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Cerebral amino acid transport in vitro during development: A kinetic analysis
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
138
(1970)
COMMUNICATIONS Cerebral
Amino
Acid Transport
During
Development:
A Kinetic
in Vifro Analysis
Changes in the transport of amino acids during the development of the brain have been reported by some authors (l-3), but no comprehensive study on this subject has been performed so far. In particular, no attempt was made to understand the mechanisms underlying the changes of cerebral amino acid transport observed during ontogenesis. The present investigation was particularly directed to the study of this problem. A kinetic analysis of the initial accumulation of five amino acids by brain slices from chick embryos and young chicks was performed. With brain slices, the mechanisms of cerebral amino acid transport can be studied at cellular level without the interference of the brain-barrier system, of bulk-flow phenomena, and of other possible factors present in the living animal (4). The amino acids chosen, cy-aminoisobutyric acid (AIB), leucine, lysine, n-glutamate, and -y-aminobutyric acid (GABA), belong to different transport classes, as characterized by substrate-specificity studies in adult mouse brain slices (5). Fertilized eggs (Livorno strain) purchased from a local hatchery were incubated in a thermostat at 38”. Slices from whole cerebrum (0.4-mm thick) were prepared by an automatic tissue chopper after removing the cerebellum, the pons, and the medulla. Since brains from g-day-old embryos are sufficiently thin to be incubated without slicing, they were divided into five parts-two hemispheres, two optic lobes, and midbrain-by an iridectomy knife. The tissue was placed in Erlenmeyer flasks containing ice-cold medium (10 mM glucose, 128 mM NaCl, 5 mM KCl, 2.7 mM CaC12, 1.2 ells MgSOd, 5 rnM NazHPO,, 50 mM Tris-HCl buffer at pH 7.4) and incubated in a shaking waterbath at 37” for 20 min. Then new medium (one fifth of the final volume) containing a given concentration of %-labeled amino acid was added, and the incubation was continued for an additional 5 min. The volume of medium was high enough to prevent any significant decrease in the amino acid concentration during the incubation time. The radioactivity of the incubated tissue and of the incubat,ion medium was measured
as already described (6). Paper chromatographic controls showed that 92-1OO~o of t.he radioactivity present in the tissue had an RF value corresponding to the amino acid originally present in the incubation medium. The uptake of amino acids was linear with time during the first 5 min of incubation. Initial accumulation rates (pmoles/ml intracellular water/min) were calculated from the known specific radioactivity of the medium, and from the radioactivity of the tissue extract, after correcting for the amount of amino acid present in the extracellular space of the tissue (7). The amino acid concentrations used ranged from 0.02 to 2.0-4.0 mM. For each stage and for each amino acid 6-9 different concentrations were used, the lowest of which differed at least 100 times from the highest. Four to eight separat,e determinations were performed for every concentration. The standard deviation of the calculated accumulation rates ranged from 2 to 6%. The data were analyzed according to the kinetics of Michaelis-Menten. The applicability of this type of kinetics to our system was tested by three different plots: (1) substrate concentration (amino acid concentration in the medium) over velocity versus substrate concentration; (2) velocity versus velocity over substrate concentration; (3) reciprocal of velocity versus reciprocal of substrate concentration [Lineweaver-Burk plot (S)]. In all plots straight lines were obtained with AIB, leucine, lysine, and n-glutamate. The results obtained with GABA will be discussed separately. Subsequent calculations were performed on the Lineweaver-Burk plots. The slopes and intercepts were calculated both graphically and by the method of least squares. The agreement between the two methods was generally excellent. In some cases slight differences in the values of amino acid influx at the lowest, amino acid concentration used (0.02 mM) brought significant changes to the intercept values calculated with the method of least squares. In these cases, in order to increase the accuracy of calculation, other experiments at amino acid concentrations somewhat higher than 0.02 mM were performed. Transport rates were not corrected for diffusion or other types of nonsaturable transport processes. In fact, the contribution of nonsaturable trans-
347
348
COMMUNICATIONS TABLE
I
APPARENT K,n VALUES AND MAXIMAL VELOCITIES OF TRANSPORT OF AMINO ACIDS IN INCUBATED BRAIN TISSUE AT DIFFERENT AGES? a-Aminoisobutyrate Age (days)
Embryos 8 14 19 Chicks 15
Leucine
n-Glutamate
Lysine
Apparent Km
vmx
Apparent Km
VI&¶,
Apparent K,
vmax
Apparent Km
V,X
2.64 2.12 2.52
0.53 1.23 1.58
0.79 0.82 0.73
0.36 0.76 0.73
0.39 0.49 0.48
0.13 0.28 0.41
1.70 1.29 1.77
0.60 1.67 2.84
2.39
1.28
0.80
0.80
0.45
0.23
1.21
1.79
Q For experimental details and methods values are given as @moles/ml intracellular
of calculation water/min.
