Changes in kinetics of amino acid uptake at the ageing ovine blood–cerebrospinal fluid barrier

Neurobiology of Aging 33 (2012) 121–133

Changes in kinetics of amino acid uptake at the ageing ovine blood–cerebrospinal fluid barrier R.L. Chen ∗ , J.E. Preston Pharmaceutical Science Division, School of Biomedical & Health Sciences, King’s College London, London SE1 1UL, UK Received 28 May 2009; received in revised form 15 January 2010; accepted 19 January 2010 Available online 5 February 2010

Abstract Amino acids (AA) in brain are precisely controlled by blood–brain barriers, which undergo a host of changes in both morphology and function during ageing. The effect of these age-related changes on AA homeostasis in brain is not well described. This study investigated the kinetics of four AA (Leu, Phe, Ala and Lys) uptakes at young and old ovine choroid plexus (CP), the blood–cerebrospinal fluid (CSF) barrier (BCB), and measured AA concentrations in CSF and plasma samples. In old sheep, the weight of lateral CP increased, so did the ratio of CP/brain. The expansion of the CP is consistent with clinical observation of thicker leptomeninges in old age. AA concentrations in old CSF, plasma and their ratio were different from the young. Both Vmax and Km of Phe and Lys were significant higher compared to the young, indicating higher trans-stimulation in old BCB. Cross-competition and kinetic inhibition studies found the sensitivity and specificity of these transporters were impaired in old BCB. These changes may be the first signs of a compromised barrier system in ageing brain leading increased AA influx into the brain causing neurotoxicity. © 2010 Elsevier Inc. All rights reserved. Keywords: Amino acid; Choroid plexus; Kinetic; Ageing; CSF

1. Introduction Amino acids (AA) have a number of roles in brain as neurotransmitters, neurotransmitter precursors and building blocks of peptides and protein (Gietzen and Rogers, 2006; Sanacora et al., 2008). The brain depends on a diverse array of AA for normal development and function. Imbalances of AA profoundly influence brain function, as shown by the irreversible mental retardation that occurs in phenylketonuria (Giovannini et al., 2007; Waisbren et al., 2007) and maple syrup urine disease (Chuang et al., 2006; PessoaPureur and Wajner, 2007) and by the neuronal degeneration and death that occurs with excessive excitotoxic AA release in hypoxia, hypoglycemia, ischaemia, and seizures (Schwarcz and Meldrum, 1985; Beal, 1992; Deutsch et al., 2001; Tannenberg et al., 2004; Tilleux and Hermans, 2007). AA are charged molecules under physiological conditions, and entry or removal from brain is controlled by AA transporters at ∗

Corresponding author. Tel.: +44 02078486161; fax: +44 02078486569. E-mail address: [email protected] (R.L. Chen).

0197-4580/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2010.01.015

brain barriers, which comprise the capillary endothelial cells of the blood–brain barrier (BBB), and the choroid plexus (CP) epithelial cells of the blood–cerebrospinal fluid (CSF) barrier (BCB) (Smith, 2000; Ohtsuki and Terasaki, 2007). AA in CSF are about one-third or less than in plasma (Hawkins et al., 2006), which is mostly caused by asystemical distribution of AA transporters on the two sides of brain barriers: AA transport from brain to blood was about 8–12 times greater than from blood to brain (Knudsen et al., 1990). This is important as many AA are neurotransmitters or neuromodulators (Xiang et al., 2003). As most neurodegenerative diseases occurred in old ages, there is a need for research on AA homeostasis in the central nervous system (CNS) during ageing to improve our understanding of disease conditions as well as ageing. However, there are few studies on whether ageing CP can handle AA correctly, i.e. maintain AA concentration in the brain/CSF. Aging is associated with decrease in sizes of CP epithelial cell layer losing about 11% in height between infancy and 88 years (Serot et al., 2000), and a reduction in the nucleus:cytoplasm ratio with the development of adaptive

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R.L. Chen, J.E. Preston / Neurobiology of Aging 33 (2012) 121–133

rearrangements (Babik, 2007). The epithelial basement membrane was coarser, thicker and more irregular into old age (Serot et al., 2001). The epithelia have cellular inclusions that increase in number with age including the ubiquitous lipofuscin age-pigment (Wen et al., 1999) and Biondi ring tangles (Eriksson and Westermark, 1986). Ageing-related reductions in Na+ –K+ -ATPase, Na+ K+ –2Cl− co-transporter (Cottrell et al., 2001), as well as in carbonic anhydrase II and aquaporin I also occur (Masseguin et al., 2005). These morphological changes in normal ageing CP might lead to alterations in CP functions, such as decreases in CSF secretion and turn-over, protein synthesis and CSF–blood barrier transport and/or clearance of substances from CSF (Preston, 2001; Preston et al., 2005; Chen et al., 2005, 2008, 2009). This study continuously investigated the ageing-related functional changes in the CP by studying kinetics of 4 AA (Leu, Phe, Ala, Lys) uptakes at in situ perfused young and old ovine CP, and explored their contributions to AA concentration in CSF. The sheep was chosen for study, because of the longer lifespan compared to rodents, the great CSF volume obtained, and the well established and validated for the AA transport methodologies (Preston et al., 1989; Segal et al., 1990; Preston and Segal, 1990, 1992).

2. Methods All procedures were within the guidelines of the Animals (Scientific procedures) Act, 1986, UK. Sixty Clun Forest strain female sheep between 1 and 10 years old were used, and divided into 2 groups: aged 1–2 years young adult (30) and 7–10 years old sheep (30). The sheep were anaesthetized with i.v. thiopentone sodium (20 mg kg−1 ) and injected with heparin (1000 U kg−1 ). CSF was collected from the cisterna magna by a needle puncture and blood was taken from jugular veins. Both CSF and blood were centrifuged at 13,000 × g for 10 min to eliminate any cells and other insoluble material. CSF contaminated with blood was discarded. Thereafter the sheep were exsanguinated via the carotid artery and the head removed. 2.1. Single pass, unidirectional uptake AA in in situ CP perfusion This technique was previously described (Segal et al., 1990). Briefly, the brain was removed from skull intactly, and the circle of Willis supplying the choroidal arteries to each lateral ventricle CP was cannulated and perfusion commenced with ringer (in mM NaCl 123, KCl 4.8, NaH2 PO4 1.22, CaCl2 2.4, MgSO4 1.22, NaHCO3 25) containing 4% bovine serum albumin (BSA) to maintain colloid osmotic pressure. The ringer was warmed to 37 ◦ C and gassed with 95% O2 and 5% CO2 . The ringer outflow was collected from the Great vein of Galen, into which the veins from each CP flow. The cerebral hemispheres were opened to gain access to the CSF side of the plexuses, and the ventricles were super-

fused with mock CSF (in mM NaCl 148, KCl 2.9, NaH2 PO4 0.25, CaCl2 2.5, MgCl2 1.8, NaHCO3 26) which was also pre-warmed to 37 ◦ C and gassed with 95% O2 /5% CO2 . In 9 sheep (4 young and 5 old), the choroidal arteries were torn when the brain was taken from the skull and the CP could not be cannulated and perfused. In the rest, when the CP was successfully perfused, the plexuses were stable and secreted CSF typically up to 5 h. After half hour isotope free perfusion, a 100-␮l bolus containing 14 C-Leu (12.3 kBq, 10.5 ␮M) or 14 C-Phe (46.25 kBq, 25.13 ␮M) or 14 C-Ala (18.5 kBq, 83.33 ␮M) or 14 C-Lys (37 kBq, 33.3 ␮M), and extra-cellular marker 3 H-mannitol (37 kBq, 0.333 ␮M) were added to the perfusion fluid. After 20–25 s, to allow for the clearance of the dead space of the tubing, a ‘run’ of 20 sequential one drop samples of venous effluent were collected over approximately 1 min. A final sample was then collected for a total of 4 min to account for any backflux of tracer and the flow rate determined by weight. Uptake of AA relative to mannitol in each drop was calculated and the maximal cellular uptake (Umax ) determined from samples where the 14 C and 3 H activities had reached peak levels. The Unet (the uptake values from all the single drops and the final 4-min collection) and any backflux (Umax − Unet ) were also calculated (Preston et al., 1989; Segal et al., 1990). For kinetic analysis of AA uptake, plexuses were perfused with different concentrations of unlabelled AA (up to 600 ␮M). Since the injectate bolus undergoes some mixing with the Ringer before reaching the plexuses, the unlabeled AA concentration reaching the CPs was estimated by calculating a dilution factor (Segal et al., 1990). The flux was calculated from: Flux = −F × ln(1 − Umax )s

(1)

where F was the perfusate flow rate (ml min−1 g−1 ) and s (␮M) was the final AA concentration. Plots of concentration versus flux were fitted by non-linear regression analysis (GraphPad Prism4, CA, USA) to calculate the following equation:   Vmax × s Flux = + Kd × s (2) Km + s where Km (␮M) was the half-saturation constant, Vmax (pmol min−1 g−1 ) was the maximal flux, Kd (ml min−1 g−1 ) was the cellular diffusion rate, and s was the final AA concentrations (␮M). For cross-competition study, the CPs were perfused with 5 mM non-metabilzed analogues 2aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH) or N-methylaminoisobutyric acid (MeAIB). For kinetic inhibition study, the CP were perfused with 0.05, 1, or 5 mM of other unlabelled AA. The integrity of radio-label was studied by applying dual labeled (both 14 C and 3 H) AA and mannitol into 100 ␮l inject bolus. The Umax , Unet , and Backflux were calculated as above.

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2.2. AA concentrations in CSF and plasma

Table 1 Ovine choroid plexus, brain and body weights.

Four pairs of young and old CSF and plasma samples were sent to Alta Bioscience, University of Birmingham for AA quantification (www.Altabioscience.bham.ac.uk). The analytical technique is based on the method first described by Spackman et al. (1958). Briefly, all samples were deproteined by adding of an equal volume of 5% trichloroacetic acid followed by centrifugation. The AAs were separated by ion exchange chromatography on a strong cation exchange resin. A series of lithium citrate buffers giving a gradient of increasing pH, were used to obtain resolution of all of the physiological AAs. Acidic AAs such as aspartic acid elute first, followed by the neutrals, then the basic amino acids such as Arg. After separation, the AAs were reacted post-column with a stream of ninhydrin reagent for 5 min at 125 ◦ C, then the absorbance of the coloured complex was detected at both 660 and 460 nm. The chromatogram was transferred to an ‘Atlas’ integration and data handling system, where the peaks were integrated and the areas compared against those from a separation of a standard calibration mixture.

Parameters

Young sheep

Age (years) Choroid plexus (g) Brain (g) Body (g) Ratio (%) (choroid plexus: brain)

1.48 0.134 106.1 68.66 0.127

2.3. Materials (11.8 GBq mmol−1 ), 14 C-Phe (18.1 GBq 14 C-Lys (5.85 GBq mmol−1 ), −1 (11.4 GBq mmol ) were obtained from GE Healthcare (Amersham Place, UK). 3 H-mannitol (740 GBq mmol−1 ) was obtained from MP Biomedicals, Inc. (Irvine, CA, USA). All other materials were from Sigma unless stated. These commercially obtained materials were ultra-pure grade. 14 C-Leu

mmol−1 ),

14 C-Ala

All values were expressed as mean ± SEM. ANOVA and unpaired t-test as appropriate were used to compare means of different age sheep. Values of p < 0.05 were considered statistically significant.

3. Results 3.1. Baseline of unidirectional uptake of Leu, Phe, Ala and Lys into the CP The lateral CPs were larger in old sheep compared to the young, while the brain and body weights were not different. Therefore the ratio of CP/brain (in weight) increased significantly in old sheep (Table 1). Flow rates were similar between the two groups, so were flow rates corrected by CP weight (Table 2). The extraction of AA was greater than that of mannitol, giving a net cellular uptake for AA. Overall the values for Unet were similar to those for Umax indicating negligible backflux. Between the 2 groups, there were no statistical difference in either the Unet or the Umax of all the 4 tested AAs (Table 2).

0.08 (8) 0.02 (8) 3.48 (8) 3.18 (8) 0.02 (8)

7.04 0.272 100.9 65.5 0.274

± ± ± ± ±

0.26 (8)* 0.03 (8)* 4.93 (8) 5.69 (8) 0.03 (8)*

* Means p < 0.05, in comparison of the young and old. Number of sheep given in parentheses.

3.2. Kinetics parameters of unidirectional uptake of Leu, Phe, Ala and Lys The uptake was then examined in the presence of unlabeled AA. The Umax decreased as the concentration of unlabeled AA increased in both young and old CP, indicating carrier-mediated AA uptake into the CP. However, in a few of experiments especially those with Phe and Lys, the increase of unlabeled AA concentration after 100 ␮M did not significantly decrease the Umax , therefore raised the flux which is in proportion to the concentration of total AA (refer to Eq. (1)). This phenomenon was more obvious in the old CP perfusion than the young. Fig. 1 is the plot of AA concentration versus the flux and shows that both Vmax and Km of Phe and Lys are significant higher in the old animals than the young, while those of Leu and Ala are not different (Fig. 1) (Table 3). Table 2 Amino acids uptake at basolateral side of ovine choroid plexus. Parameters

2.4. Data analysis

± ± ± ± ±

Old sheep

Young sheep

Old sheep

1.25 0.18 3.71 0.71

± ± ± ±

0.07 (26) 0.01 (26) 0.38 (26) 0.10 (26)

7.90 0.24 2.93 0.73

± ± ± ±

0.28 (25)* 0.01 (25)* 0.31 (25) 0.08 (25)

Umax (%) Leucine Phenylalanine Lysine Alanine

29.52 33.78 23.15 16.98

± ± ± ±

2.44 (13) 4.13 (16) 3.70 (10) 2.42 (12)

32.85 30.19 22.07 20.64

± ± ± ±

2.68 (12) 5.06 (11) 2.92 (12) 2.99 (11)

Unet (%) Leucine Phenylalanine Lysine Alanine

26.02 30.66 16.75 18.56

± ± ± ±

2.79 (11) 4.15 (14) 3.21 (10) 2.41 (10)

30.11 25.14 25.22 24.63

± ± ± ±

3.70 (11) 4.02 (10) 3.48 (12) 4.03 (10)

Backflux (%) Leucine Phenylalanine Lysine Alanine

17.26 13.28 22.31 6.72

± ± ± ±

8.44 (11) 4.82 (14) 7.87 (10) 3.37 (10)

14.81 11.12 6.25 9.27

± ± ± ±

7.51 (11) 4.54 (10) 4.71 (12) 7.17 (10)

Flux (pM min−1 g−1 ) Leucine Phenylalanine Lysine Alanine

8.37 7.88 8.57 11.63

± ± ± ±

1.14 (12) 1.55 (12) 1.54 (8) 1.19 (11)

9.23 6.36 6.69 12.7

± ± ± ±

2.16 (12) 1.44 (7) 0.87 (10) 2.79 (9)

Age (years) Choroid plexus (g) Flow rate (ml min−1 g−1 ) Flow rate (ml min−1 )

*

Means p < 0.05, in comparison of the young and old. Number of sheep or experiments given in parentheses.

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Fig. 1. Kinetics study of AA uptake into young and old CP. Phe and Lys uptakes into old CP had significant higher kinetics (both Km and Vmax ) than the young, and while Leu and Ala uptakes into old CP were similar to the young. Solid symbols were from young preparation while open symbols were from the old.

3.3. Cross-competition study

The Lys uptake was not changed with either BCH or MeALB in either age group (Table 4).

In the young CP, 5 mM BCH completely inhibited the Phe uptake, significantly reduced the Leu uptake and did not affect the Ala uptake (Table 4). MeAIB did not significantly interfere with any AA uptakes although had some inhibition on the Ala uptake (not significant). In the old CP, 5 mM BCH significantly inhibited the Phe (but not completely) and Leu uptake and did not affect the Ala uptake (Table 4). MeAIB did not interfere with any AA uptakes.

3.4. Kinetics inhibition study In the young CP, Leu significantly inhibited both Phe and Ala uptakes, while only Phe but not Ala significantly affected the Leu uptake (Fig. 2). In the old CP, Leu and Phe cross-inhibited each other but with less effect than in the young CP perfusion and there was no interaction between Leu and Ala (Fig. 2).

Table 3 Kinetic study of amino acids uptake at basolateral side of ovine choroid plexus.

Table 4 Effects of BCH and MeAIB on AA uptakes at basolateral side of ovine CP.

Parameters

Flux (pmol min−1 g−1 )

Young sheep 7.30 6.27 11.95 7.76

Young sheep

Old sheep

(nmol min−1 g−1 )

Vmax Leu Phe Ala Lys

6.54 5.03 5.30 3.43

± ± ± ±

1.11 (9) 1.27 (5) 2.27 (4) 1.47 (5)

6.04 24.39 4.75 69.77

± ± ± ±

1.41 (6) 12.82 (5)* 1.20 (4) 15.85 (4)*

Control Leucine Phenylalanine Alanine Lysine

Km (␮M) Leu Phe Ala Lysine

2.79 1.28 4.28 2.28

± ± ± ±

1.71 (9) 1.43 (3) 3.75 (4) 1.98 (5)

2.61 36.05 3.93 30.14

± ± ± ±

0.78 (6) 33.28 (6)* 3.38 (6) 14.91 (4)*

BCH (5 mM) Leucine Phenylalanine Alanine Lysine

0.88 ± 0.45 (5)* 0 (4)* 9.83 ± 4.65 (3) 5.62 ± 1.46 (3)

−0.49 0.08 −0.45 0.35

± ± ± ±

0.27 (9) 0.03 (5) 0.58 (4) 0.57 (5)

0.14 −0.02 0.47 −0.14

± ± ± ±

0.23 (6) 1.52 (6) 0.53 (6) 0.21 (4)

MeAIB (5 mM) Leucine Phenylalanine Alanine Lysine

5.45 2.86 5.55 6.20

Kd (␮l min−1 g−1 ) Leu Phe Ala Lys

* Means p < 0.05, in comparison of the young and old. Number of experiments

given in parentheses.

*

± ± ± ±

± ± ± ±

1.35 (7) 0.93 (8) 3.43 (7) 0.45 (4)

1.58 (4) 0.07 (3) 1.41 (4) 0.47 (3)

Old sheep 6.18 5.10 9.54 6.51

± ± ± ±

1.42 (3) 0.96 (8) 1.80 (6) 1.25 (8)

0.93 3.21 9.13 7.09

± ± ± ±

0.37 (3)* 0.70 (3)* 2.69 (3) 0.53 (3)

4.96 2.03 7.70 7.82

± ± ± ±

1.43 (3) 0.30 (3) 2.64 (6) 1.73 (5)

Means p < 0.05, in comparison of the control. Number of experiments given in parentheses.

R.L. Chen, J.E. Preston / Neurobiology of Aging 33 (2012) 121–133

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Fig. 2. Cross-competition of AA uptake into young and old CP. In young sheep, Leu uptake was significantly inhibited by Phe and vice versa. Ala uptake can be inhibited by 5 mM Leu. In old sheep, Leu and Phe interacted each other significantly but only at highest concentration (i.e. 5 mM). *Means p < 0.05, **indicates p < 0.01 in comparison of the control value. Solid symbols were from young preparation while open symbols were from the old. The presence of Leu, Phe, Ala, Lys were represented, respectively, by symbols (, , 䊉, .).

Lys uptake was not changed with unlabeled other 3 AAs (Leu, Phe and Ala) (Fig. 2) in either young or old CP.

Arg and Cys in plasma, and decreases of CSF/plasma ratios of Lys, Arg, Cys, Gln and Ser.

3.5. Integrity of radio-isotope 4. Discussion Fig. 3 shows an example of studies on the integrity of the radio-isotopes. There was the same extraction of 14 C versus 3 H-lysine, and 14 C versus 3 H-mannitol. Umax for 3 H-AA/14 C-mannitol, and 14 C-AA/3 H-mannitol was similar (Fig. 3). 3.6. AA concentration in CSF and plasma There are some changes in AA concentration in CSF and plasma, and the ratio of CSF/plasma between these two groups (Table 5). There were age-related decreases of Phe, Cys, Trp, Gln and Ser but increase of Glu in CSF, increases of

The AA transport across the plasma membrane of mammalian cells has been studied for several decades. Many of these studies are on intestine and kidney epithelium and few is on brain barriers (Palacin et al., 1998; Mann et al., 2003; Broer, 2008). AA transport has been characterized as saturable and stereospecific (Oldendorf, 1971, 1973), and the transport systems differ in substrate specificity, inhibition by model ligand and Na+ -dependence (Broer, 2008). In the BBB, four Na+ independent facilitative carriers were identified on the luminal membrance: L1, y+ , xG− and n (Hawkins et al., 2006). On the abluminal side, there were five Na+ dependent

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Fig. 3. An example of the recovery of 14 C-mannitol vs. 3 H-mannitol (A), or 14 C-Lys vs. 3 H-Lys (B), or 14 C-mannitol vs. 3 H-Lys (C), or 14 C-Lys vs. 3 H-mannitol (D), in one run of 20 venous samples plotted as a percentage of total radioactivity injected and against time. There were almost identical curves in A and B. The lower recovery of Lys relative to mannitol in C and D indicates cellular uptake of Lys at the basolateral face of the CP. The net uptake of Lys was not different between C and D.

facilitative carriers: A (Sanchez del Pino et al., 1992), ASC (Tayarani et al., 1987), N (Lee et al., 1998), LNAA (O’Kane and Hawkins, 2003) and EAAT (O’Kane et al., 1999), along with L1, y+ (Sanchez del Pino et al., 1995). The information of AA transport on the BCB is little. We have developed and validated an in situ ovine CP perfusion to characterize a number of AA transport from either side of the CP (Preston et al., 1989; Segal et al., 1990; Preston and Segal, 1990, 1992). This study applied this technique to investigate if there was any age-related difference in 4 AA (Leu, Phe, Lys and Ala) uptake from luminal side of ovine CP. The 4 AAs chosen represent both essential and non-essential AA utilizing different transporter systems (Broer, 2008). Leu and Phe are large essential neutral AAs (NAA) with branched or aromatic side chain, respectively, while Ala is a small non-essential NAA. Lys is a nutritionally important essential cationic AA. The movement of essential NAA from blood to brain is greater than non-essential NAA (Oldendorf, 1971), with the movement of the latter minimal (Oldendorf and Szabo, 1976). This study has 4 major findings: (1) the weight of CP increased during ageing but not the brain resulting significant higher ratio of CP/brain in ageing; (2) both Km and Vmax of Phe and Lys uptakes in the old sheep were significantly increased, suggesting higher trans-stimulation; (3) cross-competition and kinetic inhibition studies indicated the impairment of sensitivities and specificities of AA transporters in the CP during ageing; (4) there were some changes in AA concentration in old CSF, plasma and their ratio compared to the young. The weight of old CP doubled compared to the young, which is in consistent with ageing-related expanded ventric-

ular system (Gonzalez-Soriano et al., 2001) and with clinical observation of thicker leptomeninges in old age (Esiri, 2007). The expansion of the CP in old age might be caused by most frequently reported ageing-related alterations in the CP, where thickening of capillary walls and basement membrane were present in the old rat compared to the young (Masseguin et al., 2005). However, the epithelial cell layer (the main functional cells in the CP) becomes flattened in humans, losing about 11% in height by age 88 (Serot et al., 2000). We did not find ageing-related changes in brain weight in health ageing sheep, which is consistent with studies in non-human primates (Esiri, 2007). In human, it is widely accepted that brain size is decreased with increasing age (Esiri, 2007). There is a small loss of brain weight of about 0.1% per year, but more rapid loss thereafter (Anderson et al., 1983). There is now a large amount of evidence from imaging studies that adult brain shrinks with age (Ikram et al., 2008; Farrell et al., 2009; Gonoi et al., in press). Gray matter decreases steadily after adolescence, whereas white matter shows a peak around age 40 and decreases thereafter (Allen et al., 2005; Abe et al., 2008). Studies in human are complicated by the fact that most elderly brains are affected by the pathological changes of amyloid plaque and neurofibrillary tangle formation (Esiri, 2007). They are also affected by cerebrovascular disease (MRC CFAS, 2001). Sheep have been extensively used in the field of neuropathology including the neurodegenerative prion disease scrapie (Hunter, 2007) and as a model of brain trauma (Anderson et al., 2003). Sheep are one of few animals that develop neurofibrillary tangles during normal ageing (Nelson et al., 1993, 1994; Nelson and

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Table 5 AA concentrations in young and old ovine CSF, plasma and their ratios. AA Essential AA Histidine

CSF (␮M)

Plasma (␮M)

Ratio (%)

(Y) (O)

19.02 ± 2.57 14.73 ± 2.48

97.63 ± 15.79 81.92 ± 9.83

19.75 ± 1.28 17.83 ± 1.49

Isoleucine

(Y) (O)

4.03 ± 0.33 4.50 ± 0.45

93.28 ± 18.40 120.22 ± 15.50

4.67 ± 0.65 3.79 ± 0.17

Leucine

(Y) (O)

11.25 ± 0.46 12.02 ± 1.37

149.75 ± 19.09 174.00 ± 24.30

7.93 ± 1.15 7.00 ± 0.33

Lysine

(Y) (O)

23.88 ± 4.34 19.93 ± 2.00

126.05 ± 19.71 148.50 ± 16.52

19.02 ± 2.77 13.52 ± 0.76§

Methionine

(Y) (O)

1.37 ± 0.25 1.65 ± 0.53

18.52 ± 3.81 26.10 ± 4.02

8.63 ± 2.20 6.16 ± 1.73

Phenylalanine

(Y) (O)

10.95 ± 0.38 8.48 ± 0.90*

63.75 ± 8.24 58.83 ± 4.68

18.28 ± 2.87 14.47 ± 1.17

Threonine

(Y) (O)

24.17 ± 6.02 26.20 ± 4.80

74.03 ± 21.45 94.88 ± 19.59

34.15 ± 2.45 28.67 ± 3.00

Tryptophan

(Y) (O)

0.34 ± 0.02 0.21 ± 0.04*

21.63 ± 4.45 28.63 ± 2.53

0.76 ± 0.44 0.68 ± 0.17

Valine

(Y) (O)

2.98 ± 0.96 2.70 ± 0.90

216.25 ± 35.52 263.25 ± 42.49

1.44 ± 0.42 0.98 ± 0.25

(Y) (O)

27.08 ± 2.25 30.42 ± 4.16

279.25 ± 40.37 358.75 ± 45.44

10.41 ± 2.02 8.83 ± 1.34

Arginine

(Y) (O)

24.00 ± 2.82 29.68 ± 2.20

113.30 ± 15.91 213.75 ± 26.76*

21.11 ± 2.15 13.97 ± 3.67*

Asparagine

(Y) (O)

3.58 ± 0.56 4.00 ± 1.06

39.38 ± 8.49 49.93 ± 9.09

9.91 ± 1.51 7.91 ± 1.14

Aspartic acid

(Y) (O)

n/a 1.00 ± 0.58

18.92 ± 7.53 10.79 ± 1.50

n/a 4.34 ± 2.63

Cystine

(Y) (O)

0.60 ± 0.06 0.48 ± 0.02§

Glutamic acid

(Y) (O)

24.02 ± 3.35 33.63 ± 4.91§

199.50 ± 56.40 198.00 ± 42.74

14.97 ± 4.37 19.28 ± 4.16

Glutamine

(Y) (O)

338.50 ± 11.06 270.75 ± 33.42§

296.75 ± 37.20 298.75 ± 29.96

119.23 ± 14.00 91.85 ± 10.11§

Glycine

(Y) (O)

8.85 ± 1.08 6.85 ± 7.81

480.00 ± 81.91 529.50 ± 68.33

1.25 ± 0.42 1.54 ± 0.19

Ornithine

(Y) (O)

5.57 ± 0.66 5.55 ± 1.21

108.18 ± 39.14 124.63 ± 20.65

6.33 ± 1.18 4.36 ± 0.27

Proline

(Y) (O)

n/a n/a

116.95 ± 13.85 119.70 ± 24.96

n/a n/a

Serine

(Y) (O)

24.85 ± 3.37 16.02 ± 2.05*

Taurine

(Y) (O)

1.33 ± 0.12 1.53 ± 0.29

98.93 ± 9.11 130.18 ± 23.58

1.37 ± 0.17 1.28 ± 0.34

Tyrosine

(Y) (O)

12.15 ± 1.40 10.80 ± 1.03

61.80 ± 13.36 64.95 ± 6.09

21.35 ± 2.99 16.77 ± 1.17

Non-essential AA Alanine

0.75 ± 0.10 1.42 ± 0.31§

71.00 ± 12.86 73.45 ± 9.30

Data was expressed as mean ± SEM, n/a means data not available as the AA could not be detected in CSF. * p < 0.05 compared to young. § p < 0.1 compared to young.

84.37 ± 11.81 43.98 ± 15.45§

36.72 ± 4.59 22.35 ± 2.56*

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Saper, 1995, 1996; Braak et al., 1994) and have been recently used as a non-transgenic, human-like model of Alzheimer’s disease to investigate the effects of ovariectomy and subsequent estrogen replacement (Barron et al., 2009). The average life expectancy of sheep was 7.1 years and with a maximum lifespan of 12 years (Davids et al., 1966). The corresponding human data was reported to be approximately 79 years (Western Europe and USA) with a maximum lifespan of 122 years (France) (Preston, 2001). Sheep reach reproductive maturity relatively faster than humans. They can mate at 7–9 months of age (gestation is 5 months), which in human terms may be considered post-pubescent. Based on these findings, sheep ages ranging from approximately 3–6 years are considered as human equivalent of middle age and sheep ages between 7.5 and 9.5 years old may be roughly considered to be equivalent to humans aged 80–100 years. The ratio of CP/brain (in weight) is increased in old sheep, which is in the range of rat’s (Spector and Johanson, 1989). Accompanying with these morphological changes, we have observed some changes in kinetics of the uptake of 4 AA. The entries of these AA into brain needs at least one AA transport system and some by as many as three (Hawkins et al., 2006). The commonest transporters for these AA are system L and y+ (Hawkins et al., 2006). The “L” transport system is a Na+ -independent, BCH sensitive, bi-directional NAA transporter (Christensen, 1990), comprising 4 proteins identified as LAT1–4 (Palacin et al., 2005; Verrey, 2003). LAT1 and LAT2 are members of the solute carrier (SLC) 7 family (Kanai et al., 1998; Mastroberardino et al., 1998; Pineda et al., 1999; Segawa et al., 1999), containing 4F2 antigen (4F2hc, SLC3A2)/CD98 (Kanai et al., 1998; Mastroberardino et al., 1998) which is essential for their functional expression at the plasma membrane (Pineda et al., 1999; Segawa et al., 1999). In brain, LAT1 and LAT2 expression is predominantly in the endothelial cells forming the BBB (Boado et al., 1999; Duelli et al., 2000; Kageyama et al., 2000; Kido et al., 2001; Killian and Chikhale, 2001) and around the lateral edges of the BCB epithelia (Roberts et al., 2008). LAT1 is an important transport route for large NAA entry into the brain (Hawkins et al., 2006) with a high affinity for large NAA with Km values in the ␮M range (Kido et al., 2001; Omidi et al., 2008; Segawa et al., 1999). LAT2 has less affinity for large NAA with Km values in the mM range (Kido et al., 2001) but displays a broader substrate selectivity range than LAT1, accepting also some smaller NAA such as Ala and Gly (Segawa et al., 1999). LAT3 and LAT4 showing the biochemical properties of system L have a monomeric structure and do not require 4F2hc to express functional activity (Babu et al., 2003; Bodoy et al., 2005), and do not exist in either brain endothelial cells or CP epithelial cells. Leu, which is found in large quantities in cerebral proteins (Banos et al., 1973), has the highest influx into the brain of any AAs (Daniel et al., 1977), while Phe has the greatest affinity for the cerebrovascular NAA transport site (Miller et al., 1985; Smith et al., 1987; Hargreaves and Pardridge,

1988). Both Leu and Phe occupy more than half of “L” system transporters (Christensen, 1984). Branched NAA (e.g. Leu) are especially effective in completing with aromatic AA (e.g. Phe) for entry to brain. Consequently, when plasma branched NAA concentration rises, they impair the entry of aromatic AA (Fernstrom and Wurtman, 1972), most which are neurotransmitter or neurotransmitter precursors. Besides the “L” system transport, Leu was found also to utilize “ASC” and “A” system (Pardridge, 1998). Although Phe entry into other tissues might utilize “T” system (Christensen, 1990), this is not the case in brain (Friesema et al., 2008) as Phe uptake into brain is found solely to utilize “L” system (Pardridge, 1998) and Phe is used as a model AA to study “L” system function (Hawkins et al., 2006). Ala is known to be the substrate of several AA transporters including system A, ASC, L, and B0 and the Imio acid carrier (Broer, 2002). System A is distinguished from other Na+ -dependent carriers by its acceptance of MeAIB as a unique substrate (Christensen et al., 1967). Its isoforms SNAT1 and SNAT2 were found in brain including the CP (Melone et al., 2004, 2006; Gonzalez-Gonzalez et al., 2005; Weiss et al., 2005). ASC activity was measured in abluminal membranes of BBB after blocking system A with MeAIB (Tayarani et al., 1987; Hargreaves and Pardridge, 1988). Its isoforms ASCT1 (Weis et al., 2007) and ASCT2 (Broer et al., 1999) were found in brain. Ala is also well known a substrate of LAT2 (Pardridge, 1998). In primary cultures, Ala uptake into astrocytes was largely mediated by the LAT2, whereas the uptake into neurons was mediated by B0 AT2 (Broer et al., 2007). Lys is known to be mediated by 5 transport systems (y+ , + y L, b+ , b0,+ , and B0,+ ) to enter mammalian cells (Palacin et al., 1998). The system y+ is a Na+ -independent transporter for cationic AA, which interacts weakly with NAA in the presence of Na+ (Antonioli and Christensen, 1969). Later other transporters (systems y+ L, b+ , b0,+ , and B0,+ ) have been identified in cells that were previously thought to posses only system y+ activity (Deves and Boyd, 1998; Closs et al., 2006). Recently cationic AA transporters have been cloned (Closs et al., 1993; MacLeod, 1996) which present some, but not all, the features ascribed to system y+ . Lys transport across BBB is mediated exclusively by system y+ (O’Kane et al., 2006). In the CP, the uptake of Lys was significantly inhibited only by other cationic AA (Preston and Segal, 1992). The Km of the 4 AAs in the young sheep is of the order of 1–10 ␮M, consistent with our early studies (Preston et al., 1989; Segal et al., 1990; Preston and Segal, 1990, 1992) and approximately within the normal physiological plasma concentration of AA (Smith and Stroll, 1998). System L at the BBB is saturated by endogenous AA under normal conditions as the Km is smaller than the plasma concentration of NAA (Smith and Stroll, 1998). Each AA must compete for available transport site. The uniquely high affinity (low Km ) of neutral AA transport results in the physical basis of the unique sensitivity of the brain to changes in plasma AA concentration within physiological range (Pardridge, 1983). Large imbal-

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ances in the plasma concentration of one or several NAAs will lead to marked changes in transport and concentrations of all competing AA. For example, a 10-fold elevation of the plasma L-Phe concentration decrease the influxes of completing AAs into brain by >50% but does not alter Phe and other AAs uptake into other tissues such as skeletal muscle (Momma et al., 1987), as the affinity of BBB transport system for NAAs is 10–100-fold greater than that of other tissue (Pardridge, 1983). In old sheep, both Km and Vmax of Phe, Lys uptake into the CP were 2 or 3 times higher than the young. This can be explained by trans-stimulation, in which AA influx is stimulated by accumulated intracellular AA (Sweiry et al., 1991). Trans-stimulation is a characteristic feature of system y+ in a number of cells and tissues (Munck, 1980; White et al., 1982; Stieger et al., 1983; Bogle et al., 1996) and shown in the system L (Kelley and Potter, 1978; Lahoutte et al., 2004). Pan et al. (2002) have demonstrated that an increase of intracellular Ala concentration stimulates L-3 H-Ala uptake in human intestinal Caco-2 cell cultures. In our study, we found trans-stimulation at tissue level (i.e. the CP) and interestingly trans-stimulation was easier to be induced in the old CP compared to the young. To further investigate the possible involvement of multiple transport systems in the uptake process, cross-competition and kinetics inhibition studies were conducted, which showed: (1) In young CP, Phe uptake solely utilized “L” system transport as its uptake was completely inhibited by BCH, which is in a good agreement with a number of studies (Hargreaves and Pardridge, 1988; Sanchez del Pino et al., 1995; Pardridge, 1998; O’Kane and Hawkins, 2003). Phe was suggested as a representative substrate of the facilitative transport system LAT1 (Hawkins et al., 2006). (2) Leu uptake to choroidal epithelial cells appeared to be via more than one system (e.g. L system) given that BCH did not completely inhibit Leu uptake. This is consistent with observation of Partridge et al. (Hargreaves and Pardridge, 1988; Pardridge, 1998), in which Leu utilizes both “ASC” and “A” besides “L” system transport. In our study, Leu uptake was not inhibited by 5 mM MeAIB suggesting that the “A” system makes insignificant contribution to Leu uptake into the CP. (3) Ala uptake was not significantly inhibited by either MeAIB or BCH suggesting that neither “A” system nor LAT2 transports a large amount of Ala into CP, and/or BCH might not be a strong inhibitor for LAT2. (4) Lys uptake at CP utilized different AA transporters from the above-mention ones, and is consistent with the operation of transport y+ (Christensen, 1989). Lys uptake was not inhibited by BCH suggesting the possible presence of system B0,+ at CP and BBB is not significant (O’Kane and Hawkins, 2003). (5) In old CP, BCH less strongly affected Phe and Leu uptake compared to the young. The interaction between

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Leu and Ala was also abolished. These suggest the changes of specificity and sensitivity in AA transporters in old CP, which could be caused by a single nucleotide polymorphisms (Boado et al., 2003) and/or age-dependent oxidation and covalent aggregation (Sadineni and Schoneich, 2007). Moreover, LAT1 expression is regulated by aldosterone (Mastroberardino et al., 1998), argininine-vasopressin and adrenergic agents (Duelli et al., 2000). In this study, mannitol was used as the vascular space marker because it has the similar size as AA and has been used in transport and permeability studies at both the BBB (Ennis et al., 2003) and the BCB (Tenenbaum et al., 2005). Dual labeled (both 14 C and 3 H) AA and mannitol was used in the experiments to assess the integrity of radio-label, as possible breakdown of the labeled AA by enzymes in the CP, or protein incorporation during storage to liberate the 3 H and 14 C which is then taken up by the cells, might be responsible for erroneously high uptake measurements. We have not found any radio-label breakdown in these experiments. Several studies have described AA transport at the BBB during aging, and generated some controversial results. Smith et al. (1987) and Tang and Melethil (1995) found age-dependent differences in BBB transport parameters of tryptophan in rat. In humans, O’Tuama et al. (1991) reported [11 C]-L-methionine transport decreased with advancing age, however, Koeppe et al. (1990) demonstrated no significant decline in [11 C]-aminocyclohexanecarboxylate transport with age. Ito et al. (1995) also found no age-dependent changes in K1, an indicator of AA transport from the blood to the brain, in a dynamic PET study with L-(2-18 F)fluorophenyl-alanine (18 F-Phe). We found higher incidence of trans-stimulation in old CP, which occurred significantly in the AA utilizing sole transport system such as Phe and Lys rather that those utilizing multiple ones such as Leu and Ala. The concentration of AA measured in this study is in a good agreement with others in sheep (Brenton and Gardiner, 1988). AA concentration decreased in old CSF but increased in plasma, resulting significant decrease of their ratios. In rat, there was significant ageing-related decrease and strainrelated changes of CSF AAs (Takasugi et al., 2003). In human, there was ageing-related increase of CSF AA (Ferraro and Hare, 1985), and ageing-related decrease of plasma AA and essential: non-essential AA (Ravaglia et al., 2002). This study has some limitations: AA transport is a complex phenomenon affected by both hormones and neurotransmitters (Grammas et al., 1992). The transport in the CP was affected by both adrenergic and cholinergic activities, and there is evidence of progressive loss of adrenergic and cholinergic innervation of cerebral cortical capillary vessels in ageing and dementia (Mann, 1983). The in situ CP perfusion does not include the influence of nervous system. Other perfusion techniques, e.g. ventriculo-cisternal perfusion, is less invasive and keeps intact nervous system, however, cannot be applied specially on the functions of the CP (Al-Sarraf et al.,

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2000). The in situ ovine CP perfusion only perfuses the CP allowing us to apply precious radio-isotopes in the perfusate to study transport kinetics. In summary, this study has described some age-related changes in ovine lateral CP. The size of the CP was increased so was the ratio of CP/brain. The specificities of AA uptake into the CP was reduced and trans-stimulation was increased in old CP. AA concentrations in old CSF, plasma and their ratios were different from the young. This study predicts that old CP will be vulnerable to plasma hyper-aminoacidemias than the young owing to the higher Km (lower affinity) of AA transport. Transport-induced alteration in brain AA concentrations can lead to changes in neurotransmitters metabolism (Pardridge, 1983) and ultimately influence brain function.

Conflict of interest statement There is no competing interest or conflict of interest.

Disclosure statement All procedures were within the guidelines of the Animals (Scientific procedures) Act, 1986, UK.

Acknowledgement Supported by BBSRC grants G02219 and AG05138.

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