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Developmental changes in the pattern of amino acid transport at the blood-brain barrier in rats
Developmental Brain Research, 6 (1983) 175-182 Elsevier Biomedical Press
175
Developmental Changes in the Pattern of Amino Acid Transport at the Blood-Brain Barrier in Rats JEANNE-MARIE LEFAUCONNIER and RENAUD TROUVI~ 1NSERM Unitd de Toxicologie Expdrimentale, H6pital Fernand Widal, 200 Rue du Faubourg Saint Denis, 75010 Paris (France)
(Accepted May 24th, 1982) Key words: amino acids - - biological transport - - blood-brain barrier - - development
Oldendorf's method has been widely used to estimate and characterize the transport of amino acids across the blood-brain barrier in rats. However, it cannot be used with very young animals. A modification of this method (retrograde injection into the right brachial artery, instead of orthograde injection into the common carotid artery) allowed the estimation of the brain uptake index of some amino acids in 5-, 12- and 19-day-old rats, as well as the study of self- and cross-inhibition and of sodium dependency. The results obtained showed that the pattern of transport of amino acids was different in 5-day-old and in 19-day-old rats. In young rats, besides the presence of the L-system, which transported large neutral amino acids as in adult rats, the presence of another system of transport for neutral amino acids was strongly suggested. The activity of this system which transported alanine, serine, cysteine and threonine, decreased during development and it had many of the characteristics of the ASC system described by Christensen 7. In addition, the presence of a system of transport for fl-amino acids at the blood-brain barrier is suggested. INTRODUCTION It has been k n o w n for some time, that the c o m p o sition o f the free a m i n o acid p o o l o f an i m m a t u r e b r a i n differed f r o m t h a t o f a m a t u r e b r a i n (most a m i n o acids are at a higher level in i m m a t u r e t h a n in m a t u r e brain, while some are at a lower level: a s p a r tate, glutamate, glutamine, G A B A ) . This has been shown in species ranging f r o m h u m a n s 14,2° to rats 1°, 19, m i c e l A 3 guinea pigs 19 a n d chickens 17. The differences between i m m a t u r e a n d m a t u r e b r a i n have been a t t r i b u t e d to several factors which can change as the m a t u r a t i o n p r o c e e d s : the activity o f synthesis o f b r a i n - g e n e r a t e d a m i n o acids, the influx o f a m i n o acids f r o m b l o o d to brain, the effiux f r o m b r a i n or C S F to b l o o d , the sink a c t i o n o f the C S F a n d the rate o f m e t a b o l i s m . F u r t h e r m o r e , a m i n o acids have been r e p o r t e d to enter m o r e readily into the b r a i n o f i m m a t u r e r a t h e r t h a n m a t u r e animals, when injected i.v. or i.p. at a high dose 11,12,29. H o w e v e r Seta et al. 29 have shown 0165-3806/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
that the greater increase in b r a i n content o f a m i n o acids in n e w b o r n mice as c o m p a r e d to a d u l t was n o t o f the same m a g n i t u d e for all a m i n o acids : the b r a i n c o n t e n t o f serine, threonine, t r y p t o p h a n , isoleucine was significantly increased after an i.p. injection o f cold a m i n o acids, while the injection o f a s p a r t a t e a n d g l u t a m a t e caused little change. Bafios et al. 5 have m e a s u r e d the influx o f several a m i n o acids f r o m the b l o o d to the b r a i n by m e a n s o f a p r o g r a m m e d infusion, m a i n t a i n i n g a steady conc e n t r a t i o n in p l a s m a , o f a labeled a m i n o acid. T h e y have shown t h a t between 1 a n d 10 weeks after birth, thele was a decrease in the influx o f all a m i n o acids tested except for glutamic acid. In this study, influx was m e a s u r e d u n d e r physiological c o n d i t i o n s a n d its rate d e p e n d e d b o t h on the p l a s m a c o n c e n t r a t i o n o f the a m i n o acid a n d on the characteristics o f the capillary t r a n s p o r t system. T h e c a r o t i d injection technique d e v e l o p e d by O l d e n d o r f zl allows an accurate study o f the t r a n s p o r t process f r o m b l o o d to brain, as it measures the influx o f substances inde-
176 pendently of their plasma concentration. Using this technique, Lorenzo and Gewirtz as reported that in rabbits, the brain uptake of tryptophan declined with age. In the same animal, Braun et al. 6 showed that the brain uptake of arginine was 6 times higher in newborns than in adults: in contrast, the brain uptake of glutamate and phenylalanine was the same both in newborns and in adults. An adaptation of Oldendorf's technique (intracardiac instead of intracarotid injection) allowed Purdy and Bondy % to perform a study of bloodbrain transport of proline and tyrosine during development of the chick, and Sershen and Lajtha ~7 to study the blood brain transport of a number of amino acids in the newborn rat. Sershen and Lajtha (ref. 27) showed that some amino acids (glutamate, aspartate, taurine, GABA) had a low and non-inhibitable uptake, while all others had a high and inhibitable uptake. From their results it appeared that the decrease in uptake between birth and adulthood was much higher for some amino acids (arginine, serine) than for others (leucine). To our knowledge, a comparison between the influx of various amino acids at several periods in development has not been made. Therefore, it seemed that such a study could furnish information on the evolution of the patterns of amino acid transport during development, it could eventually show a correlation between changes in amino acid transport and developmental events of the post-natal period and it could perhaps lead to a better understanding of the cause of the alterations in the free amino acid pool between immature and mature brain. To perform such a study, we developed a technique which allowed us to measure the transport from blood to brain of several amino acids in 5-, 12and 19-day-old rats. This technique, which we have already briefly described 16, is a modification of the carotid injection technique described by Oldendorf for adult rats ~. In Oldendorf's technique, a bolus of a buffered solution containing the 14C-labeled test amino acid and tritiated water as a diffusible reference, is injected into the common carotid artery of a nembutal anesthetized rat, which is decapitated 5-15 s later. A brain uptake index (BUI) is defined by Oldendorf as the ratio of disintegrations/min for the 14C to 3H in the tissue divided by the same ratio for the injected
solution and expressed as a percentage: 14("
.
m brain BUI
:
aH J4C
100 .
.
.
m mjectate aH This method is valid only if a free arterial flow past the needle persists throughout the procedure. In immature rats, the smallest needle almost completely occludes the carotid artery, so that the method could be used only with animals 19-21 days-old s.~:'. We modified this method by performing a retrograde injection into the right brachial artery. This resulted in a clear bolus in the carotid artery. In spitc of some limitations, which will be detailed, this nrethod gave us information about the changes in the amino acid transport processes during development.
MATERIAL
AND
METHODS
Anhnals" Mother rats with their offspring (10 rats per nursing mother) were obtained from lffa Credo (3 All6e des Platanes, 94260 Fresnes, France). On arrival, each litter was housed in a plastic cage and the mother was fed a commercial diet and tap water ad libitum. Animals were maintained in a temperature-controlled room with a constant cycle of 12 11 light, 12 h dark. Three rats from each litter were used for a transport study at 5 days, 3 at 12 days and 3 at 19 days of age. Blood-brain transport oj" radioactive sucrose and amino acids' This was studied using the following modification in the technique described by Oldendorf z~. An injection solution was prepared of 10 mM N-2-hydroxyethylpiperazine-N'-2 ethane sulfonate (HEPES) buffered to p H 7.40 and containing: (mEq/1): N a ' , 145; K +, 5; CI , 151; Ca z~, 3 and 5 mM glucose. Rats were anesthetized with ether ; the right brachial artery was exposed and cannulated with a 30-gauge needle (in 5- and 12-day-old rats) or a 26-gauge needle (in 19-day-old rats) under a stereomicroscope. The solution, containing 1/zCi/ml of the 14Clabeled test amino acid and 5 #Ci/ml of tritiated
177 water in a volume of 0.12, 0.23 or 0.28 ml according to the age, was injected for about 1 s into the brachial artery in the retrograde direction. The injectate passed in the axillary and subclavian arteries as a bolus in the retrograde direction and in the common carotid artery in the orthograde direction. The largest portion of the injected fluid was distributed to the cervical and thoracic branches of the axillary and subclavian arteries; however, a substantial fraction reached the bifurcation of the subclavian and carotid artery and could be seen as a clear bolus in the common carotid artery. The animals were decapitated at a certain time, depending upon their age: 20 s for 5-day-old animals, 15 s for 12-dayold and 10 s for 19-day-old animals. The brain was removed from the skull, the left hemisphere put into a closed vial and later weighed, and the right hemisphere was cut into 2 pieces. Each piece was digested in a scintillation vial in l ml of a tissue solubilizer: Soluene (Packard instruments) in a water bath at 55 °C. After cooling, a scintillation mixture containing 10 ml toluene, 40 mg PPO and 1 mg dimethyl POPOP was added before counting in an Intertechnique SL 3000 scintillation spectrophotometel. Counts/min were converted to disintegrations/min by using external standardization and predetermined efficiency curves; 10/~1 of the injected solution were similarly counted. The brain uptake index (BU1) was calculated according to Oldendorf as the ratio of disintegrations/min for the 14C and 3H in the brain, divided by the same ratio for the injected solution and expressed as a percentage. All labeled amino acids were injected at the highest specific activity readily available and at a concentration of l0 raM. In competition studies, the tracer amino acid under study was added to a buffered solution containing a 10 mM concentration of the competing amino acid. Isotonicity was maintained by adjusting the concentration of sodium chloride in the injected solution. In measures made in the absence of sodium, sodium chloride was replaced by D-mannitol in equivalent osmolarity. Labeled amino acids were obtained from Amersham, labeled sucrose from New England Nuclear, unlabeled amino acids from Sigma, HEPES buffer from Calbiochem and Soluene from Packard Instruments.
RESULTS
Preliminary studies As a preliminary to the use of the technique described under Material and Methods it seemed necessary to check: (i) that the injection in the right brachial artery produced a clear bolus in the right common artery; (ii) that this injection did not cause too large an alteration in carotid pressure; (iii) in addition, the volume of the injected bolus and the time elapsed between injection and sacrifice had to be determined for every period assayed. The passage of a clear bolus in the common carotid artery after injection in the right brachial artery could be ascertained by exposition of the carotid artery before the injection. Such a bolus could be observed in 49 out of 50 rats, 12-21 daysold. It was not possible to measure the carotid pressure in animals as young as those in our experiments. Therefore the assay was done with adult rats in the following manner : a needle was inserted in the external carotid artery and pushed to the bifurcation of the common carotid artery. It was then connected to a pressure transducer. Under these conditions, it was observed that the injection of 0.3 ml of buffer into the right brachial artery produced a clear bolus in the common carotid artery, but caused only a slight increase in carotid pressure (from 1 to 5 mm Hg). As the mean weight of the hemisphere rostral to midbrain was 175 mg at 5 days of age, 380 mg at 12 days and 480 mg at 19 days, the volume of the injectate had to be adjusted to each one. It was decided that the same radioactivity in tritium/g of brain wet weight would be given, regardless of the age of the rat (between 100 and 200.103 dpm/g wet weight). The same radioactivity per g was obtained when a 2 l-day-old rat was injected into the common carotid artery with 0.1 ml of a solution containing the same quantity of tritiated water/ml. This volume was 0.12 ml at 5 days of age, 0.23 at 12 days and 0.28 at 19 days. In the brachial injection, the fraction of the injectate which reached the hemisphere was between 1.5 and 3 ~o of the injectate. It varied more here than it did in the carotid injection. As a consequence, in a few cases, only a small amount of the injectate reached the cerebral circulation. In these
178 cases we observed t h a t for a m i n o acids with a BUI between 15 and 40 °/~,,, the BUI b e c a m e inversely correlated to the a m o u n t o f tritiated water which had reached brain. The minimal a m o u n t o f radioactivity in tritium to give a c o n s t a n t BUi for these substances was 100.103 d p m / g wet weight. Consequently, e x p e r i m e n t a l a n i m a l s with less r a d i o a c t i v i t y in their right hemisphere were excluded for the analysis o f the data. Because the cerebral circulation time was not k n o w n for these y o u n g rats, the delay between injection a n d d e c a p i t a t i o n was chosen a c c o r d i n g to which gave the lowest B U I 6 a n d the lowest s t a n d a r d deviation for a n o n - p e r m e a n t substance (sucrose). This came to 20 s at 5 days, 15 at 12 days and 10 at 19 days. B U I o f amino acids injected at tracer dose at 5, 12 and 19 days after birth
F o r reasons which will be m a d e clearer in the discussion, the BUIs o f the a m i n o acids can be comp a r e d only within the same time-period. Table I allows the BUIs o f a m i n o acids injected at tracer dose to be c o m p a r e d with the B U I o f sucrose. It can be seen that at 5 days after birth, 2 clusters o f a m i n o acids could be clearly differentiated: one in which a m i n o acids had a B U ! close to that o f sucrose: taurine, glutamic acid, proline, glycine; a n o t h e r in which a m i n o acids had much higher BUIs. The
latter included neutral a m i n o acids: alaninc, cysteine, serine, threonine, leucine, methioninc, phenylalanine, t r y p t o p h a n and the basic a m i n o acid: lysine. A t 19 days, the BUIs o f the a m i n o acids with a low BUI remained close to that o f sucrose. A m o n g other a m i n o acids, 3 groups could be differe n t i a t e d : (a) neutral a m i n o acids with a BUI higher than 40: methionine, leucine, phenylalaninc, tryptop h a n : (b) neutral a m i n o acids with a BUI c o m p r i s e d between I0 and 20: alanine, serine, cysteine, threonine; (c) the basic a m i n o acid: lysine with a BUI close to that o f the second group. SelJ:inhibition
This is shown in Table 11, where the BUIs o f the a m i n o acids at the 10 m M c o n c e n t r a t i o n are given as a percentage o f their B U i s at tracer concentration. II can be seen that, in our experiments, the BUIs o f glycine and glutamic acid were not significantly decreased by the a d d i t i o n o f cold a m i n o acid. In contrast, that o f taurine, which was very low, was still decreased by a small percentage by the a d d i t i o n o f cold taurine ( - - 3 0 ~;,). This decrease was statistically significant at all time-periods studied. The 1o~ BUI o f proline was also decreased by the a d d i t i o n o f cold a m i n o acid, b u t in 5-day-old animals only. The BUIs o f all other a m i n o acids were m a r k e d l y decreased by the a d d i t i o n o f a 10 m M c o n c e n t r a t i o n of cold a m i n o acid to the injectate.