see text. Apparent
K, values are lW%;
V,.,
40. (b)
15-day-old
chicks
plots of GABA influx. l/v = reciprocal velocity of uptake FIG. 1. Lineweaver-Burk (pmoleslml intracellular water/min)-I. l/S = reciprocal concentration in incubation medium (mM-I). Rough values of apparent K, calculated for the high concentration limb of the curves of Fig. la are 0.77, 0.83, and 0.91 mM for S-, 14-, and 19-day-old embryos, respectively. The apparent K, value calculated for 15-day-old chicks is 0.72 mnn. The corresponding V,,, values are: 0.15, 1.5, 2.5, and 1.4 amoles/ml intracellular water/min, respectively. port as determined by the method described by Akedo and Christensen (9) was negligible in our experimental conditions, within the range of concentrations used. Table I shows t,he maximal velocities of transport (V,,,) and the apparent K,n values calculated for AIB, leucine, lysine, and n-glutamate. It can be seen that, according to the amino acid tested, the V,,, values increase 2-5 times during embryonic development, then remain constant (leucine) or decrease more or less sharply. In contrast, the apparent K, values do not show any significant change with age. These results suggest that during cerebral development no significant qualitative change in the mechanisms of transport t.akes place. The affinity of the t.ransport sites for
the amino acids tested seems to remain fairly constant with age; it could be inferred also that the substrate specificity of cerebral amino acid transport does not undergo any significant developmental change. The data of Table I point to the existence of only one transport system for each of the four amino acids, at least within the range of the amino acid concentrations used. It must be noted, however, that the method of analysis used cannot discriminate between two transport sites with relatively similar affinity constants. The reasons for the changes in maximal velocities of transport may obviously be of different nature. One possibility to be considered is that the amount of carriers available for transport
COMMUNICATIONS changes with age. The fact that the V,,. values change with age in a way that is characteristic for each compound (Table I) suggests that different carriers rather than different sites on the same carrier molecule are responsible for the transport of amino acids belonging to different classes. It also suggests that no direct correlation can be established between changes in transport rates and changes in the amount of energy available or in the number of cells per unit weight. The behavior of GABA differs from that of the other amino acids tested. In fact, with GABA, the Lineweaver-Burk plot (Fig. la) and the other plots mentioned give a two-limbed curve at all three embryonic ages considered. The break becomes less evident with increasing age, and it is no longer present in 15-day-old chicks (Fig. lb). Deviation from linearity of Lineweaver-Burk plots can be interpreted as evidence for the existence of at least two modes of mediated influx of widely different affinities (10, 11). The data of Fig. 1 suggest that a second mode of entry (or carrier) having a lower affinity fo< GABA is utilized at concentrations above 0.3-0.5 mrvr in the embryonic brain. An accurate calculation of V max and apparent K, values on this graph is not possible because of the partial overlapping of the two transport systems. Even with these limitations, it is interesting to note that the rough values of apparent K, calculated for the high concentration limbs of the two-limbed curves are all similar, whereas V,,, values differ significantly one from the other (legend to Fig. 1). In particular, the apparent K, calculated for t.he high concentration limb of the curve obtained with &day-old embryos, which is probably only slightly distorted since the break is very sharp, is almost identical to that calculated for the curve of 15.day-old chicks, whichshows no break (legend to Fig. 1). Therefore, at least one of the transport systems for GABA shows the same developmental behavior as those systems mediating the transport of the other amino acids tested. The second system either tends to disappear with age, or contributes so little to the overall measured rates in more mature brains that its detection becomes impossible. It must be emphasized that the data presented here result from the analysis of a highly heterogeneous cell population. Moreover, during the developmental period considered, the differentiation of nerve cells and the proliferation and maturation of glial cells determine significant
