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
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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. / Bravo, Silvina A; Nielsen, Carsten Uhd; Frokjaer, Sven; Brodin, Birger.
In: Molecular Pharmaceutics, Vol. 2, No. 2, 2005, p. 98-108.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - 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
AU - Bravo, Silvina A
AU - Nielsen, Carsten Uhd
AU - Frokjaer, Sven
AU - Brodin, Birger
PY - 2005
Y1 - 2005
N2 - 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.
AB - 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.
KW - Aminolevulinic Acid
KW - Analgesics
KW - Animals
KW - Anti-Bacterial Agents
KW - Anti-Inflammatory Agents
KW - Apoproteins
KW - Biological Transport
KW - Cell Line
KW - Cell Proliferation
KW - Cephalexin
KW - Culture Media
KW - Dexamethasone
KW - Dipeptides
KW - Diuretics, Osmotic
KW - Electric Impedance
KW - Electrophysiology
KW - Endorphins
KW - Epidermal Growth Factor
KW - Epithelium
KW - Hydrogen-Ion Concentration
KW - Immunohistochemistry
KW - Insulin
KW - Kidney
KW - Kidney Tubules, Proximal
KW - Kinetics
KW - Mannitol
KW - Microscopy, Confocal
KW - Photosensitizing Agents
KW - Rats
KW - Symporters
KW - Time Factors
KW - Transferrin
U2 - 10.1021/mp049892q
DO - 10.1021/mp049892q
M3 - Journal article
C2 - 15804184
VL - 2
SP - 98
EP - 108
JO - Molecular Pharmaceutics
JF - Molecular Pharmaceutics
SN - 1543-8384
IS - 2
ER -
ID: 37899515