Calcium Enhances In Vitro Free Radical‐Induced Damage to Brain Synaptosomes, Mitochondria, and Cultured Spinal Cord Neurons

Abstract

Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca²⁺-free Krebs buffer resulted in a 27% inhibition of synaptosomal γ-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl₂ increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 μM CaCl₂ produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca²⁺ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl₂ and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na⁺, K⁺-ATPase activity. Neither X/XO/ADP/FeCl₂ nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca²⁺ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca²⁺.