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Multidrug resistant proteins mediate extracellular ATP release in Caco2 cell monolayers
Author(s) -
Rudenkyy Sergiv,
Nakano Takanari,
Akiba Yasutada,
Kaunitz Jonathan D
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1068.23
Subject(s) - extracellular , microbiology and biotechnology , multidrug resistance associated protein 2 , hek 293 cells , transfection , atp binding cassette transporter , intracellular , caco 2 , chemistry , secretion , purinergic receptor , adenosine triphosphate , pannexin , atpase , biochemistry , cell , transporter , biology , connexin , receptor , gap junction , enzyme , gene
Epithelial ATP release is an essential component of extracellular purinergic secretory regulation; ATP transporting channels, pannexin (Panx) hemichannels and multidrug resistant proteins (MDR/MRP) have been implicated. The robust intestinal ecto‐ATPase activity present in Caco‐2 cells impairs measurements of medium ATP. We thus transfected c‐Myc‐tagged GPI‐anchored plasma membrane luciferase (pmeLuc) to Caco2 cell monolayer to monitor ATP release at the cell surface. Confluent Caco‐2 cell monolayers were transfected with pmeLuc, confirmed by immunolocalization. Although transfection efficacy into Caco2 cell is ~ 10 %, much less than in HEK293 ~ 80 %, luminescence was measurable over the background (~ 3000 in Caco‐2, ~ 30,000 in HEK293). Luminescence was stable over 40 min, suggesting constitutive ATP release. Among the pharmacological reagents reported as inhibitors for ATP release, MK571, a specific MDR/MRP inhibitor dose‐dependently inhibited ATP release. Stilbene anion transport inhibitors or Panx inhibitors had no effect. Vesicles expressing P‐glycoprotein (Mdr1) and MRP4, but not MRP2 accumulated ATP inside. MDR/MRP mediates ATP release in intestinal epithelia, a novel mechanism for transmembrane ATP transport. Regulation of ATP release may have important implications for the physiological functioning of intestinal and other secretory epithelia. NIH‐NIDDK R01 DK54221 & R21 DK081199