peptide transporter PEPT2 mediated transport system

Neuroscience Letters 271 (1999) 117±120

Interaction of kyotorphin and brain peptide transporter in synaptosomes prepared from rat cerebellum: implication of high af®nity type H 1/peptide transporter PEPT2 mediated transport system Takuya Fujita a,*, Takeshi Kishida a, Naoki Okada a, Vadivel Ganapathy b, Frederick H. Leibach b, Akira Yamamoto a a

Department of Biopharmaceutical Sciences, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607±8414, Japan b Department of Biochemistry and MolecularBiology, Medical College of Georgia, Augusta, GA 30912±2100, USA Received 8 June 1999; received in revised form 30 June 1999; accepted 1 July 1999

Abstract High-af®nity type H 1/peptide cotransporter PEPT2 is preferentially expressed in the kidney, and is responsible for reabsorption of di- and tripeptides in epithelial tubules. Interestingly, PEPT2 has been recently cloned from rat brain. However, there is very little information available on the peptide transporter activity in the brain. In the present study, we investigated the interaction of kyotorphin (L-tyrosyl-L-arginine) with the peptide transporter using synaptosomes prepared from rat cerebellum. The activity of the peptide transporter was assessed by measuring the uptake of radiolabeled glycyl-sarcosine (Gly-Sar), which is a prototypical substrate for the peptide transporter, in the presence of H 1gradient. Kyotorphin competitively inhibited the uptake of Gly-Sar with an inhibitory constant (Ki) of 30 ^ 4 mM in rat cerebellum synaptosomes. This uptake property is very close to that of PEPT2. Carnosine (b -alanyl-L-histidine) also inhibited the uptake of Gly-Sar, on the other hand, TRH did not interact with the peptide transporter. RT-PCR using speci®c primers revealed that PEPT2 mRNA exists in cerebellum in rat. Taken collectively, these results indicate that the functional peptide transport system in rat cerebellum might be the high af®nity transporter PEPT2. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Kyotorphin; Glycylsarcosine; Cerebellum; Synaptosomes; Peptide transporter; PEPT2

Proton-coupled peptide transporters, PEPT1 and PEPT2, mediate the transport of di- and tripeptides, and the transport systems are electrogenic transport driven by the presence of an inwardly directed H 1 gradient and a negative membrane potential [1,7,10]. PEPT1, the low-af®nity and high-capacity type peptide transporter, is mainly expressed in the small intestine [5]. PEPT2, the high-af®nity and low-capacity type peptide transporter, is mainly expressed in the kidney [3,4,9,13,14]. Interestingly, a full-length PEPT2 cDNA has been recently isolated from rat brain cDNA library [2,18]. Physiological role of PEPT2 in the brain, however, is unknown. In the present study, we investigated the interaction of neuropeptide kyotorphin (l-tyrosyl-l-arginine) [16,17] with * Corresponding author. Tel.: 181-75-595-4662; fax: 181-75595-4761. E-mail address: [email protected] (T. Fujita)

the brain peptide transporter using synaptosomes prepared from rat cerebellum. RT-PCR using speci®c primers indicated that PEPT2 mRNA exists in cerebellum. The results in the present study indicate that the functional peptide transport system in rat cerebellum might be the high af®nity transporter PEPT2. [1± 14C]Glycylsarcosine (Gly-Sar, speci®c radioactivity, 56.8 mCi/mmol) was obtained from Cambridge Research Biochemicals (Cleveland, UK). Kyotorphin, TRH, unlabeled dipeptides, and amino acids were from Sigma (St. Louis, MO). Carnosine (b -alanyl-l-histidine) was from Peptide Institute Inc. (Osaka, Japan). All other chemicals were of analytical grade. Animal experiments were all performed in accordance with the Guideline for Animal Experimentation in Kyoto Pharmaceutical University. Crude synaptosomes (P2 fraction) were prepared from the cerebellum of rat according to the method of Sastre [15]. In brief, male Wistar rats

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 54 0- 6

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T. Fujita et al. / Neuroscience Letters 271 (1999) 117±120

weighing 200±250 g were killed by decapitation, then the brains were quickly removed and the cerebellum was dissected on ice. The tissue was homogenized with 0.32 M sucrose containing 10 mM Tris±HCl (pH 7.5) and 1 mM EDTA, and was centrifuged at 1000 £ g for 10 min. The supernatant (S1 fraction) was further centrifuged at 32 000 £ g for 30 min to yield P2 pellet (crude synaptosomes). The pellet was resuspended in the loading buffer consisted of (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, 5 glucose, and 25 HEPES±Tris (pH 7.5) to adjust protein concentration to 2.0 mg/ml. Uptake of [ 14C] Gly-Sar was measured at 378C by a rapid ®ltration technique using Millipore ®lter (DAWP; pore size, 0.64 mm) as described previously [6]. Uptake was initiated by mixing 100 ml of synaptosomes with 400 ml of uptake buffer containing radiolabeled compound. The composition of the uptake buffer was (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, 5 glucose, and 25 Mes±Tris (pH 6.0). After 3 min incubation, the mixture was then ®ltered and the

®lter remaining membrane was washed three times with 5 ml of the ice-cold loading buffer. The washed ®lter was counted by liquid scintillation spectrometry. The non-speci®c uptake and binding was determined by measuring GlySar uptake in the presence of 20 mM of glycylglycine. The kinetic parameters, Michaelis±Menten constant (Km) and maximal velocity (Vmax), were calculated by nonlinear regression program using the software Sigma Plot version 4.0 (Jandel Scienti®c). Total RNA was isolated from cerebellum using Sepasol RNA I reagent (Nacalai Tesque, Kyoto, Japan) according to the manufacturer's instructions. Subsequently, mRNA was isolated using Oligotex-dT30 (Takara Biochemicals, Ohtsu, Japan) and was subjected to RT-PCR. The primers speci®c for rat PEPT1 were 5 0 -GTC ATC TCA GTG AGC TCA-3 0 (forward primer) and 5 0 -TGG TGA CAT TTT CGT ACA-3 0 (reverse primer) which are corresponded to nucleotide positions 340±357 and 1571±1588 of cDNA, respectively [11]. The primers speci®c for rat PEPT2 were 5 0 -GCA TCT CAT

Fig. 1. (A) Inhibition of Gly-Sar uptake by kyotorphin (X) and TRH (P) in rat cerebellum synaptosomes. [ 14C]Gly-Sar (20 mM) uptake was measured for 3 min over concentration ranges of kyotorphin and TRH of 1 mM±2.5 mM. (B) Uptake of Gly-Sar into the synaptosomes in the presence of three kinds of neuropeptides (kyotorphin, carnosine and TRH) and histidine. The uptake of [ 14C]Gly-Sar (20 mM) was measured at 378C with a 3-min incubation in the presence or absence of unlabeled inhibitors (4 mM). Control uptake in the absence of inhibitors was taken as 100% (9:15 ^ 0:7 pmol/min/mg protein). (C) Kinetics of inhibition of Gly-Sar uptake by kyotorphin in the synaptosomes. Uptake was measured for 3 min over a Gly-Sar concentration range of 0.02±1 mM in the absence (X) or presence (O) of 100 mM kyotorphin. (D) The results of (C) are given as Eadie±Hofstee plot. V, uptake (pmol/min per mg protein); s, Gly-Sar concentration. Each experiment is the mean ^ SE of at least three experiments.

T. Fujita et al. / Neuroscience Letters 271 (1999) 117±120

CGC AGA TGT-3 0 (upstream primer) and 5 0 -TGA CTG GAA TGT CCT CTG-3 0 (downstream primer) which are corresponded to nucleotide positions 1030±1047 and 1953±1970 of cDNA, respectively [14]. For detection of mRNA of the peptide/histidine transporter PHT1, an oligonucleotide (5 0 -AAG GCC AAC ATC ACA CCC T-3 0 ) corresponding to nucleotide position 540±558 of cDNA was used as forward primer, and an oligonucleotide (5 0 TTA CTC TCC AGG ATT CCT-3 0 ) corresponding to nucleotide position 1277±1294 of cDNA as reverse primer. The mRNA (0.5 mg) was reverse-transcribed and used as a template for subsequent PCR with the set of speci®c primers. PCR was performed according to the following protocol: 948C for 30 s, 568C for 30 s, and 728C for 90 s; 39 cycles. We ®rst investigated the interaction of kyotorphin and TRH with brain peptide transporter by assessing their ability to compete with Gly-Sar uptake into the synaptosomes (Fig. 1A). Gly-Sar is a prototypical and peptidase resistant dipeptide substrate widely used for functional studies of peptide transporters [4±6,18,10]. Kyotorphin was found to inhibit the Gly-Sar uptake into the synaptosomes in a concentration-dependent manner. The inhibition was about 77% at a concentration of 2.5 mM. The IC50 value for the inhibitory process was 34 ^ 5 mM. On the contrary, TRH failed to inhibit the Gly-Sar uptake even at more than 2.5 mM (Fig. 1A,B). Ueda et al. [17] have reported that kyotorphin is taken up by rat brain synaptosomes in an energy-dependent manner in the Km value of 0:13 ^ 0:01 mM. We thus studied the inhibition kinetics of Gly-Sar uptake by kyotorphin in synaptosomes (Fig. 1C,D). The Michaelis±Menten constant (Km) and maximal velocity (Vmax) for Gly-Sar uptake were 0:13 ^ 0:02 mM and 56:3 ^ 2:6 pmol/min/mg protein in the absence of kyotorphin. This kinetic property is very close to that of PEPT2. Namely, this Km value is comparable with the values obtained from a rat kidney cell line SKPT (0.07 mM) [4] and a human neuroblastoma cell line SK-N-SH transiently transfected with PEPT2 cDNA (0.08 mM) [18]. The presence of kyotorphin decreased the af®nity of brain peptide transporter for Gly-Sar without affecting the Vmax value. The kinetic parameters in the presence of 0.1 mM kyotorphin were 0:30 ^ 0:06 mM for Km and 61:5 ^ 4:8 pmol/min per mg protein for Vmax. Using this Km value obtained from kinetical analysis, the Ki values of 30 ^ 4 mM was calculated from the IC50 value. These results show that kyotorphin and Gly-Sar compete for the same substrate binding site present on the peptide transporter in the cerebellum synaptosomes. To determine whether this transport system in the cerebellum is identical to PEPT2 or not, RT-PCR experiments were performed (Fig. 2). Using mRNA from cerebellum a PEPT2-speci®c ampli®cation product with a length of ~0.94 kb could be detected (Fig. 2; lane 2). However, a PEPT1speci®c product (~1.2 kb in size) was not ampli®ed (Fig. 2; lane 1). Recently the cDNA of a new peptide transporter,

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peptide/histidine transporter PHT1, has been cloned from rat brain [19]. Interestingly, PHT is capable of transporting the free amino acid histidine with a Km value in the micromolar range as well as di- and tripeptides [19]. RT-PCR analysis also shows the expression of PHT1 (~0.75 kb in size) in the cerebellum (Fig. 2; lane 3). In our preliminary experiment, however, uptake of [ 3H]histidine (2 mM) into the cerebellum synaptosomes was not inhibited by an excess concentration of dipeptide (unpublished observation). Further, 4 mM histidine failed to inhibit the [ 14C]Gly-Sar uptake into the synaptosomes (Fig. 1B). Thus PHT1 does not appear to mediate the uptake of Gly-Sar and/or kyotorphin into the cerebellum synaptosomes. Although the present study indicates that brain peptide transporter expressed in the cerebellum might be PEPT2, further study is required to identify this brain peptide transporter to PEPT2. In addition, precious distribution of PEPT2 in the brain is still unknown. Nicolaus et al. [12] have reported that PEPT2 expression in the brain is primarily restricted to neuronal cells in the hippocampus and to epithelial cells in the choroid plexus. However, Berger and Hediger [2] show that PEPT2 mRNA is expressed in astrocytes, ependymal cells and choroid plexus epithelial cells using in-situ hybridization. In these regions, brain peptide transporter, which is presumed to be PEPT2, might function as a terminator of various neuropeptides such as kyotorphin [16,17] and carnosine [8] and remove neuropeptide fragments degraded by peptidases from extracellular ¯uid. Further study remains to clarify this hypothesis. In conclusion, the present studies demonstrate that brain peptide transporter, which recognizes Gly-Sar and kyotorphin as substrates, is functionally expressed in the

Fig. 2. Expression of PEPT1, PEPT2 and PHT1 in rat cerebellum. The mRNA (0.5 mg) was subjected to RT-PCR using primer pairs speci®c for PEPT1 (lane 1), PEPT2 (lane 2), and PHT1 (lane 3). Expression of glyceraldhyde-3-phosphate dehydrogenase (GAPDH) was determined as an internal standard (lower panel). The primers speci®c for rat GAPDH were 5 0 -CCA GTA TGA CTC TAC CCA CGG CAA-3 0 (forward primer) and 5 0 -ATA CTT GGC AGG TTT CTC CAG GCG-3 0 (reverse primer) which are corresponded to nucleotide positions 158±181 and 759±782 of cDNA, respectively. Lane M is 1 kb ladder (Gibco BRL). PCR was performed according to the following protocol: 948C for 30 s, 568C for 30 s, and 728C for 90 s; 39 cycles.

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cerebellum in rats. The physiological role of this peptide transporter in the cerebellum remains to be seen. We thank Mr. J. Kishimoto for his technical assistance. This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (11771505) from Japan Society for the Promotion of Science and by a grant from Kyoto Pharmaceutical University. [1] Adibi, S.A., Renal assimilation of oligopeptides: physiological mechanisms and metabolic importance. Am. J. Physiol., 272 (1997) E723±E736. [2] Berger, U.V. and Hediger, M.A., Distribution of peptide transporter PEPT2 mRNA in the rat nervous system. Anat. Embryol., 199 (1999) 439±449. [3] Boll, M., Herget, M., Wagener, M., Weber, W.M., Markovich, D., Biber, J., Clauss, W., Murer, H. and Daniel, H., Expression cloning and functional characterization of the kidney cortex high-af®nity proton-coupled peptide transporter. Proc. Natl. Acad. Sci. USA, 93 (1996) 284±289. [4] Brandsch, M., Brandsch, C., Prasad, P.D., Ganapathy, V., Hopfer, U. and Leibach, F.H., Identi®cation of a renal cell line that constitutive expresses the kidney-speci®c highaf®nity H 1-peptide transporter. FASEB J., 9 (1995) 1489± 1496. [5] Fei, Y.J., Kanai, Y., Nussberger, S., Ganapathy, V., Leibach, F.H., Romero, M.F., Singh, S.K., Boron, W.F. and Hediger, M.A., Expression cloning of mammalian proton-coupled oligopeptide transporter. Nature, 368 (1994) 563±566. [6] Ganapathy, M.E., Prasad, P.D., Mackenzie, B., Ganapathy, V. and Leibach, F.H., Interaction of anionic cephalosporins with the intestinal and renal peptide transporters PEPT1 and PEPT2. Biochim. Biophys. Acta, 1324 (1997) 296±308. [7] Ganapathy, V. and Leibach, F.H., Role of pH gradient and membrane potential in peptide transport in intestinal and renal brush-border membrane vesicles from the rabbit. Studies with l-carnosine and glycyl-l-proline. J. Biol. Chem., 258 (1983) 14189±14192. [8] Kanaki, K., Kawashoma, S., Kashiwayanagi, M. and Kurihara, K., Carnosine-induced inward currents in rat olfactory bulb neuron in cultured slices. Neurosci. Lett., 231 (1997) 170±187.

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