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EFFECT OF TNFalpha ON Ca²⁺ CHANNELS IN SUPERIOR MESENTERIC GANGLION

M Motagally, AE Lomax
Gastrointestinal Diseases Research Group, Queen’s University, Kingston, Ontario

Inflammatory Bowel Disease (IBD) results in altered function of the gastrointestinal tract. Recent studies have suggested that altered neural regulation of intestinal function, including changes in neurotransmitter release, contributes to this change. The entry of Ca²⁺ into nerve terminals through Ca²⁺ channels determines the amount of neurotransmitter release. Therefore, the aim of this study was to characterize the affect of TNFalpha, a cytokine that is elevated in IBD, on Ca²⁺ channels of sympathetic neurons of the superior mesenteric ganglion (SMG) which regulate colonic motility, secretion and blood flow.
To study the affect of TNFalpha, SMG neurons were enzymatically dissociated from male CD1 mice and cultured overnight in control culture medium or medium containing 1 nM TNFalpha. Using the perforated patch voltage clamp technique Ca²⁺ currents were measured during a series of command potentials from –60 to +35 mV for 100 ms in 5 mV increments (10 s intervals) from a holding potential of –100 mV. We identified which Ca²⁺ channel subtypes were present in SMG neurons by specific channel blockers (300nM omega-Conotoxin GVIA, N-type; 10µM Nifedipine, L-type; 300 µM omega-Conotoxin MVIIC N, P/Q type). Ca²⁺ imaging was also used to compare Ca²⁺ influx during a depolarization in both control and TNFalpha-treated neurons. The neurons were incubated in the ratiometric Ca²⁺ sensitive dye Fura-2 AM and depolarized by superfusion of saline with 40mM K⁺. The amplitude of Fura-2 response to the high K⁺ challenge was compared in control and treated neurons.
The Ca²⁺ current in SMG neurons is mainly via N-type (60%) and L-type (20%) channels. Incubation in TNFalpha caused a significant decrease in Ca²⁺ current (~ 50%) in the treated neurons over a voltage range of –20 to +10 mV (TNFalpha n=15; untreated n=17). A significant decrease was also observed in Ca²⁺ influx as measured with fura-2 AM. The reduction in Ca²⁺ current appears to be limited to N-type Ca²⁺ channels (control N-type current= 30.9 pA/pF; TNFalpha-treated N-type current= 10.5 pA/pF; P <0.05).
These data demonstrate the ability of pro-inflammatory cytokines to alter sympathetic neurophysiology. Any changes in Ca²⁺ channels due to elevated TNFalpha during IBD will result in altered sympathetic neurotransmitter release and thus may contribute to the symptoms associated with IBD.

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