Effect of Theanine, r-glutamylethylamide, on Brain Monoamines and Striatal Dopamine Release in Conscious Rats*

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1 Neurochemical Research, Vol. 23, No. 5, 1998, pp Effect of Theanine, r-glutamylethylamide, on Brain Monoamines and Striatal Dopamine Release in Conscious Rats* Hidehiko Yokogoshi,1,2 Miki Kobayashi,1 Mikiko Mochizuki,1 and Takehiko Terashima1 Theanine, r-glutamylethylamide, is one of the major components of amino acids in Japanese green tea. Effect of theanine on brain amino acids and monoamines, and the striatal release of dopamine (DA) was investigated. Determination of amino acids in the brain after the intragastric administration of theanine showed that theanine was incorporated into brain through blood-brain barrier via leucine-preferring transport system. The concentrations of norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole acetic acid (5HIAA) in the brain regions were unaffected by the theanine administration except in striatum. Theanine administration caused significant increases in serotonin and/or DA concentrations in the brain, especially in striatum, hypothalamus and hippocampus. Direct administration of theanine into brain striatum by microinjection caused a significant increase of DA release in a dose-dependent manner. Microdialysis of brain with calcium-free Ringer buffer attenuated the theanine-induced DA release. Pretreatment with the Ringer buffer containing an antagonist of non-nmda (N-methyl-D-aspartate) glutamate receptor, MK-801, for 1 hr did not change the significant increase of DA release induced by theanine. However, in the case of pretreatment with AP-5, (± )-2-amino-5-phosphonopentanoic acid; antagonist of NMDA glutamate receptor, the theanine-induced DA release from striatum was significantly inhibited. These results suggest that theanine might affect the metabolism and/or the release of some neurotransmitters in the brain, such as DA. KEY WORDS: Theanine; serotonin; dopamine; microdialysis; glutamate receptor; NMDA receptor. INTRODUCTION The observations relating nutrient intake to neurotransmission originated from studies of a phenomenon seemingly unrelated to the brain, i.e., daily rhythms in the metabolism of dietary amino acids. When diets are consumed, the concentrations of most amino acids in the 1 School of Food and Nutritional Sciences, The University of Shizuoka, Yada, Shizuoka 422, Japan. 2 Address reprint requests to: Hidehiko Yokogoshi, Laboratory of Nutritional Biochemistry, School of Food and Nutritional Sciences, The University of Shizuoka, 52-1 Yada, Shizuoka, 422, Japan. Tel.: , Fax.: , yokogosi@fnsl.ushizuoka-ken.ac.jp. * Special issue dedicated to Dr. Richard J. Wurtman. 667 blood change predictably in ways that depend on the foods eaten. Also the beverage containing amino acids affects the amino acid metabolism in the body. Theanine, r-glutamylethylamide, is one of the major components of amino acids in Japanese green tea (1), and is also one of the components which decides its taste. Chemical structure of theanine is shown in Fig. 1. Theanine is a derivative of glutamic acid, which is one of the neurotransmitters in the brain. It is also known that the regulation of blood pressure is very dependent on catecholaminergic and serotonergic neurons in both the brain and the peripheral nervous system (2-5). High dose of theanine to spontaneously hypertensive rats (SHR) significantly decreased the blood pressure (6) /98/ $15.00/0 C 1998 Plenum Publishing Corporation

2 668 Yokogoshi, Kobayashi, Mochizuki, and Terashima EXPERIMENTAL PROCEDURE Fig. 1. Chemical Structure of Glutamine-derivatives. In general, Japanese people take several cups of green tea daily in order to take a rest, remove a stress or chat with someone. In this study, we investigated the effect of theanine, main component in green tea extract, on brain neurotransmitters, especially dopamine (DA). There are three independent transport systems for the circulating amino acids: neutral, basic and acidic (7). The neutral carrier has a preferential affinity for the large neutral essential amino acids in blood, and is analogous to the leucine-preferring, or L-system, which is sodiumindependent and insulin-insensitive (8). The alanine-preferring, or A-system, is sodium-dependent and insulinsensitive (8), and is not