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Collecting ducts are a major site of renal production and action of both prostaglandins and nitric oxide. Experiments were undertaken to examine whether nitric oxide regulates cyclooxygenase (COX)-2 expression and [PGE.sub.2] release in cultured collecting duct cells. In mIMCD-K2 cells, sodium nitroprusside (SNP) in the 50- to 800-[micro]M range induced a marked dose- and time-dependent increase in COX-2 protein levels, determined by immunoblotting, and the induction was detectable at 4 h. This was preceded by induction of COX-2 mRNA as determined by real-time-RT-PCR. The COX-2 induction was accompanied by a significant rise in [PGE.sub.2] release as determined by enzyme immunoassay. S-nitroso-N-acetylpenicillamine (SNAP) had a similar stimulatory effect on COX-2 expression and [PGE.sub.2] release. 8-bromo-cGMP (200 [micro]M) had no effect on COX-2 expression. The SNP-stimulated COX-2 expression was not affected by the guanylyl cyclase inhibitor methylene blue or the protein kinase G inhibitor KT-5823 (2.0 [micro]M). In contrast, the SNP-stimulated COX-2 expression was significantly reduced by either the Erk1/2 inhibitor PD-98059 or the P38 inhibitor SB-203580 and was abolished by combination of the two kinase inhibitors. The stimulation was also significantly blocked by the SOD mimetic tempol. Thus we conclude that NO stimulates COX-2 expression in collecting duct cells through mechanisms involving MAP kinase and superoxide, but not cGMP. cyclooxygenase-2

It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide synthase (nNOS) are coexpressed in macula densa cells and that the expression of both enzymes is stimulated in a number of high-renin states. To further explore the role of nNOS and COX-2 in renin secretion, we. determined plasma renin activity in mice deficient in nNOS or COX-2. Plasma renin activity was significantly reduced in nNOS -/- mice on a mixed genetic background and in COX-2 -/- mice on either BALB/c or C57/BL6 congenic backgrounds. In additional studies, we accumulated evidence to show an inhibitory influence of [PGE.sub.2] on nNOS expression. In a cultured macula densa cell line, [PGE.sub.2] significantly reduced nNOS mRNA expression, as quantified by real-time RT-PCR. In COX-2 -/- mice, nNOS mRNA expression in the kidney, determined by real-time RT-PCR, was upregulated throughout the postnatal periods, ranging from postnatal day (PND) 3 to PND 60. The induction of nNOS protein expression and NOS activity in COX-2 -/- mice was localized to macula densa cells using immunohistochemistry and NADPH-diaphorase staining methods, respectively. Therefore, these findings reveal that the absence of either COX-2 or nNOS is associated with suppressed renin secretion. Furthermore, the inhibitory effect of PG[E.sub.2] on nNOS mRNA expression indicates a novel interaction between NO and prostaglandin-mediated pathways of renin regulation. juxtaglomerular apparatus; cyclooxygenase-2; neuronal nitric oxide synthase; real-time reverse transcriptase-polymerase chain reaction; plasma renin activity