Role of PEPT2 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 alpha-amino cephalosporins but was unaffected by amino acids, cephalosporins lacking an alpha-amino group, and organic anions and cations. The Michaelis constant (K(m)) of GlySar was 59.6 microM 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.
Shu
Cathaleen
C
College of Pharmacy, Department of Pharmaceutical Sciences, Upjohn Center for Clinical Pharmacology, University of Michigan, 1310 E. Catherine Street, Ann Arbor, MI 48109, USA.
Shen
Hong
H
Teuscher
Nathan S
NS
Lorenzi
Philip J
PJ
Keep
Richard F
RF
Smith
David E
DE
D000818:Animals; D001812:Blood-Brain Barrier / Q000502:physiology*; D016764:Cell Polarity; D002478:Cells, Cultured; D002831:Choroid Plexus / Q000166:cytology / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D008297:Male; D018716:Molecular Mimicry; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D027981:Symporters / Q000096:biosynthesis / Q000502:physiology*
D004151:Dipeptides; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; C004194:glycylsarcosine