TABLE l Brain uptake index of sucrose and some amino acids injected at tracer concentrations in 5-, 12- and 19-day-old rats
The values in the table are the BUls of the substances measured (as described under Material and Methods) ~ S.D. Number or" animals in parentheses, n.d., not determined. lnjected substance
lnjected concentration (raM)
5 day-old rats
12 t&y-old rats
19 day-old rat.s
Sucrose Taurine Glutamic acid Proline Glycine Alanine Cysteine Serine Threonine Methionine Leucine Phenylalanine Tryptophan Lysine
0.002 0.01 0.004 0.004 0.01 0.007 0.05 0.007 0.005 0.004 0.003 0.002 0.02 0.003
7.5 8.0 8.2 11.5 9.6 24.8 27.8 28.3 34.8 44.2 57.1 65.2 43.5 36.9
5.7 = 2.0 (5) 8.4 - 2.0 (4) 6.7 ~ 1.3 (3) 10.4 r~ 2.4 (8) 7.4 ~ 3.1 (4) 15.6 ± 0.8 (5) 22.2 ~ 4.0 (5) 17.8 ~= 1.8 (6) 29.6 ~ 2.3 (6) n.d. 48.3 ± 8.2 (6) 66.1 4 4.6 (6) n.d. 18.5 =~ 2.3 (7)
4.4 =~ 1.3 (6) 6.8 4 1.3 (8) 8.2 :~,=1.7 (71 6.8 ~: 1.4 (8) 6.4 ~ 1.6 (5) 10.5 ~ 0.6 (6) 19.1 =~: 2.2 (6) 11.4 :! 2.3 (5) 17.0 ± 2.2 (5) 39.6 2:5.2 (4) 53.6 ~ 5.5 (6) 61.9 ± 5.0 (61 43.9 :t 6.9 (6) 20.8 ~ 3.7 (6)
~ 2.3 (7) ~: 1.6 (6) :L 1.9 (7) -~ 1.1 (7) ~ 1.5 (4) :~ 6.2 (5) ~= 2.9 (5) ± 2.8 (6) ~: 3.5 (4) -~:: 5.0 (5) ~: 6.1 (5) :~= 10.0 (5) ~ 8.7 (5) t 4.8 (6)
179 TABLE II Modification o[ the BUI o f some amino acids by a 10 m M concentration o] cold amino acids
The values given are the ratios of the BUIs of the amino acids at 10 mM concentration to the BUIs at tracer concentration and expressed as a percentage. Student's t-test was used to calculate the statistical differences, n -- 4-8 animals in each group, n.d., not determined. Amino acids
Taurine Glutamic acid Proline Glycine Ala nine Cysteine Serine Threonine Methionine Leucine Phenylalanine Tryptophan Lysine
5 day-old rats
12 day-old rats
19 day-old rats
71 * n.d. 76"* 94 51 * 57* * 58** 39* ** 42"** 30*** 22*** 30* * 53***
70* 96 87 89 68" 74* 67** 36* ** n.d. 29*** 29*** n.d. 46***
68** 82 78 84 75 * * n.d. 59*** 45"** 35 § 21"** 16"** 23 *** 54***
c o n c e n t r a t i o n in the absence of competitors. Crossi n h i b i t i o n has not been tested for a m i n o acids showing no or little self-inhibition. It can be seen that a-(methylamino)-isobutyric acid (meAIB), a typical substrate for the A transport system described by Christensen 7, caused no significant i n h i b i t i o n of t r a n s p o r t of any a m i n o acid. A l a n i n e a n d serine, both inhibited by a b o u t 50 ~ the t r a n s p o r t of alanine, serine a n d threonine, but n o t that of leucine. Leucine inhibited by 20-50 ~o the t r a n s p o r t of alanine, serine a n d threonine, which was much less than its self-inhibition. P h e n y l a l a n i n e (not shown in Table III) inhibited the t r a n s p o r t of leucine by 70 ~ . Lysine a n d arginine only inhibited the t r a n s p o r t of lysine. S o d i u m dependency
It has been tested only on leucine a n d alanine t r a n s p o r t in 5-day-old rats. The B U I of leucine was n o t altered by substituting m a n n i t o l for sodium in the injectate. I n contrast, the B U I of alanine was decreased by 34 ~ (P < 0.001).
§ t-test not done (n -- 4 and 2). * P < 0.05. ** P,<0.01.
DISCUSSION
*** P < 0.001.
10 m M c o n c e n t r a t i o n are given for 5-day-old rats in
O u r experiments have been performed in rats from 5 to 19 days of age. D u r i n g this period of time, due to the development a n d m a t u r a t i o n o f the vascular network, changes in cerebral b l o o d flow a n d water permeability were expected. The B U ! is
Table III, as a percentage of the BUIs at tracer
equal to the ratio of the fractional extraction of the
Cross-inhibition
The BUIs of a few a m i n o acids at tracer concent r a t i o n in presence of some other a m i n o acids at
TABLE III Modification of the brain uptake index of some amino acids by a 10 m M concentration o f the same or of another amino acid in 5-dayold rats
The values given are the BUIs of the amino acids in the presence of a 10 mM concentration of the same or of another amino acid and expressed as a percentage of the BUIs at tracer concentration in the absence of competitors. Student's t-test was used to calculate the statistical differences, n ~ 4-8 animals in each group, n.d., not determined. Labeled amino acids at tracer concentration
Alanine Serine Threonine Leucine Lysine * P < 0.05.
** P < 0.01. *** P < 0.001.
Cold amino acids at 10 m M concentration meAIB
Alanine
Serine
Threonine
Leucine
Lysine
Arginine
85 88 n.d. 92 n,d.
51 ** 68* 57** 100 81
56"* 54** 48** 98 92
n.d. n.d. 32** n.d. n.d.
79 69* 47** 30"** 113
100 110 115 n.d. 33***
n.d. n.d. n.d. 99 42***
180 amino acid to the fractional extraction of watereL Thus, like the fractional extraction, it is dependent on blood flow :~. Consequently, it may not be valid to compare the BUIs obtained at different periods of development for the same amino acid. However, at each time-period, cerebral blood flow and water permeability were probably close for all animals, as t he conditions of experiments were the same. It is thus possible to compare, within the same time-period, the BUIs of different amino acids and to compare them with the BUI of sucrose, which is very low in adult animals el. The pattern of amino acid transport at the bloodbrain barrier has been extensively studied in adult animals. Richter and Wainer e6 have shown that there were separate systems for the transport of large neutral and basic amino acids. This finding has been confirmed by several authors using various methods a,4,'~e. The system of transport for large neutral amino acids has been identified as the L-system of transport described by Christensen a°,a2. Later Oldendorf and Szabo '~a, using a third radioactive isotope in the injected bolus to correct for incomplete washout of the bolus from the brain blood compartment, demonstrated the existence of a third system for acidic amino acids. Recently, Sershen and Lajtha es have given arguments for the existence at the blood-brain barrier, of the ASC system described by ChristensenV; this has been challenged, at least as far as cysteine is concerned, by Wade and Brady ai. Our results show several features which suggest that the immature pattern of transport of amino acids across the blood-brain barrier differs from the adult. At every time-period, 2 amino acids: glycine and glutamic acid had a low BU1 close to that of sucrose. This result agrees with that of Sershen and Lajtha 27 in newborn rats and of Braun et al. 6 in newborn rabbits. This low BUI was not inhibitable by the addition of cold amino acid. This is not in agreement with the findings of Oldendorf and Szabo 2a which demonstrated in adult rats the existence of a carrier-mediated transport of glutamic acid. As we have not used a third tracer to correct for intravascular radioactivity, our method is perhaps not sensitive enough to show the presence of this carrier mediated transport. The other possible explanation
for this discrepancy is that tile appearance oi" ti~is transport system is delayed during the development. The BUI of taurinc which was very low could bc lowered still more by the addition of cold amino acid. This suggests the existence of a carrier mediated transport system for /]-amino acids at the blood-brain barrier. Proline also had a very low BUI which could, in 5day-old rats, be decreased still more by the addition of cold amino acid: this suggests the presence in young animals of a carrier mediated transport whose activity later decreased. The pattern of transport of other neutral amino acids changed between the ages of 5 and 19 days. At 5 days there was a continuity in the values of the BUIs from 25 for alanine to 65 for phenylalanine. At 19 days, 2 different groups could be distinguished: the first, made up of methionine, leucine, phenylalanine, and tryptophan with BUIs comprised between 40 and 60, the second, which included alanine, serine, cysteine and threonine with BUIs comprised between 10 and 20. If all neutral amino acids were transported by the same system, it should be expected that the ratio of the BUIs of 2 of them be constant over the whole age course (unless the K,,, of the mediated transport was altered differently during the development according to the amino acid, which seemed unlikely). The ratio of the BUI of one amino acid of the first group to another one, e.g. phenylalanine, was in fact constant throughout the development. For leucine this was 0.85 at 5 days and 0.87 at 19 days: for tryptophan, 0.67 and 0.70: for methionine, 0.68 and 0.64. On the other hand, the ratio of the BUI of any amino acid of the second group to that of phenylalanine decreased markedly during development: alanine, 38 to 17; cysteine, 43 to 31; serine, 43 to 18: threonine, 59 to 27. This group of amino acids was much less homogeneous than the first one as there was no constant ratio in the BUIs of 2 amino acids of this group. Competition experiments showed that the transport of leucine was inhibited only by an amino acid of the same group, while that of alanine, serinc or threonine was also inhibited but to a lesser extent by an amino acid of the other group. Finally no sodium dependency was shown for the transport of leucine, while a substantial one was shown for the transport of alanine.
181 All these results strongly suggest that at least two systems of transport of neutral amino acids exist during development. The first, made up of phenylalanine, leucine, tryptophan and methionine, is characterized by: (1) a high BUI at every timeperiod of development; (2) the absence of inhibition by the methylated amino acids; (3) the absence of inhibition by the amino acids of the other group; (4) a sodium independency. Therefore, it is similar to the L-system of transport described in adult rats2a,30, ae. The second group includes: alanine, serine, cysteine, threonine and perhaps proline. It is characterized by: (1) a significant decrease in activity during the development as compared to the first group ; (2) the absence of statistically significant inhibition by the methylated amino acids; (3) the presence of inhibition via amino acids of the same group, likewise, but to a lesser extent via amino acids of the other group; (4) a substantial decrease in activity in the absence of sodium. It is thus very tempting to attribute the transport of the amino acids of this group to the ASC system of Christensen (ref. 7) with which it shares many of the characteristics. However, the transport of cysteine and serine in adult animals has been attributed to the carrier system transporting phenylalanine and other large neutral amino acids by Oldendorf and Szabo ~3. That of cysteine has also been attributed to the L-system by Wade and Brady at, while Sershen and Lajtha zs have credited the transport of serine and threonine to the ASC system. We have observed that during development, the ratios of the BUIs of 2 amino acids of the second group were not constant. Therefore, it is possible that some of these amino acids are transREFERENCES 1 AgrawaI, H. C., Davis, J. M. and Himwich, W. A., Developmental changes in mouse brain: weight, water content and free amino acids, J. Neurochem., 15 (1968) 917-923. 2 Antonioli, J. A. and Christensen, H. N., Differences in schedules of regression of transport systems during reticuIocytes maturation, J. biol. Chem., 244 (1969) 15051509. 3 Bafios, G., Daniel, P. M., Moormouse, S. R. and Pratt, O. E., The influx of amino acids into the brain of the rat in vivo: the essential compared with some non-essential amino acids, Proc. roy. Soc. B, 183 (1973) 5%70. 4 Bafios, G., Daniel, P. M. and Pratt, O. E., Saturation of a shared mechanism which transports L-arginine and L-
ported by 2 different systems, as is nearly always the rule in most cells s. The predominant system would depend on age and perhaps on some other unknown factors. More detailed studies are thus needed to delineate better the role of each system in the transport of the amino acids we have placed in the second group. The basic amino acid lysine was transported, as in adult animals, by the basic amino acid transport system alone as its transport was inhibited by no other amino acid except arginine. However, the activity of its transport decreased in relationship to phenylalanine between day 5 and 19 after birth. This agrees with the result of Braun et al. 6 which showed that in rabbits, the BU1 of arginine was 6 times higher in newborn than in adult animals. The changes observed during development at the blood-brain barrier bear some similarity to those observed in the maturation of the reticulocyte of the rabbit. During that maturation Antonioli and Christensen 2 have shown that the transport of alanine and lysine decreased, as we observed at the blood-brain barrier. The decrease in transport of some amino acids during development can contribute, probably along with other factors, to the decrease in the brain content of some amino acids, at least those of the second group, as maturation proceeds. ACKNOWLEDGEMENT This study was supported by Contract 56-78-88 of the Institut National de la Sant6 et de la Recherche M6dicale.
lysine into the brain of the living rat, J. Physiol. (Lond.), 236 (1974) 29-41. 5 Bafios, G., Daniel, P. M. and Pratt, O. E., The effect of age upon the entry of some amino acids into the brain, and their incorporation into cerebral proteins, Develop. Med. Child Neurol., 20 (1978) 335-346. 6 Braun, L. D., Cornford, E. M. and Oldendorf, W. H., Newborn rabbit blood-brain barrier is selectively permeable and differs substantially from the adult, J. Neurochem., 34 (1980) 147-152. 7 Christensen, H. N., Recognition sites for material transport and information transfer. In A. Kleinzellzer and F. Bronner (Eds.), Current Topics in Membranes and Transport, Vol, 6, Academic Press, NY, 1975, pp. 227-258. 8 Cremer, J. E., Braun, L. D. and Oldendorf, W. H.,
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18
19
(hanges during development in transport processes of the Nood brain barrier, Biochim. biop/~ys. Acta, 448 (1976) 633 637. Crone, C., The permeability of capillaries in various organs as determined by use of the 'Indicator Diffusion" method, Acta physiol, stand., 58 (1963) 292 305. Fando, J. L., Dominguez, F. and Herrera, E., Tryptophan overload in the pregnant rat: effect on brain amino acids levels and in vitro protein synthesis, J. Neurochem., 37 (19813 824-829. Himwich, W. A., Petersen, J. C. and Allen, M. L., Hematoencephalic exchange as a function of age, Neurology, 7 (19573 705-710. Kutmer, R., Sims, J. A. and Gordon, M. W., The uptake of a metabolically inert amino acid by brain and other organs, J. Neurochem., 6 (1961) 311-317. kajtha, A. and Toth, J., Perinatal changes in the free amino acid pool of the brain in mice, Brain Res., 55 (19733 238-241. Lefauconnier, J. M., Portemer, C. and Chatagner, F., Free amino acids and related substances in human glial tumours and in fetal brain: comparison with normal adult brain, Brain Res., 117 (19763 105 113. Lefauconnier, J. M., Lavielle, E., Terrien, N., Bernard, G. and Fournier, E., Effect of various lead doses on some cerebral capillary functions in the suckling rat, Toxicol. appl. Pharmacol., 55 (19803 467 476. Lefauconnier, J. M. and Trouve, R., Comparaison du transport hdmatoencdphalique de divers acides aminds chez le rat 5, 12 et 21 jours apr6s la naissance, C.R. Acad. Sci. (Paris), 293 (19813 821-824. Levi, G. and Morisi, G., Free amino acids and related compounds in chick brain during development, Brain Res., 26 (19713 131-140. Lorenzo, A. V. and Gewirtz, M., Inhibition of [iqC] tryptophan transport into brain of lead exposed neonatal rabbits, Brain Res., 132 (19773 386-392. Oja, S. S., Uusitalo, A. J., Vahvelainen, M. L. and Piha, R. S., Changes in cerebral and hepatic amino acids in the rat and guinea pig during development, Brain Res., 11 (19683 655 661.
20 Okumura, N., Otsuki, S. and Kameyama, A,, Studies ~n free amino acids in human brain, d. Biochem., 47 (1960} 315-320. 21 Oldendorf, W. H., Measurement of brain uptake of radiolabeled substances using a tritiated water internal standard, Brain Res., 24 (19703 372 376. 22 Oldendorf, W. H., Brain uptake of radiolaheled amino acids, amines, and hexoses after arterial injection, Amer. J. Physiol., 221 (1971) 1629 1639. 23 Oldendorf, W. H. and Szabo, J,, Amino acid assignment to one of three blood-brain barrier amino acid carriers, Amer. J. Physiol., 230 (1976) 94-98. 24 Pardridge, W. M. and Oldendorf, W. H., Kinetic analysis of blood-brain barrier transport of amino acids, Biochim. biophys. Acta, 401 (19753 128-136. 25 Purdy, J. L. and Bondy, S. C., Blood--brain barrier: selective changes during maturation, Neuroscience, 1 (1976) 125-129. 26 Richter, J. J. and Wainer, A., Evidence for separate system for the transport of neutral and basic amino acids across the blood brain barrier, J. Neurochem., 18 (19713 613-620. 27 Sershen, H. and Lajtha, A., Capillary transport of amino acids in the developing brain, Exp. Neurol., 53 (1976) 465-474. 28 Sershen, H. and Lajtha, A., Inhibition pattern by analogs indicates the presence of ten or more transport systems for amino acids in brain cells, J. Neurochem., 32 (19793 719-726. 29 Seta, K., Sershen, H. and Lajtha, A., Cerebral amino acid uptake in vivo in newborn mice, Brain Res., 47 (19723 415425. 30 Wade, L. A. and Katzman, R., Synthetic amino acids and the nature of L-dopa transport at the blood brain barrier, J. Neurochem., 25 (1975) 837-842. 31 Wade, L A. and Brady, H. M., Cysteine and cystine transport at the blood-brain barrier, J. Neurochem., 37 (1981) 730-734. 32 Yudilevitch, D. L., De Rose, N. and Sepulveda, E. V., Facilitated transport of amino acids through the bloodbrain barrier of the dog studied in a single capillary circulation, Brain Res., 44 (1972) 569-578.
175
Developmental Changes in the Pattern of Amino Acid Transport at the Blood-Brain Barrier in Rats JEANNE-MARIE LEFAUCONNIER and RENAUD TROUVI~ 1NSERM Unitd de Toxicologie Expdrimentale, H6pital Fernand Widal, 200 Rue du Faubourg Saint Denis, 75010 Paris (France)
(Accepted May 24th, 1982) Key words: amino acids - - biological transport - - blood-brain barrier - - development
Oldendorf's method has been widely used to estimate and characterize the transport of amino acids across the blood-brain barrier in rats. However, it cannot be used with very young animals. A modification of this method (retrograde injection into the right brachial artery, instead of orthograde injection into the common carotid artery) allowed the estimation of the brain uptake index of some amino acids in 5-, 12- and 19-day-old rats, as well as the study of self- and cross-inhibition and of sodium dependency. The results obtained showed that the pattern of transport of amino acids was different in 5-day-old and in 19-day-old rats. In young rats, besides the presence of the L-system, which transported large neutral amino acids as in adult rats, the presence of another system of transport for neutral amino acids was strongly suggested. The activity of this system which transported alanine, serine, cysteine and threonine, decreased during development and it had many of the characteristics of the ASC system described by Christensen 7. In addition, the presence of a system of transport for fl-amino acids at the blood-brain barrier is suggested. INTRODUCTION It has been k n o w n for some time, that the c o m p o sition o f the free a m i n o acid p o o l o f an i m m a t u r e b r a i n differed f r o m t h a t o f a m a t u r e b r a i n (most a m i n o acids are at a higher level in i m m a t u r e t h a n in m a t u r e brain, while some are at a lower level: a s p a r tate, glutamate, glutamine, G A B A ) . This has been shown in species ranging f r o m h u m a n s 14,2° to rats 1°, 19, m i c e l A 3 guinea pigs 19 a n d chickens 17. The differences between i m m a t u r e a n d m a t u r e b r a i n have been a t t r i b u t e d to several factors which can change as the m a t u r a t i o n p r o c e e d s : the activity o f synthesis o f b r a i n - g e n e r a t e d a m i n o acids, the influx o f a m i n o acids f r o m b l o o d to brain, the effiux f r o m b r a i n or C S F to b l o o d , the sink a c t i o n o f the C S F a n d the rate o f m e t a b o l i s m . F u r t h e r m o r e , a m i n o acids have been r e p o r t e d to enter m o r e readily into the b r a i n o f i m m a t u r e r a t h e r t h a n m a t u r e animals, when injected i.v. or i.p. at a high dose 11,12,29. H o w e v e r Seta et al. 29 have shown 0165-3806/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
that the greater increase in b r a i n content o f a m i n o acids in n e w b o r n mice as c o m p a r e d to a d u l t was n o t o f the same m a g n i t u d e for all a m i n o acids : the b r a i n c o n t e n t o f serine, threonine, t r y p t o p h a n , isoleucine was significantly increased after an i.p. injection o f cold a m i n o acids, while the injection o f a s p a r t a t e a n d g l u t a m a t e caused little change. Bafios et al. 5 have m e a s u r e d the influx o f several a m i n o acids f r o m the b l o o d to the b r a i n by m e a n s o f a p r o g r a m m e d infusion, m a i n t a i n i n g a steady conc e n t r a t i o n in p l a s m a , o f a labeled a m i n o acid. T h e y have shown t h a t between 1 a n d 10 weeks after birth, thele was a decrease in the influx o f all a m i n o acids tested except for glutamic acid. In this study, influx was m e a s u r e d u n d e r physiological c o n d i t i o n s a n d its rate d e p e n d e d b o t h on the p l a s m a c o n c e n t r a t i o n o f the a m i n o acid a n d on the characteristics o f the capillary t r a n s p o r t system. T h e c a r o t i d injection technique d e v e l o p e d by O l d e n d o r f zl allows an accurate study o f the t r a n s p o r t process f r o m b l o o d to brain, as it measures the influx o f substances inde-
176 pendently of their plasma concentration. Using this technique, Lorenzo and Gewirtz as reported that in rabbits, the brain uptake of tryptophan declined with age. In the same animal, Braun et al. 6 showed that the brain uptake of arginine was 6 times higher in newborns than in adults: in contrast, the brain uptake of glutamate and phenylalanine was the same both in newborns and in adults. An adaptation of Oldendorf's technique (intracardiac instead of intracarotid injection) allowed Purdy and Bondy % to perform a study of bloodbrain transport of proline and tyrosine during development of the chick, and Sershen and Lajtha ~7 to study the blood brain transport of a number of amino acids in the newborn rat. Sershen and Lajtha (ref. 27) showed that some amino acids (glutamate, aspartate, taurine, GABA) had a low and non-inhibitable uptake, while all others had a high and inhibitable uptake. From their results it appeared that the decrease in uptake between birth and adulthood was much higher for some amino acids (arginine, serine) than for others (leucine). To our knowledge, a comparison between the influx of various amino acids at several periods in development has not been made. Therefore, it seemed that such a study could furnish information on the evolution of the patterns of amino acid transport during development, it could eventually show a correlation between changes in amino acid transport and developmental events of the post-natal period and it could perhaps lead to a better understanding of the cause of the alterations in the free amino acid pool between immature and mature brain. To perform such a study, we developed a technique which allowed us to measure the transport from blood to brain of several amino acids in 5-, 12and 19-day-old rats. This technique, which we have already briefly described 16, is a modification of the carotid injection technique described by Oldendorf for adult rats ~. In Oldendorf's technique, a bolus of a buffered solution containing the 14C-labeled test amino acid and tritiated water as a diffusible reference, is injected into the common carotid artery of a nembutal anesthetized rat, which is decapitated 5-15 s later. A brain uptake index (BUI) is defined by Oldendorf as the ratio of disintegrations/min for the 14C to 3H in the tissue divided by the same ratio for the injected
solution and expressed as a percentage: 14("
.
