title: Characterization of rPEPT2-mediated Gly-Sar transport parameters in the rat kidney proximal tubule cell line SKPT-0193 cl.2 cultured in basic growth media.
abstract: The rat proximal kidney tubule cell line SKPT-0193 cl.2 (SKPT) expresses the di-/tripeptide transporter PEPT2 (rPEPT2) and has been used to study PEPT2-mediated transport. Traditionally, SKPT cells have been cultured in growth media supplemented with epidermal growth factor (EGF), apotransferrin, dexamethasone, and insulin. It was recently demonstrated that omission of EGF from the culture media caused a drastic increase in the expression of rPEPT2. The hypothesis was therefore that the SKPT cell line might be able to differentiate and express rPEPT2 in the absence of the four agonists traditionally added. The aim of the study was thus to characterize Gly-Sar transport parameters in SKPT cells cultured in basic growth media (conventional media without added agonists). Morphology was studied using confocal laser scanning microscopy (CLSM) and immunohistochemistry. Monolayer integrity was evaluated using transepithelial electrical resistance (TEER) measurements and [(3)H]-mannitol permeabilities. Di-/tripeptide transporter activity was studied using [(14)C]-glycylsarcosine ([(14)C]-Gly-Sar). SKPT cells grown in basic media for 4 days formed confluent monolayers with a TEER of 5.03 +/- 0.33 kOmega.cm(2) (n = 5). Apical Gly-Sar uptake peaked after 3-6 days in culture. Uptake at day 4 was 5.89 +/- 0.30 pmol.cm(-2).min(-1) (n = 3). Di-/tripeptide uptake displayed an optimum at approximately pH 6. Affinity values for cephalexin, kyotorphin, and delta-aminolevulinic acid were comparable to those obtained in other PEPT2-expressing model systems. It can be concluded that SKPT cells grown in the absence of the agonists traditionally added to the culture media retain all necessary properties for PEPT2-mediated peptide uptake studies. Furthermore, the absence of the agonists might facilitate studies of hormonal regulation of PEPT2 expression and transport activity.
authors.affiliation: Molecular Biopharmaceutics, Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Silvina A
authors.initials: SA
authors.lastname: Bravo
authors.forename: Carsten Uhd
authors.initials: CU
authors.lastname: Nielsen
authors.forename: Sven
authors.initials: S
authors.lastname: Frokjaer
authors.forename: Birger
authors.initials: B
authors.lastname: Brodin
mesh_terms: D000622:Aminolevulinic Acid / Q000378:metabolism; D000700:Analgesics / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001059:Apoproteins / Q000494:pharmacology; D001692:Biological Transport; D002460:Cell Line; D049109:Cell Proliferation; D002506:Cephalexin / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004151:Dipeptides / Q000737:chemistry / Q000493:pharmacokinetics*; D004234:Diuretics, Osmotic / Q000494:pharmacology; D017097:Electric Impedance; D004594:Electrophysiology; D004723:Endorphins / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007328:Insulin / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000378:metabolism; D007700:Kinetics; D008353:Mannitol / Q000737:chemistry; D018613:Microscopy, Confocal; D017319:Photosensitizing Agents / Q000494:pharmacology; D051381:Rats; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014168:Transferrin / Q000494:pharmacology
chemical_list: D000700:Analgesics; D000900:Anti-Bacterial Agents; D000893:Anti-Inflammatory Agents; D001059:Apoproteins; D003470:Culture Media; D004151:Dipeptides; D004234:Diuretics, Osmotic; D004723:Endorphins; D007328:Insulin; D017319:Photosensitizing Agents; D027981:Symporters; D014168:Transferrin; C028284:apotransferrin; C093694:hydrogen-coupled oligopeptide transporter PepT2; C019448:kyotorphin; C004194:glycylsarcosine; D008353:Mannitol; D004815:Epidermal Growth Factor; D003907:Dexamethasone; D000622:Aminolevulinic Acid; D002506:Cephalexin