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Am J Physiol Renal Physiol 281: F123-F132, 2001;
0363-6127/01 $5.00
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Vol. 281, Issue 1, F123-F132, July 2001

Effect of COX-2 inhibitor NS-398 on expression of PGE2 receptor subtypes in M-1 mouse CCD cells

Rania Nasrallah1, Odette Laneuville2, Shawn Ferguson1, and Richard L. Hébert1

Departments of 1 Cellular and Molecular Medicine and Kidney Research Centre and of 2 Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5

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 µM 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.

adenosine 3',5'-cyclic monophosphate measurements; cyclooxygenase-2; E-prostanoid receptor subtypes; semiquantitative reverse transcriptase-polymerase chain reaction; Western blotting; cortical collecting duct; prostaglandin E2


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