Regulation of cytosolic COX-2 and prostaglandin E2 production by nitric oxide in activated murine macrophages

Murine macrophages (IWW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and similar to 10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 x g indicates that the COX-2 is distributed both in the 100,000 x 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 less than or equal to 80% is inadequate to augment PGE, production. However, inhibition of nitrite accumulation by greater than or equal to 85% with higher concentrations of L-NMMA shows 1) up-regulation of PGE, production, 2) accumulation of COX-2 protein in the 100,000 x 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 PGE, production in response to LPS in RAW 264.7 cells, This inhibition is, in part, due to decreased expression of cytosolic COX-2 protein.