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J Am Soc Nephrol 10:2261-2271, 1999
© 1999 American Society of Nephrology

NS-398 Upregulates Constitutive Cyclooxygenase-2 Expression in the M-1 Cortical Collecting Duct Cell Line

SHAWN FERGUSON*, RICHARD L. HÉBERT*,{dagger} and ODETTE LANEUVILLE{ddagger}

* Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada.
{dagger} Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
{ddagger} Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.

Correspondence to Dr. Odette Laneuville, Department of Biochemistry, Microbiology, and Immunology, 451 Smyth Road, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5. Phone: 613-562-5800, extension 8402; Fax: 613-562-5440; E-mail: olaneuv{at}danis.med.uottawa.ca

Abstract. The cortical collecting duct (CCD) is a major site of intrarenal prostaglandin E2 (PGE2) synthesis. This study examines the expression and regulation of the prostaglandin synthesizing enzymes cyclooxygenase-1 (COX-1) and -2 in the CCD. By indirect immunofluorescence using isoform-specific antibodies, COX-1 and -2 immunoreactivity was localized to all cell types of the murine M-1 CCD cell line. By immunohistochemistry, both COX-1 and COX-2 were localized to intercalated cells of the CCD on paraffin-embedded mouse kidney sections. When COX enzyme activity was measured in the M-1 cells, both indomethacin (COX-1 and -2 inhibitor) and the specific COX-2 inhibitor NS-398 effectively blocked PGE2 synthesis. These results demonstrate that COX-2 is the major contributor to the pool of PGE2 synthesized by the CCD. By Western blot analysis, COX-2 expression was significantly upregulated by incubation with either indomethacin or NS-398. These drugs did not affect COX-1 protein expression. Evaluation of COX-2 mRNA expression by Northern blot analysis after NS-398 treatment demonstrated that the COX-2 protein upregulation occurred independently of any change in COX-2 mRNA expression. These studies have for the first time localized COX-2 to the CCD and provided evidence that the intercalated cells of the CCD express both COX-1 and COX-2. The results also demonstrate that constitutively expressed COX-2 is the major COX isoform contributing to PGE2 synthesis by the M-1 CCD cell line. Inhibition of COX-2 activity in the M-1 cell line results in an upregulation of COX-2 protein expression.




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