m brain BUI
:
aH J4C
100 .
.
.
m mjectate aH This method is valid only if a free arterial flow past the needle persists throughout the procedure. In immature rats, the smallest needle almost completely occludes the carotid artery, so that the method could be used only with animals 19-21 days-old s.~:'. We modified this method by performing a retrograde injection into the right brachial artery. This resulted in a clear bolus in the carotid artery. In spitc of some limitations, which will be detailed, this nrethod gave us information about the changes in the amino acid transport processes during development.
MATERIAL
AND
METHODS
Anhnals" Mother rats with their offspring (10 rats per nursing mother) were obtained from lffa Credo (3 All6e des Platanes, 94260 Fresnes, France). On arrival, each litter was housed in a plastic cage and the mother was fed a commercial diet and tap water ad libitum. Animals were maintained in a temperature-controlled room with a constant cycle of 12 11 light, 12 h dark. Three rats from each litter were used for a transport study at 5 days, 3 at 12 days and 3 at 19 days of age. Blood-brain transport oj" radioactive sucrose and amino acids' This was studied using the following modification in the technique described by Oldendorf z~. An injection solution was prepared of 10 mM N-2-hydroxyethylpiperazine-N'-2 ethane sulfonate (HEPES) buffered to p H 7.40 and containing: (mEq/1): N a ' , 145; K +, 5; CI , 151; Ca z~, 3 and 5 mM glucose. Rats were anesthetized with ether ; the right brachial artery was exposed and cannulated with a 30-gauge needle (in 5- and 12-day-old rats) or a 26-gauge needle (in 19-day-old rats) under a stereomicroscope. The solution, containing 1/zCi/ml of the 14Clabeled test amino acid and 5 #Ci/ml of tritiated
177 water in a volume of 0.12, 0.23 or 0.28 ml according to the age, was injected for about 1 s into the brachial artery in the retrograde direction. The injectate passed in the axillary and subclavian arteries as a bolus in the retrograde direction and in the common carotid artery in the orthograde direction. The largest portion of the injected fluid was distributed to the cervical and thoracic branches of the axillary and subclavian arteries; however, a substantial fraction reached the bifurcation of the subclavian and carotid artery and could be seen as a clear bolus in the common carotid artery. The animals were decapitated at a certain time, depending upon their age: 20 s for 5-day-old animals, 15 s for 12-dayold and 10 s for 19-day-old animals. The brain was removed from the skull, the left hemisphere put into a closed vial and later weighed, and the right hemisphere was cut into 2 pieces. Each piece was digested in a scintillation vial in l ml of a tissue solubilizer: Soluene (Packard instruments) in a water bath at 55 °C. After cooling, a scintillation mixture containing 10 ml toluene, 40 mg PPO and 1 mg dimethyl POPOP was added before counting in an Intertechnique SL 3000 scintillation spectrophotometel. Counts/min were converted to disintegrations/min by using external standardization and predetermined efficiency curves; 10/~1 of the injected solution were similarly counted. The brain uptake index (BU1) was calculated according to Oldendorf as the ratio of disintegrations/min for the 14C and 3H in the brain, divided by the same ratio for the injected solution and expressed as a percentage. All labeled amino acids were injected at the highest specific activity readily available and at a concentration of l0 raM. In competition studies, the tracer amino acid under study was added to a buffered solution containing a 10 mM concentration of the competing amino acid. Isotonicity was maintained by adjusting the concentration of sodium chloride in the injected solution. In measures made in the absence of sodium, sodium chloride was replaced by D-mannitol in equivalent osmolarity. Labeled amino acids were obtained from Amersham, labeled sucrose from New England Nuclear, unlabeled amino acids from Sigma, HEPES buffer from Calbiochem and Soluene from Packard Instruments.
RESULTS
Preliminary studies As a preliminary to the use of the technique described under Material and Methods it seemed necessary to check: (i) that the injection in the right brachial artery produced a clear bolus in the right common artery; (ii) that this injection did not cause too large an alteration in carotid pressure; (iii) in addition, the volume of the injected bolus and the time elapsed between injection and sacrifice had to be determined for every period assayed. The passage of a clear bolus in the common carotid artery after injection in the right brachial artery could be ascertained by exposition of the carotid artery before the injection. Such a bolus could be observed in 49 out of 50 rats, 12-21 daysold. It was not possible to measure the carotid pressure in animals as young as those in our experiments. Therefore the assay was done with adult rats in the following manner : a needle was inserted in the external carotid artery and pushed to the bifurcation of the common carotid artery. It was then connected to a pressure transducer. Under these conditions, it was observed that the injection of 0.3 ml of buffer into the right brachial artery produced a clear bolus in the common carotid artery, but caused only a slight increase in carotid pressure (from 1 to 5 mm Hg). As the mean weight of the hemisphere rostral to midbrain was 175 mg at 5 days of age, 380 mg at 12 days and 480 mg at 19 days, the volume of the injectate had to be adjusted to each one. It was decided that the same radioactivity in tritium/g of brain wet weight would be given, regardless of the age of the rat (between 100 and 200.103 dpm/g wet weight). The same radioactivity per g was obtained when a 2 l-day-old rat was injected into the common carotid artery with 0.1 ml of a solution containing the same quantity of tritiated water/ml. This volume was 0.12 ml at 5 days of age, 0.23 at 12 days and 0.28 at 19 days. In the brachial injection, the fraction of the injectate which reached the hemisphere was between 1.5 and 3 ~o of the injectate. It varied more here than it did in the carotid injection. As a consequence, in a few cases, only a small amount of the injectate reached the cerebral circulation. In these
178 cases we observed t h a t for a m i n o acids with a BUI between 15 and 40 °/~,,, the BUI b e c a m e inversely correlated to the a m o u n t o f tritiated water which had reached brain. The minimal a m o u n t o f radioactivity in tritium to give a c o n s t a n t BUi for these substances was 100.103 d p m / g wet weight. Consequently, e x p e r i m e n t a l a n i m a l s with less r a d i o a c t i v i t y in their right hemisphere were excluded for the analysis o f the data. Because the cerebral circulation time was not k n o w n for these y o u n g rats, the delay between injection a n d d e c a p i t a t i o n was chosen a c c o r d i n g to which gave the lowest B U I 6 a n d the lowest s t a n d a r d deviation for a n o n - p e r m e a n t substance (sucrose). This came to 20 s at 5 days, 15 at 12 days and 10 at 19 days. B U I o f amino acids injected at tracer dose at 5, 12 and 19 days after birth
F o r reasons which will be m a d e clearer in the discussion, the BUIs o f the a m i n o acids can be comp a r e d only within the same time-period. Table I allows the BUIs o f a m i n o acids injected at tracer dose to be c o m p a r e d with the B U I o f sucrose. It can be seen that at 5 days after birth, 2 clusters o f a m i n o acids could be clearly differentiated: one in which a m i n o acids had a B U ! close to that o f sucrose: taurine, glutamic acid, proline, glycine; a n o t h e r in which a m i n o acids had much higher BUIs. The
latter included neutral a m i n o acids: alaninc, cysteine, serine, threonine, leucine, methioninc, phenylalanine, t r y p t o p h a n and the basic a m i n o acid: lysine. A t 19 days, the BUIs o f the a m i n o acids with a low BUI remained close to that o f sucrose. A m o n g other a m i n o acids, 3 groups could be differe n t i a t e d : (a) neutral a m i n o acids with a BUI higher than 40: methionine, leucine, phenylalaninc, tryptop h a n : (b) neutral a m i n o acids with a BUI c o m p r i s e d between I0 and 20: alanine, serine, cysteine, threonine; (c) the basic a m i n o acid: lysine with a BUI close to that o f the second group. SelJ:inhibition
This is shown in Table 11, where the BUIs o f the a m i n o acids at the 10 m M c o n c e n t r a t i o n are given as a percentage o f their B U i s at tracer concentration. II can be seen that, in our experiments, the BUIs o f glycine and glutamic acid were not significantly decreased by the a d d i t i o n o f cold a m i n o acid. In contrast, that o f taurine, which was very low, was still decreased by a small percentage by the a d d i t i o n o f cold taurine ( - - 3 0 ~;,). This decrease was statistically significant at all time-periods studied. The 1o~ BUI o f proline was also decreased by the a d d i t i o n o f cold a m i n o acid, b u t in 5-day-old animals only. The BUIs o f all other a m i n o acids were m a r k e d l y decreased by the a d d i t i o n o f a 10 m M c o n c e n t r a t i o n of cold a m i n o acid to the injectate.
