We have used quantitative electron microscope autoradiography to study uptake and distribution of arachidonate in HSDMIC1 murine fibrosarcoma cells and in EPU-1B, a mutant HSDMtC1 line defective in high affinity arachidonate uptake. Cells were labeled with [3H]arachidonate for 15 min, 40 min, 2 h, or 24 h. Label was found almost exclusively in cellular phospholipids; 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. All incorporated radioactivity was found to be associated with cellular membranes. Endoplasmic reticulum (ER) contained the bulk of [3H]arachidonate at all time points in both cell types, while mitochondria, which contain a large portion of cellular membrane, were labeled slowly and to substantially lower specific activity. Plasma membrane (PM) also labeled slowly, achieving a specific activity only one-sixth that of ER at 15 rain in HSDM~Ct cells (6% of total label) and one-third of ER in EPU-1B (10% of total label). Nuclear membrane (NM) exhibited the highest specific activity of labeling at 15 min in HSDM~C1 cells (twice that of ER) but was not preferentially labeled in the mutant. Over 24 h, PM label intensity increased to that of ER in both cell lines. However, NM activity diminished in HSDM~C1 cells by 24 h to a small fraction of that in ER. In response to agonists, HSDM~C~ cells release labeled arachidonate for icosanoid synthesis most readily when they have been labeled for short times. Our results therefore suggest that NM and ER, sites of cyclooxygenase in murine fibroblasts, are probably sources for release of [3H]arachidonate, whereas PM and mitochondria are unlikely to be major sources of icosanoid precursors.Arachidonic acid (C20:4~5.8.1 u4) is the major polyunsaturated fatty acid of most mammalian cells and occurs primarily esterified in the sn-2 position of cellular phospholipids (11). In addition to serving as a structural membrane component, arachidonate is the primary substrate for the synthesis of icosanoids. These oxygenated metabolites, including prostaglandins, leukotrienes, thromboxane, and related compounds, modulate important biological processes including inflammation, allergy, and hemostasis (30). Icosanoid precursor fatty acids (chiefly arachidonate) are used by specific metabolic pathways, distinct from those used by other fatty acids, in cells that make these mediators. These cell types avidly accumulate arachidonate from their surroundings and esterify it into cellular lipids by virtue of an acyl-CoA synthetase specific for icosanoid precursor fatty acids (20, 34). A mutant cell line that lacks this enzyme is defective both in high affinity arachidonate uptake and its subsequent release in response to the agonist bradykinin (19).Upon appropriate stimulation, icosanoid synthesis is initiated by the selective release of arachidonate from phospholipids, chiefly phosphatidylinositol and phosphatidylcholine. In every case studied thus far, the availability of unesterified arachidonate, and hence this specific release ...

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.

We have used quantitative electron microscopic autoradiography to characterize the subcellular distribution of arachidonoyl phospholipids following brief (5 minutes) exposure of unstimulated human platelets to [3H]arachidonic acid. Labeled arachidonate was taken up rapidly and incorporated into phospholipids. Phospholipid radioactivity was preserved and spatially fixed during tissue processing for electron microscopy. Analysis of autoradiographs showed that following a brief exposure to 750 nmol/L [3H]arachidonate, there is selective labeling of an internal membrane compartment composed of the dense tubular system and the open canalicular system. The plasma membrane, platelet granules, and nonmembranous cytoplasm were not labeled. Since the open canalicular system is continuous with the plasma membrane and since phospholipids in continuous membranes are freely diffusible, our observations indicate that [3H]arachidonate was incorporated into phospholipids within the dense tubular system and not the open canalicular system. Thus, the dense tubular system, known to contain cyclooxygenase activity, incorporates arachidonate selectively following brief exposure to this fatty acid, presumably to concentrate it in proximity to enzymes for icosanoid synthesis.