Brevetoxin‐Induced Autocrine Excitotoxicity Is Associated with Manifold Routes of Ca2+ Influx

Abstract: Real‐time alterations in intracellular Ca2+ ([Ca2+]i) were monitored in fluo‐3‐loaded cerebellar granule neurons (CGNs) exposed to the brevetoxin PbTx‐1. [Ca2+]i was measured using a fluorescent plate reader (FLIPR), which measures simultaneously the mean intracellular Ca2+ change in a population of cultured cells in each well of a 96‐well plate. PbTx‐1 produced rapid and concentration‐dependent increases in neuronal [Ca2+]i with a potency nearly identical to that determined previously for PbTx‐1‐induced neurotoxicity. The NMDA receptor antagonists MK‐801, dextrorphan, and D(‐)‐2‐amino‐5‐phosphonopentanoic acid, and tetanus toxin, an inhibitor of Ca2+‐dependent exocytotic neurotransmitter release, effected significant reductions in both the integrated fluo‐3 fluorescence response and excitatory amino acid release and protected CGNs against PbTx‐1 neurotoxicity. The L‐type Ca2+ channel antagonist nifedipine produced a modest reduction in the fluo‐3 response but reduced substantially the plateau phase of the PbTx‐1 increment in [Ca2+]i when combined with MK‐801. When nifedipine and MK‐801 were combined with the Na+/Ca2+ exchanger (reversed mode) inhibitor KB‐R7943, the PbTx‐1 increment in [Ca2+]i was nearly completely attenuated. These data show that Ca2+ entry into PbTx‐1‐exposed CGNs occurs through three primary routes: NMDA receptor ion channels, L‐type Ca2+ channels, and reversal of the Na+/Ca2+ exchanger. There was a close correlation between reduction of the integrated fluo‐3 fluorescence response and the level of neuroprotection afforded by blockers of each Ca2+ entry pathway; however, simultaneous blockade of L‐type Ca2+ channels and the Na+/Ca2+ exchanger, although reducing the integrated [Ca2+]i response to a level below that provided by NMDA receptor blockade alone, failed to completely attenuate PbTx‐1 neurotoxicity. This finding suggests that in addition to total [Ca2+]i load, neuronal vulnerability is governed principally by the NMDA receptor Ca2+ influx pathway.