Tumour Necrosis Factor Alpha Activates Nuclear Factor Kappa B Signalling To Reduce N-Type Voltage-Gated Ca2+ Current In Postganglionic Sympathetic Neurons

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 alpha (TNF alpha; 1 nm) on VGCCs in dissociated neurons from mouse superior mesenteric ganglia (SMG). Voltage-gated Ca2+ currents (I-Ca) 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: omega-conotoxin GVIA (300 nm; N-type), nifedipine (10 mu m; L-type), and omega-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 TNF alpha-treated neurons. TNF receptor and VGCC mRNA expression were measured using PCR, and channel alpha subunit (CaV2.2) was localized with immunohistochemistry. Incubation in TNF alpha significantly decreased I-Ca amplitude and depolarization-induced Ca2+ influx. The reduction in I-Ca was limited to omega-conotoxin GVIA-sensitive N-type Ca2+ channels. Depletion of glial cells by incubation in cytosine arabinoside (5 mu m) did not affect I-Ca inhibition by TNF alpha. Preincubation of neurons with SC-514 (20 mu m) or BAY 11-7082 (1 mu m), which both inhibit nuclear factor kappa B signalling, prevented the reduction in I-Ca by TNF alpha. Inhibition of N-type VGCCs following TNF alpha incubation was associated with a decrease in CaV2.2 mRNA and reduced membrane localization of CaV2.2 immunoreactivity. These data suggest that TNF alpha inhibits I-Ca in SMG neurons and identify a novel role for NF-kappa B in the regulation of neurotransmitter release during inflammatory conditions with elevated circulating TNF alpha, such as Crohn's disease and Guillain-BarrE syndrome.