Role of PEPT 2 in Peptide / Mimetic Trafficking at the Blood-Cerebrospinal Fluid Barrier : Studies in Rat Choroid Plexus Epithelial Cells in Primary Culture

Recent studies have established the functional and molecular presence of a high-affinity peptide transporter, PEPT2, in whole tissue rat choroid plexus. However, the precise membrane location and directionality of PEPT2-mediated transport is uncertain at present. In this study, we examined the transport kinetics of a model dipeptide, glycylsarcosine (GlySar), along with the protein expression of PEPT2 using primary cell cultures of choroidal epithelium from neonatal rats. GlySar accumulation and transepithelial transport were 3 to 4 times higher when introduced from the apical as opposed to the basal side of the monolayers. GlySar apical uptake was also stimulated by an inwardly directed proton gradient. The uptake of GlySar was inhibited by di/tripeptides, carnosine, and -amino cephalosporins but was unaffected by amino acids, cephalosporins lacking an -amino group, and organic anions and cations. The Michaelis constant (Km) of GlySar was 59.6 M for apical uptake and 1.4 mM for basal uptake; this is consistent with the high-affinity properties of PEPT2 at the apical membrane. Immunoblot analyses and immunofluorescent confocal microscopy demonstrated the presence of PEPT2, but not PEPT1, in rat choroid plexus epithelial cells. Moreover, PEPT2 was present in the apical and subapical regions of the cell but was absent in the basolateral membrane. These findings demonstrate, for the first time, that PEPT2 protein is present at the apical membrane of choroidal epithelial cells and that it is functionally active at this membrane surface. The results suggest that PEPT2 may have a role in the efflux of peptides and/or mimetics from cerebrospinal fluid to the blood. Proton-coupled peptide transporters, PEPT1 and PEPT2, mediate the uptake of diand tripeptides via an electrogenic transport system driven by the presence of an inwardly directed proton gradient and negative membrane potential (Fei et al., 1994; Liu et al., 1995; Nussberger and Hediger, 1995; Leibach and Ganapathy, 1996). PEPT1, a low-affinity and high-capacity transporter, is mainly expressed in the small intestine and at low levels in the proximal tubule of the kidney (Terada et al., 1997; Shen et al., 1999). PEPT2, a high-affinity and low-capacity transporter, is mainly expressed in the kidney (Shen et al., 1999). These transporters serve important physiological and pharmacological functions since small peptides and peptidomimetic drugs are absorbed in the intestine or reabsorbed in the kidney via these carriers. More recently, two peptide/histidine transporters, PHT1 (Yamashita et al., 1997) and PHT2 (Sakata et al., 2001), were cloned from rat brain and shown to transport histidine and small peptides with high affinity and in a proton gradientdependent manner. Whereas PHT1 is expressed strongly in the brain and eye, PHT2 is expressed primarily in the lymphatic system and detected faintly in the brain. PHT1 and PHT2 share an amino acid identity of 49%, but homology to either rat PEPT1 or PEPT2 is less than 25%. More importantly, their physiological role in the brain has yet to be