Subcellular localization of prostaglandin-forming cyclooxygenase in Swiss mouse 3T3 fibroblasts by electron microscopic immunocytochemistry.

Abstract

Swiss mouse 3T3 fibroblasts were grown in tissue culture, fixed with lysine-paraformaldehyde-periodate solutions containing 0 to 0.1% Tween 20, and then stained for cyclooxygenase antigenicity using rabbit anti-cyclooxygenase IgG in the peroxidase anti-peroxidase procedure. When examined by light microscopy, those cells fixed in the presence of 0.03 to 0.1% Tween 20 exhibited staining throughout the cytoplasm and around the nucleus but not on the cell surface. No staining occurred when either preimmune IgG or anti-cyclooxygenase IgG adsorbed with purified enzyme was substituted for the immune IgG. Electron microscopic examination of cells treated with fixative containing 0.05% Tween 20 and then stained for cyclooxygenase antigenicity revealed electron-dense deposits on the endoplasmic reticulum and nuclear membrane but not the mitochondrial or plasma membranes. No staining was seen in cells treated with control sera. Agents such as angiotensin II, bradykinin, antidiuretic hormone, and thrombin interact, apparently with the 3T3 cell surface to cause a release of arachidonic acid and prostaglandin E2 formation (Pong, S.S., Hong, S. L., and Levine, L. (1977) J. Biol. Chem. 252, 1408-1413). Our results establish that conversion of arachidonic acid to the prostaglandin endoperoxide precursor of PGE2 actually takes place on the endoplasmic reticulum and the nuclear envelope.