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.

The intestinal H+/peptide symporter PEPT1: structure-affinity relationships.

Peptide transporter 1, PEPT1, of the mammalian enterocyte is presently under intense investigation in many laboratories because of its nutritional importance in the absorption of protein hydrolysis products and because more recent studies have shown that many drugs and prodrugs gain entry into the systemic circulation via PEPT1. Until the exact structural features of the substrate binding site of PEPT1 become available, for example by X-ray crystallography, determination of affinities followed by proof of actual membrane translocation will have to suffice when testing for possible new substrates for PEPT1. Affinity constants reflect the strength of their interaction with the binding site of the transporter. A review of the literature shows a wide range of affinity constants between 2 microM and 30 mM. We consider affinity constants for substrates or inhibitors of PEPT1 lower than 0.5 mM as high affinity, between 0.5 and 5.0 mM as medium affinity and above 5 mM as low affinity. Values above 15 mM we consider with great caution. In this mini-review we discuss affinities and structural determinants which affect affinities of a variety of substrates for PEPT1.