INHIBITION OF BIOENERGETICS ALTERS INTRACELLULAR CALCIUM, MEMBRANE-COMPOSITION, AND FLUIDITY IN A NEURONAL CELL-LINE

The effect of inhibited bioenergetics and ATP depletion on membrane co mposition and fluidity was examined in cultured neuroblastoma-glioma h ybrid NG108-15 cells. Sodium cyanide (CN) and 2-deoxyglucose (2-DG) we re used to block oxidative phosphorylation and anaerobic glycolysis, r espectively. Endoplasmic reticulum (ER) Ca2+-pump activity measured by Ca-45(2+) uptake was >92% inhibited in intact cells incubated with CN (1 mM) and 2-DG (20 mM) for 30 min. In addition, exposure of cells to CN and 2-DG caused a 134% increased release of isotopically labeled a rachidonic acid (H-3-AA) or arachidonate-derived metabolites from memb ranes. Removal of Ca2+ from the incubation medium ablated the CN/2-DG induced release of H-3-AA or its metabolites. Membrane fluidity of int act cells was measured by electron spin resonance spectroscopy using t he spin label 12-doxyl stearic acid. The mean rotational correlation t ime (tau(c)) of the spin label increased 49% in CN/2-DG exposed cells compared to controls, indicating a decrease in membrane fluidity. Thes e results Show that depletion of cellular ATP results in inhibition of the ER Ca2+-pump, loss of AA from membranes, and decreased membrane f luidity. We propose that impaired bioenergetics can increase intracell ular Ca2+ as a result of Ca2+-pump inhibition and thereby activate Ca2 +-dependent phospholipases causing membrane effects. Since neurons der ive energy predominantly from oxidative metabolism, ATP depletion duri ng brain hypoxia may initiate a similar cytotoxic mechanism.