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The green tea amino acid theanine is abundant in green tea rather than black and oolong teas, which are all made of the identical tea plant “Chanoki” (Camellia sinensis). Theanine has a molecular structure close to glutamine (GLN) compared to glutamic acid (Glu), in terms of the absence of a free carboxylic acid moiety from the gamma carbon position. Theanine efficiently inhibits [3H]GLN uptake without affecting [3H]Glu uptake in rat brain synaptosomes. In contrast to GLN, however, theanine markedly stimulates the abilities to replicate and to commit to a neuronal lineage following prolonged exposure in cultured neural progenitor cells (NPCs) prepared from embryonic and adult rodent brains. Upregulation of transcript expression is found for one of the GLN transporter isoforms, Slc38a1, besides the promotion of both proliferation and neuronal commitment along with acceleration of the phosphorylation of mechanistic target of rapamycin (mTOR) and relevant downstream proteins, in murine NPCs cultured with theanine. Stable overexpression of Slc38a1 similarly facilitates both cellular replication and neuronal commitment in pluripotent embryonic carcinoma P19 cells. In P19 cells with stable overexpression of Slc38a1, marked phosphorylation is seen for mTOR and downstream proteins in a manner insensitive to further additional phosphorylation by theanine. Taken together, theanine would exhibit a novel pharmacological property to up-regulate Slc38a1 expression for activation of the intracellular mTOR signaling pathway required for neurogenesis after sustained exposure in undifferentiated NPCs in the brain. In this review, a novel neurogenic property of the green tea amino acid theanine is summarized for embryonic and adult neurogenesis with a focus on the endogenous amino acid GLN on the basis of our accumulating evidence to date.

Hepatic encephalopathy (HE) is a neuropsychiatric disorder that occurs due to acute and chronic liver diseases, the hallmark of which is the increased levels of ammonia and subsequent alterations in glutamine synthesis, i.e. conditions associated with the pathophysiology of HE. Under physiological conditions, glutamine is fundamental for replenishment of the neurotransmitter pools of glutamate and GABA. The different isoforms of glutamine transporters play an important role in the transfer of this amino acid between astrocytes and neurons. A disturbance in the GABA biosynthetic pathways has been described in bile duct ligated (BDL) rats, a well characterized model of chronic HE. Considering that glutamine is important for GABA biosynthesis, altered glutamine transport and the subsequent glutamate/GABA-glutamine cycle efficacy might influence these pathways. Given this potential outcome, the aim of the present study was to investigate whether the expression of the glutamine transporters SAT1, SAT2, SN1 and SN2 would be affected in chronic HE. We verified that mRNA expression of the neuronal glutamine transporters SAT1 and SAT2 was found unaltered in the cerebral cortex of BDL rats. Similarly, no changes were found in the mRNA level for the astrocytic transporter SN1, whereas the gene expression of SN2 was increased by two-fold in animals with chronic HE. However, SN2 protein immuno-reactivity did not correspond with the increase in gene transcription since it remained unaltered. These data indicate that the expression of the glutamine transporter isoforms is unchanged during chronic HE, and thus likely not to participate in the pathological mechanisms related to the imbalance in the GABAergic neurotransmitter system observed in this neurologic condition.

We have reported previously that creatine is biosynthesized from glycine and l -arginine in Muller cells, but the mechanism responsible for glycine and l -arginine uptake by Muller cells remains elusive. To explore this issue, [14C]glycine and [3H] l -arginine uptake by Muller cells was characterized using a conditionally immortalized rat Muller cell line (TR-MUL5 cells). [14C]Glycine uptake by TR-MUL5 cells was Na+- and Cl−-dependent, and a saturable process with Michaelis–Menten constants of 48.6 μM and 4.53 mM, and inhibited by glycine transporter 1 (GlyT1) and system A substrates. Under Cl−-free conditions, the high-affinity process was abolished. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA1 and ATA2) mRNA are expressed in TR-MUL5 cells. These uptake studies suggest that GlyT1 and system A are involved in [14C]glycine uptake for the high- and low-affinity processes, respectively, in TR-MUL5 cells. [3H] l -Arginine uptake by TR-MUL5 cells was primarily an Na+-independent and saturable process with Michaelis–Menten constants of 15.0 and 403 μM. This process was inhibited by substrates of cationic amino acid transporter (CAT)s, such as l -arginine, l -lysine, and l -ornithine. The expression of CAT1 protein was detected in TR-MUL5 cells. These results suggest that CAT1 is at least involved in [3H] l -arginine uptake by TR-MUL5 cells. In conclusion, GlyT1 and CAT1 most likely mediate glycine and l -arginine uptake, respectively, by Muller cells and are expected to play an important role in supplying precursors for creatine biosynthesis in Muller cells.

The relative roles of the three sodium-dependent transport systems: A, ASC and N in the uptake of [ 3 H ]Gln, and the compatibility of the uptake characteristics with the expression of mRNAs coding for the Gln transporting molecules, were examined in primary cultures of astrocytes and neurons derived from mouse cerebellum, a glutaminergic system-enriched structure, and in cerebral cortex. Gln uptake activity (Vmax) was higher in cerebellar astrocytes or neurons than in their cerebral cortical counterparts. The N-methylamino-isobutyric acid (MeAiB)- and pH-sensitive, system A-mediated component of the uptake, and the uptake of [ 14 C ]MeAiB itself, was much more active in neurons than in astrocytes derived from either region. Also, the expression of mRNA for GlnT (SAT1), a system A isoform specific for Gln, was only expressed in neurons derived from both structures, while an alanine (Ala)-preferring system A transporter, SAT2, was expressed in neurons and astrocytes from either region. System ASC-mediated Gln uptake and expression of ASCT2 mRNA were in both structures more pronounced in astrocytes than in neurons, consistent with the postulated role of ASCT2 in the efflux of de novo synthesized Gln from astrocytes. System N-mediated (threonine+MeAiB-inhibitable) Gln uptake showed comparable activities in all four types of cells, which is compatible with the ubiquitous expression of NAT2 mRNA—a mouse brain-specific N-system isoform.

We have previously demonstrated the functional expression of glutamine (Gln) transporter (GlnT) believed to predominate in neurons for the neurotransmitter glutamate pool by rat neocortical astrocytes devoid of neuronal marker expression, with exacerbated vulnerability to oxidative stress after transient overexpression. To evaluate molecular mechanisms underlying the exacerbation, we established stable GlnT transfectants in rat astrocytic C6 glioma cells. In two different clones of stable transfectants with increased intracellular Gln levels, exposure to hydrogen peroxide (H(2)O(2)) and A23187, but not to tunicamycin or 2,4-dinitrophenol, led to significant exacerbation of the cytotoxicity compared to cells with empty vector (EV). Stable GlnT overexpression led to a significant increase in heme oxygenase-1 protein levels in a manner sensitive to H(2)O(2), whereas H(2)O(2) was significantly more effective in increasing NO(2) accumulation and reactive oxygen species (ROS) generation in stable GlnT transfectants than in EV cells. Moreover, exposure to A23187 led to a more effective increase in the generation of ROS in stable GlnT transfectants than in stable EV transfectants. These results suggest that GlnT may play a role in the mechanisms underlying the determination of cellular viability in astrocytes through modulation of intracellular ROS generation.

Although glycine plays a pivotal role in neurotransmission and neuromodulation in the retina and is present in high concentration in the retina, the source of retinal glycine is still unclear. The purpose of the present study was to investigate glycine transport across the inner blood-retinal barrier (inner BRB). [(14)C]Glycine transport at the inner BRB was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) as an in vitro model of the inner BRB and in vivo vascular injection techniques. [(14)C]Glycine uptake by TR-iBRB2 cells was Na(+)- and Cl(-)-dependent, and concentration-dependent with Michaelis-Menten constants of 55.4 microM and 8.02 mM, and inhibited by glycine transporter 1 (GlyT1) and system A inhibitors. These uptake studies suggest that GlyT1 and system A are involved in [(14)C]glycine uptake by TR-iBRB2 cells. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA 1 and ATA2) mRNA are expressed in TR-iBRB2 cells. An in vivo study suggested that [(14)C]glycine is transported from blood to the retina whereas [(14)C]alpha-methylaminoisobutyric acid, a selective substrate for system A, is not. In conclusion, GlyT1 most likely mediates glycine transport at the inner BRB and is expected to play an important role in regulating the glycine concentration in the neural retina.

The system N glutamine (Gln) transporter SN1(SNAT3) is overexpressed in human malignant glioma cells in situ as compared to the adjacent brain tissue or metastases from different organs [Sidoryk, M., Matyja, E., Dybel, A., Zielinska, M., Bogucki, J., Jaskolski, D.J., Liberski, P.P., Kowalczyk, P., Albrecht, J., 2004]. Increased expression of a glutamine transporter SNAT3 is a marker of malignant gliomas. NeuroReport 15, 575-578], but its role in tumor growth as compared to the other Gln transporters is unknown. One of the profound, growth-promoting effects of glial tumor in situ is acidification of the extracellular space. In the kidney SN1(SNAT3) mRNA participates in the adaptation to acidosis. In this study therefore, expression of mRNAs coding for SN1(SNAT3) and other Gln transporters was measured in human (T98G) and rat (C6) glioma cells incubated for 4h in an acidic medium (AI) (pH 6.5). MTT assay revealed no cell loss in AI cells, and intracellular pH (pHi) as measured by a fluorescent probe (BCECF-AM) was slightly alkaline in C6 and T98G cells, indicating that the cells have adapted to AI. AI significantly decreased the SN1(SNAT3) mRNA expression in C6 (a 60% decrease) and T98G cells (a 50% decrease). The decrease retreated in C6 cells 4h after transferring them back to the neutral medium. The expression of ASCT2 mRNA (system ASC), ATA1 mRNA (system A) and SN2(SNAT5) mRNA (system N) were not affected by AI in either of the cell lines. [(3)H]Gln uptake in C6 or T98G cells grown in neutral medium was mainly mediated by system ASCT2: system N contributed to only approximately 7% of the uptake. AI did not affect the total Gln uptake, and only slightly decreased the system N-mediated component of the uptake. Hence, SN1(SNAT3) does not seem to be involved in the adaptation of cultured glioma cells to acidic millieu.