present on the lumenal side of the blood-brain barrier (9). Another sodium-dependent neutral amino acid transport system has a preference for alanine, serine and cysteine, thus called the ASC-system (8), and this system is not present on the blood-brain barrier (10). We first examined whether or from which transport system theanine is incorporated into brain via bloodbrain barrier, then we investigated the effects of theanine on the brain monoamine concentrations, and the activity of dopaminergic neurons. Experimental Design. Experimental rats (SLC, Hamamatsu, Japan) were housed in individual wire cages in a temperature- and humidity-controlled room (24 C and 55% relative humidity) with a 12-hr cycle of light and dark. Theanine was obtained from Taiyo Kagaku Co., Ltd. (Yokkaichi, Japan). Male Wistar rats(about 150 g body weight) were intragastrically administered with various levels of theanine (0, 1000, 2000, or 4000 mg/kg body weight), and the concentrations of amino acids, theanine and monoamines in the brain were determined 2 hrs after theanine administration (experiment 1). Dopamine release from the striatum in unanesthetized rats was determined after a microinjection of theanine by using in vivo microdialysis (Eicom Microdialysis system) (experiment 2) (11). To examine whether calcium (Ca) ion may participate in the increase of DA release induced by theanine, Ca-free mobile buffer, the microinjection of glutamate diethyl ester hydrochloride (GDEE), a glutamate receptor blocker (experiment 2), or antagonists of glutamate receptor such as MK-801 and AP-5 (experiment 3) was introduced in the microdialysis system. The experimental procedures used in this study met the guidelines of the Animal Care and Use Committee of the University of Shizuoka. Amino Acids and Monoamines Analyses in Serum and/or Brain. Amino acids including theanine in serum and brain were determined by an automatic amino acid analyzer (L-8500, Hitachi, Japan). Blood was collected by cardiac puncture under light anesthesia with ethylether. The brain was perfused with saline from the carotid artery by a cannulated syringe. After decapitation, the brain of each animal was immediately removed, frozen on dry ice and stores at 70 C until assays. The concentrations of monoamines in various brain regions, i.e., locus coeruleus, hippocampus, amygdala, striatum, cerebral cortex, cerebellum, brain stem and hypothalamus, of normal rats were determined by the electrochemical detector following the separation by high performance liquid chromatography (HPLC) (12). DA Release by Microdialysis. The guide cannula of the microdialysis probe into the striatum was stereotaxically implanted 0.2mm posterior to the bregma, 3.0mm lateral to the midline, and 3.5mm ventral to the cortical surface according to the atlas of Paxinos and Watson (13). After one day of the operation, the dummy cannula was removed from the guide cannula, and the dialysis probe was inserted. This dialysis probe (MI-A-I-8-03, Eicom, Japan) had a dialysis membrane (at molecular weight cut-off 5,000) and a side tube in order to microinject the theanine or GDEE from outside. The probe was perfused with a Ringer solution (147 mm Na + 4 mm K +, 2.3 mm Ca 2+, and mm Cl -, ph 6.0) at the rate of 2 ul/min (EP-60; Eicom, Japan). Using samples collected for 2 hrs before injecting the theanine, the basal levels of releasable DA and its metabolites (3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)) were measured. The releasable DA and its metabolites were separated by reverse-phase HPLC using MA-ODS column (5 um particles, 4.6 x 250 mm; Eicom, Japan), and detected with an electrochemical detector (CB-100; Eicom, Japan). On the other hand, in order to examine the mechanism of DA release induced by theanine from striatum, each antagonist (500 umol) of glutamatergic neurons ((+)-MK-801, dizocilpine maleate, non-competitive NMDA receptor antagonist; AP-5, (± )2-amino-5-phosphonopentanoic acid, NMDA receptor antagonist) was dissolved in Ringer buffer and pre-perfused for 1 hr before perfusion of both of theanine and its antagonist for 1 hr (then dialysis probe, MI-A-I-8, Eicom, Japan). These antagonists were purchased from Research Biochemicals International (Natick, MA). The mobile phase was composed of 0.1M citric acid buffer, ph 3.9, containing 12% methanol, 20 mm EDTA and 160 mg/1 sodium-1-octane-sulfate. The graphite electrode (WE-3G; Eicom, Japan) was set at V