349
changes in the anatomical and chemical architecture of the brain. In spite of these marked changes, a well-defined and consistent pattern could be obtained for the transport of each of the amino acids tested. In particular, it is noteworthy that the kinetics of GABA accumulation differs from that of the other amino acids t,ested during early stages of brain maturation, and becomes similar only in more advanced stages of differentiation. ACKNOWLEDGMENTS This work was supported in part by the Consiglio Nasionale delle Ricerche, Rome. I thank Dr. R. Levi-Montalcini for stimulating discussion, Dr. M. G. Lattes for assistance, and Miss M. T. Ciotti for technical help. REFERENCES 1. LAJTHA, A., LAHIRI, S., AND TOTH, J. J. Neurochem. 10, 765 (1963). 2. LEVI, G., KANDERA, J., AND LAJTHA, A. Arch. Biochem. Biophys. 119, 303 (1967). 3. VAHVELAINEN, M. L., AND OJA, S. S. Brain Res. 13. 227 (1969). 4. LAJTHA, A., BLASBERG, R., AND LEVI, G. in “Protein Nutrition and Free Amino Acid Patterns” (J. H. Leathem, ed.), p. 187. Rutgers Univ. Press, New Brunswick, New Jersey (1968). 5. BLASBERG, R., AND LAJTIIA, A. Brain Res. 1, 86 (1966). 6. LEVI, G., AND AMADUCCI, L. J. Neurochem. 16, 459 (1968). 7. LEVI, G., AND LATTES, M. G. J. Neurochem. in press. 8. LINEWEAVER, H., AND BURK, D. J. Amer. Chem. Sot. 66, 658 (1934). 9. AKEDO, H., AND CHRISTENSEN, H. N. J. Biol. Chem. 33’7, 118 (1962). 10. WINTER, C. G., AND CHRISTENSEN, H. N. J. Biol. Chem. 240, 3594 (1965). 11. HILLMAN, R. E., ALBRECHT, I., AND ROSENBERG, L. E. J. Biol. Chem. 243, 5566 (1968). GIULIO LEVI Laboratorio di Chimica BioEogica Istituto Superiore di Sanitd and Laboratorio di Biologia Cellulare de1 CNR, Rome, Italy Received January 16, 1970; accepted February 10, 1970
OF
BIOCHEMISTRY
AND
BIOPHYSICS
138
(1970)
COMMUNICATIONS Cerebral
Amino
Acid Transport
During
Development:
A Kinetic
in Vifro Analysis
Changes in the transport of amino acids during the development of the brain have been reported by some authors (l-3), but no comprehensive study on this subject has been performed so far. In particular, no attempt was made to understand the mechanisms underlying the changes of cerebral amino acid transport observed during ontogenesis. The present investigation was particularly directed to the study of this problem. A kinetic analysis of the initial accumulation of five amino acids by brain slices from chick embryos and young chicks was performed. With brain slices, the mechanisms of cerebral amino acid transport can be studied at cellular level without the interference of the brain-barrier system, of bulk-flow phenomena, and of other possible factors present in the living animal (4). The amino acids chosen, cy-aminoisobutyric acid (AIB), leucine, lysine, n-glutamate, and -y-aminobutyric acid (GABA), belong to different transport classes, as characterized by substrate-specificity studies in adult mouse brain slices (5). Fertilized eggs (Livorno strain) purchased from a local hatchery were incubated in a thermostat at 38”. Slices from whole cerebrum (0.4-mm thick) were prepared by an automatic tissue chopper after removing the cerebellum, the pons, and the medulla. Since brains from g-day-old embryos are sufficiently thin to be incubated without slicing, they were divided into five parts-two hemispheres, two optic lobes, and midbrain-by an iridectomy knife. The tissue was placed in Erlenmeyer flasks containing ice-cold medium (10 mM glucose, 128 mM NaCl, 5 mM KCl, 2.7 mM CaC12, 1.2 ells MgSOd, 5 rnM NazHPO,, 50 mM Tris-HCl buffer at pH 7.4) and incubated in a shaking waterbath at 37” for 20 min. Then new medium (one fifth of the final volume) containing a given concentration of %-labeled amino acid was added, and the incubation was continued for an additional 5 min. The volume of medium was high enough to prevent any significant decrease in the amino acid concentration during the incubation time. The radioactivity of the incubated tissue and of the incubat,ion medium was measured
