Tumour necrosis factor α activates nuclear factor κB signalling to reduce N-type voltage-gated Ca2+ current in postganglionic sympathetic neurons

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Abstract

Inflammation has profound effects on the innervation of affected tissues, including altered neuronal excitability and neurotransmitter release. As Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) is a critical determinant of excitation-secretion coupling in nerve terminals, the aim of this study was to characterize the effect of overnight incubation in the inflammatory mediator tumour necrosis factor α (TNFα; 1 nM) on VGCCs in dissociated neurons from mouse superior mesenteric ganglia (SMG). Voltage-gated Ca2+ currents (ICa) were measured using the perforated patch clamp technique and the VGCC subtypes present in SMG neurons were estimated based on inhibition by selective VGCC blockers: ω-conotoxin GVIA (300 nM; N-type), nifedipine (10 μM; L-type), and ω-conotoxin MVIIC (300 nM; N-, P/Q-type). We used intracellular Ca2+ imaging with Fura-2 AM to compare Ca2+ influx during depolarizations in control and TNFα-treated neurons. TNF receptor and VGCC mRNA expression were measured using PCR, and channel α subunit (CaV2.2) was localized with immunohistochemistry. Incubation in TNFα significantly decreased ICa amplitude and depolarization-induced Ca2+ influx. The reduction in ICa was limited to ω-conotoxin GVIA-sensitive N-type Ca2+ channels. Depletion of glial cells by incubation in cytosine arabinoside (5 μM) did not affect ICa inhibition by TNFα. Preincubation of neurons with SC-514 (20 μM) or BAY 11-7082 (1 μM), which both inhibit nuclear factor κB signalling, prevented the reduction in ICa by TNFα. Inhibition of N-type VGCCs following TNFα incubation was associated with a decrease in CaV2.2 mRNA and reduced membrane localization of CaV2.2 immunoreactivity. These data suggest that TNFα inhibits ICa in SMG neurons and identify a novel role for NF-κB in the regulation of neurotransmitter release during inflammatory conditions with elevated circulating TNFα, such as Crohn's disease and Guillain-Barré syndrome.

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