NS-398 upregulates constitutive cyclooxygenase-2 expression in the M-1 cortical collecting duct cell line

The cortical collecting duct (CCD) is a major site of intrarenal prostaglan din E-2 (PGE(2)) synthesis. This study examines the expression and regulati on of the prostaglandin synthesizing enzymes cyclooxygenase-1 (COX-1) and - 2 in the CCD. By indirect immunofluorescence using isoform-specific antibod ies, COX-1 and -2 immunoreactivity was localized to all cell types of the m urine 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 kidn ey sections. When COX enzyme activity was measured in the M-1 cells, both i ndomethacin (COX-1 and -2 inhibitor) and the specific COX-2 inhibitor NS-39 8 effectively blocked PGE(2) synthesis. These results demonstrate that COX- 2 is the major contributor to the pool of PGE(2) synthesized by the CCD. By Western blot analysis, COX-2 expression was significantly upregulated by i ncubation with either indomethacin or NS-398. These drugs did not affect CO X-1 protein expression. Evaluation of COX-2 mRNA expression by Northern blo t analysis after NS-398 treatment demonstrated that the COX-2 protein upreg ulation occurred independently of any change in COX-2 mRNA expression. Thes e studies have for the first time localized COX-2 to the CCD and provided e vidence 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 m ajor COX isoform contributing to PGE(2) synthesis by the M-1 CCD cell line. Inhibition of COX-2 activity in the M-1 cell line results in an upregulati on of COX-2 protein expression.