TABLE l Brain uptake index of sucrose and some amino acids injected at tracer concentrations in 5-, 12- and 19-day-old rats
The values in the table are the BUls of the substances measured (as described under Material and Methods) ~ S.D. Number or" animals in parentheses, n.d., not determined. lnjected substance
lnjected concentration (raM)
5 day-old rats
12 t&y-old rats
19 day-old rat.s
Sucrose Taurine Glutamic acid Proline Glycine Alanine Cysteine Serine Threonine Methionine Leucine Phenylalanine Tryptophan Lysine
0.002 0.01 0.004 0.004 0.01 0.007 0.05 0.007 0.005 0.004 0.003 0.002 0.02 0.003
7.5 8.0 8.2 11.5 9.6 24.8 27.8 28.3 34.8 44.2 57.1 65.2 43.5 36.9
5.7 = 2.0 (5) 8.4 - 2.0 (4) 6.7 ~ 1.3 (3) 10.4 r~ 2.4 (8) 7.4 ~ 3.1 (4) 15.6 ± 0.8 (5) 22.2 ~ 4.0 (5) 17.8 ~= 1.8 (6) 29.6 ~ 2.3 (6) n.d. 48.3 ± 8.2 (6) 66.1 4 4.6 (6) n.d. 18.5 =~ 2.3 (7)
4.4 =~ 1.3 (6) 6.8 4 1.3 (8) 8.2 :~,=1.7 (71 6.8 ~: 1.4 (8) 6.4 ~ 1.6 (5) 10.5 ~ 0.6 (6) 19.1 =~: 2.2 (6) 11.4 :! 2.3 (5) 17.0 ± 2.2 (5) 39.6 2:5.2 (4) 53.6 ~ 5.5 (6) 61.9 ± 5.0 (61 43.9 :t 6.9 (6) 20.8 ~ 3.7 (6)
~ 2.3 (7) ~: 1.6 (6) :L 1.9 (7) -~ 1.1 (7) ~ 1.5 (4) :~ 6.2 (5) ~= 2.9 (5) ± 2.8 (6) ~: 3.5 (4) -~:: 5.0 (5) ~: 6.1 (5) :~= 10.0 (5) ~ 8.7 (5) t 4.8 (6)
179 TABLE II Modification o[ the BUI o f some amino acids by a 10 m M concentration o] cold amino acids
The values given are the ratios of the BUIs of the amino acids at 10 mM concentration to the BUIs at tracer concentration and expressed as a percentage. Student's t-test was used to calculate the statistical differences, n -- 4-8 animals in each group, n.d., not determined. Amino acids
Taurine Glutamic acid Proline Glycine Ala nine Cysteine Serine Threonine Methionine Leucine Phenylalanine Tryptophan Lysine
5 day-old rats
12 day-old rats
19 day-old rats
71 * n.d. 76"* 94 51 * 57* * 58** 39* ** 42"** 30*** 22*** 30* * 53***
70* 96 87 89 68" 74* 67** 36* ** n.d. 29*** 29*** n.d. 46***
68** 82 78 84 75 * * n.d. 59*** 45"** 35 § 21"** 16"** 23 *** 54***
c o n c e n t r a t i o n in the absence of competitors. Crossi n h i b i t i o n has not been tested for a m i n o acids showing no or little self-inhibition. It can be seen that a-(methylamino)-isobutyric acid (meAIB), a typical substrate for the A transport system described by Christensen 7, caused no significant i n h i b i t i o n of t r a n s p o r t of any a m i n o acid. A l a n i n e a n d serine, both inhibited by a b o u t 50 ~ the t r a n s p o r t of alanine, serine a n d threonine, but n o t that of leucine. Leucine inhibited by 20-50 ~o the t r a n s p o r t of alanine, serine a n d threonine, which was much less than its self-inhibition. P h e n y l a l a n i n e (not shown in Table III) inhibited the t r a n s p o r t of leucine by 70 ~ . Lysine a n d arginine only inhibited the t r a n s p o r t of lysine. S o d i u m dependency
It has been tested only on leucine a n d alanine t r a n s p o r t in 5-day-old rats. The B U I of leucine was n o t altered by substituting m a n n i t o l for sodium in the injectate. I n contrast, the B U I of alanine was decreased by 34 ~ (P < 0.001).
§ t-test not done (n -- 4 and 2). * P < 0.05. ** P,<0.01.
DISCUSSION
*** P < 0.001.
10 m M c o n c e n t r a t i o n are given for 5-day-old rats in
O u r experiments have been performed in rats from 5 to 19 days of age. D u r i n g this period of time, due to the development a n d m a t u r a t i o n o f the vascular network, changes in cerebral b l o o d flow a n d water permeability were expected. The B U ! is
Table III, as a percentage of the BUIs at tracer
equal to the ratio of the fractional extraction of the
Cross-inhibition
The BUIs of a few a m i n o acids at tracer concent r a t i o n in presence of some other a m i n o acids at
TABLE III Modification of the brain uptake index of some amino acids by a 10 m M concentration o f the same or of another amino acid in 5-dayold rats
The values given are the BUIs of the amino acids in the presence of a 10 mM concentration of the same or of another amino acid and expressed as a percentage of the BUIs at tracer concentration in the absence of competitors. Student's t-test was used to calculate the statistical differences, n ~ 4-8 animals in each group, n.d., not determined. Labeled amino acids at tracer concentration
Alanine Serine Threonine Leucine Lysine * P < 0.05.
