Regulation of Cytosolic COX-2 and Prostaglandin E2 Production by Nitric Oxide in Activated Murine Macrophages

Abstract

Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and ∼10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 × g indicates that the COX-2 is distributed both in the 100,000 × g soluble fraction and membrane fraction. Stimulation of RAW 264.7 cells with LPS in the presence of inducible nitric oxide synthase inhibitor l-NMMA at concentrations that inhibit nitrite accumulation by ≤80% is inadequate to augment PGE2 production. However, inhibition of nitrite accumulation by ≥85% with higher concentrations of l-NMMA shows 1) up-regulation of PGE2 production, 2) accumulation of COX-2 protein in the 100,000 × g soluble and membrane fractions of the cytosolic fraction, and 3) with no significant effects on the accumulation of COX-2 mRNA. These experiments suggest that low concentrations of nitric oxide (10–15% of the total) attenuate PGE2 production in response to LPS in RAW 264.7 cells. This inhibition is, in part, due to decreased expression of cytosolic COX-2 protein.