Glycyl-L-glutamine disposition in rat choroid plexus epithelial cells in primary culture: role of PEPT2.
OBJECTIVE
The purpose of this research was to determine the polarity and directionality of the PEPT2-mediated uptake and transepithelial transport of the neuropeptide glycyl-L-glutamine (GlyGln) in choroid plexus.


METHODS
The transport kinetics of [3H]GlyGln was studied in neonatal rat choroid plexus epithelial cells in primary culture grown on laminin-coated Transwell filter inserts. Using a bicarbonate artificial cerebrospinal fluid (CSF) buffer (pH 7.4) at 37 degrees C, GlyGln studies were performed as a function of time, substrate concentration, and the presence of potential inhibitors (at 1 mM).


RESULTS
GlyGln (2 microM) accumulation was about three to four times greater when introduced from the apical (CSF-facing) as opposed to the basal (blood-facing) side of the cell monolayer, and transepithelial transport was about two times greater in the apical-to-basal direction. The apical uptake of radiolabeled GlyGln (2 microM) was inhibited significantly by dipeptides (i.e., unlabeled GlyGln and cysteinylglycine) and some neuropeptides (i.e., carnosine, N-acetylaspartylglutamate, kyotorphin), but was unaffected by amino acids (i.e., glycine, glutamine) as well as by [D-Arg2]-kyotorphin and glutathione. The concentration-dependent apical uptake of GlyGln (2-1000 microM) was characterized by a high-affinity process (i.e., Vmax of 72 pmol/mg/min; Km of 136 microM), consistent with the properties of PEPT2. The intracellular hydrolysis of GlyGln was extensive, however, with only 40% of the dipeptide remaining intact after 1 h.


CONCLUSIONS
The results demonstrate that PEPT2 plays an important role in regulating the apical uptake of GlyGln at the blood-CSF interface. Once inside the cell, GlyGln is rapidly degraded to its constitutive amino acids for further processing.
Hu
Yongjun
Y
Department of Pharmaceutical Sciences, The University of Michigan, Ann Arbor, Michigan 48109, USA.
Ocheltree
Scott M
SM
Xiang
Jianming
J
Keep
Richard F
RF
Smith
David E
DE
D000818:Animals; D000831:Animals, Newborn; D002336:Carnosine / Q000378:metabolism; D002478:Cells, Cultured; D002555:Cerebrospinal Fluid / Q000378:metabolism; D002831:Choroid Plexus / Q000166:cytology / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D051381:Rats; D049031:Sarcoglycans / Q000378:metabolism; D027981:Symporters / Q000378:metabolism*
D004151:Dipeptides; D049031:Sarcoglycans; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; C039788:glycylglutamine; D008353:Mannitol; D002336:Carnosine