heterogeneity of steady state accumulation is mostly due to regional differences in exit, since the influx of this amino acid is essentially the same in all the areas except white matter. It can be seen that the degree of amino acid accumulation at steady state is lower in those areas where exit is higher, and vice-versa. In white matter all influx, efflux, and steady stateare lower than average. Finding regional differences in the exit of D-glutamate and lysine, but not of AIB, is in agreement with other experiments in which the exit of these amino acids was studied with slices from whole brain (Levi et al., 1965; Levi et a/., 1966). In these studies it was shown that the exit rates of D-glutamate and lysine depend not only on the intracellular concentration of the amino acid, but also on the experimental time at which exit is measured. In contrast, no time-dependence of AIB exit could be demonstrated. To explain these findings it was suggested that exit of D-glutamate and that of lysine occur from different intracellular pools having different exit rates. The regional heterogeneity of D-glutamate and lysine exit shown in Table I1 could partly reflect this compartmentation. TABLE IV COMPARISON O F AMINO ACID ACCUMULATION

in vitro T O A M I N O A C I D L E V E L in vivo I N

DIFFERENT AREAS O F T H E R A T

Leucine Area

Hemisphere Caudate nucleus Midbrain Cerebellum Pons-medulla Total areas analysed

CNS

Lysine

Taurine

Relative Relative Relative Relative Relative Relative accumulation distribution accumulation * distribution* * accumulation distribution

79

115

78

135

83

129 143

266 35 121

66 109 35

100 (4.6)

100 (0.28)

100 (13)

100 (5.0)

86

101

107

75

106

100

106

104

125 100

100 96

91

100 (3.3)

100 (0.10)

In parenthesis values for total areas analysed are expressed as pmoles/g. Relative values are calculated taking the average of total areas as 100. * Recalculated from Levi and Lajtha, 1965. ** From Lajtha, Blasberg and Levi, 1966.

REGIONAL DIFFERENCES I N CEREBRAL A M I N O ACID TRANSPORT

223

While the picture of regional amino acid transport in vitro is relatively clear, the significance that this may have in the living brain is not quite clear. If accumulation in vitro is an indirect measure of the availability of carriers, the question arises whether or not carrier availability is the limiting factor in determining the amino acid levels in the living brain. In previous experiments we noted that amino acids that are accumulated to a high level in vitro have a high concentration in the living brain, whereas amino acids that are accumulated to lower levels have lower in vivo concentrations. We concluded that the transport capacitl- for a particular amino acid as measured by brain slices may be one of the factors determining its concentration in the living brain (Lajtha e t a / . , 1965). It seemed therefore logical to compare the relative accumulation of amino acids by different brain areas in vitro to their regional distribution in vivo. Table IV shows this comparison for leucine, lysine, and taurine. It is apparent that no parallelism exists between relative accumulation and relative distribution of any of the three amino acids presented. Indeed, with lysine and taurine, areas having higher levels in vivo tend to accumulate less in vitro, and areas that accumulate more in vitro tend to have lower levels in vivo. Although with other amino acids (such as alanine and glycine) there seems to be a better parallelism (Levi et a/., in preparation), it has to be concluded that factors other than the transport capacity as measured with brain slices are operative to regulate and control the regional distribution of a particular amino acid in the living brain. In order to investigate this problem further, the uptake and the exchange of some amino acids were studied in experiments in vivo. Table V shows the results obtained with lysine. It can be seen that the regional TABLE V RELATIVE D I S T R I B U T I O N O F L Y S I N E I N D I F F E R E N T A R E A S O F T H E R A T

Area

Hemispheres Midbrain Cerebellum Pons-medulla Total areas analysed

Control

distribution'

Distribution after loading2

Distribution of tracer3

CNS t+4

75 104 129 143

74 95 136 157

88 97 119 123

86 108 111 117

I00 (0.28 / t M / g )

100

(0.34 pM/g)

100 (750 cts/g)

(34 min)

loo

I . Plasma concentration: 0.44 /rmoles/g. 2. Plasma concentration: 3.4 pmoles/g. The animals had an intraperitoneal injection of lysine (90 mg in 1 ml) and were killed 10 min later. 3. Plasma activity: 1 I 800 cts/g. The animals had an intraperitoneal injection of [14C]lysine(3 p C in 1 ml) and were killed 5 min later. 4. Calculated according to the following: t+

