hPEPT1 is responsible for uptake and transport of Gly-Sar in the human bronchial airway epithelial cell-line Calu-3.
The purpose of this work was to investigate the apical uptake and transepithelial transport of Gly-Sar along with the expression of the di-/tripeptide transporters hPEPT1 and hPEPT2 in human Calu-3 bronchial epithelial cells. The apical Gly-Sar uptake rate in Calu-3 cells followed Michaelis-Menten kinetics with a Km value of 1.3 +/- 0.3 mM and a Vmax value of 0.60 +/- 0.06 nmol cm(-2) min(-1). Transepithelial apical to basolateral transport of 50 microM [3H]-labelled Gly-Sar across the Calu-3 cell monolayer was pH-dependent. The Gly-Sar flux was significantly reduced in the presence of delta-aminolevulinic acid (2.5 mM), cephalexin (25 mM), and captopril (25 mM; p < 0.05, n = 3). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed the presence of both hPEPT1 and hPEPT2 mRNA in the Calu-3 cells. These findings were confirmed in healthy human bronchial cDNA. Restriction-endonuclease analysis identified hPEPT2 in Calu-3 cells to be the hPEPT2*1 haplotype. Western blotting demonstrated expression of the hPEPT1 protein (approximately 80 kDa), and the immunolabel was mainly localized in the apical membrane as judged by immunolocalization studies using confocal laser scanning microscopy (CLSM). This work presents for the first time hPEPT1 and hPEPT2*1 expression in human Calu-3 cells. Surprisingly, the results indicate that Gly-Sar uptake and transport in Calu-3 cells are hPEPT1-mediated rather than hPEPT2-mediated.
Søndergaard
Helle Bach
HB
Department of Pharmaceutics and Analytical Chemistry, The Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
Brodin
Birger
B
Nielsen
Carsten Uhd
CU
D000622:Aminolevulinic Acid / Q000494:pharmacology; D015153:Blotting, Western; D001980:Bronchi / Q000187:drug effects / Q000378:metabolism*; D002216:Captopril / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002506:Cephalexin / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006239:Haplotypes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007700:Kinetics; D018613:Microscopy, Confocal; D000074011:Peptide Transporter 1; D012333:RNA, Messenger / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004151:Dipeptides; D000074011:Peptide Transporter 1; D012333:RNA, Messenger; C082186:SLC15A1 protein, human; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; C004194:glycylsarcosine; D000622:Aminolevulinic Acid; D002216:Captopril; D002506:Cephalexin