Activation of a calcium entry pathway by sodium pyrithione in the bag cell neurons of Aplysia

The ability of sodium pyrithione (NaP), an agent that produces delayed neuropathy in some species, to alter neuronal physiology was accessed using ratiometric imaging of cytosolic free Ca 2 + concentration ([Ca 2 + ] i ) infura PE-filled cultured Aplysia bag cell neurons. Bath-application of NaP evoked a [Ca 2 + ] i elevation in both somata and neurites with an EC 5 0 of 300 nM and a Hill coefficient of 1. The response required the presence of external Ca 2 + , had an onset of 3-5 min, and generally reached a maximum within 30 min. 2-Methyl-sulfonylpyridine, a metabolite and close structural analog of NaP, did not elevate [Ca 2 + ] i . Under whole-cell current-clamp recording, NaP produced a 14 mV depolarization of resting membrane potential that was dependent on external Ca 2 + . These data suggested that NaP stimulates Ca 2 + entry across the plasma membrane. To minimize the possibility that a change in cytosolic pH was the basis for NaP-induced Ca 2 + entry, bag cell neuron intracellular pH was estimated with the dye 2',7'-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester. Exposure of the neurons to NaP did not alter intracellular pH. The slow onset and sustained nature of the NaP response suggested that a cation exchange mechanism coupled either directly or indirectly to Ca 2 + entry could underlie the phenomenon. However, neither ouabain, a Na + /K + ATPase inhibitor, nor removal of extracellular Na + , which eliminates Na + / Ca 2 + exchanger activity, altered the NaP-induced [Ca 2 + ] i elevation. Finally, the possibility that NaP gates a Ca 2 + -permeable ion channel in the plasma membrane was examined. NaP did not appear to activate two major forms of bag cell neuron Ca 2 + -permeable ion channels, as Ca 2 + entry was unaffected by inhibition of voltage-gated Ca 2 + channels using nifedipine or by inhibition of a voltage-dependent, nonselective cation channel using a high concentration of tetrodotoxin. In contrast, two potential store-operated Ca 2 + entry current inhibitors, SKF-96365 and Ni 2 + , attenuated NaP-induced Ca 2 + entry. We conclude that NaP activates a slow, persistent Ca 2 + influx in Aplysia bag cell neurons.