as already described (6). Paper chromatographic controls showed that 92-1OO~o of t.he radioactivity present in the tissue had an RF value corresponding to the amino acid originally present in the incubation medium. The uptake of amino acids was linear with time during the first 5 min of incubation. Initial accumulation rates (pmoles/ml intracellular water/min) were calculated from the known specific radioactivity of the medium, and from the radioactivity of the tissue extract, after correcting for the amount of amino acid present in the extracellular space of the tissue (7). The amino acid concentrations used ranged from 0.02 to 2.0-4.0 mM. For each stage and for each amino acid 6-9 different concentrations were used, the lowest of which differed at least 100 times from the highest. Four to eight separat,e determinations were performed for every concentration. The standard deviation of the calculated accumulation rates ranged from 2 to 6%. The data were analyzed according to the kinetics of Michaelis-Menten. The applicability of this type of kinetics to our system was tested by three different plots: (1) substrate concentration (amino acid concentration in the medium) over velocity versus substrate concentration; (2) velocity versus velocity over substrate concentration; (3) reciprocal of velocity versus reciprocal of substrate concentration [Lineweaver-Burk plot (S)]. In all plots straight lines were obtained with AIB, leucine, lysine, and n-glutamate. The results obtained with GABA will be discussed separately. Subsequent calculations were performed on the Lineweaver-Burk plots. The slopes and intercepts were calculated both graphically and by the method of least squares. The agreement between the two methods was generally excellent. In some cases slight differences in the values of amino acid influx at the lowest, amino acid concentration used (0.02 mM) brought significant changes to the intercept values calculated with the method of least squares. In these cases, in order to increase the accuracy of calculation, other experiments at amino acid concentrations somewhat higher than 0.02 mM were performed. Transport rates were not corrected for diffusion or other types of nonsaturable transport processes. In fact, the contribution of nonsaturable trans-
347
348
COMMUNICATIONS TABLE
I
APPARENT K,n VALUES AND MAXIMAL VELOCITIES OF TRANSPORT OF AMINO ACIDS IN INCUBATED BRAIN TISSUE AT DIFFERENT AGES? a-Aminoisobutyrate Age (days)
Embryos 8 14 19 Chicks 15
Leucine
n-Glutamate
Lysine
Apparent Km
vmx
Apparent Km
VI&¶,
Apparent K,
vmax
Apparent Km
V,X
2.64 2.12 2.52
0.53 1.23 1.58
0.79 0.82 0.73
0.36 0.76 0.73
0.39 0.49 0.48
0.13 0.28 0.41
1.70 1.29 1.77
0.60 1.67 2.84
2.39
1.28
0.80
0.80
0.45
0.23
1.21
1.79
Q For experimental details and methods values are given as @moles/ml intracellular
of calculation water/min.
see text. Apparent
K, values are lW%;
V,.,
40. (b)
15-day-old
chicks
plots of GABA influx. l/v = reciprocal velocity of uptake FIG. 1. Lineweaver-Burk (pmoleslml intracellular water/min)-I. l/S = reciprocal concentration in incubation medium (mM-I). Rough values of apparent K, calculated for the high concentration limb of the curves of Fig. la are 0.77, 0.83, and 0.91 mM for S-, 14-, and 19-day-old embryos, respectively. The apparent K, value calculated for 15-day-old chicks is 0.72 mnn. The corresponding V,,, values are: 0.15, 1.5, 2.5, and 1.4 amoles/ml intracellular water/min, respectively. port as determined by the method described by Akedo and Christensen (9) was negligible in our experimental conditions, within the range of concentrations used. Table I shows t,he maximal velocities of transport (V,,,) and the apparent K,n values calculated for AIB, leucine, lysine, and n-glutamate. It can be seen that, according to the amino acid tested, the V,,, values increase 2-5 times during embryonic development, then remain constant (leucine) or decrease more or less sharply. In contrast, the apparent K, values do not show any significant change with age. These results suggest that during cerebral development no significant qualitative change in the mechanisms of transport t.akes place. The affinity of the t.ransport sites for
the amino acids tested seems to remain fairly constant with age; it could be inferred also that the substrate specificity of cerebral amino acid transport does not undergo any significant developmental change. The data of Table I point to the existence of only one transport system for each of the four amino acids, at least within the range of the amino acid concentrations used. It must be noted, however, that the method of analysis used cannot discriminate between two transport sites with relatively similar affinity constants. The reasons for the changes in maximal velocities of transport may obviously be of different nature. One possibility to be considered is that the amount of carriers available for transport
COMMUNICATIONS changes with age. The fact that the V,,. values change with age in a way that is characteristic for each compound (Table I) suggests that different carriers rather than different sites on the same carrier molecule are responsible for the transport of amino acids belonging to different classes. It also suggests that no direct correlation can be established between changes in transport rates and changes in the amount of energy available or in the number of cells per unit weight. The behavior of GABA differs from that of the other amino acids tested. In fact, with GABA, the Lineweaver-Burk plot (Fig. la) and the other plots mentioned give a two-limbed curve at all three embryonic ages considered. The break becomes less evident with increasing age, and it is no longer present in 15-day-old chicks (Fig. lb). Deviation from linearity of Lineweaver-Burk plots can be interpreted as evidence for the existence of at least two modes of mediated influx of widely different affinities (10, 11). The data of Fig. 1 suggest that a second mode of entry (or carrier) having a lower affinity fo< GABA is utilized at concentrations above 0.3-0.5 mrvr in the embryonic brain. An accurate calculation of V max and apparent K, values on this graph is not possible because of the partial overlapping of the two transport systems. Even with these limitations, it is interesting to note that the rough values of apparent K, calculated for the high concentration limbs of the two-limbed curves are all similar, whereas V,,, values differ significantly one from the other (legend to Fig. 1). In particular, the apparent K, calculated for t.he high concentration limb of the curve obtained with &day-old embryos, which is probably only slightly distorted since the break is very sharp, is almost identical to that calculated for the curve of 15.day-old chicks, whichshows no break (legend to Fig. 1). Therefore, at least one of the transport systems for GABA shows the same developmental behavior as those systems mediating the transport of the other amino acids tested. The second system either tends to disappear with age, or contributes so little to the overall measured rates in more mature brains that its detection becomes impossible. It must be emphasized that the data presented here result from the analysis of a highly heterogeneous cell population. Moreover, during the developmental period considered, the differentiation of nerve cells and the proliferation and maturation of glial cells determine significant
349
changes in the anatomical and chemical architecture of the brain. In spite of these marked changes, a well-defined and consistent pattern could be obtained for the transport of each of the amino acids tested. In particular, it is noteworthy that the kinetics of GABA accumulation differs from that of the other amino acids t,ested during early stages of brain maturation, and becomes similar only in more advanced stages of differentiation. ACKNOWLEDGMENTS This work was supported in part by the Consiglio Nasionale delle Ricerche, Rome. I thank Dr. R. Levi-Montalcini for stimulating discussion, Dr. M. G. Lattes for assistance, and Miss M. T. Ciotti for technical help. REFERENCES 1. LAJTHA, A., LAHIRI, S., AND TOTH, J. J. Neurochem. 10, 765 (1963). 2. LEVI, G., KANDERA, J., AND LAJTHA, A. Arch. Biochem. Biophys. 119, 303 (1967). 3. VAHVELAINEN, M. L., AND OJA, S. S. Brain Res. 13. 227 (1969). 4. LAJTHA, A., BLASBERG, R., AND LEVI, G. in “Protein Nutrition and Free Amino Acid Patterns” (J. H. Leathem, ed.), p. 187. Rutgers Univ. Press, New Brunswick, New Jersey (1968). 5. BLASBERG, R., AND LAJTIIA, A. Brain Res. 1, 86 (1966). 6. LEVI, G., AND AMADUCCI, L. J. Neurochem. 16, 459 (1968). 7. LEVI, G., AND LATTES, M. G. J. Neurochem. in press. 8. LINEWEAVER, H., AND BURK, D. J. Amer. Chem. Sot. 66, 658 (1934). 9. AKEDO, H., AND CHRISTENSEN, H. N. J. Biol. Chem. 33’7, 118 (1962). 10. WINTER, C. G., AND CHRISTENSEN, H. N. J. Biol. Chem. 240, 3594 (1965). 11. HILLMAN, R. E., ALBRECHT, I., AND ROSENBERG, L. E. J. Biol. Chem. 243, 5566 (1968). GIULIO LEVI Laboratorio di Chimica BioEogica Istituto Superiore di Sanitd and Laboratorio di Biologia Cellulare de1 CNR, Rome, Italy Received January 16, 1970; accepted February 10, 1970