Regulation of cytosolic COX-2 and prostaglandin E2 production by nitric oxide in activated murine macrophages.
Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and approximately 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 </=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 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 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.
Patel
R
R
Department of Rheumatology, Hospital for Joint Diseases, New York 10003, USA.
Attur
M G
MG
Dave
M
M
Abramson
S B
SB
Amin
A R
AR
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D051546:Cyclooxygenase 2; D003600:Cytosol / Q000201:enzymology*; D015232:Dinoprostone / Q000096:biosynthesis*; D004790:Enzyme Induction / Q000276:immunology; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D009569:Nitric Oxide; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone