Cyclooxygenase-2 expression and function in the medullary thick ascending limb

The medullary thick ascending limb (MTAL) metabolizes arachidonic acid (AA) via cytochrome P-450 (CyP450)- and cyclooxygenase (COX)dependent pathways. In the present study, we demonstrated that the COX-2-selective inhibitor, NS-398, prevented tumor necrosis factor-alpha (TNF)- and phorbol myristate acetate (PMA)mediated increases in PGE(2) production by cultured MTAL cells. Accumulation of COX-2, but not COX-1, mRNA increased when cells were challenged with TNF (1 nM) or PMA (1 mu M). Pretreatment of cells for 30 min with actinomycin D (AcD, 1 mu M) had little effect on COX-2 mRNA accumulation in unstimulated cells or in cells challenged with either TNF or PMA. Moreover, a posttranscriptional mechanism(s) appears to contribute significantly to COX-2 mRNA accumulation as pretreatment for 15 min with cycloheximide (CHX, 1 mu M) caused a superinduction of COX-2 mRNA accumulation in unstimulated cells as well as in cells challenged with either TNF or PMA. Expression of COX-2 protein in unstimulated MTAL cells was attenuated by preincubation for 2 h with dexamethasone (Dex, 2 mu M); however, Dex had little or no effect on COX-2 expression in cells challenged with either PMA or TNF. The time-dependent inhibition of (86)Rb uptake by MTAL cells challenged with TNF was diminished by pretreating cells with NS-398. These data suggest that TNF-mediated induction of COX-2 protein expression accounted for the lag-time required for this cytokine to inhibit (86)Rb uptake in MTAL cells.