SWELLING, ACIDOSIS, AND IRREVERSIBLE DAMAGE OF GLIAL-CELLS FROM EXPOSURE TO ARACHIDONIC-ACID IN-VITRO

Swelling and damage of C6 glioma cells and of primary cultured astrocy tes were analyzed in vitro during incubation with arachidonic acid (AA ; 20:4). The cells were suspended in a physiological medium supplement ed with AA at concentrations of 0.001-1.0 mM. Cell swelling was quanti fied by flow cytometry with hydrodynamic focusing. Flow cytometry was also utilized for assessment of cell viability by exclusion of the flu orescent dye propidium iodide and for measurement of the intracellular pH (pH(i)) by 2',7'-bis-(2-carboxyethyl)-5(and -6)carboxyfluorescein. Administration of AA caused an immediate dose-dependent swelling of C 6 glioma cells, even at a concentration of 0.01 mM. At this level cell volume increased within 20 min to 105.0% of control, at 0.1 mM to 111 .0%, while at 1.0 mM to 123.7%. Following a phase of rapid cell volume increase, swelling leveled off during the subsequent observation peri od of 70 min. Viability of the C6 glioma cells was 90% under control c onditions. It remained unchanged after raising AA concentrations to 0. 1 mM. At 0.5 mM, however, cell viability fell to 72.8%, and at 1.0 mM to 32.7%. pH(i) of the glioma cells was 7.3 under control conditions. In parallel with the early swelling phase, AA led to a dose-dependent decrease of the intracellular pH and an elevated lactate production of the cells. During incubation with 0.1 mM AA, pH(i) decreased In addit ion, swelling-inducing properties of linoleic (18:2) or stearic (18:0) acid were analyzed for evaluation of the specificity of glial swellin g induced by AA. Whereas stearic acid (0.1 mM) failed to induce a swel ling response, linoleic acid (0.1 mM) was found to be effective. The v olume increase of the glial cells, however, was only half of that foun d during exposure to AA at the same concentration. Further, glial swel ling from AA or linoleic acid was completely inhibited by the aminoste roid U-74389F, an antagonist of lipid peroxidation. Finally, omission of Na+ ions in the suspension medium with replacement by choline led a lso to inhibition of the cell volume increase by AA. Experiments using astrocytes from primary culture confirmed the swelling-inducing prope rties of AA at a quantitative level, whereas vulnerability of the cell s to AA was increased. The present results demonstrate an important ro le of AA in cytotoxic swelling and irreversible damage of glial cells at concentrations that occur in vivo in cerebral ischemia or trauma. T he damaging potential of AA might be enhanced by a concurrently evolvi ng intracellular acidosis, stimulating the formation of oxygen-derived free radicals and lipid peroxidation.