Bupivacaine Indirectly Potentiates Glutamate-induced Intracellular Calcium Signaling in Rat Hippocampal Neurons by Impairing Mitochondrial Function in Cocultured Astrocytes.

Abstract

Bupivacaine induces central neurotoxicity at lower blood concentrations than cardiovascular toxicity. However, central sensitivity to bupivacaine is poorly understood. The toxicity mechanism might be related to glutamate-induced excitotoxicity in hippocampal cells. The intracellular free Ca concentration ([Ca]i), mitochondrial membrane potential, and reactive oxygen species generation were measured by fluorescence and two-photon laser scanning microscopy in fetal rat hippocampal neurons and astrocytes. In astrocyte/neuron cocultures, 300 μM bupivacaine inhibited glutamate-induced increases in [Ca]i in astrocytes by 40% (P < 0.0001; n = 20) but significantly potentiated glutamate-induced increases in [Ca]i in neurons by 102% (P = 0.0007; n = 10). Ropivacaine produced concentration-dependent effects similar to bupivacaine (0.3 to 300 μM). Tetrodotoxin did not mimic bupivacaine's effects. In pure cell cultures, bupivacaine did not affect glutamate-induced increases in [Ca]i in neurons but did inhibit increased [Ca]i in astrocytes. Moreover, bupivacaine produced a 61% decrease in the mitochondrial membrane potential (n = 20) and a 130% increase in reactive oxygen species generation (n = 15) in astrocytes. Cyclosporin A treatment suppressed bupivacaine's effects on [Ca]i, mitochondrial membrane potential, and reactive oxygen species generation. When astrocyte/neuron cocultures were incubated with 500 μM dihydrokainic acid (a specific glutamate transporter-1 inhibitor), bupivacaine did not potentiate glutamate-induced increases in [Ca]i in neurons but still inhibited glutamate-induced increases in [Ca]i in astrocytes. In primary rat hippocampal astrocyte and neuron cocultures, clinically relevant concentrations of bupivacaine selectively impair astrocytic mitochondrial function, thereby suppressing glutamate uptake, which indirectly potentiates glutamate-induced increases in [Ca]i in neurons.