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Changes in the kinetics of the acidic amino acid brain and CSF uptake during development in the rat
Developmental Brain Research 102 Ž1997. 127–134
Research report
Changes in the kinetics of the acidic amino acid brain and CSF uptake during development in the rat Hameed Al-Sarraf 1, Jane E. Preston ) , Malcolm B. Segal Sherrington School of Physiology, UMDS St. Thomas’ Campus, Lambeth Palace Road, London SE1 7EH, UK Accepted 30 April 1997
Abstract Using a bilateral in situ brain perfusion technique, the rate of influx of the acidic amino acids, aspartate and glutamate, into both brain and CSF, were measured in the rat. The kinetic constants for uptake of these amino acids across the blood–brain and blood–CSF barriers in neonatal Ž1-week-old. and adult Ž7–10 weeks-old. rats were calculated; the half saturation constant Ž K m . at both barriers did not change with age, whereas the maximal transport Ž Vmax . at both barriers was greater in the younger age group, and reduced by more than 50% with maturity. The diffusion constant K d at the blood–brain barrier was not different from zero at either age, although at the blood–CSF barrier there was some diffusion at both ages, which did not change with maturity. The entry of these amino acids into the neonatal brain shown in our previous study can be explained by a greater maximal transport in the neonates which, coupled with the elevated plasma amino acid concentrations of the young animal, would result in higher blood-to-brain and blood-to-CSF flux in the neonate. q 1997 Elsevier Science B.V. Keywords: Aspartate; Glutamate; Blood–brain barrier; Cerebrospinal fluid; Mannitol; Age
1. Introduction The entry of acidic amino acids into brain from blood is at a lower rate compared to the essential amino acids w2,25,19x. This may reflect the fact that acidic amino acids can be synthesised by brain; however, the mechanisms controlling blood-to-brain transport are of major importance, since these compounds are also excitatory neurotransmitters w13,18,32x and high plasma levels can result in brain tissue toxicity w24x. In addition, the immature brain is found to be more vulnerable to high levels of derivatives of aspartate and glutamate such as NMDA and monosodium glutamate than the adult brain w23,28,29x. As with the other amino acids, the acidic amino acid transporter is not unique to neuronal tissue. It is present in non-neuronal tissues such as fibroblasts, hepatocytes, isolated microvessels and also at the blood–brain barrier ŽBBB. w5,8,17,22,25x. Since the BBB is a tight endothe-
lium, the rate at which lipid-insoluble molecules, such as aspartate and glutamate, penetrate the brain will depend on the kinetic characteristics of the carrier systems involved in their transport across the BBB. By using a bilateral in situ brain perfusion technique and extending the time of perfusion up to 30 min, a measurable uptake of radiolabelled neurotransmitter amino acids from blood into brain has been shown w1,26x. In our previous study we have shown that the immature blood– brain and blood–cerebrospinal fluid barriers are more permeable than adult barriers to the acidic amino acids, aspartate and glutamate w1x. This uptake was shown to be via a saturable carrier-mediated transport system into both brain and cerebrospinal fluid ŽCSF.. The aim of this investigation is to clarify the characteristics of the acidic amino acid transporter at the brain barriers during development.
2. Materials and methods ) Corresponding author. Dept. Gerontology, King’s College London, Cornwall House, Waterloo Road, London SE1 8WA, UK. Fax: Ž44. Ž171. 872-3235; E-mail: [email protected] 1 Present address: Dept. Physiology, Faculty of Medicine, Kuwait University, PO Box 24923, Safat-13110, State of Kuwait.
0165-3806r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 7 . 0 0 0 8 9 - 8
2.1. The perfusion fluid A Ringer plasma substitute containing protein was used to perfuse the brain w26x, containing Žin mM.: Naq 142,
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H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134
Kq 5.7, Cly 127, Ca2q 2.5, Mg 2q 1.21, HCOy 25, 3 H 2 PO4y 1.2, SO42y 1.21, glucose 10%, and 4% bovine serum albumin. After 30 min perfusion, no change in brain water was detected w26x. Evans Blue-labelled albumin was added in trace amounts for visualisation. The perfusate was contained in a reservoir and gassed with 95% O 2 and 5% CO 2 , with a PO 2 of 650 to 720 mmHg and a pH of 7.4 ŽInstrumentation Laboratories Gas Analyser 1304.. The perfusate was warmed Ž378C. and passed through a filter and bubble trap, using a Watson–Marlow peristaltic pump. The perfusion pressure and temperature were monitored continuously. To study amino acid flux, serial concentrations of 0.001, 0.01, 0.025, 0.05, 0.1, 0.25 or 0.5 mM of unlabelled aspartate or glutamate were prepared in the perfusion medium. 2.2. Animals and anaesthesia Rats of either sex were obtained from Bantin and Kingman ŽUK.. Suckling rats of 1 week postnatal age Ž12–14 g. and adults 7–9 weeks old Ž200–225 g. were used. Rats were anaesthetised by intraperitoneal administration of fentanyl citraterfluanisone Ž0.3 mlrkg. and midazolam hydrochloride Ž2 mgrkg. and then were heparinised Ž100 000 Urkg., according to the Animals ŽScientific Procedures. Act 1986, UK. Rats were maintained on a heated pad throughout the experiment. Adult rat rectal temperature was monitored continuously. For the neonatal rats, a thermometer was placed under the skin using the dissection site for entry. 2.3. Brain perfusion technique The whole brain was perfused using the bilateral in situ brain perfusion technique detailed in Preston et al. w26x, which describes how the integrity of the brain was assessed in a separate group of rats by comparing perfusate with and without red blood cells, and by measuring brain mannitol space, water content and ATP levels. The brain was perfused via both common carotid arteries. During cannulation of the arteries perfusion was always maintained in the contralateral carotid artery, and the artery being cannulated was ligated for less than 30 s before perfusion was re-established. There was minimal contribution from the systemic circulation since the perfusion pressure was slightly greater than systemic. Perfusion pressure was maintained between 60 and 110 mmHg depending on the age of the rat w1,26x. w 14 CxGlutamate or w 14 Cxaspartate were introduced to the perfusion circuit through a side arm with w 3 Hxmannitol, using a Harvard slow-drive syringe ŽType 22., to give a final concentration of aspartate 0.13 m M, glutamate 0.10 m M, and mannitol 30 m M. The concentration of isotopes in the perfusate was constant throughout the perfusion time. The radiolabelled compounds were 98–99.4% pure determined by descending paper chromatography following manufacturer’s instructions ŽWhatman No. 1 paper; solvent systems n-
b u ta n o lr a c e tic a c id r w a te r 5 : 1 : 4 , o r n butanolrethanolrwater 50 : 32 : 18.. At the end of each perfusion period Ž20 min. a sample of CSF Ž20–100 m l depending on the size of the rat. was taken by cisternal puncture with a glass micropipette, followed by immediate decapitation and removal of the brain. 2.4. Capillary depletion analysis To separate brain capillary endothelium from the brain parenchyma, a dextran density centrifugation technique was used for capillary depletion w31x. The brain was homogenised in a glass homogeniser Ž8–10 strokes. with HEPES buffer at 48C, in a ratio of 1 : 5, brain : buffer. A dextran solution ŽSigma clinical grade 70 000. was then added to a final concentration of 13% and further briefly homogenised Ž3 strokes.. An aliquot of homogenate was taken for liquid scintillation counting and the remainder centrifuged. The supernatant was carefully separated from the pellet which contained the brain vasculature w31x. The degree of supernatant contamination with blood vessels was assessed by measuring the activity of the vascular enzyme alkaline phosphatase, using Sigma Assay Kit ŽLS 108.. 2.5. Sample preparation and scintillation counting Samples of homogenised brain Žapproximately 70 mg., supernatant Ž0.5 ml., pellet Ž10–15 mg. and CSF Ž20–100 m l. were weighed using a Cahn Microbalance, and samples of perfusion fluid Ž100 m l. collected from each cannula. Tissue solublizer, Soluene-350 Ž0.5 ml., was added to all samples, and left overnight. After addition of scintillation fluid, Uniscint BD Ž3.5 mlrsample., the activities of w 14 Cxamino acids and w 3 Hxmannitol in each sample were measured using a liquid scintillation b-counter ŽRackbeta, Spectral 1219.. 2.6. Calculations Radioisotope uptake into brain and CSF after 20 min perfusion was measured and termed R: R brain Ž ml gy1 . s Ž Brain radioactivity dpm gy1 . r Ž Perfusate radioactivity dpm mly1 .
Ž 1. R CSF Ž ml mly1 . s Ž CSF radioactivity dpm mly1 . r Ž Perfusate radioactivity dpm mly1 .
Ž 2. We have previously shown that uptake over this time is unidirectional w1x. w 3 HxMannitol was included in the perfusion since it penetrates the blood–brain barrier only very slowly and thus gives an estimate of vascular space over the time course of these experiments w26x.
H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134
The unidirectional transfer constant ŽKin. for 14 Clabelled aspartate or glutamate into brain or CSF was measured as follows: Brain Kin Ž ml miny1 gy1 . s Ž 14 C R brain y3 H R brain . rPerfusion time
Ž 3.
14
CSF Kin Ž ml miny1 mly1 . s C R CSFrPerfusion time
Ž 4. The kinetics characteristics of 14 C-labelled acidic amino acid transfer at blood–brain and blood–CSF barriers were investigated by challenging the uptake with unlabelled amino acid at increasing concentrations. The unidirectional flux, Õ, obtained at each concentration was calculated from: Õ Ž nmol miny1 gy1 . s Kin = w s x
Ž 5.
where wsx is the concentration of the unlabelled amino acid in mM w30x. A plot of concentration against flux was constructed and the line of best fit was calculated using weighted non-linear regression analysis ŽIBM program, Biosoft Inc., UK., which followed Michaelis–Menten parameters: Õ s Ž Vmax = w s x rK m q s . q K d = w s x
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0.019 " 0.008 ml gy1 in neonates, which agrees with our previous findings w1x. This space was taken to represent the vascular space, and was accounted for in the calculations for brain tissue flux Žsee Section 2.. w 3 HxMannitol entry into CSF, R CSF , represents the permeability of the blood– CSF barrier to mannitol, rather than a ‘‘space’’. R CSF declined significantly with age from 0.023 " 0.004 ml mly1 in the neonate to 0.012 " 0.005 ml mly1 for the adult Ž P - 0.05, n s 4–6., which might reflect tightening of the blood–CSF barrier with maturity. 3.2. Amino acid flux into brain The acidic amino acid fluxes into capillary depleted brain Žsupernatant. and endothelium compartments Žpellet. were plotted separately against the serial concentration of amino acid in the perfusion medium. Fig. 1 shows w 14 Cxaspartate flux; Fig. 2 shows w 14 Cxglutamate flux. Flux was proportional to perfusate amino acid concentration at low values, then approached steady state with higher concentrations. For both amino acids and age groups, flux into supernatant was greater than into pellet. Of the radioactivity in the pellet, only 60–68% was due to the labelled amino acid, while in the supernatant more than 80% of
Ž 6.
where Vmax is the maximum flux Žnmol miny1 gy1 .; K m is the half saturation constant ŽmM.; and K d is the diffusion constant Žml miny1 gy1 .. Difference between means were analysed for statistical significance using two-way analysis of variance followed by t-tests using pooled sample variance. Difference from zero was confirmed using Wilcoxon signed rank test. Statistical significance was taken as - 0.05. 2.7. Materials Radiolabelled compounds: L-wU- 14 Cxaspartic acid Ž224.8 mCirmmol., L-wU- 14 Cxglutamic acid Ž281.4 mCirmmol., and D-w1- 3 H ŽN.xmannitol Ž26.4 mCirmmol. were obtained from Dupont New England Nuclear Corp. ŽUK.. Bovine serum albumin Žfraction V., HEPES, and dextran were obtained from Sigma Chemical Co. ŽUK.. Tissue solubilizing reagent, Soluene-350, was purchased from Packard company, and scintillation fluid ŽUniscint BD. was obtained from National Diagnostics ŽUK.. All other non-radioactive chemicals used in the perfusion solution were obtained from BDH Laboratory Supplies.
3. Results 3.1. [ 3 H]Mannitol as a baseline for amino acid uptake After 20 min in situ brain perfusion the mannitol space, R brain , ranged from 0.013 " 0.006 ml gy1 in adults to
Fig. 1. w 14 CxAspartate fluxes into Ža. capillary depleted supernatant, and Žb. endothelium containing pellet for rats aged 1 week Žfilled squares. and adults Žopen squares.. Values are means"S.E.M., ns 4–6 for each point. ) P - 0.05, ) ) P - 0.01 significantly different from adult using ANOVA.
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H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134
Fig. 2. Differential w 14 Cxglutamate fluxes into Ža. capillary depleted supernatant, and Žb. endothelium containing pellet for rats aged 1 week Žfilled squares. and adults Žopen squares.. Values are means"S.E.M., ns 3–5 for each point. ) P - 0.05, ) ) P - 0.01 significantly different from adult.
Fig. 3. CSF fluxes for w 14 Cxaspartate and w 14 Cxglutamate in 1-week-old Žfilled squares. and adult rats Žopen squares.. Values are mean"S.E.M., ns 4–5 for each point. ) ) P - 0.01 significantly different from adult.
activity was labelled amino acid, as determined by ascending paper chromatography w1x. At amino acid concentrations above 0.05 mM, the 20 min flux into neonatal brain was significantly greater than flux into the adult brain for both glutamate and aspartate Ž P - 0.05..
ŽFig. 3.. Flux into neonatal CSF was greater than into the adult at the highest amino acid concentrations used Ž P 0.05., similar to supernatant uptake. 80–85% of activity was due to the labelled amino acid. However, unlike brain supernatant, a steady-state flux was not approached in these experiments.
3.3. Amino acid flux into CSF
3.4. Kinetic constants at the blood–brain barrier
w 14 CxAmino acids fluxes into CSF were calculated as described, and plotted against amino acid concentration
Values for half saturation constant Ž K m ., maximal transport Ž Vmax ., and diffusion constant Ž K d . were esti-
Table 1 Kinetic constants for w 14 Cxaspartate in whole brain homogenate, capillary depleted supernatant, and pellet containing endothelium
Homogenate Žwhole brain. Supernatant Žbrain parenchyma. Pellet Žendothelium.
Age
Km ŽmM.
Vmax Žnmol miny1 gy1 .
Kd Žml miny1 gy1 .
Adult Neonate Adult Neonate Adult Neonate
0.101 " 0.051 0.083 " 0.045 0.092 " 0.019 0.078 " 0.013 0.068 " 0.020 0.060 " 0.019
0.135 " 0.021 0.260 " 0.027 0.158 " 0.013 0.220 " 0.012 0.071 " 0.014 0.084 " 0.012
0.09 " 0.10 0.10 " 0.20 0.16 " 0.21 y0.039 " 0.93 y0.11 " 0.38 y0.34 " 0.53
)
)
Values are mean " S.E.M., n s 32. K m s half saturation constant, Vmax s maximal transport, and K d s diffusional constant in whole brain homogenate, capillary depleted supernatant, and pellet containing endothelium. ) P - 0.01, significant difference from adult values. K d was not significantly different from zero in any compartment or either age Ž P ) 0.05..
H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134 Table 2 Kinetic constants for
14
131
C-glutamate in whole brain homogenate, capillary depleted supernatant, and pellet containing endothelium
Homogenate Žwhole brain. Supernatant Žbrain parenchyma. Pellet Žendothelium.
Age
Km ŽmM.
Vmax Žnmol miny1 gy1 .
Kd Žml miny1 gy1 .
Adult Neonate Adult Neonate Adult Neonate
0.024 " 0.016 0.031 " 0.013 0.061 " 0.034 0.054 " 0.023 0.056 " 0.041 0.056 " 0.025
0.215 " 0.025 0.382 " 0.031 0.219 " 0.024 0.358 " 0.045 0.077 " 0.021 0.092 " 0.021
0.11 " 0.17 0.15 " 0.22 y0.29 " 0.36 y0.11 " 0.24 y0.05 " 0.13 0.22 " 0.041
))
)
Values are mean " S.E.M., n s 28. ) P - 0.01, ) ) P - 0.001, significant difference from adult values. K d was not significantly different from zero in any compartment or either age Ž P ) 0.05..
mated from the line of best fit using Eq. Ž6.. These are shown in Table 1 Žaspartate. and Table 2 Žglutamate.. For both amino acids, whole brain K m did not change with maturity Ž P ) 0.05., whereas Vmax reduced by approximately 50% with age Ž P - 0.01. from neonatal values of 0.260 nmol miny1 gy1 for aspartate and 0.382 nmol miny1 gy1 for glutamate. The diffusion constant, K d , was not significantly different from zero Ž P ) 0.05., suggesting insignificant diffusion of these amino acids across the BBB at either age. Kinetic parameters were then calculated separately for the endothelium containing pellet and the capillary depleted supernatant Žbrain parenchyma.. As can be seen in Tables 2 and 3, the supernatant kinetics showed the same pattern as whole brain. The calculated K m values were the same in all compartments including pellet Ž P ) 0.05., and in the two age groups. However, the supernatant Vmax showed age related change, being higher in the neonate than the adult Ž P - 0.01. for both amino acids. In addition, the glutamate Vmax into supernatant and whole brain was greater than that for aspartate for both age groups Ž P 0.05.. The pellet Vmax was significantly smaller than the supernatant Vmax for both amino acids Ž P - 0.001. and did not change with age Ž P ) 0.05., at around 0.07–0.09 nmol miny1 gy1 for both amino acids and ages. 3.5. Kinetic constants at the blood–CSF barrier The Kinetic parameters calculated for the entry of these amino acids into CSF are shown in Table 3. As was seen
for brain, there was no significant change in K m with age Ž P ) 0.05., but the Vmax declined with maturity by 50% for aspartate Žfrom 0.271 nmol miny1 gy1 . and by 65% for glutamate Žfrom 0.670 nmol miny1 gy1 . Ž P - 0.001.. In the neonate, but not the adult, glutamate Vmax was greater than aspartate Vmax Ž P - 0.01.. Unlike brain, CSF K d was significant for both amino acids, and higher for glutamate than aspartate Ž P - 0.05.. However, this did not change with age Ž P ) 0.05..
4. Discussion w 3 HxMannitol was used as a vascular space marker to correct for perfusate trapping in the vascular lumen after perfusion. It also acts a probe to monitor the passive permeability of the BBB, situated at the level of the brain capillary endothelium, and the blood–CSF barrier, situated at the choroid plexus epithelium. We have previously shown that BBB permeability to mannitol does not change with age using multiple time point data to calculate Kin for mannitol w26x and this is in agreement with the present studies and those of Daniel et al. w6x. The permeability of the blood–CSF barrier, however, did change with age in these studies, and R CSF reduced significantly from 0.023 to 0.012 ml gy1 with maturity. This reduction may represent ‘‘tightening’’ of the blood–CSF barrier, although there is little morphological evidence for this from other studies w19x. More likely it represents reduced surface area or pore number relative to the size of the brain w14x.
Table 3 Amino acid kinetics for neonate and adult CSF uptake
w 14 CxAspartate w 14 CxGlutamate
Age
Km ŽmM.
Vmax Žnmol miny1 mly1 .
Kd Žml miny1 mly1 .
Adult Neonate Adult Neonate
0.045 " 0.031 0.059 " 0.014 0.040 " 0.021 0.070 " 0.019
0.134 " 0.050 0.271 " 0.045 0.202 " 0.032 0.670 " 0.105
0.281 " 0.094 0.417 " 0.089 0.587 " 0.068 0.744 " 0.165
)
))
Values are mean " S.E.M., n s 27–32. ) P - 0.05, ) ) P - 0.001, difference from adult. K d , was significantly different from zero Ž P - 0.001. for both aspartate and glutamate.
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H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134
However, using in situ perfusion incorporating w 3 Hxmannitol, it was possible to separate and correct for these passive permeability properties when studying amino acid transport. There are numerous data on the kinetic constants for acidic amino acid transport into various CNS preparations. Kinetically, the transport systems for glutamate and for aspartate tend to be similar, for example in synaptosomes K m and Vmax values are around 10 m M and 10 nmol miny1 mgy1 protein w10x. Astrocytes, which regulate the brain interstitial amino acid composition, also have high affinity uptake of neurotransmitter amino acids w7,12,15,16x. Depending on species and type of amino acid transport system investigated, the kinetic constants at the BBB show a variety of developmental responses. For example, the K m for tryptophan is not different in young adult rats compared to neonates, but the capacity of transport declines with age w27x. K m values are similar when calculated for neonatal and adult arginine transport in rabbits w3x, but neutral amino acid transport demonstrates age-related changes w4x. The kinetic constants for aspartate and glutamate transport into the brain are shown in Tables 1 and 2 for both adults and neonates. The half saturation constants for brain uptake were not different with age for either amino acid, and in addition, did not differ according to the brain compartment studied. With plasma aspartate levels of approximately 0.02 mM in adult and 0.03 mM in neonates w9x, the carrier would be working at less than half saturation at both ages, and increasing plasma aspartate would result in greater transport into brain. Glutamate uptake would be more than half saturated at normal plasma concentrations of G 0.1 mM. Because of this, there may be greater competition for uptake by glutamate resulting in more blood to brain transport compared to aspartate, at both ages. Unlike the K m values, the transport capacity Ž Vmax . into whole brain was significantly reduced with age, and adult Vmax was approximately half that of the neonate. The greater capacity for transport combined with higher plasma levels of amino acid in the neonate would result in greater uptake into young brains compared to adults. Most of the uptake would be into brain parenchyma since the Vmax of the endothelial portion of brain was smaller than that for the supernatant. It is of interest that the pellet Vmax did not change with age, supporting the finding that amino acid sequestration by capillary endothelial cells does not account for the greater brain uptake in the neonate w1x. The diffusion constants K d , for aspartate and glutamate into brain were not significantly different from zero, and did not change with age consistent with the mannitol data. Eq. Ž6. can be applied to predict amino acid flux into the brain from blood under ideal conditions, with no competing amino acid. For comparison by age, this approach has been applied for both neonatal and adult rats
Table 4 Combined rates of acidic amino acid flux, available to brain tissue directly via the BBB and indirectly across the blood–CSF barrier
Adult Neonate
Brain CSF Brain CSF
Weight Žg.