3 Theanine and Dopaminergic Neurons 669 Table I. Brain Amino Acid Concentrations after Theanine Administration (experiment 1) 0 Theanine ND Leucine - preferring transport system Phe ± b Tyr ± b Leu ± 0.028" Ile ± b Val ± b His ± b Met ± c Thr ± Theanine (mg/100g body weight) 100 u mol/g brain ± a ± a ± a ± a ± a ± a ± a ± b ± Alanine- preferring transport system Ala Gly Ser Thr ± 0.218" ± a ± ± ± ab ± ab ± ± ± a ± a ± 0.004" ± ± 0.004" ± 0.006" ± 0.005" ± a ± ± 0.037" ± 0.162" ± ± Alanine, Serine and Cysteine - preferring transport system Ala Ser Cys ± 0.218" ± ± bc ± ab ± ± ab ± a ± ± 0.009" Others Tau Glu Asp Lys Car Arg ± b ± a ± 0.354" ± b ± b ± b ± ab ± 0.664" ± bc ± ab ± 0.010" ± a ± 0.270" ± ab ± ab ± 0.047" ± ± 0.015" ± 0.100" ± a ± a ± a ± a ± 0.006" ± 0.012" ± ab ± ± ab ± 0.307" ± ± ± ab ± ± c ± a ± 0.642" ± c ± 0.027" ± ab ± 0.015" Means ± SEM for six rats per group. Means within a same row not sharing the same letter are significantly different (Duncan's multiple range test, p < 0.05). ND = not detectable. (vs. Ag/AgCl). The perfusate from the striatum (40 ul) was injected into the HPLC every 20 min via an automatic injector (AS-19; Eicom, Japan). After completion of the experiment, the position of the microdialysis probe was histologically examined. The values of releasable monoamines were expressed as % change from the averaged basal values (DA, 97.1 ± 1.8; DOPAC, ± 865.6; HVA, ± 218.9; respectively, pg/40 ul perfusate/20 min) 2 hours before chemical administration. Statistics. Duncan's multiple range test (14) was used to compare the differences of means after they were analyzed by ANOVA. P- values of less than 0.05 are considered to be of statistical significance. RESULTS Effect of Theanine on Brain Amino Acid and Monoamine Concentrations in Normal Rats (Experiment 1). The concentrations of theanine in the brain increased in a dose-dependent manner after the administration of theanine (Table I). On the contrary, the concentrations of large neutral amino acids such as phenylalanine, tryptophan, threonine, tyrosine and branched-chain amino acids, which were incorporated via L-system, decreased significantly by a high dose of theanine. The concentrations of other amino acids such as alanine, serine, glycine, aspartic acid and glutamic acid were unchanged by the theanine administration. Therefore, theanine may be competitively incorporated in the brain via L-system at the blood brain barrier. The concentrations of monoamines (catecholamine and 5-hydroxyindole) in various brain regions were shown in Fig. 2-A, B, C, D and E. The concentrations of norepinephrine and DOPAC was unchanged by the administration of theanine. However, the concentration of DA in striatum was significantly elevated following the administration of theanine. On the other hand, the concentrations of serotonin in the brain regions tended to increase following the administration of theanine; they were significantly elevated, in hippocampus, striatum and hypothalamus. However, 5HIAA was unaffected by the administration of theanine. Effect of Theanine on DA Release from Brain Striatum in Rats (Experiment 2). When theanine was directly

4 670 Yokogoshi, Kobayashi, Mochizuki, and Terashima Fig. 3. Dose-dependent Theanine Stimulation of Dopamine Striatal Release (experiment 2). Theanine (0, 5 or 10 umol/20 ul) was microinjected into striatum. The values were measured by means of in vivo microdialysis, and expressed as % change from the averaged basal values 60 min before administration. * Significantly different from the basal value perfused with Ringer solution (P < 0.05). Fig. 2. Monoamine Concentrations Norepinephrine (A), DA (B), DOPAC (C), Serotonin (D) and 5-HIAA (E) in Brain Regions of Normal Rats Administered with Saline (Hi) or Theanine (ts3) (experiment 1). The vertical bars on each column represent the means ± SEM. * Significantly different from the control group (saline) (P < 0.05). LC; locus coeruleus, Hip; hippocampus, Amy; amygdala, ST; striatum, CC; cerebral cortex, Ce; cerebellum, Bs; brain stem, Hyp; hypothalamus. microinjected into brain striatum, the DA release significantly and dose-dependently increased during the first 20min of the administration of theanine, and then it gradually decreased (Fig. 3). To