hPEPT1 is responsible for uptake and transport of Gly-Sar in the human bronchial airway epithelial cell-line Calu-3

Abstract

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.

Drug Transporter Protein Quantification of Immortalized Human Lung Cell Lines Derived from Tracheobronchial Epithelial Cells (Calu-3 and BEAS2-B), Bronchiolar–Alveolar Cells (NCI-H292 and NCI-H441), and Alveolar Type II-like Cells (A549) by Liquid Chromatography–Tandem Mass Spectrometry

“…On the contrary, MDR1 protein expressions were not observed in tested five immortalized lung cell lines except A549 in this study, whereas MDR1 expression in the lung has been previously reported by RT‐PCR, and MDR1 has been found to be functionally active in Calu‐3 cells and in ex vivo rodent lungs . Furthermore, PEPT2 was reported to be responsible for the uptake and transport Gly‐Sar in Calu‐3 monolayers, but this study showed no detection in PEPT2 protein expression. This finding suggested a detection limit in mass‐based assays compared with immunoassays and PCR.…”

Drug transporters in the lung—do they play a role in the biopharmaceutics of inhaled drugs?

“…100 Gene microarrays revealed PEPT1 is expressed in the lungs although to a lower extent than PEPT2 23 and PEPT1 mRNA was detected very recently in the human bronchi of healthy adults. 101 The recent discovery of PEPT1 in human lung implies the early assumption that the transporter does not affect pulmonary drug disposition might need reappraisal.…”

Inhaled chemotherapy in lung cancer: future concept of nanomedicine

“…221 PEPT 2 and more recently PEPT 1 were detected first in the airway epithelium and then in the bronchial epithelium. 222,223 These two transporters have also been found in animals and cell lines. 224,225 However, how they interact with drug formulations and their activity in respiratory diseases 226 has not been fully investigated.…”