L-Serine is considered a functional amino acid in the central nervous system, and induces sedation and hypnotic effects in some animal models of acute and chronic stress. Accordingly, while L-serine is a candidate anti-stress factor, the central mechanism of L-serine is not clear. The present study clarifies the action of L-serine using acute chick brain slices. We investigated the changes in some extracellular fluid amino acid concentrations in response to L-serine perfusion. Taurine concentration decreased while L-alanine concentration increased following L-serine perfusion. To examine the involvement of the taurine transporter, the effect of L-serine on the taurine concentration in the presence and absence of Na<sup>+</sup> was also investigated. Na<sup>+</sup> had no effect on taurine concentration induced by L-serine perfusion. These results suggest that L-serine has an ability to promote L-alanine synthesis facilitating the catabolism of taurine. In conclusion, L-serine modifies the metabolism of taurine and L-alanine in the extracellular space in chick brain.
L-Serine, a non-essential amino acid, is an important metabolic precursor in the synthesis of proteins, sphingolipids, other amino acids and nucleotides [
It has been reported that externally supplemented Lserine has anti-stress effects in animals. Asechi et al. conducted a series of experiments to investigate the relationship between L-serine in the CNS and stress-related behavior of animals using the chick separation-stress paradigm [
In the mammalian CNS, most of the L-serine is synthesized via a route known as the phosphorylated L-serine pathway [9-12]. In this pathway, L-serine is converted from L-phosphoserine by phosphoserine phosphatase [
Brain slice preparations are well established models for a wide spectrum of in vitro investigations in neuroscience. The action of cells can be observed in real time using brain slice cultures. Therefore, to further investigate the role of L-serine in the chick brain, we examined the effect of L-serine on the external environment of cells using acute brain slices.
One-day-old male layer chicks (Julia) purchased from a local hatchery (Murata Hatchery, Fukuoka, Japan) were maintained in a windowless room at a constant temperature of 30˚C ± 1˚C. Lighting was provided continuously. Chicks were given free access to a commercial starter diet (Toyohashi Feed and Mills Co. Ltd., Aichi, Japan) and water. This experiment was performed at 7 or 8 daysof-age. Experimental procedures followed the guidance for Animal Experiments in the Faculty of Agriculture and in the Graduate Course of Kyushu University and the Law (No. 105) and Notification (No. 6) of the Japanese Government. During the experiments, all efforts were made to minimize animal suffering.
The standard artificial cerebrospinal fluid (ACSF) was composed of 125 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 1.5 mM MgSO4, 1.6 mM CaCl2, 26 mM NaHCO3, 10 mM glucose, 500 μM ascorbic acid, and 1-5 μM taurine (1 μM for Experiment 1; 5 μM for Experiments 2 and 3), prepared with ultrapure water and bubbled with 95% O2/5% CO2. Based on previous studies investigating the effects of L-serine in chicks exposed to acute stressful condition [7,16], ACSF containing 84 mM L-serine was used as the L-serine solution in Experiments 1 and 2.
In Experiment 3, ACSF containing 8.4 mM L-serine was used to confirm whether the effect of L-serine occurred at lower doses. The osmotic pressure was equalized between standard ACSF and the L-serine solution. A Na+-free solution was made by substituting NaCl and NaHCO3 with choline chloride and NaH2PO4 with KH2PO4 in ACSF.
The derivatizing solution (othophthaldialdehyde/2-mercaptoethanol (OPA/2-ME) solution) contained 27 mg of OPA (Wako, Tokyo, Japan) dissolved in 1.0 ml of absolute ethanol. After the addition of 20 μl of 2-ME (Wako, Tokyo, Japan) the solution was diluted to 10 ml with 0.1 mol/l carbonate buffer (pH 9.5). Further, this solution was diluted with 0.1 mol/l carbonate buffer (1:4) before use.
Chicks were decapitated after cervical dislocation. The brains were quickly removed and placed in ice-cold ACSF. Vertical telencephalic slices 500 μm in thickness (2,500 - 3,000 μm from anterior end of telencephalon) were immediately prepared using a Leica VT1000S microtome® (Leica, Nussloch, Germany), and kept in an interfacetype holding chamber at 38˚C for at least 30 min in ACSF before applying each treatment solution.
After a 30 min recovery period, brain slices were exposed to each solution during a recording period and the perfusate was collected for 20 min. The L-serine solution was perfused for 5 - 10 min in this period. The flow rate was set at 1.5 ml/min. In Experiments 1 and 3, amino acid concentrations of collected perfusate were analyzed. In Experiment 2, the free amino acid content of telencephalic slices was measured in slices collected and preserved in liquid nitrogen after the recording period.
