Preliminary investigation into the expression of proton-coupled oligopeptide transporters in neural retina and retinal pigment epithelium (RPE): lack of functional activity in RPE plasma membranes.
OBJECTIVE
To determine the expression and functional activity of proton-coupled oligopeptide transporters (POT) in retinal pigment epithelial (RPE) cells.


METHODS
RT-PCR was used to probe the presence of POT mRNA in freshly isolated bovine RPE (BRPE) and human RPE (HRPE) cells, a human RPE cell line (ARPE-19), and human and bovine neural retina. [14C]GlySar uptake was used to characterize POT activity in cultured ARPE-19 cells and freshly isolated BRPE cell sheet suspensions.


RESULTS
PHT1 mRNA was expressed in BRPE, HRPE, ARPE-19, and bovine and human neural retina. In contrast, PEPT2 and PHT2 were expressed only in bovine and human retina, and PEPT1 could not be detected. GlySar exhibited a linear uptake over 6 h at pH values of 6.0 and 7.4, with greater uptake at pH 7.4 (p < 0.01). GlySar uptake did not exhibit saturability (5-2000 microM) and was unchanged when studied in the presence of 1 mM L-histidine. In contrast, GlySar uptake was significantly decreased when studied at 4 degrees C or in the presence of endocytic inhibitors at 37 degrees C (p < 0.01). Studies in BRPE cell sheet suspensions validated the results obtained in ARPE-19 cells and strongly suggested the absence of POT on the apical and basolateral membranes of RPE.


CONCLUSIONS
PHT1 mRNA is present in native bovine and human RPE and a human RPE cell line. However, the data argue against PHT1 being expressed on plasma membranes of RPE. Overall, GlySar appears to be taken up by RPE cells via a low-affinity, endocytic process.
Department of Pharmaceutical Sciences, The University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
Scott M
SM
Ocheltree
Richard F
RF
Keep
Hong
H
Shen
Dongli
D
Yang
Bret A
BA
Hughes
David E
DE
Smith
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology*; D002478:Cells, Cultured; D004151:Dipeptides / Q000378:metabolism; D006639:Histidine / Q000494:pharmacology; D006801:Humans; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D009474:Neurons / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D000074011:Peptide Transporter 1; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters / Q000096:biosynthesis / Q000235:genetics; D013997:Time Factors
D002352:Carrier Proteins; D004151:Dipeptides; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D000074011:Peptide Transporter 1; D012333:RNA, Messenger; C082186:SLC15A1 protein, human; C491359:SLC15A4 protein, human; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; C004194:glycylsarcosine; D006639:Histidine