examine the effect of Ca on DA release stimulated by theanine, the Ca-free Ringer buffer was perfused. Perfusion of the Ca-free buffer did not induce DA release from the striatum. Under this condition, the microinjection of theanine into striatum did not induce DA release (Data were not shown). Since the glutamatergic neurons co-exist with DA neurons in the striatum and the entry of Ca into DA neurons is also regulated by NMDA or non-nmda receptors, we determined in the next study the DA release from antagonist-pretreated striatum by perfusion of Ringer buffer containing theanine with or without antagonist. Pretreatment with GDEE inhibited the increase of DA release from striatum induced by theanine (Fig. 4). Effect of Microperfusion of Glutamatergic Neuron's Antagonist on Striatal DA Release Induced by Theanine (Experiment 3). We examined the effect of NMDA- or non-nmda receptor antagonist on DA release induced by theanine. Theanine perfusion induced Fig. 4. Time Course of the Effect of GDEE on the Release of Dopamine from the rat striatum caused by Theanine (experiment 2). The values were measured by means of in vivo microdialysis, and expressed as % change from the averaged basal values 60 min before administration. The arrow indicates the time when the sample (theanine, 10 umol; GDEE, 1 nmol) was microinjected. * Significantly different from the basal value perfused with Ringer solution (P < 0.05). the significant increase of striatal DA release (4.29 fold, Fig. 5) like microinjection of theanine in experiment 2. In the case of pretreatment of MK-801, non-competitive NMDA receptor antagonist, DA release was significantly enhanced by theanine (4.35 fold), as the same manner without MK-801 (Fig. 6). AP-5 is a potent and selective antagonist of the NMDA receptors. NMDA receptor involved in modulation of striatal DA release was corroborated by blockage of the stimulatory effect of NMDA

5 Theanine and Dopaminergic Neurons 671 Fig. 5. Effect of Theanine on the Release of Dopamine, DOPAC, and HVA from the Rat Striatum (experiment 3). Theanine (500 mmol, 2 ul/min) was perfused into striatum for 1 hour. The values were measured by means of in vivo microdialysis, and expressed as % change from the averaged basal values 2 hours before administration. * Significantly different from the basal value perfused with Ringer solution (P < 0.05). Fig. 7. Effect of AP-5 on Theanine-induced Dopamine Release from the Rat Striatum (experiment 3). AP-5 (500 umol, 2 ul/min) was perfused into striatum for 1 hour, thereafter Ringer buffer containing AP- 5 and theanine was perfused for 1 hour. The values were measured by means of in vivo microdialysis, and expressed as % change from the averaged basal values 2 hours before administration. * Significantly different from the basal value perfused with Ringer solution (P < 0.05). DISCUSSION Fig. 6. Effect of MK-801 on Theanine-induced Dopamine Release from the Rat Striatum (experiment 3). MK-801 (500 umol, 2 ul/min) was perfused into striatum for 1 hour, thereafter Ringer buffer containing MK-801 and theanine was perfused for 1 hour. The values were measured by means of in vivo microdialysis, and expressed as % change from the averaged basal values 2 hours before administration. * Significantly different from the basal value perfused with Ringer solution (P < 0.05). by AP-5. Pretreatment of AP-5, NMDA receptor antagonist, significantly inhibited the increase of DA release (2.32 fold) from striatum induced by theanine (Fig. 7). First increase of DA release observed by pretreatment of AP-5 may be due to the stimulation of Ca entry into synaptosomes by changing the Ca permeability (15,16). Thus, theanine action for the increase of DA release may be due to the enhancement of the utilization of Ca via NMDA receptors. Many pathways of brain nutrient metabolism are influenced as much by the availability of precursor nutrients inside of brain cells as by the activity of cellular nutrient-metabolizing enzymes. Nutrient supply in the brain can affect cerebral metabolism, because the ratelimiting enzymes for a variety of metabolic pathways are normally unsaturated by the existing concentrations of precursor nutrients in the brain (14-21). Theanine is one of the amino acids present in Japanese green tea. Its chemical structure resembles glutamic acid, which is one of the important