Key role for constitutive cyclooxygenase-2 of MDCK cells in basal signaling and response to released ATP.
Madin-Darby canine kidney (MDCK) cells release ATP upon mechanical or biochemical activation, initiating P2Y receptor signaling that regulates basal levels of multiple second messengers, including cAMP (J Biol Chem 275: 11735--11739, 2000). Data shown here document inhibition of cAMP formation by Gd(3+) and niflumic acid, channel inhibitors that block ATP release. cAMP production is stimulated via Ca(2+)-dependent activation of cytosolic phospholipase A(2), release of arachidonic acid (AA), and cyclooxygenase (COX)-dependent production of prostaglandins, which activate prostanoid receptors coupled to G(s) and adenylyl cyclase. In the current investigation, we assessed the expression and functional role of the two known isoforms of COX, COX-1 and COX-2. Treatment of cells with either a COX-1-selective inhibitor, SC-560, or COX-2-selective inhibitors, SC-58125 or NS-398, inhibited basal and UTP-stimulated cAMP levels. COX inhibitors also decreased forskolin-stimulated cAMP formation, implying this response is in part attributable to an action of AA metabolites. These findings imply an important role for the inducible form of COX, COX-2, under basal conditions. Indeed, COX-2 expression was readily detectable by immunoblot, and treatments that induce or reduce COX-2 expression in other cells (interleukin-1beta, tumor necrosis factor-alpha, phorbol ester, or dexamethasone) had minimal or no effect on the levels of COX-2 immunoreactivity. RT-PCR using isoform-specific primers detected COX-2 mRNA. We conclude that COX-2 is constitutively expressed in MDCK-D(1) cells and participates in basal and P2Y(2)-mediated signaling, implying a key role for COX-2 in regulation of epithelial cell function.
Ostrom
R S
RS
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636, USA.
Gregorian
C
C
Drenan
R M
RM
Gabot
K
K
Rana
B K
BK
Insel
P A
PA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002460:Cell Line; D000242:Cyclic AMP / Q000037:antagonists & inhibitors; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009578:Nitrobenzenes / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000502:physiology*; D011720:Pyrazoles / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009578:Nitrobenzenes; D011720:Pyrazoles; D012333:RNA, Messenger; C115461:SC 560; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; C090859:1-((4-methylsulfonyl)phenyl)-3-trifluoromethyl-5-(4-fluorophenyl)pyrazole; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases