Uptake of [3H]l -serine in rat brain synaptosomal fractions

Brain Research 983 (2003) 36–47 www.elsevier.com / locate / brainres

Research report

Uptake of [ 3 H]L-serine in rat brain synaptosomal fractions Takeshi Takarada, Vladimir J. Balcar 1 , Katsuhiro Baba, Akiko Takamoto, Gabriela B. Acosta 2 , Katsura Takano, Yukio Yoneda* Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Technology, 13 -1 Takara-machi, Kanazawa, Ishikawa 920 -0934, Japan Accepted 13 May 2003

Abstract Accumulation of [ 3 H]L-serine in crude synaptosomal fractions freshly prepared from rat brain has been found to be temperaturesensitive and to consist of both Na 1 -dependent and Na 1 -independent components. The accumulation of [ 3 H]L-serine measured at submicromolar concentrations had a distinct substrate selectivity, different from the uptake of [ 3 H]L-proline, [ 3 H]L-glutamate and [ 3 H]GABA. It was fully inhibited by L-glutamine, L-asparagine, L-cysteine, L-alanine, L-leucine, L-isoleucine, L-tyrosine, L-phenylalanine, L-threonine and by the synthetic marker for the large neutral amino acid transport systems 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, but not influenced by b-alanine, taurine, glycine nor was it inhibited by the marker for the A system, L-2-methylamino isobutyric acid. D-Serine at 1 mM concentration produced no significant inhibition of the accumulation of 10 nM [ 3 H]L-serine. We conclude that 1 L-serine uptake observed in the present study is mediated by at least two distinct transport systems: a Na -dependent one of lower affinity 1 (Km in mM range) and a Na -independent system of higher affinity (Km ¯20–100 mM). Characteristics of [ 3 H]L-serine accumulation displayed at low substrate concentrations suggest that it was mediated neither by the typical ‘A’, nor by the ‘large neutral’, amino acid transport systems but predominantly by transporters belonging to the recently identified LAT ( L-amino acid transporter) family.  2003 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Uptake and transporters Keywords: Small neutral amino acid; Large neutral amino acid; Transport; System A; System L; LAT

1. Introduction The small neutral amino acid L-serine is synthesized from the glycolytic intermediate 3-phosphoglycerate dehydrogenase (3-PGDH) and used for synthesis of proteins, amino acids, membrane lipids and nucleotides. L-Serine is neither an essential amino acid, nor does it seem to act as a neurotransmitter in the mammalian central nervous system (CNS). In a recent study, however, L-serine is shown to have strong trophic actions on cerebellar Purkinje neurons

*Corresponding author. Tel. / fax: 181-76-234-4471. E-mail address: [email protected] (Y. Yoneda). 1 On leave from Department of Anatomy and Histology, The University of Sydney, Australia. 2 On leave from Instituto de Investigaciones Farmacologicas, Consejo Nacional de Investigaciones Cientificas y Tecnicas, Buenos Aires, Argentina. 0006-8993 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03024-5

in vitro [21]. The Bergman glia appears to be the main source of L-serine to Purkinje neurons, because in the cerebellar cortex, Bergman glia expresses 3-PGDH but Purkinje neurons do not [21]. An inborn error in the biosynthesis of L-serine has been implicated as a causal factor in a serious congenital disorder (3-PGDH deficiency) characterized by microcephaly, psychomotor retardation and seizures [17]. While some of the pathological manifestations of the disorder might be explained by inadequate synthesis of proteins and lipids resulting from insufficient supply of L-serine, the nature of the disorder may reflect more specific actions of L-serine in the function of the CNS, perhaps also its role as a precursor for glycine biosynthesis [16]. For example, low micromolar concentrations of L-serine have been shown to exert a potent effect on the morphology of the primary sensory neurons in culture [44] and may also be involved in the development of neurons in the hippocampus [34] and cerebellum

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[21]. Furthermore, L-serine is the substrate of serine racemase, a glia-located enzyme producing D-serine that can be released into the extracellular space to act as a chemical messenger [24,46,55]. L-Serine may also play a role in the activation of microglia [48]. Several of the small neutral amino acids, particularly g-aminobutyrate (GABA), glycine, b-alanine and taurine have been shown to be neuroactive [13]. While GABA and glycine fulfil criteria for inhibitory neurotransmitters [13], glycine and D-serine [26] are involved in the excitatory synaptic transmission mediated by NMDA receptors. GABA, glycine, L-proline, taurine and b-alanine are taken up by central nervous tissue via specific Na 1 -dependent transport systems that are mediated by specialized transporter molecules [1,2,5,6,19,26,28,30]. The remaining small neutral amino acids L-serine, L-alanine and L-cysteine are not normally classed as ‘neuroactive’ nor are they considered as putative neurotransmitters despite some of them having been shown to produce weak inhibitory actions when applied to central neurons [14]. It is also less clear how these amino acids cross cell membranes in the central nervous tissue. Particularly L-serine presents a potentially complex case. It could be taken up via the small amino acid transport system (referred to as system A in brain tissue) [10,11] but also handled by ASC (alanine serine cysteine) transporters, which mediates Na 1 -dependent transport of neutral amino acids [8]. Furthermore, L-serine could be taken up by a specific transport system together with glycine and alanine, at least in parts of the avian brain [25]. Consequently, despite being a nonessential amino acid without direct inhibitory or excitatory actions in the CNS, L-serine appears to be indispensable for the normal development of brain and could play an important—if indirect—role in signaling between glia and neurons. Moreover, concentrations of L-serine in the cerebrospinal fluid (CSF) have been reported only about an order of magnitude greater than the concentrations of the neuroactive amino acids glutamate, aspartate and GABA [45] and significantly lower than the serine concentrations in plasma [44]. This suggests that extracellular L-serine in the CNS is tightly controlled, perhaps, like in the case of the neuroactive amino acids, by means of specific ‘high affinity’ transport systems. Amino acid transport systems are distinguished primarily by substrate specificity and ionic requirements. In almost all mammalian cells, small neutral amino acids, including L-serine, are transported predominately by the Na 1 -dependent transport system ASC and system A as well as by the Na 1 -independent transport system asc. However, little is known about L-serine transport system in the mammalian CNS to date. In the present study we have examined, using crude synaptosomal fractions and synaptic membranes prepared from the rat brain, characteristics of [ 3 H]L-serine uptake such as ionic requirements, temperature dependence, kinetic constants and substrate selectivity. All parameters

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measured for [ 3 H]L-serine uptake and, in some experiments, [ 3 H]L-serine binding, were compared to the corresponding parameters obtained for the accumulation and binding of [ 3 H]L-glutamate, [ 3 H]GABA and [ 3 H]L-proline, measured under the same conditions in parallel. High affinity transport systems would be beneficial for L-serine to act as a neurotrophic factor in the CNS. To analyze the presence of such a transporter with high affinity, we used [ 3 H]L-serine at low micromolar concentrations in this study. The chief aim of the study was to establish which specific amino acid transport system(s), known to exist in the CNS, would be, on the basis of their pharmacology, ionic requirements and kinetic characteristics, most likely to handle L-serine when it is present in the extracellular space at low micromolar concentrations.

2. Materials and methods

2.1. Materials Choline chloride (ChCl), potassium acetate, L-serine, glycine, GABA, b-alanine, taurine, L-cysteine, L-cystine, glutathione, L-alanine, L-asparagine, L-leucine, L-phenylalanine and L-tyrosine were purchased from Wako (Japan). Glutamine, L-2-methylamino isobutyric acids ( L-2MeAIB) came from Sigma (St. Louis, MO, USA) and BCH originated from Calbiochem. [ 3 H]L-Glutamate, [ 3 H]L-GABA, [ 3 H]L-proline, and [ 3 H]L-serine were purchased from Perkin-Elmer (NEN, Boston, MA, USA) (spec. activity; L-[3,4- 3 H]-glutamate: 43 Ci / mmol, g-[2,33 H(N)]-aminobutyric acid: 30 Ci / mmol, L-[2,3- 3 H]-proline: 25 Ci / mmol, L-[ 3 H(G)]-serine: 23 Ci / mmol). D-serine,

2.2. Preparation of tissue samples Male Wistar rats, 8–10 weeks old, were decapitated, and the brains were removed from the cranial cavity and homogenized with a glass–PTFE homogenizer in 15 volumes of 0.32 M sucrose. The homogenates were centrifuged at 800 g for 10 min, and the supernatant was centrifuged at 20 000 g for 20 min. The pellet (P25crude synaptosomal fraction) was suspended with a glass–PTFE homogenizer in fresh 0.32 M sucrose and again centrifuged at 20 000 g for 20 min. The procedure was repeated three times, and the resulting pellet was suspended and the suspension was used in uptake experiments within 5 h after preparation. In order to prepare crude synaptic membrane fractions for receptor binding assays, P2 was suspended with a Physcotron homogenizer in ice-cold distilled water and, 30 min later, centrifuged at 8000 g for 30 min. The floating loose pellet was combined with supernatant, followed by centrifugation at 50 000 g for 30 min and subsequent washing three-times by suspending and centrifuging at 50 000 g for 30 min in ice-cold 50 mM

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Tris–acetate buffer (pH 7.4). The final pellet was suspended in 0.32 M sucrose for storage at 280 8C until use in binding experiments.

Table 1 Effect of various amino acids on the uptake of 10 nM [ 3 H]L-serine in the presence of Na 1 IC 50 6S.E.M. (mM)

n H 6S.E.M.

n

(a) Small neutral amino acids L-Serine 36.667.0 L-Cysteine 84.3614.2 L-Alanine 64.4611.5

0.5760.06 ††† 0.6760.07 †† 0.6160.06 †††

40 40 20

(b) Glutamine and asparagine L-Glutamine 27.665.0 L-Asparagine 34.365.4

0.6060.06 ††† 0.5660.05 †††

20 20

a

2.3. Uptake and binding experiments Uptake experiments were carried out using fresh synaptosomal fractions originating from 20 mg of tissue (wet weight) per 1 ml of incubation medium. This consisted of 125 mM NaCl, 3.5 mM KCl, 1.5 mM CaCl 2 and 1.2 mM MgSO 4 , 1.25 mM KH 2 PO 4 , 25 mM NaHCO 3 , 10 mM HEPES and 10 mM D-glucose, pH adjusted to 7.4. In some experiments NaCl was replaced with equimolar concentrations of ChCl, KCl and potassium acetate. The tissue was first incubated for 5 min at 2 8C or 30 8C, followed by the addition of 10 nM radiolabeled substrate ([ 3 H]L-glutamate, [ 3 H]L-proline, [ 3 H]GABA and [ 3 H]L-serine) and subsequent incubation for 1–30 min (in time course studies) or for 5 min (in all other studies). The incubation was terminated by vacuum-filtration through Whatman glass fiber-filters of type D and rapid washing, three times, with isotonic saline at 2–4 8C). The radioactivity on the filter was measured using liquid scintillation counting. Parallel experiments were always performed without any incubation as 0 time to obtain radioactivity not specifically taken up into brain preparations for all radiolabeled substrates used. Binding experiments were carried out in a similar fashion, except that Na 1 -free 50 mM Tris–acetate buffer (pH 7.4) was used as a medium, both for the incubation and for washing, using crude synaptic membrane fractions extensively washed and treated with a low concentration of Triton X-100 as described previously [38]. In binding studies, incubations were extended in the presence of 10 nM radiolabeled substrates ([ 3 H]L-glutamate, [ 3 H]L-proline, [ 3 H]GABA and [ 3 H]L-serine) for up to 60 min as needed. Nonspecific binding was determined in the presence of excess unlabeled ligands at 0.1 mM and subtracted from each experimental value to obtain specific binding. Protein was determined either by the technique of Lowry et al. [33] or by the Bio-Rad protein assay [7]. The Bio-Rad method was used in all kinetic studies.

The values of IC 50 were computed from normalized data (control5100) using GraphPad PRISM software. The data, obtained at five inhibitor concentrations: 0.1, 1.0, 10.0, 100 and 1000 mM, two to four points per each concentration, were fitted to two equations [12,49]: y 5 100 2 h100 / [1 1 10 3 (log (IC 50 ) 2 x)]j and y 5 100 2 100 / h1 1 10 3 [n H ? (log (IC 50 ) 2 x)]j and compared by F-test [35,36]. The F-test indicated that the more complex equation (with n H ± 1) represented a statistically significant improvement over the simpler form (n H 5 1) at least at †† P, ,0.01 or ††† , P,0.001. The value of n H was, therefore, considered significantly smaller that unity in such cases. In addition, similar statistical analysis (comparing the equations y 5 100 2 100 / h1 1 10 3 [n H ? (log (IC 50 ) 2 x)]j and y 5 100 2 A / h1 1 10 3 [n H ? (log (IC 50 ) 2 x)]j indicated that the inclusion of constant A (representing the maximum portion of [ 3 H]L-serine uptake that could be inhibited) did not significantly (P,0.05) improve the fit thus indicating that all uptake was sensitive to inhibitors (cf. Fig. 7 and Table 2). a No effect at 100 mM: D-Serine, glycine, GABA, b-alanine, taurine, cystine, glutathione (whether reduced or not), L-2-methylamino isobutyric acid ( L-2-MeAIB).

2.5. Data processing Statistical evaluation of the data including curve fitting, and preparation of the figures was carried out using either Microsoft EXCEL software (Figs. 1–4) or PRISM 3.1 (GraphPad, San Diego, CA, USA) for the computation of, respectively, the inhibition constants IC 50 and n H in Tables 1 and 2 [49] and the kinetic constants Km , Vmax and k diff in Figs. 5 and 6. Equations with and without additional constants corresponding to either n H or a term for a component of the uptake that was sensitive to the inhibition (i.e. to establish whether such component was smaller than the total uptake at the given substrate concentration; see legend of Table 1 for the form of the equations) were compared using statistical tests [35,36].

2.4. Pharmacology and regional distribution 3. Results In pharmacological studies, test compounds were introduced into the medium at the beginning of the incubation before the addition of radiolabeled substrate. The IC 50 values were computed from plots of uptake, normalized as percentage of control, versus logarithm of inhibitor concentration [49]. The regional distribution was determined using crude synaptosomal fractions freshly prepared from brain regions isolated according to Glowinski and Iversen [22]. All other preparations used were obtained from whole rat brain including cerebellum.

3.1. Time course, temperature dependence and ionic requirements The uptake of [ 3 H]L-serine was strongly temperature dependent in synaptosomal fractions freshly prepared from rat brain irrespective of the presence of Na 1 and Cl 2 ions; slow uptake observed at 2 8C was essentially linear and probably corresponded to passive diffusion (Fig. 1). Substitution of K 1 for Na 1 reduced [ 3 H]L-serine uptake to

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Table 2 Effect of large amino acids on the uptake of 10 nM [ 3 H] L-serine Na 1 present

L-Isoleucine L-Leucine L-Phenylalanine L-Threonine L-Tyrosine

BCH

Na 1 replaced by Ch 1

IC 50 6SEM (mM)

n H 6SEM

n

IC 50 6SEM (mM)

34.268.7 20.164.8 18.962.7 23.462.0 63.4610.7* 91.8618.9***

0.6160.09 † 0.5560.07 †† 0.6160.05 ††† 0.6060.03 ††† 0.7060.08 † 0.6960.09 †

14 15 14 20 15 15

19.363.0 25.863.9 21.964.1 36.166.0 63.265.8***

n H 6SEM

0.6760.06 †† 0.7860.06 † 0.6560.07 †† not tested 0.6260.06 ††† 0.8160.06 †

Na 1 replaced by K 1 n

IC 50 6SEM (mM)

10 14 15

11.261.5 24.862.8 17.964.3

15 15

39.167.3 59.867.3**

n H 6SEM

0.7460.07 † 0.7660.06 †† 0.7160.11 not tested 0.7660.06 0.7860.07 †

n 13 15 14 15 15

Different from the remaining values of IC 50 at *P,0.05, **P,0.01 or ***P,0.001. Different from unity at † P,0.05, †† P,0.01 or ††† P,0.001. In the last column, IC 50 for L-isoleucine was also different from that for L-tyrosine at P,0.01. BCH stands for 2-aminobicyclo[2,2,1]heptane-2-carbocxylic acid, the remaining details same as in the legend for Table 1.

about half of that observed at 125 mM Na 1 . Replacement of Na 1 with choline ion or of Cl 2 with acetate ion caused an additional decrease in [ 3 H]L-serine uptake but even under these conditions, there remained substantial temperature-sensitive component (Fig. 1). In all cases uptake reached a plateau by approximately 10 min. Uptake of 10 nM [ 3 H]L-serine followed a time course similar to that displayed by the uptake of [ 3 H]L-glutamate, [ 3 H]GABA and [ 3 H]L-proline under similar experimental conditions and remained linear with time for about 5 min (Fig. 2). There were, however, clear differences between [ 3 H]L-serine uptake and the uptake of the other three radiolabeled substrates in terms of ionic requirements (Fig. 2). Replacement of Na 1 with K 1 led to almost complete inhibition of temperature-dependent uptake of [ 3 H]L-glutamate and [ 3 H]L-proline, with a concomitant drastic reduction of portion of that of [ 3 H]GABA. However, substitution of Na 1 by K 1 kept the temperature-dependent portion of [ 3 H]L-serine uptake.

3.2. Regional distribution Of central structures examined, the uptake of [ 3 H]Lserine was most avid in the forebrain regions such as cerebral neocortex, hippocampus and striatum in the presence of high concentrations of Na 1 but it appeared less efficient in the olfactory bulb and in the cerebellum (Fig. 3). The pattern of the regional distribution was similar to that produced by the uptake of [ 3 H]L-glutamate, [ 3 H]GABA and [ 3 H]L-proline (Fig. 3) and might have reflected relative proportions of the gray matter in the individual regions. This would hold also for the cerebellum, which, in the present preparation, includes the white medullary center i.e. it contains a significant proportion of white matter.

3.3. Binding to synaptic membranes When synaptic membrane preparations were incubated

Fig. 1. Time course of [ 3 H]L-serine uptake. Time course of uptake of 10 nM [ 3 H]L-serine by synaptosomal fractions at 2 (closed triangles) and 30 8C (closed circles) was measured either in the presence of 125 mM NaCl, or, when either Na 1 , Cl 2 or both were replaced by equimolar concentrations of the other ions as indicated. Values are the mean6S.E. in three separate experiments done in triplicate, while S.E. was within the symbols in the figure.

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Fig. 2. Comparison of uptake of [ 3 H]L-glutamate, [ 3 H]L-proline, [ 3 H]GABA and [ 3 H]L-serine. Synaptosomes were incubated with [ 3 H]L-glutamate, [ 3 H]L-proline, [ 3 H]GABA and [ 3 H]L-serine at 10 nM substrate concentrations, for time course measured over 30 min in the presence of (a) 125 mM Na 1 or (b) when 125 mM was replaced by equimolar concentrations of K 1 , at 2 (closed triangles) and 30 8C (closed circles). Values are the mean6S.E. in four separate experiments done in triplicate, while S.E. was within the symbols in the figure.

in Na 1 -free, Tris–acetate buffered media, containing either 10 nM [ 3 H]L-glutamate or 10 nM [ 3 H]GABA, there was an easily detectable specific binding of both radioligands in a manner inversely dependent on the temperature. Under the same conditions, neither [ 3 H]L-proline nor [ 3 H]L-serine, both tested at 10 nM concentrations, were retained to any significant degree by the synaptic membranes even after 60 min incubation (Fig. 4). Neither was any binding detected when the temperature was decreased to 2 8C.

3.4. Kinetics of [ 3 H] L-serine uptake Direct plot of the initial rate of transport (v) versus [ 3 H]L-serine concentrations ([S]) was indicative of a saturable transport with Km between 200 and 300 mM (Fig. 5A). Two-component model was rejected by the F-test

analysis [35,36,49] and using the Hill equation [45] indicated apparent n H 50.7 and only a marginally statistically significant (P50.033) improvement of the fit. Introduction of a simple nonsaturable (linear) component (i.e. diffusion) resulted in a fit that was ‘better’ (by the F-test criterion) than the single-component saturable model at P50.06. Neither the value of Km nor the resolution into two components (P50.06 cannot be considered as statistically significant) could, therefore, be accepted as satisfactory without further analyses. The lack of statistical significance does not, however, necessarily imply the presence of only a single component (cf [49]. for more discussion). It is evident that in a simple v versus [S] analysis, the statistical impact of the points obtained at low concentrations could be underestimated (Fig. 5B). In order to

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Fig. 3. Regional variations of uptake of [ 3 H]L-glutamate, [ 3 H]L-proline, [ 3 H]GABA and [ 3 H]L-serine. Each brain structure was dissected for preparation of synaptosomal fractions, followed by incubation with [ 3 H]L-glutamate, [ 3 H]L-proline, [ 3 H]GABA and [ 3 H]L-serine at 10 nM substrate concentrations in the presence of normal (125 mM) Na 1 , for 5 min at 30 8C. Values are the mean6S.E. in four separate experiments done in triplicate. Abbreviations: CX, cerebral neocortex; HC, hippocampus; ST, striatum; CL, cerebellum; OB, olfactory bulb.

overcome this problem we used an Eadie–Hofstee type transformation (v versus v / [S], where v is the initial rate of uptake). Furthermore, Eadie–Hofstee equation used to analyze the data was modified so as to include a nonsaturable component corresponding to that in Fig. 5B. Specifically, the simple Eadie–Hofstee analysis: v 5Vmax 2 Km ? (v / [S]), was modified by deriving an analogous equation from a Michaelis–Menten relationship extended to include a linear (‘nonsaturable’) component: v 5Vmax ? [S] /(Km 1 [S]) 1 k diff ? [S] [4]. The rearrangement resulted in the following form: v 5 hVmax 1 Km ? (k diff 2 (v / [S])) /

h1 2 k diff /(v / [S])j, where k diff is an apparent ‘diffusion’ coefficient [3]. By comparing fits to the two equations, the data could be clearly resolved (P,0.001 by the F-test) into at least two components (Fig. 5C). Further inspection of the points in the region of v , 1,500 pmol / mg / min (Fig. 5C) would seem to suggest that the data might have been better fitted by introducing (an) additional component(s) but the F-test analysis indicated that this would not be justified at P,0.05 level of statistical significance. Computations using the points corresponding to v , 1,500 pmol / mg / min (i.e. disregarding—though not correcting

Fig. 4. Comparison of binding of [ 3 H]L-glutamate, [ 3 H]L-proline, [ 3 H]GABA and [ 3 H]L-serine. Synaptic membrane preparations were incubated in the presence of 10 nM substrates for up to 1 h at 2 (closed triangles) and 30 8C (closed circles), using a Na 1 -free 50 mM Tris–acetate buffered medium. Values the mean6S.E. in three separate experiments done in triplicate.

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Fig. 5. Uptake of [ 3 H]L-serine in the presence of Na 1 . Initial rate of uptake was measured for 5 min at 0.01, 0.1, 0.9, 2.7, 9.0, 18.0, 27.0, 54.0, 90.0, 180, 270 and 450 mM substrate concentrations in the presence of normal Na 1 concentrations. The points in (A) and (B) are the means6S.E. of three measurements, the points in (C) and (D) are single measurements.

for—the ‘nonsaturable’ part of uptake) resulted in a single set of parameters Km 528.562.7 mM and Vmax 51.5060.11 nmol / mg / min (Fig. 5C, dashed line). We have not pursued Eadie–Hofstee analysis further; instead, we have computed the kinetic parameters, using the Woolf–Hanes transformation [13,45], modified so as to include either a nonsaturable component: [S] /v 5 hVmax /(Km 1 [S]) 1 k diff j 21 , or two saturable affinities: [S] /v 5 h 1Vmax /( 1 Km 1 [S]) 1 2 Vmax /( 2 Km 1 [S])j 21 . The F-test analysis preferred the former equation to the simple one at P,0.0001 (Fig. 5D) but rejected the presence of the model involving two saturable components (P.0.05). Similar computations performed with the data obtained in the absence of Na 1 (NaCl replaced by equimolar concentration of ChCl) were consistent with the presence of only one component (Fig. 6), with kinetic parameters similar to those of the ‘saturable’ portion of [ 3 H]L-serine uptake observed in the presence of Na 1 . In other words, using a Na 1 -free medium resulted in the disappearance of the apparently ‘nonsaturable’ component (Figs. 5 and 6).

3.5. Substrate selectivity L-Asparagine, L-glutamine, L-cysteine and L-alanine caused complete inhibition of [ 3 H]L-serine uptake with IC 50 values within 40–90 mM (Table 1). There was no inhibition by a-methylamino isobutyric acid ( L-2-

Fig. 6. Uptake of [ 3 H]L-serine in the absence of Na 1 . Initial rate of uptake was measured for 5 min at 0.01, 0.1, 1.0, 3.0, 10.0, 20.0, 30.0, 60.0, 100.0, 200, 300 and 500 mM substrate concentrations; Na 1 was replaced by equimolar concentrations of K 1 . The points in (A) are means6S.E. of three measurements, while the points in (B) are single measurements.

methylamino isobutyric acid, L-2-MeAIBA, a typical substrate for the amino acid transport system A; Table 1). Moreover, the data in Table 1 point to the strong stereoselectivity of [ 3 H]L-serine uptake and, also, demonstrate that [ 3 H]L-serine uptake under the conditions employed in the present study could not be mediated by glycine transporters (100 mM glycine produced no significant inhibition) nor is there any overlap with the b-system as evidenced by the lack of effect of both taurine and b-alanine, even at 1 mM concentrations (Table 1). [ 3 H]LSerine uptake was, however, inhibited by both L-cysteine and L-alanine (Table 1) with IC 50 values similar to those for the other inhibitors (Tables 1 and 2). The IC 50 values for L-serine, L-alanine, and BCH when determined against uptake of [ 3 H]L-serine at 1 mM in the presence of NaCl were not significantly different from those observed at 10 nM [ 3 H]L-serine (data not shown). In addition, D-serine and

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Fig. 7. Inhibition of 10 nM [ 3 H]L-serine uptake by L-leucine, L-tyrosine and BCH. Uptake (normalized as % control) in the presence of various concentrations of inhibitors was plotted against logarithm of inhibitor concentration (log [I]). The values of constants are the means6S.E. For further details of curve-fitting and statistical analyses cf. legends of Table 1.

ineffective as inhibitors at 10 nM [ 3 H]Lserine had no effect at 1 mM [ 3 H]L-serine, either. Furthermore, statistical analysis (legend of Table 1) indicated no significant component of [ 3 H]L-serine uptake that could not be inhibited by the inhibitors shown in Tables 1 and 2 (cf. also Fig. 7). It should be pointed out that [ 3 H]L-serine uptake was inhibited by several typical substrates of the large neutral amino acid transport system (system L), particularly by L-threonine, L-leucine, L-isoleucine, L-phenylalanine, Ltyrosine and, also, by BCH (2-aminobicyclo[2,2,1]heptane2-carboxylic acid, a typical substrate for the amino acid transport system L), the synthetic marker for the L system. The value of IC 50 for BCH was, however, significantly higher than the corresponding values for the inhibitions by the other large neutral amino acids, both in the presence and absence of Na 1 (Table 2). L-2-MeAIBA,

4. Discussion Given the recently proposed roles of L-serine in the normal morphological development and function of the CNS [24,43,46,55] as well as the possible involvement of L-serine deficiency in a serious neurological disorder, it seems of great importance to understand the intercellular traffic in L-serine, particularly because of the uneven distribution of its synthetic enzymes at a cellular level. The essential importance of the present findings is that [ 3 H]Lserine accumulation was predominantly mediated by LAT family in rat brain synaptosomal fractions. Experiments using immunocytochemistry indicated that, in the hippocampus and the cerebellar neocortex, L-serine was stored mainly in astrocytes while in the brain stem it was found primarily in neurons [57]. It has further been suggested that such variations may reflect specific roles of L-serine in the particular regions: trophic factor in the cerebellum and

the hippocampus [21,34] and the substrate of serine racemase in the brain stem [24,46]. There could even be an alternative explanation for the regional variations in the L-serine storage pattern, one that is related to the glycinergic neurotransmission; while in the forebrain, glycine acts as a co-transmitter on NMDA receptors [28], the presence of the high affinity Na 1 -dependent glycine uptake was only detected in the caudal regions of the CNS such as brain stem and spinal cord, where glycine is thought to act as an inhibitory synaptic transmitter [14,29]. Thus the neuronal presence of L-serine in the brain stem might reflect its local role as a possible precursor (via L-serine hydroxymethyl transferase) [16] of the inhibitory neurotransmitter glycine. In a previous study using in situ hybridization technique, mRNA for the key enzyme of L-serine biosynthesis, 3-PGDH, was highly expressed in the cerebellum and olfactory bulb. The possibility that regional variation of [ 3 H]L-serine uptake might be at least in part affected by endogenous L-serine, therefore, is not entirely excluded. High levels of L-serine in astrocytes found in the forebrain regions may be the result of the action of 3PGDH that is known to be present in astrocytes [20]. In this context it should be stressed that the synaptosomal preparations are known to contain not only nerve endings but also particles of glial origin [4,15]. The present experiments may, therefore, reflect the presence of transport systems in either neurons or glia, perhaps both. Such transport systems could either help to supplement the astrocyte stores of L-serine or it could be a ‘regulatory’ part of the mechanism of the trophic action exerted onto neurons by L-serine released from glial cells. Multiple functions of L-serine may thus be paralleled by the multiplicity of L-serine uptake systems. In the mammalian CNS, there is no doubt that Lglutamate and GABA are an excitatory and inhibitory amino acid neurotransmitter, respectively. However, neither proline nor serine has been recognized as a neuro-

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transmitter although there are some reports that these amino acids are neuroactive [14]. The fact that specific binding was detected with both [ 3 H]L-glutamate and [ 3 H]GABA, but not with [ 3 H]L-proline and [ 3 H]L-serine, in brain synaptic membranes, thus, gives support to an idea that radioactivity trapped on the filter would be really derived from [ 3 H]serine incorporated into synaptosomal fractions but not from binding to membranes. As replacement of NaCl with KCl undoubtedly depolarizes synaptosomal fractions freshly prepared from rat brains, however, the possibility that particular endogenous substances released during depolarization may modulate the apparent uptake of four different radioactive substrates in the presence of KCl at a high concentration is not ruled out. Kinetic analysis of [ 3 H]L-serine uptake has indicated the presence of at least two components—low and high affinity. Trophic activity has been detected at micromolar concentrations of L-serine [43] indicating that it is a transport system capable of operating at very low concentrations (characterized by low Km or high affinity) that should be of primary interest. The experiments studying the substrate selectivity, ionic requirements and temperature dependence have been carried out at submicromolar substrate concentrations thus favoring, in theory, the high affinity component of uptake. Even under those conditions, however, uptake of [ 3 H]L-serine seems to be mediated by more than one transport system as indicated by its ionic requirements. In fact, it cannot be concluded that the apparently nonsaturable Na 1 -dependent component of [ 3 H]L-serine represents solely a passive ‘diffusion’. It seems much more likely that this Na 1 -dependent component corresponds to a saturable, low-affinity (Km .1 mM), transport system. The lack of effect of b-alanine, taurine and glycine rules out any contribution either from the taurine- and b-alaninepreferring transport systems [1,6] or from any of the glycine-preferring transporters including those mediating the hypothetical CGA transport system that might exist in the avian brain [25]. Moreover no effect of D-serine is suggestive of the absence of contribution of alanine-insensitive D-serine transporter [27] from [ 3 H]L-serine uptake demonstrated in this study. There are, however, many more Na 1 -dependent small amino acid transport systems that could be involved in L-serine transport: small amino acid transport systems or SAT (sodium-coupled amino acid transporter) [42,47,52,53,56]. Particularly, the recently cloned transporters SAT1 and SAT2 are probably present in brain and have been studied in cultured neurons. The problem is that the typical substrate for these transporters, also referred to as A system, L-2-MeAIBA [54], did not interact with [ 3 H]L-serine uptake (Table 1) thus ruling out any contribution from the typical SAT systems to the [ 3 H]L-serine uptake observed in the present experiments. The family of SN (system N) transporters that are not only Na 1 -dependent but also pH-dependent could also be considered. SN1 has been studied in cell cultures derived

from hippocampus and has been found to be present in astrocytes [9]. There is as yet little precise information about the characteristics of this class of transporters [23] and they may not handle the large neutral amino acids such as L-threonine or BCH [18,50] nor do they interact with L-cysteine making them improbable candidates for the major mediators of L-serine uptake observed in our studies. Possible contribution from ASCT that belongs to the transport system ASC looks, on the first approach, somewhat intriguing because the family of transporters includes Na 1 -dependent ASCT1 and ASCT2 as well as a related Na 1 -independent Asc transporter. These transporters display preference not only for L-serine, L-cysteine and Lalanine which have all been found to inhibit [ 3 H]L-serine uptake, they are also insensitive, just as [ 3 H]L-serine uptake in the present studies, to L-2-MeAIBA [8]. It has, however, been reported that ASCT2, the only known glutamine sensitive ASC transporter, is not expressed in the adult brain [51] while neither the Na 1 -independent Asc nor the ubiquitous ASCT1 seem to interact with L1 glutamine [8]. Moreover, Na -independent transporter Asc is capable of transporting D-serine [37]. Although there is no reason to assume that all known system ASC transporters have been cloned, their participation in [ 3 H]L-serine uptake observed in the present study is also very improbable. It should be emphasized that the discussion of pharmacological characteristics of [ 3 H]L-serine uptake observed in the present experiments is based on data obtained at very low substrate concentration (0.01 mM). Using kinetic parameters (for example those from Fig. 5B) to estimate contributions from the high Km (low affinity) Na 1 -dependent uptake suggests that this component can account for no more that 2% of [ 3 H]L-serine uptake at 0.01 mM concentration, the proportion would rise to 15 and 18% at 1 and 10 mM, respectively, reaching about 33% at 100 mM substrate concentration. This means that the system with the characteristics described here would remain dominant at extracellular concentrations of L-serine that are most likely to exist in brain under physiological conditions (as indicated by serine concentrations in the CSF [39],). The above calculations also imply that the decrease in the 0.01 mM [ 3 H]L-serine uptake in the absence of Na 1 cannot be explained by the elimination of the Na 1 -dependent low affinity component. It is more probable that the high affinity component is not entirely Na 1 -independent. Rather, it could have marginally lower affinity (by |50%) for [ 3 H]L-serine. Such a small difference would not be easily detected by conventional kinetic computations but it is consistent with the result of Woolf–Hanes analysis, which suggests doubling in the value of Km (P,0.02 by Student’s t-test) but no significant change in Vmax of the Na 1 -independent component in the absence of Na 1 . In this kinetic analysis, thus, high affinity [ 3 H]L-serine uptake would be mediated by Na 1 -independent system. The presence of the temperature-sensitive, Na 1 -indepen-

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dent [ 3 H]L-serine uptake in synaptosomal fractions derived from the rat brain is thus difficult to be accounted for while considering only those systems that are well known and well-characterized in brain tissue. The transport system that takes up [ 3 H]L-serine at low concentrations used in our experiments must be able to handle both large neutral and small neutral amino acids, and also L-asparagine and Lglutamine but should not have any significant affinity for glycine, b-alanine, taurine and GABA. It should interact with the synthetic L-system marker BCH but not with the synthetic A-system marker L-2-MeAIBA. It seems that the recently identified transporters belonging to the LAT ( Lamino acid transporter) family are the best candidates. LAT family is comprised of either LAT1 or LAT2 to exhibit the activity of system L. System L is a widespread mediator of the Na 1 -independent transport of branched chain and bulky neutral amino acids such as leucine, phenylalanine and tyrosine. LAT1 exhibits transport properties consistent with system L, and is capable of transporting L-DOPA across the blood–brain barrier [31]. In contrast to LAT1, LAT2 has been shown to have a broadspectrum substrate selectivity that includes both the large neutral amino acids as well as some small ones such as L-serine and L-alanine. In a previous study using the LAT2 system expressed in oocytes, [ 3 H]L-leucine uptake was significantly inhibited by L-alanine, L-serine, L-threonine, L-cysteine, L-leucine, L-isoleucine, L-phenylalanine, Ltyrosine and BCH in the absence of sodium ion. Moreover, LAT2 mRNA was detected in brain [32,40]. Thus on the basis of the present data it is plausible to suggest that the LAT2 or (a) LAT2-like transporter(s) is / are chiefly responsible for the uptake of L-serine under the presently used experimental conditions. In this context, it is interesting to note that a hypothetical L-2-MeAIBA-insensitive neutral amino acid transport system has recently been proposed to be of crucial importance for the transport of glutamine into the synaptosomal compartment in guinea pig cerebral cortex under depolarizing conditions (high extracellular concentration of K 1 ) in vitro [41]. Results of the present study suggest that L-serine could share the same transport system. Low micromolar concentrations of L-serine in brain extracellular space would, therefore, inhibit such uptake, reducing glutamine / glutamate cycling as a consequence [41] and could thus influence the availability of synaptic glutamate under the conditions of high neuronal activity. In conclusion, [ 3 H]L-serine has been found to enter synaptosomal fractions derived from rat brain via two kinetically distinct transport system. The system with Km in low micromolar region (high affinity) was Na 1 -independent while the low affinity system (detectable only as an apparently nonsaturable diffusion in the kinetic studies) was Na 1 -dependent. The maximum rate of transport by the latter, low affinity, Na 1 -dependent, system was probably much higher than that of the former, high affinity, Na 1 independent system, because both Na 1 -dependent (Km .1

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mM) and Na 1 -independent (Km ,0.1 mM) uptake functioned at low [ 3 H]L-serine concentrations. The substrate specificity of both components was very similar and involved large as well as small neutral amino acids. The sum of the characteristics of [ 3 H]L-serine uptake by synaptosomal fractions prepared from rat brain strongly suggests that it is mediated by a system consisting of recently identified transporters of LAT family.

Acknowledgements The authors wish to acknowledge Ms. Hiroko Saiki and Ms. Ayame Sano for their excellent technical assistance. VJB wishes to thank to the Japanese Ministry of Education, Science, Sports, Culture and Technology for funding his position at Kanazawa University. GBA was supported by The Japan Society for Promotion of Science (JSPS). This work was supported in part by Grants-in-Aids for Scientific Research to YY and VJB from the Ministry of Education, Science, Sports, Culture and Technology, Japan.

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