Brevetoxin-induced autocrine excitotoxicity is associated with manifold routes of Ca2+ influx

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 lever 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.