title: Tumour necrosis factor alpha activates nuclear factor kappaB signalling to reduce N-type voltage-gated Ca2+ current in postganglionic sympathetic neurons.
abstract: Inflammation has profound effects on the innervation of affected tissues, including altered neuronal excitability and neurotransmitter release. As Ca(2+) influx through voltage-gated Ca(2+) 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 (TNFalpha; 1 nM) on VGCCs in dissociated neurons from mouse superior mesenteric ganglia (SMG). Voltage-gated Ca(2+) 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 microM; L-type), and omega-conotoxin MVIIC (300 nM; N-, P/Q-type). We used intracellular Ca(2+) imaging with Fura-2 AM to compare Ca(2+) influx during depolarizations in control and TNFalpha-treated neurons. TNF receptor and VGCC mRNA expression were measured using PCR, and channel alpha subunit (CaV2.2) was localized with immunohistochemistry. Incubation in TNFalpha significantly decreased I(Ca) amplitude and depolarization-induced Ca(2+) influx. The reduction in I(Ca) was limited to omega-conotoxin GVIA-sensitive N-type Ca(2+) channels. Depletion of glial cells by incubation in cytosine arabinoside (5 microM) did not affect I(Ca) inhibition by TNFalpha. Preincubation of neurons with SC-514 (20 microM) or BAY 11-7082 (1 microM), which both inhibit nuclear factor kappaB signalling, prevented the reduction in I(Ca) by TNFalpha. Inhibition of N-type VGCCs following TNFalpha incubation was associated with a decrease in CaV2.2 mRNA and reduced membrane localization of CaV2.2 immunoreactivity. These data suggest that TNFalpha inhibits I(Ca) in SMG neurons and identify a novel role for NF-kappaB in the regulation of neurotransmitter release during inflammatory conditions with elevated circulating TNFalpha, such as Crohn's disease and Guillain-Barré syndrome.
authors.affiliation: Department of Physiology, Queen's University, Kingston, Ontario, Canada.
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Motagally
authors.forename: Mark K
authors.initials: MK
authors.lastname: Lukewich
authors.forename: Susan P
authors.initials: SP
authors.lastname: Chisholm
authors.forename: Shadia
authors.initials: S
authors.lastname: Neshat
authors.forename: Alan E
authors.initials: AE
authors.lastname: Lomax
mesh_terms: D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D007150:Immunohistochemistry; D018836:Inflammation Mediators / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism; D018408:Patch-Clamp Techniques; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017779:Sympathetic Fibers, Postganglionic / Q000187:drug effects / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: C500963:Cacna1b protein, mouse; D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D018836:Inflammation Mediators; D016328:NF-kappa B; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha