Interaction of kyotorphin and brain peptide transporter in synaptosomes prepared from rat cerebellum: implication of high affinity type H+/peptide transporter PEPT2 mediated transport system.
High-affinity type H+/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+-gradient. Kyotorphin competitively inhibited the uptake of Gly-Sar with an inhibitory constant (Ki) of 30 +/- 4 microM in rat cerebellum synaptosomes. This uptake property is very close to that of PEPT2. Carnosine (beta-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 specific 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 affinity transporter PEPT2.
Fujita
T
T
Department of Biopharmaceutical Sciences, Kyoto Pharmaceutical University, Japan. fujita@mb.kyoto-phu.ac.jp
Kishida
T
T
Okada
N
N
Ganapathy
V
V
Leibach
F H
FH
Yamamoto
A
A
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002531:Cerebellum / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D004723:Endorphins / Q000378:metabolism*; D008297:Male; D000074011:Peptide Transporter 1; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D027981:Symporters; D013574:Synaptosomes / Q000378:metabolism*
D002352:Carrier Proteins; D004151:Dipeptides; D004723:Endorphins; D000074011:Peptide Transporter 1; D012333:RNA, Messenger; C489940:Slc15a1 protein, rat; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; C019448:kyotorphin; C004194:glycylsarcosine