Effect of COX-2 inhibitor NS-398 on expression of PGE2 receptor subtypes in M-1 mouse CCD cells.
Our present study has investigated the effect of cyclooxygenase-2 (COX-2) inhibition on prostaglandin E2 (PGE2) receptor expression in M-1 cortical collecting duct cells and measured their response to PGE2. Using a semiquantitative titration analysis method, we show that following the addition of the COX-2-specific inhibitor NS-398, E-prostanoid receptor subtype (EP3 and EP4) mRNA expression was found to increase threefold each vs. the vehicle-treated control. We also observed that EP1 but not EP2 is expressed in M-1 cells and EP2 levels are not induced by NS-398. To determine the status of the PGE2 response on exposure to NS-398, we measured cAMP levels in cells after stimulation with varying concentrations of PGE2, then pretreated the cells with 10 microM NS-398 before PGE2 exposure and found a significant rise in the stimulatory effect of PGE2 on cAMP production. Finally, Western blot analysis of the levels of the EP4 receptor protein in control vs. NS-398-treated cells revealed an induction in protein levels in these cells, correlating with the induction in EP4 mRNA. We conclude that NS-398 upregulates the expression of EP3 and EP4 mRNA in M-1 cells. Also, EP4 protein levels are increased, resulting in an increased stimulation of cAMP production by PGE2.
Nasrallah
R
R
Department of Cellular and Molecular Medicine and Kidney Research Centre, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5.
Laneuville
O
O
Ferguson
S
S
Hébert
R L
RL
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D000242:Cyclic AMP / Q000032:analysis; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D007685:Kidney Tubules, Collecting / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009578:Nitrobenzenes / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D018078:Receptors, Prostaglandin E / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013449:Sulfonamides / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009578:Nitrobenzenes; D020033:Protein Isoforms; D012333:RNA, Messenger; D018078:Receptors, Prostaglandin E; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D000242:Cyclic AMP; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone