Comparative effects of sodium pyrithione evoked intracellular calcium elevation in rodent and primate ventral horn motor neurons.

Oral administration of sodium pyrithione (NaP) causes hindlimb weakness in rodents, but not in primates. Previous work using Aplysia neurons has demonstrated that NaP produces a persistent influx of Ca(2+) ions across the plasma membrane. To determine whether this also occurs in mammalian neurons and whether this could underlie the inter-species difference between rodents and primates, we have tested the effects of NaP on intracellular Ca(2+) levels ([Ca(2+)](i)) in rat and monkey motor neurons in vitro. Motor neurons present in spinal cord slices from rhesus monkey embryos (E37 and 56) and from rat E16 were dissected and cultured on glass coverslips. Following 2 weeks (rhesus) or 2-3 days (rat) in culture, neurons were loaded with fura-PE3/AM, and examined for [Ca(2+)](i) changes in response to NaP. Rhesus motor neurons were identified by immunostaining for Islet-1 (MN specific antigen) and neuron specific enolase (NSE). Motor neurons from both species exhibited dose-dependent NaP-evoked increases in [Ca(2+)](i) However, the dose-response curve for the Rhesus motor neurons was significantly shifted to the right of the rat dose-response curve, whereas the overall amplitude of the Ca(2+) rise was similar in both species. As shown previously for the Aplysia neurons, the action of NaP is attenuated by SKF 96365, an inhibitor of store-operated calcium entry. In contrast the action of NaP is unaffected by nifedipine and tetrodotoxin, blockers of voltage-dependent Ca(2+) and Na(+) channels, respectively, or by ouabain, an inhibitor of the plasma membrane Na(+)/K(+) ATPase. Our results indicate that the NaP-induced increase in [Ca(2+)](i) is conserved across species and suggest that the toxicological sensitivity of rodent over primate to pyrithione could be due to the enhanced sensitivity of rodent motor neurons to NaP-evoked intracellular Ca(2+) elevation.

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)) in fura 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(50) of approximately 300 nM and a Hill coefficient of approximately 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 approximately 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.