Uptake and subcellular distribution of [3H]arachidonic acid in murine fibrosarcoma cells measured by electron microscope autoradiography.

Abstract

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 ...

Modulation of muscle rNav1.4 Na+ channel isoform by arachidonic acid and its non‐metabolized analog

“…Moreover, ETYA, the non-metabolized analog of AA showed a similar modulatory effect on rNa V 1.4 currents but was more potent than AA, suggesting that the modulation by extracellular AA may be direct and not mediated indirectly by oxygenated metabolites. A previous report on the uptake of 3H-AA from extracellular fluid into cells using electron microscope autoradiography demonstrated that the incorporated radioactivity associated with the plasma membrane was labeled more slowly (Neufeld et al, 1985), which also supports our hypothesis that AA might be responsible for the inhibitory effects on rNa V 1.4 channels.…”

Brain metabolism of nutritionally essential polyunsaturated fatty acids depends on both the diet and the liver

“…Unesterified DHA also can be lost by β-oxidation in mitochondria or peroxisomes, or by other pathways (not shown). The endoplasmic reticulum compartment is in very rapid equilibrium with unesterified plasma DHA that has been dissociated from circulating albumin, whereas the synaptic compartment does not exchange with plasma DHA 70 . This allows injecting radiolabeled DHA* intravenously and determining the incorporation rates J in (circled), a critical parameter, of unesterified unlabeled plasma DHA into individual membrane phospholipids, as well as DHA turnover rates and half-lives in those phospholipids.…”

Control of inositol polyphosphate‐mediated calcium mobilization by arachidonic acid in pancreatic acinar cells of rats.

“…In view of this, it is interesting to speculate whether AA contributes to Ca2+ oscillations (Berridge & Irvine, 1989;Berridge, 1990;Harootonian, Kao, Paranjape & Tsien, 1991;Marty, 1991) or spatial waves (Kasai & Augustine, 1990;Lechleiter, Girard, Peralta & Clapham, 1991;Toescu, Lawrie, Petersen & Gallacher, 1992). AA liberation and incorporation (deacylation and reacylation) can occur in intramembrane systems, including nuclear and endoplasmic reticulum membranes in fibrosarcoma cells (Neufeld, Majerus, Krueger & Saffitz, 1985;Capriotti, Furth, Arrasmith & Laposata, 1988). This may also be the case in pancreatic acinar cells, 25-2 743 where the first rise in [Ca2+]i evoked by acetylcholine (Kasai & Augustine, 1990;Toescu et al 1992) and probably by InsP, (Marty, 1991) takes place in the luminal pole or the granular region.…”