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 [C-14]glycylsarcosine (Gly-Sar), a substrate for H+/oligopeptide transporters PEPT1 and PEPT2, and [H-3]histidine, a substrate for peptide/histidine transporters PHT1 and PHT2, was measured at 37 degreesC by a rapid filtration technique. The uptake of [14 C]Gly-Sar into synaptosomes was stimulated by an inwardly directed W-gradient. The uptake system exhibited a Michaelis-Menten constant (K-t) of 110 +/- 20 muM 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 [C-14]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-Saruptake with the K-i value of 29 +/- 14 muM. These uptake properties of the brain peptide transporter (i.e., the K-t value for Gly-Sar uptake and the Ki 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. (C) 2003 Elsevier B.V. All rights reserved.