** P < 0.01. *** P < 0.001.
Cold amino acids at 10 m M concentration meAIB
Alanine
Serine
Threonine
Leucine
Lysine
Arginine
85 88 n.d. 92 n,d.
51 ** 68* 57** 100 81
56"* 54** 48** 98 92
n.d. n.d. 32** n.d. n.d.
79 69* 47** 30"** 113
100 110 115 n.d. 33***
n.d. n.d. n.d. 99 42***
180 amino acid to the fractional extraction of watereL Thus, like the fractional extraction, it is dependent on blood flow :~. Consequently, it may not be valid to compare the BUIs obtained at different periods of development for the same amino acid. However, at each time-period, cerebral blood flow and water permeability were probably close for all animals, as t he conditions of experiments were the same. It is thus possible to compare, within the same time-period, the BUIs of different amino acids and to compare them with the BUI of sucrose, which is very low in adult animals el. The pattern of amino acid transport at the bloodbrain barrier has been extensively studied in adult animals. Richter and Wainer e6 have shown that there were separate systems for the transport of large neutral and basic amino acids. This finding has been confirmed by several authors using various methods a,4,'~e. The system of transport for large neutral amino acids has been identified as the L-system of transport described by Christensen a°,a2. Later Oldendorf and Szabo '~a, using a third radioactive isotope in the injected bolus to correct for incomplete washout of the bolus from the brain blood compartment, demonstrated the existence of a third system for acidic amino acids. Recently, Sershen and Lajtha es have given arguments for the existence at the blood-brain barrier, of the ASC system described by ChristensenV; this has been challenged, at least as far as cysteine is concerned, by Wade and Brady ai. Our results show several features which suggest that the immature pattern of transport of amino acids across the blood-brain barrier differs from the adult. At every time-period, 2 amino acids: glycine and glutamic acid had a low BU1 close to that of sucrose. This result agrees with that of Sershen and Lajtha 27 in newborn rats and of Braun et al. 6 in newborn rabbits. This low BUI was not inhibitable by the addition of cold amino acid. This is not in agreement with the findings of Oldendorf and Szabo 2a which demonstrated in adult rats the existence of a carrier-mediated transport of glutamic acid. As we have not used a third tracer to correct for intravascular radioactivity, our method is perhaps not sensitive enough to show the presence of this carrier mediated transport. The other possible explanation
for this discrepancy is that tile appearance oi" ti~is transport system is delayed during the development. The BUI of taurinc which was very low could bc lowered still more by the addition of cold amino acid. This suggests the existence of a carrier mediated transport system for /]-amino acids at the blood-brain barrier. Proline also had a very low BUI which could, in 5day-old rats, be decreased still more by the addition of cold amino acid: this suggests the presence in young animals of a carrier mediated transport whose activity later decreased. The pattern of transport of other neutral amino acids changed between the ages of 5 and 19 days. At 5 days there was a continuity in the values of the BUIs from 25 for alanine to 65 for phenylalanine. At 19 days, 2 different groups could be distinguished: the first, made up of methionine, leucine, phenylalanine, and tryptophan with BUIs comprised between 40 and 60, the second, which included alanine, serine, cysteine and threonine with BUIs comprised between 10 and 20. If all neutral amino acids were transported by the same system, it should be expected that the ratio of the BUIs of 2 of them be constant over the whole age course (unless the K,,, of the mediated transport was altered differently during the development according to the amino acid, which seemed unlikely). The ratio of the BUI of one amino acid of the first group to another one, e.g. phenylalanine, was in fact constant throughout the development. For leucine this was 0.85 at 5 days and 0.87 at 19 days: for tryptophan, 0.67 and 0.70: for methionine, 0.68 and 0.64. On the other hand, the ratio of the BUI of any amino acid of the second group to that of phenylalanine decreased markedly during development: alanine, 38 to 17; cysteine, 43 to 31; serine, 43 to 18: threonine, 59 to 27. This group of amino acids was much less homogeneous than the first one as there was no constant ratio in the BUIs of 2 amino acids of this group. Competition experiments showed that the transport of leucine was inhibited only by an amino acid of the same group, while that of alanine, serinc or threonine was also inhibited but to a lesser extent by an amino acid of the other group. Finally no sodium dependency was shown for the transport of leucine, while a substantial one was shown for the transport of alanine.
181 All these results strongly suggest that at least two systems of transport of neutral amino acids exist during development. The first, made up of phenylalanine, leucine, tryptophan and methionine, is characterized by: (1) a high BUI at every timeperiod of development; (2) the absence of inhibition by the methylated amino acids; (3) the absence of inhibition by the amino acids of the other group; (4) a sodium independency. Therefore, it is similar to the L-system of transport described in adult rats2a,30, ae. The second group includes: alanine, serine, cysteine, threonine and perhaps proline. It is characterized by: (1) a significant decrease in activity during the development as compared to the first group ; (2) the absence of statistically significant inhibition by the methylated amino acids; (3) the presence of inhibition via amino acids of the same group, likewise, but to a lesser extent via amino acids of the other group; (4) a substantial decrease in activity in the absence of sodium. It is thus very tempting to attribute the transport of the amino acids of this group to the ASC system of Christensen (ref. 7) with which it shares many of the characteristics. However, the transport of cysteine and serine in adult animals has been attributed to the carrier system transporting phenylalanine and other large neutral amino acids by Oldendorf and Szabo ~3. That of cysteine has also been attributed to the L-system by Wade and Brady at, while Sershen and Lajtha zs have credited the transport of serine and threonine to the ASC system. We have observed that during development, the ratios of the BUIs of 2 amino acids of the second group were not constant. Therefore, it is possible that some of these amino acids are transREFERENCES 1 AgrawaI, H. C., Davis, J. M. and Himwich, W. A., Developmental changes in mouse brain: weight, water content and free amino acids, J. Neurochem., 15 (1968) 917-923. 2 Antonioli, J. A. and Christensen, H. N., Differences in schedules of regression of transport systems during reticuIocytes maturation, J. biol. Chem., 244 (1969) 15051509. 3 Bafios, G., Daniel, P. M., Moormouse, S. R. and Pratt, O. E., The influx of amino acids into the brain of the rat in vivo: the essential compared with some non-essential amino acids, Proc. roy. Soc. B, 183 (1973) 5%70. 4 Bafios, G., Daniel, P. M. and Pratt, O. E., Saturation of a shared mechanism which transports L-arginine and L-
ported by 2 different systems, as is nearly always the rule in most cells s. The predominant system would depend on age and perhaps on some other unknown factors. More detailed studies are thus needed to delineate better the role of each system in the transport of the amino acids we have placed in the second group. The basic amino acid lysine was transported, as in adult animals, by the basic amino acid transport system alone as its transport was inhibited by no other amino acid except arginine. However, the activity of its transport decreased in relationship to phenylalanine between day 5 and 19 after birth. This agrees with the result of Braun et al. 6 which showed that in rabbits, the BU1 of arginine was 6 times higher in newborn than in adult animals. The changes observed during development at the blood-brain barrier bear some similarity to those observed in the maturation of the reticulocyte of the rabbit. During that maturation Antonioli and Christensen 2 have shown that the transport of alanine and lysine decreased, as we observed at the blood-brain barrier. The decrease in transport of some amino acids during development can contribute, probably along with other factors, to the decrease in the brain content of some amino acids, at least those of the second group, as maturation proceeds. ACKNOWLEDGEMENT This study was supported by Contract 56-78-88 of the Institut National de la Sant6 et de la Recherche M6dicale.
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