Modulation of the Fluoroacetate Effects on Calcium Balance and Mitochondrial Potential in Primary Culture of Rat Hippocampal Cells

Fluoroacetate (FA) is a metabolic poison whose toxic effect is caused by the inhibition of mitochondrial aconitase, leading to the accumulation of citrate in cells and target organs, primarily in the brain, spinal cord, heart, and kidneys. Previously, during the development of a treatment regimen for acute FA poisoning, a positive effect of methylene blue (MB) on the survival of experimental animals was revealed. The mechanism of action of MB in acute FA intoxication has not been established; however, based on the physicochemical characteristics and effects of MB described in the literature, we hypothesized that MB, as an artificial electron acceptor, promotes citrate utilization via cytoplasmic aconitase and isocitrate dehydrogenase, which leads to a decrease in citrate levels and restoration of Ca2+ ion balance. The aim of this study is to identify the effects of FA on calcium balance and mitochondrial potential in astrocytes and hippocampal neurons of rats under in vitro conditions, as well as the nature of the modulation of these effects by methylene blue, the transmembrane electron transport inhibitor 4-hydroxycoumarin (4HC), the Ca2+ chelator BAPTA, and the ryanodine receptor inhibitor dantrolene. It was found that FA causes death of astrocytes, but not neurons, in primary hippocampal cultures, which is associated with an increase in intracellular calcium ion levels ([Ca2+]i). Citrate has no toxic effect on astrocytes but potentiates the drop in mitochondrial membrane potential in response to FA. 4-Hydroxycoumarin suppresses the calcium response in neurons and has virtually no effect on astrocytes. However, 4HC suppresses changes in the mitochondrial potential of astrocytes. In response to the addition of FA in the presence of citrate, dantrolene prevents an increase in [Ca2+]i, especially in neurons. At the same time, MB neutralizes the effect of FA, preventing a decrease in the rate of mitochondrial potential change in both neurons and astrocytes. Thus, the effects of MB on astrocytes may be associated with the inhibition of transmembrane electron transport, a reduction in the level of reactive oxygen species, and the modulation of ryanodine receptor sensitivity. The observed effects support our hypothesis regarding the molecular mechanism of action of MB in astrocytes; however, further experiments are needed to obtain direct evidence.