functional substances in the brain. In previous studies, we sought physiological functions of theanine for brain. As one of the behavioral indexes for brain function, blood pressure was determined (6). Increase in the dose of theanine resulted in a concomitant decrease in blood pressure, a significant decrease being observed with the high doses. The systolic blood pressure of SHR was especially affected by the administration of theanine. This antihypertensive action of theanine was specific to SHR and was not observed in WKY. Glutamine, which resembles theanine in structure, did not exhibit an antihypertensive action on SHR. Therefore, this antihypertensive action of theanine was specific for theanine (6). To characterize the mode for intestinal absorption of theanine, the ionic dependency and kinetic properties of the theanine- and glutamineevoked transmural electrical potential difference changes were investigated in vitro. We found that the intestinal

6 672 Yokogoshi, Kobayashi, Mochizuki, and Terashima absorption of theanine and glutamine was mediated by a common Na + -coupled co-transporter in the brush-border membrane (25). In this study, we first investigated whether theanine was incorporated into brain. Theanine was accumulated in a dose-dependent manner into brain after theanine administration. In general, each circulating amino acid is competitively incorporated into brain via a respective transport system such as L-, A- or ASC-system (26). Comparing with the concentrations of serum amino acids, the concentrations of almost all amino acids transported via L-system were significantly decreased in the brain by the administration of theanine. This result suggests that theanine is incorporated into brain via L-system. In the next study, the effect of administered theanine on the monoamines in brain regions was also investigated. We observed some alterations in catecholamines and 5-hydroxyindoles levels in each brain region following the theanine administration. Especially in striatum, where dopaminergic neurons are concentrated, the contents of DA were significantly greater after the intragastric administration of theanine than those after saline administration. As DA concentration was changed by theanine administration, we examined the effect of DA release induced by theanine using a brain microdialysis technique. When theanine was directly microinjected into brain striatum, the DA release was significantly enhanced, and its response was dose-dependent. Other investigators showed with a similar method that glutamatergic neurons co-existed with DA neurons in the striatum, and that glutamate induced DA release from the striatum in freely moving rats (27). The receptors of glutamatergic neurons, especially NMDA(N-methyl-D-aspartate)-receptors, are known to be coupled with Ca entry, and DA release is regulated by this Ca utilization. Therefore, we further examined whether the stimulation of DA release caused by theanine occurred under the Ca-free Ringer buffer. Results showed that theanine did not increase the DA release from striatum under the Ca-free condition (Data were not shown). The pretreatment (microinjection) of glutamate receptor blocker, GDEE, diminished theanine-induced stimulation of DA release from striatum. Therefore, we used other antagonists dissolved in Ringer's solution to clarify the mechanism of theanineinduced DA release from striatum. NMDA receptor antagonist, AP-5, inhibited the enhancement of DA release induced by theanine administration, however, non- NMDA receptor antagonist, MK-801, did not affect DA release. These results suggested that the stimulating action of theanine on DA release from striatum might be caused by the utilization of Ca via NMDA receptors. However, there are still many questions regarding the mechanism of the stimulation of DA release caused by theanine; that is, whether a direct stimulation of DA neurons, or a concomitant activation of interneurons which promotes the glutamate release, resulting in DA release. It is also unclear whether magnesium ion can be related to this theanine-induced DA release through the changes in NMDA receptors (28,29). Further investigations on these physiological functions of theanine are needed. REFERENCES 1. Sakato, Y Studies on the chemical constituent of tea. Part III. On a new amide theanine. Nippon Nogeikagaku Kaishi 23: Kuhn, D. M., Wolf, W. A., and Lovenberg, W Review of the role of the central serotonergic neuronal system in blood pressure regulation. Hypertension 2: Bresnahan, M. R., Hatzinikolaw, P., Brunner, H. R., and Gavras, H Effect of tyrosine infusion in normotensive and hypertensive rats. Am. J. Physiol. 