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Cl − secretion in ATP‐treated renal epithelial C7–MDCK cells is mediated by activation of P 2Y1 receptors, phospholipase A 2 and protein kinase A
Author(s) -
Akimova A. Olga,
Bourcier Nathalie,
Taurin Sebastien,
Bundey Richard A.,
Grygorczyk Konrad,
Gekle Michael,
Insel Paul A.,
Dulin Nickolai O.,
Orlov Sergei N.
Publication year - 2005
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2005.094375
Subject(s) - thapsigargin , protein kinase a , protein kinase c , chemistry , phospholipase c , receptor , microbiology and biotechnology , endocrinology , medicine , kinase , biology , intracellular , biochemistry
This study examines the mechanism of P 2Y ‐induced Cl − secretion in monolayers of C7–Madin–Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current ( I SC ). Both ATP‐induced arachidonic acid (AA) synthesis and I SC in ATP‐treated cells were abolished by the phosholipase A 2 (PLA 2 ) inhibitor, AACOCF 3 . The cyclo‐oxygenase inhibitor indomethacin decreased I SC and cAMP production in ATP‐treated cells with an IC 50 of ∼0.3 μ m . ATP led to rapid activation of cAMP‐dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator‐stimulated phosphoprotein. PKA activity and I SC evoked by ATP, as well as by prostaglandin E 1 (PGE 1 ), were diminished in the presence of the PKA inhibitor H‐89 or an adenovirus‐mediated expression of PKA‐inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and I SC triggered by ATP and AA, but did not affect PKA activation and I SC detected with PGE 1 . The kinetics of [Ca 2 + ] i elevation in ATP‐ and thapsigargin‐treated cells were similar and suppressed by the Ca i 2+ chelator BAPTA. Neither baseline nor maximal increment of ATP‐induced I SC was affected by thapsigargin and BAPTA. Real‐time PCR showed that C7 cells express more mRNA for P 2Y1 and P 2Y2 than for other P 2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP ≫ UTP) indicates that P 2Y1 rather than P 2Y2 receptors contribute to PKA and I SC activation. Viewed collectively, these data show that Cl − secretion in C7–MDCK monolayers treated with basolateral ATP is triggered by P 2Y1 receptors and is mediated by subsequent [Ca 2 + ] i ‐independent activation of PLA 2 and PKA.