Functional characterization of brain peptide transporter in rat cerebral cortex: identification of the high-affinity type H+/peptide transporter PEPT2.
In this report, we studied the functional characteristics of a brain peptide transporter using synaptosomes prepared from rat cerebral cortex. Crude synaptosomes (P(2) fraction) were prepared from cerebral cortices in male Wistar rats. Uptake of [14C]glycylsarcosine (Gly-Sar), a substrate for H(+)/oligopeptide transporters PEPT1 and PEPT2, and [3H]histidine, a substrate for peptide/histidine transporters PHT1 and PHT2, was measured at 37 degrees C by a rapid filtration technique. The uptake of [14C]Gly-Sar into synaptosomes was stimulated by an inwardly directed H(+)-gradient. The uptake system exhibited a Michaelis-Menten constant (K(t)) of 110+/-20 microM for Gly-Sar. This value is comparable to the K(t) value for Gly-Sar uptake via the high-affinity H(+)/peptide transporter PEPT2. The H(+)-dependent uptake of [14C]Gly-Sar into synaptosomes was inhibited by di- and tripeptides and beta-lactam antibiotics, but was unaffected by amino acids glycine and histidine. In particular, kyotorphin (Tyr-Arg) completely inhibited Gly-Sar uptake with the K(i) value of 29+/-14 microM. These uptake properties of the brain peptide transporter (i.e., the K(t) value for Gly-Sar uptake and the K(i) value of kyotorphin for Gly-Sar uptake) are very similar to those of PEPT2. RT-PCR and Western blotting analyses revealed that PEPT2 is actually expressed in the cerebral cortex in rat. These results indicate that a H(+)-coupled high affinity peptide transport system is functionally expressed in the cerebral cortex and that this transport system is identical to PEPT2.
Fujita
Takuya
T
Department of Biochemical Pharmacology, Kyoto Pharmaceutical University, Yamashina, Kyoto 607-8414, Japan. fujita@mb.kyoto-phu.ac.jp
Kishida
Takeshi
T
Wada
Miyuki
M
Okada
Naoki
N
Yamamoto
Akira
A
Leibach
Frederick H
FH
Ganapathy
Vadivel
V
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport; D015152:Blotting, Northern; D015153:Blotting, Western; D002247:Carbon Isotopes / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D000074011:Peptide Transporter 1; D011522:Protons; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013379:Substrate Specificity; D027981:Symporters / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014316:Tritium / Q000378:metabolism
D000900:Anti-Bacterial Agents; D002247:Carbon Isotopes; D002352:Carrier Proteins; D004151:Dipeptides; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D000074011:Peptide Transporter 1; D011522:Protons; D012333:RNA, Messenger; C489940:Slc15a1 protein, rat; C429144:Slc15a3 protein, rat; C105521:Slc15a4 protein, rat; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; D014316:Tritium; C004194:glycylsarcosine; D006639:Histidine