239: Lown, B., Temte, J. V., and Reich, P Basis for recurring ventricular fibrillation in the absence of coronary heart disease and its management. New Eng. J. Med. 294: Sved, A. F., Fernstrom, J. D., and Wurtman, R. J Tyrosine administration reduces blood pressure and enhances brain norepinephrine release in spontaneously hypertensive rats. Proc. Natl. Acad. Sci., U.S.A. 76: Yokogoshi, H., Kato, Y., Sagesaka, Y., Matsuura, T., Kakuda, T., and Takeuchi, N Reduction effect of theanine on blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats. Biosci. Biotech. Biochem. 59: Oldendorf, W. H., and Szabo, J Amino acid assignment to one of three blood-brain barrier amino acid carriers. Am. J. Physiol. 230: Christensen, H. N Developments in amino acid transport, illustrated for the blood-brain barrier. Biochem. Pharmacol. 28: Sershen, H., and Lajtha, A Inhibition pattern by analogs indicates the presence of ten or more transport systems for amino acids in brain cells. J. Neurochem. 32: Wade, L. A., and Brady, H. M Cysteine and cystine transport at the blood-brain barrier. J. Neurochem. 37: Horie, K., Morita, A., and Yokogoshi, H Endothelin-1 and endothelin-3 modulate dopaminergic neurons through different mechanisms. Life Sci. 57: Yokogoshi, H Effect of dietary level of protein or methionine and threonine on the amino acids and catecholamines in brain of rats fed a high tyrosine diet. J. Nutr. Sci. Vitaminol. 31: Paxinos, G., and Watson, C The rat brain in stereotaxic coordinates, Academic Press, New York. 14. Duncan, D. B Multiple-range tests for correlated and heteroscedastic means. Biometrics 13: Pastuszko, A., Yee, D. K., and Wilson, D. F Regulation of calcium uptake in synaptosomes from rat brain by D,L-2-amino- 5-phosphonovaleric acid. FEBS Lett. 218: Yee, D. K., Pastuszko, A., Nelson, D., and Wilson, D. F Effects of DL-2-amino-5-phosphonovalerate on metabolism of catecholamines in synaptosomes from rat brain. J. Neurochem. 52:

7 Theanine and Dopaminergic Neurons Drewes, L. R., and Gilboe, D. D Glycolysis and the permeation of glucose and lactate in the isolated, perfused dog brain during anoxia and postanoxic recovery. J. Biol. Chem. 248: Fernstrom, J. D., and Wurtman, R. J Brain serotonin content: physiological dependence on plasma tryptophan levels. Science 173: Rubin, R. A., Ordonez, L. A., and Wurtman, R. J Physiological dependence of brain methionine and S-adenosylmethionine concentrations on serum amino acid pattern. J. Neurochem. 23: Cohen, E. L., and Wurtman, R. J Brain acetylcholine: Control by dietary choline. Science 191: Gibson, C. J., and Wurtman, R. J Physiological control of brain norepinephrine synthesis by brain tyrosine concentration. Life Sci. 22: Yokogoshi, H., and Wurtman, R. J Meal composition and plasma amino acid ratios: Effect of various proteins or carbohydrates, and of various protein concentrations. Metabolism 35: Yokogoshi, H., Iwata, T., Ishida, K., and Yoshida, A Effect of amino acid supplementation to low protein diet on brain and plasma levels of tryptophan and brain 5-hydroxyindoles in rats. J. Nutr. 117: Yokogoshi, H., Hayase, K., and Yoshida, A The quality and quantity of dietary protein affect brain protein synthesis in rats. J. Nutr. 122: Kitaoka, S., Hayashi, H., Yokogoshi, H., and Suzuki, Y Transmural potential changes associated with the in vitro absorption of theanine in the guinea pig intestine. Biosci. Biotech. Biochem. 60: Pardridge, W. M Regulation of amino acid availability to the brain. Pages , in Wurtman, R. J., and Wurtman, J. J. (eds.), Nutrition and the brain, Raven Press, New York. 27. Shimizu, N., Duan, S., Hori, T., and Oomura, Y Glutamate modulates dopamine release in the striatum as measured by brain microdialysis. Brain Res. Bull. 25: Mayer, M. L., Westbrook, G. L., and Guthrie, P. B Voltagedependent block by Mg 2+ of NMDA responses in spinal cord neurons. Nature 309: Nowak, L., Bregestovski, P., Ascher, P., Herbert, A., and Prochiantnz, A Magnesium gates glutamate-activated channels in mouse central neurons. Nature 307: