Mass quantitation of agonist-induced arachidonate release and icosanoid production in a fibrosarcoma cell line. Effect of time of agonist stimulation, amount of cellular arachidonate, and type of agonist

The mass of total arachidonate released from phospholipids upon agonist stimulation of the cell and the fraction of released arachidonate which is converted to icosanoids are two parameters of arachidonate metabolism which have been difficult to quantitate because the mass of arachidonate released upon cell stimulation is very low. We have been able to quantitate both of these parameters under a variety of experimental conditions using a unique essential fatty acid-deficient mouse fibrosarcoma cell line (EFD-1), which when repleted with arachidonate, produces prostaglandin E₂ (PGE₂). Because there is no endogenous pool of arachidonate in these cells, the specific activity of exogenous arachidonate does not change upon incorporation into cells, an advantage which permits mass determination of very small quantities of arachidonate directly from radioactive counts. EFD-1 cells were incubated with various concentrations of [¹⁴C]arachidonate (for release studies) or unlabeled arachidonate (for PGE₂ radioimmunoassays) for 24 h and then stimulated with bradykinin. The time courses for arachidonate release and PGE₂ production demonstrated that free arachidonate was rapidly converted to PGE₂ with plateau levels attained for both parameters within 240 s of agonist exposure for 2 μM and for 10 μM arachidonate-repleted cultures. There was a linear relationship (r = 0.94) between the mass of arachidonate in the cell and the mass of arachidonate released upon stimulation, up to a cellular concentration of 11 nmol of arachidonate/10⁶ cells, a concentration 10-20% above normal for the parent mouse fibrosarcoma cell line (HSDM₁C₁) which is not essential fattyacid-deficient. Importantly, the percent of released arachidonate which was converted to PGE₂ decreased from 90 to 15% with increasing concentrations of cellular arachidonate, because PGE₂ production plateaued at ≥6 nmol of arachidonate/10⁶ cells, but total arachidonate release continued to rise. Finally, we demonstrated that agonist stimulation with thrombin, A23187, and bradykinin all showed the same percent conversion of released arachidonate to PGE₂, implying that the determination of this fraction is not a function of the mechanism of release. These studies with our unique cell line indicate that, when the concentration of arachidonate in the cell is not elevated above amounts normally found in our HSDM₁C₁ cell line, released arachidonate is rapidly and almost quantitatively converted to PGE₂, independent of the agonist used to stimulate the cells. However, when the arachidonate concentration in the cell rises slightly above normal, substantial quantities of arachidonate released from phospholipids are not converted to icosanoids and can exit the cell.