Acetate, the Metabolite of Ethanol, Increases Cytosolic Calcium and mRNA Expression Levels of EGR1 and TNFα in Dopaminergic Like PC12 Cells

Abstract

We have previously reported that acetate activates NMDAR in vivo and in vitro, increases pro‐inflammatory cytokine mRNA expression, and cytosolic calcium levels, all of which may contribute to neurotoxicity. Neural growth factor (NGF) derived PC12 cells are a reasonable model used in substances of abuse and neurodegeneration studies as they are primarily dopaminergic in nature. Neurotransmitter release is governed in part from rises in cytosolic calcium levels which facilitate loading of synaptic vesicles and exocytosis from the synaptic cleft. We hypothesized that acetate may have similar effects in NGF derived PC12 cells which may facilitate increased dopamine release contributing to alcohol dependence. NGF derived PC12 cells exposed to (2 mM) acetate had a significant (p<0.05) increase in cytosolic calcium relative to baseline, measured in real‐time with a fluorescent calcium probe (Fluo‐4AM). Additionally, acetate (2 mM) significantly (p<0.05) upregulated mRNA expression levels of a calcium dependent protein, early growth response 1 (EGR1) at 15 and 30 minutes post acetate treatment, 5 and 6 fold respectively relative to control. EGR1 is an early response protein implicated to alterations in synaptic plasticity and also the immune response. Furthermore, 30 minute treatment with acetate also significantly (p<0.05) upregulated mRNA expression of tumor necrosis factor alpha (TNFα), 2 fold compared to control. This data suggests that physiological circulating levels of acetate (2 mM) following ethanol consumption and metabolism lead to increased cytosolic calcium levels, and mRNA expression levels of EGR1 and TNFα in dopaminergic like PC12 cells. These in combination may contribute to the development of alcohol dependence and alcohol associated neurotoxicity.Support or Funding Information(Funding support: AHA 16PRE27780121, Andrew Chapp, R15‐HL129213 Zhiying Shan; R15‐HL122952, Qinghui Chen).