=

Tissue concentration x In2

The first two columns are taken from Lajtha et al., 1966. Rrferenres pp. 227-228

flux

224

C . LEV1

distribution of lysine is not significantly changed when the brain level of lysine is increased after a large intraperitoneal injection. In other words, lysine is taken up more by areas having higher lysine concentration. The third column of the Table shows that also the distribution of tracer, 5 min after an intraperitoneal injection of [Wllysine, closely parallels the relative concentration in the various areas. Higher exchange rates of lysine in areas with higher physiological level had already been shown (Lajtha and Mela, 1961). The fourth column shows that in spite of a higher rate of exchange, the half-life time of the pool of free lysine is higher in those areas where lysine level is higher. When the same type of experiment was done with leucine, whose physiological level in the various areas is very homogeneous, no significant regional difference in either uptake or exchange was found (Levi et al., in preparation). These findings suggest that at least with lysine and leucine there may be a rather close correlation between regional transport in vivo and regional physiological levels. The same type of experiment was repeated with taurine. This amino acid was chosen in view of the large differences in its concentration in the various areas. It was thought that taurine transport as well might differ widely from one area to the other and that a clearer correlation might appear between transport and concentration. Table VI shows that when the physiological plasma level of taurine is increased over 100 times, its brain concentration does not show any significant increase. The only region in which a relatively high increase of taurine level occurs is pons-medulla; this suggests the existence of regional differences in the blood-brain barrier to this amino T A B L E VI RELATIVE DISTRIBUTION OF TAURINE IN DIFFERENT AREAS OF THE RAT

Areas Hemisphere Caudate nucleus Midbrain Cerebellum Pons-medulla Total areas analysed

CNS

ti4

Control disisrributionl

Distribution after loading2

Distribution of tracer3

115 135 66 109 35

119 125 63 98 46

98 81 117

109

113 159 54 104 31

I00

100 (530 cts/g)

100 (74 hours)

100

(5.0 / W g )

(5.3 PM/i3)

100

1. Plasma concentration: 0.20 pmoles/g. 2. Plasma concentration : 24 ,umoles/g. The animals had two intraperitoneal injections of taurine (270 mg in 3 ml), one at time 0, and one after 10 min. They were killed 20 min after the first injection. 3. Plasma activity: 13 000 cts/g. The animals had an intraperitoneal injection of [14C]taurine(3 pC in I ml) and were killed 10 min later.

Tissue concentration x In2 flux Relative values are calculated taking the average of total areas as 100.

4. Calculated according to the following: tf

=

REGIONAL DIFFERENCES I N

CEREBRAL AMINO ACID T R A N S P O R T

225

acid. Nevertheless, the relative distribution pattern remains essentially the same. The third column shows that the exchange of taurine between plasma and brain is quite slow and rather homogeneous. In contrast with lysine, it tends to be somewhat higher in areas with lower physiological level and vice versa. The half-life time of the taurine pool, whose average value is 74 h, closely parallels the pattern of taurine concentration in the various areas. I n view of these results i10 clear correlation seems to exist between transport and regional distribution of this amino acid. DISCUSSION

The data on amino acid transport in slices from different regions of the CNS confirm previous studies with slices from whole brains (Levi et a/., 1966; Lajtha et a/., 1966) in that the steady state or equilibrium level reached by the amino acids in the tissue upon incubation is mainly determined by the relative extent of the processes of accumulation and exit. Other factors, such as homoexchange, or the metabolism of the compounds studied, do not interfere significantly (Blasberg and Lajtha, 1965). The results presented seem to rule out the possibility that the regional differences of amino acid accumulation are determined primarily by factors such as regional differences in the number of cells per unit of tissue weight, or differences in the energy sources available for transport. In fact, if these were the limiting factors of amino acid accumulation in the various areas, the regional pattern of accumulation would be expezted to be the same with all the amino acids tested. It is interesting to note that although none of the areas studied has a homogeneous cell population, the histological composition of the various areas is quite different. It seems, therefore, that several constituents of the nervous tissue are responsible for the high amino acid accumulation of brain slices, and it is likely that the affinities of different amino acids or classes of amino acids for the various cell types are not the same. This may have a functional significance, and may reflect different metabolic requirements of different types of cells. It has been recently shown that neurons and glial cells do not have the same permeability to ions (Reed and Woodbury, 1963; Pappius, 1965). The problem becomes more complex if we try to correlate regional transport in vitro with regional amino acid concentrations and transport in vivo. In the living brain the physiological concentrations of free amino acids can be considered to be in dynamic equilibrium with those of plasma and cerebrospinal fluid. If brain concentrations were determined mainly by the transport capacity of the cells, a correlation would be expected between steady state accumulation in brain slices and in vivo brain concentrations. In agreement with this we have shown that in most cases amino acids that are accumulated to high levels in vitro have high in vivo concentrations, and vice-versa. This correlation was shown with mouse whole brain (Lajtha, Blasberg and Levi, 1966) and also with whole brains of other species (Levi et al., 1967). In contrast, the present report shows no clear correlation between regional pattern of amino acid accumulation in vitro and regional distribution in vivo, sugRc/c.rmces pp. 227-2211

226

G . LEV1

gesting that in the living brain other factors, such as regional differences in metabolic rates, may be operating. Also, there might be quantitative differences of the processes of influx and efflux between in vitro and in vivo conditions, as suggested by Lajtha, Lahiri and Toth (1963). According to these authors transport processes may act in vivo mostly to exclude substances from the brain, and in vitro may act mostly to accumulate substances in the slices. In other words, the exit process from brain tissue would not be as active in vitro as it is in vivo, where amino acids can be excluded from the brain also against a concentration gradient of elevated plasma levels (Lajtha and Toth, 1962). This hypothesis would explain why amino acid net accumulation into the living brain is much restricted. Regional differences in the activity of the exit process in vivo could therefore be an important factor in determining amino acid concentrations in the various areas of the brain under physiological circumstances and after plasma loading, and could explain the lack of correlation with the in vitro accumulations. In the opinion of the writer a high activity of the exit process in the living brain could be explained on the basis of the unique anatomical connections of nervous tissue cells, and of glial cells in particular. It is widely accepted that glial cells are in close contact with the basal membrane of blood capillaries (De Robertis and Gerschenfeld, 1961), and it is also accepted by most authors that there is a discrete extracellular space in the brain (Rall el al., 1962; Reed et al., 1964; Van Harreveld et al., 1965). A number of considerations would suggest that the extracellular fluid is in equilibrium with the cerebrospinal fluid, and that no barrier exists to the passage of substances from the former to the latter (Davson and Bradbury, 1965). Glial cells could take up blood amino acids from the small space existing between the capillary endothelium and the glial end feet. This space might well be occupied by a simple cdpillary filtrate (Davson and Bradbury, 1965) or by a fluid whose chemical composition is determined by the permeability properties of the capillary endothelium. Amino acids taken up would tend to exit preferentially into the extracellular space, since the concentration gradient in that direction is more favorable. Extracellular amino acids could then diffuse to the cerebrospinal fluid and could be rapidly eliminated through the arachnoid villi or by other routes. Thus the extracellular concentration would be kept low, with a mechanism similar to that described by Davson (1963) as “sink-action” of the cerebrospinal fluid; and the facilitated exit from the intracellular compartment would prevent the intracellular accumulation of amino acids. According to this model the cerebral concentration of those amino acids that are not produced or are not rapidly metabolized in the CNS would be mainly determined by the permeability properties of capillary endothelia and glial cells. Regional differences in amino acid concentration and uptake in vivo could be at least partly determined by regional differences in the size of the extracellular space, and in the readiness with which amino acids can diffuse to the cerebrospinal fluid. Since these mechanisms cannot be present in incubated brain slices, the lack of a close parallelism between regional transport i n vitro and regional distribution and uptake in vivo is not surprising.

REGIONAL DIFFERENCES I N C E R E B R A L A M I N O ACID TRANSPORT

227

SUMMARY

Amino acid transport in different areas of the CNS was studied both in vitro and in vivo. The transport capacity of different areas of the CNS as measured in vitro

varies from one area to the other, and also varies with the amino acid tested. Regional differences in the degree of amino acid accumulation at steady state seem to depend mostly on differences of accumulation rates with AIB, of exit rates with lysine, and on regional differences of both these processes with D-glutamate. It is suggested that the regional heterogeneity of cerebral amino acid accumulation and exit may be determined by differences in the permeability properties of the various cell types of the nervous tissue. The pattern of amino acid accumulation in slices from various brain areas does not correspond to the pattern of regional uptake or exchange in the living brain. This suggests the existence of regional differences in the restriction of amino acid transport in the living brain. In most cases no apparent correlation seems to exist between regional amino acid transport in vitro and regional amino acid concentration in vivo. Jnstead, the regional in vivo transport of some amino acids, such as lysine and leucine, tends to parallel the pattern of regional concentrations. The importance of transport pro:esses in the regulation of the level of cerebral amino acids may therefore vary from one case to the other. The possible reasons for the restriction of amino acid transport in the living brain, and for the low correlation between in vitro and in vivo findings are briefly discussed. ACKNOWLEDGEMENTS

The experimental work was done at the New York State Research Institute for Neurochemistry and Drug Addiction. 1 want to thank Dr. A. Lajtha for his hospitality and his advice. The cooperation of Dr. J. Kandera, Dr. A. Cherayil, and Mr. J. Toth is also gratefully acknowledged. The investigation was supported in part by Grant No. N B 04360 from the U S . Public Health Service (to Dr. A. Lajtha).

REFERENCES P. N . AND SCHOLEFIELD, P. G. (1962) Amino acid transport in brain cortex slices. 11. AHADOM, Competition between amino acids. Canad J . Biochem., 40, 1591-1602. BART,B., LOGOTETHIS, J., AELONY, Y. A N D Bovrs, M. (1962) Quantitative profile of freeaminoacids in various areas of cerebral cortex in normal guinca pigs. Exptl. Neurol., 5, 519-524. H. (1958) Determination of glutamic acid, glutamine, glutathione and BERL,S. A N D WAELSCH, y-aminobutyric acid and their distribution in brain tissue. J . Neurochem., 3, 161-169. BLASBERG, R. A N D LAJTHA, A. (1965) Substrate specificity of steady state amino acid transport in mouse brain slices. Arch. Biochem. Biophys., 112, 361-377. - (1966) Heterogeneity of the mediated transport systems of amino acid uptake in brain. Brain Res., I, 86-104. DAVSON, H. (1963) The cerebrospinal fluid. Ergeb. Physiol. biol. Cheni. exptl. Pharmacol., 52,21-73. DAVSON, H. AND BRADBURY, M. (1965) Theextracellular space of the brain. Progressin Brairi Research, E. D. P. De Robertis and R. Carrea (Eds.). Elsevier Publishing Company. Amsterdam. Vol. 15 (p. 124-134).

228

G . LEV1

DEROBERTIS, E. D. P. AND GERSCHENFELD, H. M. (1961) Submicroscopic morphology and function of glial cell. Intern. Rev. Neurobiol., 3, 1-65. GUROFF, G., KING, w.AND UDENFRIEND, s. (1961) The uptake Of tyrosine by rat brain in vitro. J .

biol. Chem., 236, 1773 -1777. KETY,S. S. AND ELKES,J., Editors (1961) Regional Newochertiisfry. Pergamon Press, Oxford. LAHiRI, S. A N D LAJTHA,A. (1964) Cerebral amino acid transport in vitro. I. Some requirements and properties of uptakc. J. Nertrochem., 11, 77-86. LAJTHA,A. (1961) Exchange rates of amino acids betwcen plasma and brain in different parts of the brain. Regional Neitrochemistry. S. S. Kety and J. Elkes, Editors. Pergamon Press, Oxford. (p. 19-24). LAJTHA,A. A N D MELA,P. (1961) The Brain-Barrier System. I. The exchange of free amino acids between plasma and brain. J. Neurochern., 7 , 210-217. LAJTHA, A. AND TOTH,J. (1962) The Brain-Barrier System. I I I.Theefflux of intracerebrally administered amino acid from the brain. J. Neurocheni., 9, 199-212. LAJTHA, A., LAHIRI, S. AND TOTH,J. (1963) The Brain-Barrier System. IV. Cerebral amino acid uptake in different classes. J. Neurocheni., 10, 765-773. LAJTHA, A.. BLASBERG, R. A N D LEVI,G. (1966) Control of cerebral amino acid concentrations. Signif icance of changes in plasma amino a c i d p a t t e r m New Brunswick, New Jersey. Rutgers University Press. In press. LtvI, G. A N D LAJTHA,A. (1965) Cerebral amino acid transport in vitro. 11. Regional differences in amino acid uptake by slices from the central nervous system of the rat. J. Neurochem., 12,639-648. LEw, G . , CHERAYIL, A. A N D LAJTHA,A. (1965) Cerebral amino acid transport in vitro. 111. Heterogeneity of exit. J. Nertrochem., 12, 757-770. LEVI,G., BLASBERG, R. AND LAJTHA,A. (1966) Substrate specificity of cerebral amino acid exit in vitro. Arch. Biochein. Biophys., 114, 339-351. LEVI,G., KANDERA, J. AND LAJTHA,A. (1967) Control of cerebral metabolite levels. I. Amino acid uptake and levels in various species. Arch. Biochem. Biophys., 119,303-31 1. NAKAMURA, R. A N D NAGAYAMA, M. (1966) Amino acid transport by slices from various regions of the brain. J . Neurochem., 13, 305-31 3. NEAME, K. D. (1962) Uptake of L-histidine, L-proline, L-tyrosine and L-ornithine by brain, intcstinal mucosa, testis, kidney, splcen, liver, heart muscle, skeletal muscle and erythrocytes of the rat in vitro. J. Physiol., 162, 1-12. OKUMURA, N., OTSUKI,S. AND FUKAI, N. (1959) Amino acid concentration in different parts of the dog brain. Acta M e d . Okayama, 13, 27-30. PAPPIUS, H. M., (1965) The distribution of water in brain tissue swollen in vitro and in vivo. Progress in Brain Research. E. D. P. De Robertis and R. Carrea, Editors. Elsevier Publishing Company, Amsterdam. Vol. 15 (p. 135-154). RALL,D. P., OPPELT,W. W. A N D PATLACK, C. S. (1962) Extracellular space of brain as dctermined by diffusion of inulin from the ventricular system. f i f e Sci., 2, 4 3 4 8 . RAMlREZ DE GUGLIELMONE, A. AND GOMEZ,c. J . (1966) Free amino acids in different arcas of rat brain. Ac/a Physiol. I atinoamer., 16, 26-37. REED,D. J., WOODBURY, D. M. AND HOLTZER, R. L. (1964) Brain edema, electrolytes, and extracellular space. Arch. Neirrol., 10, 604616. SINGH,S. 1. A N D MALHOTRA, C. L. (1964) Amino acid content of monkey brain: effects of reserpine on some amino acids of certain regions of monkey brain. J. Neurocliem., 11, 865-872. SHAW,R. K., AND HEINE, J. D. (1965) Ninhydrin positive substances present in different areas of normal rat brain. J. Nertrochem., 12, 151-155. STERN, J. R., ECCLESTON, L. V.. HEMS,R. AND KREBS,H. A. (1949) Accumulation of glutamic acid in isclated brain tissue. Biochenr. J., 44, 410418. TSUKADA, Y., NAGATA, Y.,HIRANO, S. A N D MATSUTANI, T. (1963) Active transport of amino acid into cerebral cortex slices. J. Neurochem., 10, 241-256. VANHARREVELD, A., CROWELL, J. A N D MALHOTRA, S. K. (1965) A study of extracellular space in central nervous system by freeze-substitution. J. Cell Biol.,25, 117-137.

REGIONAL DIFFERENCES I N CEREBRAL AMINO A C I D T R A N S P O R T

229

DISCUSSION

D. H. FORD:How much are the regional differences due to the differences in cell population, that is to the density of the cells per unit area? If you take, e.g., the uptake of almost any amino acid by the hypothalamus, one finds that the highest accumulation of label is near the arcuate nucleus, which has almost the highest nerve cell population density in the hypothalamus. Thus, the degree of accumulation of labeled compomds appears to be, in part at least, related to the number of neurons present in a particular region. G. LEVI:I did not measure the density of the cells in the different areas.

D. H. FORD: In your preparation there are cells mixed with white matter, all mixed up together. Anatomically it would be difficult to define this as cerebral cortex. G. LEVI:The preparations that I used are not anatomically pure, in the sense that in no case are we dealing only with neurons or glia or white matter. Nevertheless the cellular composition of the various areas that I used is very different. Now, what I found is that the regional pattern of transport is not the same with all amino acids. In other words, none of the areas has the highest accumulation with all of the amino acids, and since the cell population of the various areas is different, the structures involved in the transport of amino acids must also be different. Therefore, according to the type of structure and to the type of amino acid we may have different results.

D. H. FORD:I don’t think I would want to say that the structures involved in transport would necessarily be different in an anatomical sense because we are still dealing with the problem of the capil-

lary: glia: neuronal relationship, which probably occurs in a similar manner in most areas of the brain. The difference in accumulation in different regions may again be closely related to the population density. I t makes a tremendous difference in the amount, and perhaps the rate with which a compound would be accumulated.

G. LEVI:I would like to emphasize that the conditions are different in vivo and in vitro: in brain slices there is no longer a capillary: glia barrier and the various cell types are in direct contact with the slibstances present in the medium. A. LAJTHA: There was some indication of a greater exit of glutamic acid by slices from areas richer in neurons, but since this was not true for other amino acids, we can’t say that there is a single pattern of neurons extruding all amino acids more than glia.