Flux Žnmol miny1 gy1 . Aspartate
Glutamate
2.12"0.07 0.228"0.043 0.58"0.02 0.072"0.009
0.03
0.23
0.09
0.41
Fluxes were calculated using the kinetic constant ŽTable 1Table 2Table 3. based on plasma amino acid concentrations of glutamate Ž0.1 mM. and aspartate Ž0.02 and 0.03 mM.. Values for brain weights and CSF volumes Žassuming 1 mls1 g. are means"S.E.M., ns18–22.
ŽTable 4.. The predicted influx for aspartate was calculated in the adult, 0.022 nmol miny1 gy1 , and in the neonate 0.074 nmol miny1 gy1 . The corresponding values for glutamate were 0.210 and 0.361 nmol miny1 gy1 for adult and neonate. Thus, the calculated possible influx for both amino acids is greater in the neonate. These adult flux values compare well with previous data for aspartate brain uptake in the rat, 0.144 nmol miny1 gy1 w3x, even though the earlier measurements were made in the presence of competing amino acids and without the same correction for vascular space as we have used. K m values at the blood–CSF barrier were similar to those at the BBB ŽTable 3. and did not change with age. The Vmax values were also similar, with the exception of neonatal glutamate Vmax which was nearly twice the brain Vmax value. However, the developmental changes seen were the same at both barriers, and Vmax fell by more than 50% with maturity. There was, however, a striking difference in the diffusion constants Ž K d . for the two barriers, being not different to zero at the BBB, but significant at the blood–CSF barrier. There was some indication that K d at the blood–CSF barrier might reduce with maturity, but any change with age failed to reach significance. Taking into account the diffusional component, fluxes for aspartate and glutamate into CSF were calculated ŽEq. Ž6.., and exceeded those into brain ŽTable 4.. The predicted fluxes were 0.04 and 0.11 nmol miny1 gy1 for aspartate in adult and neonate CSF, respectively, and 0.20 and 0.53 nmol miny1 gy1 for glutamate. Although acidic amino acids have much lower influx rates into brain compared to the neutral and basic amino acids ŽTable 5., by characterising the kinetics of this group of amino acids during development we have shown that there is a substantial entry across the blood–CSF barrier, especially in the immature brain, in addition to entry across the BBB. Factors which may affect amino acid entry into the brain are the rate of cerebral blood flow, the rate of brain parenchymal cell uptake, the plasma concentration of amino acids and competitors, and the blood to brain permeability. Although cerebral blood flow rates increase with age w26x
H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134 Table 5 BBB amino acid transport systems measured in the adult rat Amino acid transport system
Neutral Basic Acidic a
Representative substrate
Phenylalanine a Lysine a Aspartate Glutamate
Km
Vmax
ŽmM.
Žnmol miny1 gy1 .
0.12 0.10 0.10 0.02
30 6 0.02 0.22
Values obtained from Oldendorf, 1971 w21x.
this is unlikely to affect brain uptake for these slowly penetrating acidic amino acids. Brain parenchymal cell uptake, estimated in glial and synaptosomal preparations w10,11,20x show Vmax values more than 1000 times greater than the Vmax at the BBB in both neonates and adults suggesting that BBB transport is the rate-limiting step for brain accumulation of these amino acids, and not capacity of brain cell uptake. The greatest contributors to high neonatal brain amino acid levels are therefore likely to be elevated plasma concentrations plus greater Vmax . As the brain and choroid plexuses grow, they may not be accompanied by an increase in the number of transporters, so resulting in a lower number of carriers per unit mass in the adult compared to the neonate and accounting for the low Vmax in the adult. The possibility of structural changes in these carrier systems appears remote since the carrier operates with a similar affinity in both the immature and the mature brains.
Acknowledgements We gratefully acknowledge the support of the Wellcome Trust, and sponsorship for HA-S by the Kuwait Foundation for Science.
References w1x H. Al-Sarraf, J.E. Preston, M.B. Segal, The entry of acidic amino acids into brain and CSF during development using in situ brain perfusion in the rat, Dev. Brain Res. 90 Ž1995. 151–158. w2x G. Banos, ˜ P.M. Daniel, O.E. Pratt, The influx of amino acids into the brain of the rat in vivo: the essential compared with some non-essential amino acids, Proc. R. Soc. London B 183 Ž1973. 59–70. w3x G. Banos, ˜ P.M. Daniel, O.E. Pratt, The effect of age upon the entry of some amino acids into the brain, and their incorporation into cerebral protein, Dev. Med. Child Neurol. 20 Ž1978. 335–346. w4x L.D. Braun, E.M. Cornford, W.H. Oldendorf, Newborn rabbit blood–brain barrier is selectively permeable and differs substantially from the adult, J. Neurochem. 34 Ž1980. 147–152. w5x H.N. Christensen, M. Makowske, Recognition chemistry of anionic amino acids for hepatocyte transport and for neurotransmitter action compared, Life Sci. 33 Ž1983. 2255–2267.
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w6x P.M. Daniel, D.K.C. Lam, O.E. Pratt, Comparison of the vascular permeability of the brain and spinal cord to mannitol and inulin in rats, J. Neurochem. 45 Ž1985. 647–649. w7x L.P. Davies, G.A.R. Johnston, Uptake and release of D- and Laspartate by rat brain slices, J. Neurochem. 26 Ž1976. 1007–1014. w8x H. Davson, M.N. Lipovac, J.B. Mackic, J.E. Preston, M.B. Segal, G. Tang, B.V. Zlokovic, Kinetics of L-glutamic acid uptake by the luminal side of the blood–brain barrier studied in an in situ perfused brain of the anaesthetised guinea-pig, J. Physiol. 423 Ž1990. 36. w9x H. Davson, K. Welch, M.B. Segal, The Physiology and Pathophysiology of the Cerebrospinal Fluid, Churchill Livingstone, Edinburgh, 1987. w10x E.A. Debler, A. Lajtha, High-affinity transport of g-aminobutyric acid, glycine, taurine, L-aspartic acid, and L-glutamic acid in synaptosomal ŽP2. tissue: a kinetic and substrate specificity analysis, J. Neurochem. 48 Ž1987. 1851–1856. w11x E.A. Debler, A. Lajtha, Amino acid uptake in synaptosomal preparations, in: L. Rakic, D.J. Begley, B.V. Zlokovic ŽEds.., Peptide and Amino Acid Transport Mechanisms in the Central Nervous System, MacMillan Press, London, 1988, pp. 195–208. w12x I. Drejer, O.M. Larsson, A. Schousboe, Characterisation of uptake and release processes for D- and L-aspartate in primary cultures of astrocytes and cerebral granule cells, Neurochem. Res. 8 Ž1983. 231–243. w13x F. Fonnum, Glutamate: a neurotransmitter in mammalian brain, J. Neurochem. 42 Ž1984. 1–11. w14x M.D. Habgood, G.W. Knott, K.M. Dziegielewska, N.R. Saunders, The nature of the decrease in blood–cerebrospinal fluid barrier exchange during postnatal brain development in the rat, J. Physiol. 468 Ž1993. 73–83. w15x E. Hansson, P. Eriksson, M. Nilsson, Amino acid and monoamine transport in primary astroglial cultures from defined brain regions, Neurochem. Res. 10 Ž1985. 1335–1341. w16x L. Hertz, A. Schousboe, Interactions between neurons and astrocytes in the turnover of GABA and glutamate: GABA neurotransmission, Brain Res. Bull. 52 Ž1980. 389–395. w17x H.T. Hutchinson, H.M. Eisenberg, B. Haber, High-affinity transport of glutamate in rat brain microvessels, Exp. Neurol. 87 Ž1985. 260–269. w18x K. Krnjevic, Glutamate and g-aminoisobutyric acid in brain, Nature 228 Ž1970. 119–124. w19x K. Møllgard, ˚ D.H. Malinowska, N.R. Saunders, Lack of correlation between tight junction morphology and permeability properties in developing choroid plexus, Nature 264 Ž1976. 293–294. w20x M. Nilsson, E. Hansson, L. Ronnback, Interactions between val¨ ¨ proate, glutamate, aspartate and GABA with respect to uptake in astroglial primary cultures, Neurochem. Res. 17 Ž1992. 327–332. w21x W.H. Oldendorf, Brain uptake of radiolabelled amino acids, amines, and hexoses after arterial injection, Am. J. Physiol. 221 Ž1971. 1629–1639. w22x W.H. Oldendorf, J. Szabo, Amino-acid assignment to one of three blood–brain barrier amino acid carriers, Am. J. Physiol. 230 Ž1976. 940–948. w23x J.W. Olney, Brain lesions, obesity and other disturbances in mice treated with monosodium glutamate, Science 164 Ž1969. 719–721. w24x J.W. Olney, Brain damage and oral intake of certain amino acids, Adv. Exp. Med. Biol. 69 Ž1976. 497–506. w25x W.M. Pardridge, Regulation of amino acid availability to brain: selective control mechanisms for glutamate, in: L.J. Filer Jr. et al. ŽEds.., Glutamic Acid: Advances in Biochemistry and Physiology, Raven Press, New York, 1979, pp. 125–137. w26x J.E. Preston, H. Al-Sarraf, M.B. Segal, Permeability of the developing blood–brain barrier to 14 C-mannitol using the rat in situ brain perfusion technique, Dev. Brain Res. 87 Ž1995. 69–76. w27x G.S. Sarna, M.D. Tricklebank, B.D. Kantamaneni, Effect of age on variables influencing the supply of tryptophan to the brain, J. Neurochem. 39 Ž1982. 1283–1290.
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w28x D.D. Schoepp, A.Y. Gamble, C.R. Salhoff, B.G. Johnson, P.L. Ornstein, Excitatory amino acid-induced convulsions in neonatal rats mediated by distinct receptor subtypes, Eur. J. Pharmacol. 182 Ž1990. 421–427. w29x K. Seta, H. Sershen, A. Lajtha, Cerebral amino acid uptake in vivo in newborn mice, Brain Res. 47 Ž1972. 415–425. w30x Y. Takasato, S.I. Rapoport, Q.R. Smith, An in situ brain perfusion
technique to study cerebrovascular transport in the rat, Am. J. Physiol. 247 Ž1984. Ž1984. H484–H493. w31x D. Triguero, J. Buciak, W.M. Pardridge, Capillary depletion method for the quantification of blood–brain barrier transport of circulating peptides and plasma proteins, J. Neurochem. 54 Ž1990. 1882–1888. w32x J.C. Watkins, R.H. Evans, Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol. 21 Ž1981. 165–204.
Research report
Changes in the kinetics of the acidic amino acid brain and CSF uptake during development in the rat Hameed Al-Sarraf 1, Jane E. Preston ) , Malcolm B. Segal Sherrington School of Physiology, UMDS St. Thomas’ Campus, Lambeth Palace Road, London SE1 7EH, UK Accepted 30 April 1997
Abstract Using a bilateral in situ brain perfusion technique, the rate of influx of the acidic amino acids, aspartate and glutamate, into both brain and CSF, were measured in the rat. The kinetic constants for uptake of these amino acids across the blood–brain and blood–CSF barriers in neonatal Ž1-week-old. and adult Ž7–10 weeks-old. rats were calculated; the half saturation constant Ž K m . at both barriers did not change with age, whereas the maximal transport Ž Vmax . at both barriers was greater in the younger age group, and reduced by more than 50% with maturity. The diffusion constant K d at the blood–brain barrier was not different from zero at either age, although at the blood–CSF barrier there was some diffusion at both ages, which did not change with maturity. The entry of these amino acids into the neonatal brain shown in our previous study can be explained by a greater maximal transport in the neonates which, coupled with the elevated plasma amino acid concentrations of the young animal, would result in higher blood-to-brain and blood-to-CSF flux in the neonate. q 1997 Elsevier Science B.V. Keywords: Aspartate; Glutamate; Blood–brain barrier; Cerebrospinal fluid; Mannitol; Age
1. Introduction The entry of acidic amino acids into brain from blood is at a lower rate compared to the essential amino acids w2,25,19x. This may reflect the fact that acidic amino acids can be synthesised by brain; however, the mechanisms controlling blood-to-brain transport are of major importance, since these compounds are also excitatory neurotransmitters w13,18,32x and high plasma levels can result in brain tissue toxicity w24x. In addition, the immature brain is found to be more vulnerable to high levels of derivatives of aspartate and glutamate such as NMDA and monosodium glutamate than the adult brain w23,28,29x. As with the other amino acids, the acidic amino acid transporter is not unique to neuronal tissue. It is present in non-neuronal tissues such as fibroblasts, hepatocytes, isolated microvessels and also at the blood–brain barrier ŽBBB. w5,8,17,22,25x. Since the BBB is a tight endothe-
lium, the rate at which lipid-insoluble molecules, such as aspartate and glutamate, penetrate the brain will depend on the kinetic characteristics of the carrier systems involved in their transport across the BBB. By using a bilateral in situ brain perfusion technique and extending the time of perfusion up to 30 min, a measurable uptake of radiolabelled neurotransmitter amino acids from blood into brain has been shown w1,26x. In our previous study we have shown that the immature blood– brain and blood–cerebrospinal fluid barriers are more permeable than adult barriers to the acidic amino acids, aspartate and glutamate w1x. This uptake was shown to be via a saturable carrier-mediated transport system into both brain and cerebrospinal fluid ŽCSF.. The aim of this investigation is to clarify the characteristics of the acidic amino acid transporter at the brain barriers during development.
2. Materials and methods ) Corresponding author. Dept. Gerontology, King’s College London, Cornwall House, Waterloo Road, London SE1 8WA, UK. Fax: Ž44. Ž171. 872-3235; E-mail: [email protected] 1 Present address: Dept. Physiology, Faculty of Medicine, Kuwait University, PO Box 24923, Safat-13110, State of Kuwait.
0165-3806r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 7 . 0 0 0 8 9 - 8
2.1. The perfusion fluid A Ringer plasma substitute containing protein was used to perfuse the brain w26x, containing Žin mM.: Naq 142,
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Kq 5.7, Cly 127, Ca2q 2.5, Mg 2q 1.21, HCOy 25, 3 H 2 PO4y 1.2, SO42y 1.21, glucose 10%, and 4% bovine serum albumin. After 30 min perfusion, no change in brain water was detected w26x. Evans Blue-labelled albumin was added in trace amounts for visualisation. The perfusate was contained in a reservoir and gassed with 95% O 2 and 5% CO 2 , with a PO 2 of 650 to 720 mmHg and a pH of 7.4 ŽInstrumentation Laboratories Gas Analyser 1304.. The perfusate was warmed Ž378C. and passed through a filter and bubble trap, using a Watson–Marlow peristaltic pump. The perfusion pressure and temperature were monitored continuously. To study amino acid flux, serial concentrations of 0.001, 0.01, 0.025, 0.05, 0.1, 0.25 or 0.5 mM of unlabelled aspartate or glutamate were prepared in the perfusion medium. 2.2. Animals and anaesthesia Rats of either sex were obtained from Bantin and Kingman ŽUK.. Suckling rats of 1 week postnatal age Ž12–14 g. and adults 7–9 weeks old Ž200–225 g. were used. Rats were anaesthetised by intraperitoneal administration of fentanyl citraterfluanisone Ž0.3 mlrkg. and midazolam hydrochloride Ž2 mgrkg. and then were heparinised Ž100 000 Urkg., according to the Animals ŽScientific Procedures. Act 1986, UK. Rats were maintained on a heated pad throughout the experiment. Adult rat rectal temperature was monitored continuously. For the neonatal rats, a thermometer was placed under the skin using the dissection site for entry. 2.3. Brain perfusion technique The whole brain was perfused using the bilateral in situ brain perfusion technique detailed in Preston et al. w26x, which describes how the integrity of the brain was assessed in a separate group of rats by comparing perfusate with and without red blood cells, and by measuring brain mannitol space, water content and ATP levels. The brain was perfused via both common carotid arteries. During cannulation of the arteries perfusion was always maintained in the contralateral carotid artery, and the artery being cannulated was ligated for less than 30 s before perfusion was re-established. There was minimal contribution from the systemic circulation since the perfusion pressure was slightly greater than systemic. Perfusion pressure was maintained between 60 and 110 mmHg depending on the age of the rat w1,26x. w 14 CxGlutamate or w 14 Cxaspartate were introduced to the perfusion circuit through a side arm with w 3 Hxmannitol, using a Harvard slow-drive syringe ŽType 22., to give a final concentration of aspartate 0.13 m M, glutamate 0.10 m M, and mannitol 30 m M. The concentration of isotopes in the perfusate was constant throughout the perfusion time. The radiolabelled compounds were 98–99.4% pure determined by descending paper chromatography following manufacturer’s instructions ŽWhatman No. 1 paper; solvent systems n-
b u ta n o lr a c e tic a c id r w a te r 5 : 1 : 4 , o r n butanolrethanolrwater 50 : 32 : 18.. At the end of each perfusion period Ž20 min. a sample of CSF Ž20–100 m l depending on the size of the rat. was taken by cisternal puncture with a glass micropipette, followed by immediate decapitation and removal of the brain. 2.4. Capillary depletion analysis To separate brain capillary endothelium from the brain parenchyma, a dextran density centrifugation technique was used for capillary depletion w31x. The brain was homogenised in a glass homogeniser Ž8–10 strokes. with HEPES buffer at 48C, in a ratio of 1 : 5, brain : buffer. A dextran solution ŽSigma clinical grade 70 000. was then added to a final concentration of 13% and further briefly homogenised Ž3 strokes.. An aliquot of homogenate was taken for liquid scintillation counting and the remainder centrifuged. The supernatant was carefully separated from the pellet which contained the brain vasculature w31x. The degree of supernatant contamination with blood vessels was assessed by measuring the activity of the vascular enzyme alkaline phosphatase, using Sigma Assay Kit ŽLS 108.. 2.5. Sample preparation and scintillation counting Samples of homogenised brain Žapproximately 70 mg., supernatant Ž0.5 ml., pellet Ž10–15 mg. and CSF Ž20–100 m l. were weighed using a Cahn Microbalance, and samples of perfusion fluid Ž100 m l. collected from each cannula. Tissue solublizer, Soluene-350 Ž0.5 ml., was added to all samples, and left overnight. After addition of scintillation fluid, Uniscint BD Ž3.5 mlrsample., the activities of w 14 Cxamino acids and w 3 Hxmannitol in each sample were measured using a liquid scintillation b-counter ŽRackbeta, Spectral 1219.. 2.6. Calculations Radioisotope uptake into brain and CSF after 20 min perfusion was measured and termed R: R brain Ž ml gy1 . s Ž Brain radioactivity dpm gy1 . r Ž Perfusate radioactivity dpm mly1 .
Ž 1. R CSF Ž ml mly1 . s Ž CSF radioactivity dpm mly1 . r Ž Perfusate radioactivity dpm mly1 .
Ž 2. We have previously shown that uptake over this time is unidirectional w1x. w 3 HxMannitol was included in the perfusion since it penetrates the blood–brain barrier only very slowly and thus gives an estimate of vascular space over the time course of these experiments w26x.
H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134
The unidirectional transfer constant ŽKin. for 14 Clabelled aspartate or glutamate into brain or CSF was measured as follows: Brain Kin Ž ml miny1 gy1 . s Ž 14 C R brain y3 H R brain . rPerfusion time
Ž 3.
14
CSF Kin Ž ml miny1 mly1 . s C R CSFrPerfusion time
Ž 4. The kinetics characteristics of 14 C-labelled acidic amino acid transfer at blood–brain and blood–CSF barriers were investigated by challenging the uptake with unlabelled amino acid at increasing concentrations. The unidirectional flux, Õ, obtained at each concentration was calculated from: Õ Ž nmol miny1 gy1 . s Kin = w s x
Ž 5.
where wsx is the concentration of the unlabelled amino acid in mM w30x. A plot of concentration against flux was constructed and the line of best fit was calculated using weighted non-linear regression analysis ŽIBM program, Biosoft Inc., UK., which followed Michaelis–Menten parameters: Õ s Ž Vmax = w s x rK m q s . q K d = w s x
129
0.019 " 0.008 ml gy1 in neonates, which agrees with our previous findings w1x. This space was taken to represent the vascular space, and was accounted for in the calculations for brain tissue flux Žsee Section 2.. w 3 HxMannitol entry into CSF, R CSF , represents the permeability of the blood– CSF barrier to mannitol, rather than a ‘‘space’’. R CSF declined significantly with age from 0.023 " 0.004 ml mly1 in the neonate to 0.012 " 0.005 ml mly1 for the adult Ž P - 0.05, n s 4–6., which might reflect tightening of the blood–CSF barrier with maturity. 3.2. Amino acid flux into brain The acidic amino acid fluxes into capillary depleted brain Žsupernatant. and endothelium compartments Žpellet. were plotted separately against the serial concentration of amino acid in the perfusion medium. Fig. 1 shows w 14 Cxaspartate flux; Fig. 2 shows w 14 Cxglutamate flux. Flux was proportional to perfusate amino acid concentration at low values, then approached steady state with higher concentrations. For both amino acids and age groups, flux into supernatant was greater than into pellet. Of the radioactivity in the pellet, only 60–68% was due to the labelled amino acid, while in the supernatant more than 80% of
Ž 6.
where Vmax is the maximum flux Žnmol miny1 gy1 .; K m is the half saturation constant ŽmM.; and K d is the diffusion constant Žml miny1 gy1 .. Difference between means were analysed for statistical significance using two-way analysis of variance followed by t-tests using pooled sample variance. Difference from zero was confirmed using Wilcoxon signed rank test. Statistical significance was taken as - 0.05. 2.7. Materials Radiolabelled compounds: L-wU- 14 Cxaspartic acid Ž224.8 mCirmmol., L-wU- 14 Cxglutamic acid Ž281.4 mCirmmol., and D-w1- 3 H ŽN.xmannitol Ž26.4 mCirmmol. were obtained from Dupont New England Nuclear Corp. ŽUK.. Bovine serum albumin Žfraction V., HEPES, and dextran were obtained from Sigma Chemical Co. ŽUK.. Tissue solubilizing reagent, Soluene-350, was purchased from Packard company, and scintillation fluid ŽUniscint BD. was obtained from National Diagnostics ŽUK.. All other non-radioactive chemicals used in the perfusion solution were obtained from BDH Laboratory Supplies.
3. Results 3.1. [ 3 H]Mannitol as a baseline for amino acid uptake After 20 min in situ brain perfusion the mannitol space, R brain , ranged from 0.013 " 0.006 ml gy1 in adults to
Fig. 1. w 14 CxAspartate fluxes into Ža. capillary depleted supernatant, and Žb. endothelium containing pellet for rats aged 1 week Žfilled squares. and adults Žopen squares.. Values are means"S.E.M., ns 4–6 for each point. ) P - 0.05, ) ) P - 0.01 significantly different from adult using ANOVA.
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H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134
Fig. 2. Differential w 14 Cxglutamate fluxes into Ža. capillary depleted supernatant, and Žb. endothelium containing pellet for rats aged 1 week Žfilled squares. and adults Žopen squares.. Values are means"S.E.M., ns 3–5 for each point. ) P - 0.05, ) ) P - 0.01 significantly different from adult.
Fig. 3. CSF fluxes for w 14 Cxaspartate and w 14 Cxglutamate in 1-week-old Žfilled squares. and adult rats Žopen squares.. Values are mean"S.E.M., ns 4–5 for each point. ) ) P - 0.01 significantly different from adult.
activity was labelled amino acid, as determined by ascending paper chromatography w1x. At amino acid concentrations above 0.05 mM, the 20 min flux into neonatal brain was significantly greater than flux into the adult brain for both glutamate and aspartate Ž P - 0.05..
ŽFig. 3.. Flux into neonatal CSF was greater than into the adult at the highest amino acid concentrations used Ž P 0.05., similar to supernatant uptake. 80–85% of activity was due to the labelled amino acid. However, unlike brain supernatant, a steady-state flux was not approached in these experiments.
3.3. Amino acid flux into CSF
3.4. Kinetic constants at the blood–brain barrier
w 14 CxAmino acids fluxes into CSF were calculated as described, and plotted against amino acid concentration
Values for half saturation constant Ž K m ., maximal transport Ž Vmax ., and diffusion constant Ž K d . were esti-
Table 1 Kinetic constants for w 14 Cxaspartate in whole brain homogenate, capillary depleted supernatant, and pellet containing endothelium
Homogenate Žwhole brain. Supernatant Žbrain parenchyma. Pellet Žendothelium.
Age
Km ŽmM.
Vmax Žnmol miny1 gy1 .
Kd Žml miny1 gy1 .
Adult Neonate Adult Neonate Adult Neonate
0.101 " 0.051 0.083 " 0.045 0.092 " 0.019 0.078 " 0.013 0.068 " 0.020 0.060 " 0.019
0.135 " 0.021 0.260 " 0.027 0.158 " 0.013 0.220 " 0.012 0.071 " 0.014 0.084 " 0.012
0.09 " 0.10 0.10 " 0.20 0.16 " 0.21 y0.039 " 0.93 y0.11 " 0.38 y0.34 " 0.53
)
)
Values are mean " S.E.M., n s 32. K m s half saturation constant, Vmax s maximal transport, and K d s diffusional constant in whole brain homogenate, capillary depleted supernatant, and pellet containing endothelium. ) P - 0.01, significant difference from adult values. K d was not significantly different from zero in any compartment or either age Ž P ) 0.05..
H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134 Table 2 Kinetic constants for
14
131
C-glutamate in whole brain homogenate, capillary depleted supernatant, and pellet containing endothelium
Homogenate Žwhole brain. Supernatant Žbrain parenchyma. Pellet Žendothelium.
Age
Km ŽmM.
Vmax Žnmol miny1 gy1 .
Kd Žml miny1 gy1 .
Adult Neonate Adult Neonate Adult Neonate
0.024 " 0.016 0.031 " 0.013 0.061 " 0.034 0.054 " 0.023 0.056 " 0.041 0.056 " 0.025
0.215 " 0.025 0.382 " 0.031 0.219 " 0.024 0.358 " 0.045 0.077 " 0.021 0.092 " 0.021
0.11 " 0.17 0.15 " 0.22 y0.29 " 0.36 y0.11 " 0.24 y0.05 " 0.13 0.22 " 0.041
))
)
Values are mean " S.E.M., n s 28. ) P - 0.01, ) ) P - 0.001, significant difference from adult values. K d was not significantly different from zero in any compartment or either age Ž P ) 0.05..
mated from the line of best fit using Eq. Ž6.. These are shown in Table 1 Žaspartate. and Table 2 Žglutamate.. For both amino acids, whole brain K m did not change with maturity Ž P ) 0.05., whereas Vmax reduced by approximately 50% with age Ž P - 0.01. from neonatal values of 0.260 nmol miny1 gy1 for aspartate and 0.382 nmol miny1 gy1 for glutamate. The diffusion constant, K d , was not significantly different from zero Ž P ) 0.05., suggesting insignificant diffusion of these amino acids across the BBB at either age. Kinetic parameters were then calculated separately for the endothelium containing pellet and the capillary depleted supernatant Žbrain parenchyma.. As can be seen in Tables 2 and 3, the supernatant kinetics showed the same pattern as whole brain. The calculated K m values were the same in all compartments including pellet Ž P ) 0.05., and in the two age groups. However, the supernatant Vmax showed age related change, being higher in the neonate than the adult Ž P - 0.01. for both amino acids. In addition, the glutamate Vmax into supernatant and whole brain was greater than that for aspartate for both age groups Ž P 0.05.. The pellet Vmax was significantly smaller than the supernatant Vmax for both amino acids Ž P - 0.001. and did not change with age Ž P ) 0.05., at around 0.07–0.09 nmol miny1 gy1 for both amino acids and ages. 3.5. Kinetic constants at the blood–CSF barrier The Kinetic parameters calculated for the entry of these amino acids into CSF are shown in Table 3. As was seen
for brain, there was no significant change in K m with age Ž P ) 0.05., but the Vmax declined with maturity by 50% for aspartate Žfrom 0.271 nmol miny1 gy1 . and by 65% for glutamate Žfrom 0.670 nmol miny1 gy1 . Ž P - 0.001.. In the neonate, but not the adult, glutamate Vmax was greater than aspartate Vmax Ž P - 0.01.. Unlike brain, CSF K d was significant for both amino acids, and higher for glutamate than aspartate Ž P - 0.05.. However, this did not change with age Ž P ) 0.05..
4. Discussion w 3 HxMannitol was used as a vascular space marker to correct for perfusate trapping in the vascular lumen after perfusion. It also acts a probe to monitor the passive permeability of the BBB, situated at the level of the brain capillary endothelium, and the blood–CSF barrier, situated at the choroid plexus epithelium. We have previously shown that BBB permeability to mannitol does not change with age using multiple time point data to calculate Kin for mannitol w26x and this is in agreement with the present studies and those of Daniel et al. w6x. The permeability of the blood–CSF barrier, however, did change with age in these studies, and R CSF reduced significantly from 0.023 to 0.012 ml gy1 with maturity. This reduction may represent ‘‘tightening’’ of the blood–CSF barrier, although there is little morphological evidence for this from other studies w19x. More likely it represents reduced surface area or pore number relative to the size of the brain w14x.
Table 3 Amino acid kinetics for neonate and adult CSF uptake
w 14 CxAspartate w 14 CxGlutamate
Age
Km ŽmM.
Vmax Žnmol miny1 mly1 .
Kd Žml miny1 mly1 .
Adult Neonate Adult Neonate
0.045 " 0.031 0.059 " 0.014 0.040 " 0.021 0.070 " 0.019
0.134 " 0.050 0.271 " 0.045 0.202 " 0.032 0.670 " 0.105
0.281 " 0.094 0.417 " 0.089 0.587 " 0.068 0.744 " 0.165
)
))
Values are mean " S.E.M., n s 27–32. ) P - 0.05, ) ) P - 0.001, difference from adult. K d , was significantly different from zero Ž P - 0.001. for both aspartate and glutamate.
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H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134
However, using in situ perfusion incorporating w 3 Hxmannitol, it was possible to separate and correct for these passive permeability properties when studying amino acid transport. There are numerous data on the kinetic constants for acidic amino acid transport into various CNS preparations. Kinetically, the transport systems for glutamate and for aspartate tend to be similar, for example in synaptosomes K m and Vmax values are around 10 m M and 10 nmol miny1 mgy1 protein w10x. Astrocytes, which regulate the brain interstitial amino acid composition, also have high affinity uptake of neurotransmitter amino acids w7,12,15,16x. Depending on species and type of amino acid transport system investigated, the kinetic constants at the BBB show a variety of developmental responses. For example, the K m for tryptophan is not different in young adult rats compared to neonates, but the capacity of transport declines with age w27x. K m values are similar when calculated for neonatal and adult arginine transport in rabbits w3x, but neutral amino acid transport demonstrates age-related changes w4x. The kinetic constants for aspartate and glutamate transport into the brain are shown in Tables 1 and 2 for both adults and neonates. The half saturation constants for brain uptake were not different with age for either amino acid, and in addition, did not differ according to the brain compartment studied. With plasma aspartate levels of approximately 0.02 mM in adult and 0.03 mM in neonates w9x, the carrier would be working at less than half saturation at both ages, and increasing plasma aspartate would result in greater transport into brain. Glutamate uptake would be more than half saturated at normal plasma concentrations of G 0.1 mM. Because of this, there may be greater competition for uptake by glutamate resulting in more blood to brain transport compared to aspartate, at both ages. Unlike the K m values, the transport capacity Ž Vmax . into whole brain was significantly reduced with age, and adult Vmax was approximately half that of the neonate. The greater capacity for transport combined with higher plasma levels of amino acid in the neonate would result in greater uptake into young brains compared to adults. Most of the uptake would be into brain parenchyma since the Vmax of the endothelial portion of brain was smaller than that for the supernatant. It is of interest that the pellet Vmax did not change with age, supporting the finding that amino acid sequestration by capillary endothelial cells does not account for the greater brain uptake in the neonate w1x. The diffusion constants K d , for aspartate and glutamate into brain were not significantly different from zero, and did not change with age consistent with the mannitol data. Eq. Ž6. can be applied to predict amino acid flux into the brain from blood under ideal conditions, with no competing amino acid. For comparison by age, this approach has been applied for both neonatal and adult rats
Table 4 Combined rates of acidic amino acid flux, available to brain tissue directly via the BBB and indirectly across the blood–CSF barrier
Adult Neonate
Brain CSF Brain CSF
Weight Žg.
Flux Žnmol miny1 gy1 . Aspartate
Glutamate
2.12"0.07 0.228"0.043 0.58"0.02 0.072"0.009
0.03
0.23
0.09
0.41
Fluxes were calculated using the kinetic constant ŽTable 1Table 2Table 3. based on plasma amino acid concentrations of glutamate Ž0.1 mM. and aspartate Ž0.02 and 0.03 mM.. Values for brain weights and CSF volumes Žassuming 1 mls1 g. are means"S.E.M., ns18–22.
ŽTable 4.. The predicted influx for aspartate was calculated in the adult, 0.022 nmol miny1 gy1 , and in the neonate 0.074 nmol miny1 gy1 . The corresponding values for glutamate were 0.210 and 0.361 nmol miny1 gy1 for adult and neonate. Thus, the calculated possible influx for both amino acids is greater in the neonate. These adult flux values compare well with previous data for aspartate brain uptake in the rat, 0.144 nmol miny1 gy1 w3x, even though the earlier measurements were made in the presence of competing amino acids and without the same correction for vascular space as we have used. K m values at the blood–CSF barrier were similar to those at the BBB ŽTable 3. and did not change with age. The Vmax values were also similar, with the exception of neonatal glutamate Vmax which was nearly twice the brain Vmax value. However, the developmental changes seen were the same at both barriers, and Vmax fell by more than 50% with maturity. There was, however, a striking difference in the diffusion constants Ž K d . for the two barriers, being not different to zero at the BBB, but significant at the blood–CSF barrier. There was some indication that K d at the blood–CSF barrier might reduce with maturity, but any change with age failed to reach significance. Taking into account the diffusional component, fluxes for aspartate and glutamate into CSF were calculated ŽEq. Ž6.., and exceeded those into brain ŽTable 4.. The predicted fluxes were 0.04 and 0.11 nmol miny1 gy1 for aspartate in adult and neonate CSF, respectively, and 0.20 and 0.53 nmol miny1 gy1 for glutamate. Although acidic amino acids have much lower influx rates into brain compared to the neutral and basic amino acids ŽTable 5., by characterising the kinetics of this group of amino acids during development we have shown that there is a substantial entry across the blood–CSF barrier, especially in the immature brain, in addition to entry across the BBB. Factors which may affect amino acid entry into the brain are the rate of cerebral blood flow, the rate of brain parenchymal cell uptake, the plasma concentration of amino acids and competitors, and the blood to brain permeability. Although cerebral blood flow rates increase with age w26x
H. Al-Sarraf et al.r DeÕelopmental Brain Research 102 (1997) 127–134 Table 5 BBB amino acid transport systems measured in the adult rat Amino acid transport system
Neutral Basic Acidic a
Representative substrate
Phenylalanine a Lysine a Aspartate Glutamate
Km
Vmax
ŽmM.
Žnmol miny1 gy1 .
0.12 0.10 0.10 0.02
30 6 0.02 0.22
Values obtained from Oldendorf, 1971 w21x.
this is unlikely to affect brain uptake for these slowly penetrating acidic amino acids. Brain parenchymal cell uptake, estimated in glial and synaptosomal preparations w10,11,20x show Vmax values more than 1000 times greater than the Vmax at the BBB in both neonates and adults suggesting that BBB transport is the rate-limiting step for brain accumulation of these amino acids, and not capacity of brain cell uptake. The greatest contributors to high neonatal brain amino acid levels are therefore likely to be elevated plasma concentrations plus greater Vmax . As the brain and choroid plexuses grow, they may not be accompanied by an increase in the number of transporters, so resulting in a lower number of carriers per unit mass in the adult compared to the neonate and accounting for the low Vmax in the adult. The possibility of structural changes in these carrier systems appears remote since the carrier operates with a similar affinity in both the immature and the mature brains.
Acknowledgements We gratefully acknowledge the support of the Wellcome Trust, and sponsorship for HA-S by the Kuwait Foundation for Science.
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