The concentrations of 8 free amino acids including Laspartate, L-glutamate, L-glutamine, glycine, L-serine, taurine, L-alanine, and GABA were determined by the method described below. The tissue samples were homogenized in ice-cold 0.2 M perchloric acid solution containing 0.01 mM EDTA 2Na and left for deproteinization on ice. After 30 min, the mixtures were centrifuged at 18,000 x g for 15 min at 0˚C and the resultant supernatants then adjusted to pH 7 with 1 M sodium hydroxide. These solutions were diluted by 0.5% with 10% methanol. The diluted samples were filtered through a 0.22 μm filter (Millipore, Bedford, USA) and were applied for high-performance liquid chromatography (HPLC) after the derivation as described below. The pellets were used for protein assay using a commercially available kit (BCA Protein Assay Kit®, Thermo Fisher Scientific Inc, Rockford, USA).
Collected perfusate samples were centrifuged with a centrifuge-filtration unit (Millipore) at 10,000 g for 5 min at 0˚C. A 60 μl aliquot of filtrate was mixed with 20 μl of the OPA/2-ME solution and then the samples were incubated at room temperature for 2.5 min. The mixture (30 μl) was applied to an HPLC system (Eicom, Kyoto, Japan) with a 150 × 2.1 mm octadecyl silane column (SC-5ODS®, Eicom) with an electrochemical detector (ECD-300®, Eicom) at an applied potential of +600 mV versus Ag/AgCl reference analytical electrode. Changes in electric current (nA) were recorded in a computer using an interface system Power Chrom® (ver 2.3.2.j, AD Instruments, Tokyo, Japan). The mobile phase consisted of 0.1 mol/l monosodium phosphate buffer (pH6.0), methanol (7:3) containing 5 mg/l EDTA2Na at a flow rate of 0.23 ml/min.
The perfusate data were analyzed using a paired t-test in Experiment 1, or two-way repeated measure ANOVA in Experiment 3. A Tukey-Kramer test was done as a post hoc test. The data for slice samples were analyzed using a t-test. Values are presented as means±S.E.M. Statistical analysis was conducted using a commercially available package StatView® (version 5, SAS Institute, Cary, USA, 1998).
The effects of L-serine on the concentration of taurine and L-alanine in the perfusate are shown in
The addition of L-serine significantly decreased the concentration of taurine (P < 0.0001) and increased the concentration of L-alanine (P < 0.0001 in the perfusate. Other amino acids including aspartate, glutamate, and glycine did not change (data not shown). GABA concentrations were below the level of detection.
The effects of L-serine on the concentration of free taurine in the telencephalic slices exposed to 84 mM Lserine were determined. The taurine concentration (control, 86.559 ± 7.637 nmol/mg protein; L-serine, 100.367 ± 6.161 nmol/mg protein, respectively) did not change significantly in the slices after exposure to L-serine (P > 0.05).
Using a lower concentration of L-serine (8.4 mM, also decreased [F(2, 36) = 36.819, P < 0.0005] the concentration of taurine in the perfusate (
According to Hájos et al. [
tion of taurine in telencephalic slices was not increased by exposure to L-serine.
Secondly, we examined whether taurine was decreased in the perfusate when Na+ was deleted because it is known that the uptake of taurine via the taurine transporter is a Na+-dependent system [
taurine via the Na+ dependent taurine transporter from the perfusate into slices.
It is known that taurine plays an important role in the regulation of cell volume of neurons and glia when the osmolality between intracellular fluid and extracellular fluid is different. Taurine is transported via the cell membrane by the taurine transporter depending on the change in osmotic pressure [
In the present study, we also observed increased Lalanine concentrations in the perfusate when the brain slice was exposed to 84 mM L-serine (
These facts suggest that L-serine metabolism is tightly regulated in response to the abundance of L-serine to prevent a lack of L-serine, and it is reasonable to hypothesize that the catabolism process of L-serine is promoted when L-serine is abundant in chick brain. L-alanine synthesis, which occurs downstream of L-serine metabolism (
It was reported that L-serine and L-alanine were rapidly released into the culture medium whereas other amino acids (glycine, L-aspartic acid, L-asparagine, Lproline) were not changed when the culture of hippocampal astrocytes of mammal was maintained for 7 days [
In considering the mechanism by which L-serine promotes taurine catabolism, the pathway of taurine metabolism in the brain is important. For many years, taurine was known to be an end product of the metabolism of sulphur-containing amino acids, although physiological effects were not well understood. However, taurine catabolism was indicated by the finding of isethionic acid in the rat brain [
It was known that taurine injection induces an antianxiety-like effect in mice, and the GABAergic system may be involved in this effect. However, El Idrissi et al. reported that chronic supplementation of taurine was anxiogenic in the elevated plus maze whereas acute injection of taurine suppressed anxiety in mice [
L-Serine markedly changes some amino acid concentrations in the extracellular space. These changes may be involved in the mechanisms of the anti-stress effects of L-serine observed in some animal models [7-8].
This work was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science.