D. H.FORD: The incorporation may vary from amino acid to amino acid. You are getting entirely different curves, depending on what your amino acid is.

P. MANDEL:I have two questions. The first is: have you performed any tests concerning changes occurring in slices after 70 minutes of incubation in vitro? The second question is a general one: I should like to know something about the role of taurine in the brain. Maybe somebody has a hypothesis upon this? G. LEVI:I can easily answer the first question. We did experiments just to check the condition of the slices after periods even longer than 70 minutes. Both the accumulation and the exit remained unchanged after periods of - if I remember correctly - about two hours. I don’t have an answer to the second question, unfortunately. N. M. VAN GELDER: I just wanted to ask: could you give any indication of how reproduceable these figures are; how many experiments are represented by each figure, and what was the standard deviation?

G. LEVI:Yes, with in vitro experiments there was an average of 4 to 6 experiments, with a standard deviation of less than 10%. The number of in vivo experiments was approximately four, and the standard deviation was somewhat higher, being about I5 ?{.

230

G. LEV1

K. A. C. ELLIOTT:I may have missed something in both these last papers. Did you ascertain that these were the actual amino acids that were taken up and incorporated, or was it only radio-active carbon derived from them that appeared in these various spaces? G. LEVI:In our investigation, these amino acids were in the so called free amino acid pool. The radio-activity in the PCA-soluble fractions was from 85 to 90% in the form of the original amino acid provided, as determined by paper chromatography.

D. H. FORD:We have always done parallel paper chromatographic experiments with our radioautographic and accumulation studies, and feel reasonably sure that the radioautographic localizations shown after injecting lysine is due primarily to lysine. With the glutamine study, much of the labeled material in the tissue was present as glutamic acid as well as glutamine. D. B. TOWER: I did not quite understand your distinction a t the end between the in vivo and in v i m levels, because certainly amino acids (at least in vitro) achieve the in vivo levels during incubation, even if you have depleted them temporarily by some means. I am referring particularly to glutamate, aspartate, and so on, which are generated primarily by conversion from glucose. Thus, I think you have to make a distinction as to which amino acid you are talking about in this regard, since accumulation of these amino acids may not necessarily have anything to do with their transport. G . LEVI:The in vilro accumulation of the amino acids that were used in this investigation is due to transport and not to other factors. In fact the amino acids used were either not metabolized or metabolized very slowly. For example the D-form and not the readily metabolized L-form of glutamate was used. Anyhow factors such as the conversion from glucose to glutamate or to other amino acids would be negligible as compared to the transport rates in our experimental conditions; glutamate reach-s an intracellular level of over 50 mM in about half an hour when brain slices are incubated in presence of 2 mM glutamate.

GENERAL DISCUSSION

23 1

GENERAL D I S C U S S ION P. L. IPATA: Dr. Mandel, you have shown that the nucleoside enters the nerve cell and is incorporated into RNA molecules. We must therefore assume that it is phosphorylated; for instance, adenosine must give rise to AMP inside the cell in order to be incorporated into RNA. We have shown that the dephosphorylation of AMP inside the cell is strongly inhibited by ATP and other nucleoside triphosphates. This would facilitate the further phosphorylation of AMP to ADP and ATP for RNA synthesis.

P. MANDEL: We know that in several tissues the uptake of nucleosides and the synthesis of RNA from nucleosides is much faster than for purine and pyrimidine bases. In free nuclei with nucleosides incubated in vitro we have found a quite high synthesis of RNA, and a much lower synthesis of adenine, which means that the cells, or the nuclei, where the synthesis of RNA occurs, are able to produce ATP or nucleoside diphosphate from the nucleosides. P. L. IPATA:It is therefore meaningfull that the dephosphorylation of AMP is strongly inhibited inside the cell. Adenosine enters the cell, and is transformed into AMP; the dephosphorylation of AMP, however, is strongly inhibited by nucleoside triphosphates. I will show later that the inhibition is of the allosteric nature.

