Hydrogen Peroxide (H 2 O 2 ) Augments Intracellular Ca ++ via Voltage‐Gated Ca ++ Channels in Isolated Nucleus Tractus Solitarii (nTS) Neurons

Abstract

Central neuronal activity and its modulation via 2nd messengers within the nTS is fundamental for cardiorespiratory control. We have previously shown that H2O2 in the nTS augments action potential discharge. Here we examined the role of H2O2 on Ca++ homeostasis in primary nTS cultures using the ratiometric dye Fura 2-AM. H2O2 dose dependently (100-500µM H2O2, 1 min) augmented intracellular Ca++. The majority of nTS cells (79% of 24 cells) responded to 200µM H2O2 with an increase in cytosolic Ca++ of 16.0 ±2.8% (p蠄0.01 vs. vehicle). To identify the source for the Ca++ rise we tested 200µM H2O2 in the presence of Thapsigargin (TG; 1µM) to deplete internal Ca++ stores. In the presence of TG, there was a reduction in the number of H2O2 responding cells (45% of 46 cells), yet those that did respond increased cytosolic Ca++ by 72.5 ±21.7% (p蠄0.05 vs. vehicle & 200µM H2O2 alone). Removing extracellular Ca++ (0 Ca++ with 1mM EDTA) strongly decreased the number of H2O2 responders (10% of 23) as well as the augmentation of Ca++ in the remaining responders. To examine the potential Ca++ channel responsible we included specific Ca++ channel blockers during H2O2. The number of responders to H2O2 was reduced to 40%, 40%, 34% and 20% by blocking L-type (5µM Nifedipine), N-Type (0.1µM ω-conotoxin GVIA), T-type (5µM Mibefradil) and P/Q/N-type (ω-conotoxin MVIIIC) Ca++ channels, respectively (N=20-28). Altogether, elevations in cytosolic Ca++ in nTS cells by H2O2 likely occurs through several available Ca++ channels that contribute to the cellular hyperexcitability. Supported by: RO1 HL098602, AHA 12POST11670002