H. KOENIG: Pyrimidine nucleotides serve two major metabolic roles: ( I ) they are precursors of RNA and DNA; and (2) as coenzymes they participate in intermediary metabolism and biosynthetic reactions of lipids and carbohydrates. My interest in pyrimidine nucleotides in the central nervous system was stimulated by observations that neural tissues actively incorporate [I4C]orotic acid into neuronal and glial RNA (Koenig. H., 1958, Proc. Soc. Exper. Biol. Med., 97, 255; J . Biophys. Biochem. Cyrol., 4, 241 ; 1959, I n “Prog. in Neurobiol.” S. Korey. Ed., 4, 241, Hoeber, New York). Later studies revealed that the pyrimidine analogs 5-fluoroorotic acid and 5-fluorouridine (Koenig, H., 1960, AMA Arch. Neurol., 2: 463; 1962, 111 “Response of the Nervous System to Ionizing Radiation”, Haley, T., and Snider. R. (Eds.), p. 109, Academic Press, New York), and 6-azauracil and 6-azauridine (1961, Koenig, H., Young, 1. J., Wells, W., and Gaines, D., Trans. Am. Neurol. Ass., p. 219; 1963, Wells, W., Gaines, D., and Koenig, H., J . Nerrrochenz., 10: 709) are neurotoxic when administered intrathecally. These studies, which have recently been reviewed (1967, Koenig, H., International Review of Neurobiology, 10, Academic Press, New York), prompted an investigation of the entry and uptake of a number of natural and unnatural pyrimidines into brain and liver in rat. The experimental details and results are summarized in Table 1. A number of interesting findings are apparent. [14C]Orotic acid and [’4C])fluorooroticacid, both highly ionized, traversed the bloodbrain barrier to a limited extent, whereas [14CC]azauraciland [14CC]uridinewere 3 to 4 fold more effective in this regard, as judged by the specific activity of the acid soluble fraction. The intracerebral injection route gave much greater uptake of pyrimidines into the acid soluble fraction and RNA than the intravenous route of injection. Despite this, incorporation into brain RNA, as measured by the relative specific activity, was comparable for both routes of administration. [I4C]uridine was most actively taken up into RNA. [lJC]Oroticacid, 5-fluoroorotic acid and 5-fluorouracil were also well utilized. [I4C]Uracilwas incorporated to a lesser degree, while [14C]azauracilwas incorporated minimally into RNA. Liver differed significantly from brain in a number of respects. [*4C]Oroticacid and [14C]5-fluor~ orotic acid were extensively incorporated into the acid soluble fraction of liver, even when these wer: administered intracerebrally. The other pyrimidines, [14C]uracil, [14C]azauracil, [~~C]5-fluorouracil and [‘JCIuridine, appeared in the acid soluble fraction of liver to a limited degree. Without exception these pyrimidines, as measured by the R.S.A., were incorporated into brain R N A to a greater extent than into liver RNA. From these data it seems that RNA turnover in brain is considerably more active than RNA turnover in liver. The extent to which brain can utilize preformed pyrimidines conveyed by the blood-stream naturally depends upon the facility with which these traverse the bloodbrain barrier. Of the pyrimidines tested, uridine was most actively transported into brain and was also most extensively incorporated into brain RNA.

232

G E N E R A L DISCUSSION

TABLE 1 U P T A K E OF N A T U R A L A N D U N N A T U R A L P Y R I M I D I N E S I N T O R A T B R A I N A N D L I V E R

[I4C]Orotic acid Intravenous Intracerebral [W2]5-Fluoroorotic acid Intravenous lntracerebral [ W ] Uracil Intravenous Intracerebral [14C]6-Azauracil Intravenous Intracerebral [14C]5-Fluorouracil Intravenous Intracerebral [14C]Uridine Intravenous Intracerebral

** +

S.A.* of A.S.F.

BRAIN S.A.** of RNA

R.S.A.+

S.A.* of A.S.F.

LIVER S.A.** of RNA

355 67,500

I .2 378.0

3.4 x 10-3 5.7 x 10-3

199,000 34,800

190.0 42.0

1.0 x 10 1.2 x 10-3

428 80,000

1.7 349.0

4.1 x 10-3 4.3 x 10-3

132,000 34,800

203.0 42.0

1 . 6 x 10-3 1.2 x 10-3

198 27.500

0.5 66.0

2.3 x 10-3 2.5 x 1 0 - 3

566 869

0.3 0.2

4.8 x 10 1.6 x 10

1,339 10,622

0.4 2.3

3.0 x 1 0 - 4 2.1 x 1 0 - 4

1,567 568

0.2 0.2

1.5

-

0.1

3.2 x

0.1

0.9 x 10-3 1 . 4 x 10-3

-

34,400

193.0

5.6 x 10-3

253

947 43,000

6.7 359.0

6.5 x 10-3 8.3 x 10-3

111.3 64.7

0.I

R.S.A.+

X

10

1.9 x 10-4

Specific activity (S.A.) of acid-soluble fraction (A.S.F.) = counts per minute (C.P.M.)/g fresh weight. S.A. of RNA = C.P.M. per optical density unit at 260 mp (ODZEO). Relative specific activity (RSA)

=

S.A. of R N A S.A. of A.S.F.

Experinienral Derails: All isotopes (S.A. = 2.4 mC/mMole) were injected intravenously into rat in a dose of 1 pC/100 g, or intracerebrally in a dose of 0.5 pC/100 g. After a 4 hour exchange period, heads were plunged into liquid nitrogen, frozen brains removed with a chisel and homogenized in ice cold 1076 trichloracetic acid to yield an acid soluble fraction. After water and lipid extraction, the protein residue was hydrolyzed in 0.3 M NaOH at 37’C for 1 h., neutralized with HCI, and DNA and protein precipitated by adding cold TCA to a concentration of 10%. RNA was measured in the acid-soluble supernatant by ultraviolet spectrophotometry. Radioactivity was assayed in an automatic gas flow Geiger counter.

P. MANDEL:I think that we cannot assume that you have a higher incorporation in liver than in brain, since you relate the relative specific activity to the whole RNA, and d o not know the specific activity of RNA in the various types of cells. In the neurons you have about the same concentration of RNA as in the liver, but the apparent synthesis is lower in the liver. But I am not sure that this is so since, for example, in measuring the RNA polymerase activity, you find it either in a similar, or in a lower quantity in the brain than in the liver. H. KOENIG:RNA polymerase activity gives only the potential for RNA synthesis, as RNA synthesis depends on the amount of available template, and the availability of precursors, etc.. In brain, of course, the various cell types differ greatly in the rate of R N A synthesis. Large cells show the greatest RNA synthesis. These have the same amount of template DNA as glia. However, their chromatin is largely in a diffuse state, and is therefore metabolically active in the RNA polymerase reaction. The difference between a big nerve cell and a small nerve cell with respect to RNA synthesis is great, and still greater for glia. So this is a picture of the “average” nerve cell,

GENERAL DISCUSSION

233

P. MANDEL: We measured template independent RNA polymerase.

D. H. FORD:I t is very interesting to hear that your uptakes of adenine in the liver and brain were so comparable. We have just completed an in vivo study in rats where we have injected [3H]adenine and studied the uptake per unit wcight of ventral horn neuron, using a modified Hyden procedure to isolate the neurons. Our data indicate that nerve cells accumulate slightly more than liver cells, which emphasizes the tremendous affinity which the neuron has to have for various substrates to maintain its high metabolic needs. What the biologic nature is of the labeled material in the neurons from these preparations can only be inferred from what can be determined for blocks of grey matter from the same animals analyzed chromatographically. Such data suggest that most of the labeled material present in the neurons is probably associated with the high energy phosphates. P. MANDEL:Here again the problem is whether you do short-time experiments or longer-time experiments. If you d o longer-time experiments this is quite different. We mainly did very short-time experiments to look at the diffusion in the brain or in the liver.

D. H. FORD:Ours were for a half hour to 24 hours. Ours were from 2 minutes. P. MANDEL: P. L. IPATA: Can you answer the question if, in your experimental conditions, adenosine is deaminated to inosine in the brain? And furthermore; what is the ratio of the radioactivity among the four bases in the RNA? P. MANDEL:I cannot say if it is deaminated or not. I did not control this. But the deamination of adenosine in brain is more artifact, because if you kill the animal in good conditions you have a very low amount of inosine, and if you kill it in bad conditions you have a high amount of inosine. R . KATZMAN: One would assume from your data that polynucleotides would not enter the brain. Do you have direct evidence on this? P. MANDEL:Polynucleotides are something else. Polynucleotides might enter into the brain by pinocytosis. This may be low. I t is possible that by pinocytosis macromolecules can enter another site, but I have no evidence.

P. G . SCHOLEFIELD: Have you any evidence on how the ribosides are converted to the nucleotides? Do they go down to the base? I don't know. If you incubate the nucleotides the radioactivity is not decreased by the P. MANDEL: addition of thc adenine. And if you had to pass through the adenine step you should have a decrease. P. G. SCHOLEFIELD: If you use ascites cells under similar conditions you can get an almost equivalent quantity of lactic acid from ribosides. P. MANDEL:Yes, but that is a peculiar phenomenon, I think, for ascites cells, and perhaps also for red blood cells, which use for energy metabolism the ribose of nucleosides.