title: 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.
authors.affiliation: Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens 30602, USA.
authors.forename: F W
authors.initials: FW
authors.lastname: Berman
authors.forename: T F
authors.initials: TF
authors.lastname: Murray
mesh_terms: D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D000814:Aniline Compounds; D000818:Animals; D019898:Autocrine Communication / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D003917:Dextrorphan / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000378:metabolism; D008387:Marine Toxins / Q000494:pharmacology*; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D035964:Oxocins; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology; D014966:Xanthenes; D000095702:Polyether Toxins
chemical_list: D015763:2-Amino-5-phosphonovalerate; D000814:Aniline Compounds; D002118:Calcium; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D003917:Dextrorphan; D016291:Dizocilpine Maleate; D018691:Excitatory Amino Acid Antagonists; D005456:Fluorescent Dyes; D018698:Glutamic Acid; D008387:Marine Toxins; D009498:Neurotoxins; D009543:Nifedipine; D035964:Oxocins; D016194:Receptors, N-Methyl-D-Aspartate; D015222:Sodium Channels; D013890:Thiourea; D014966:Xanthenes; C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; C059715:Fluo-3; C053342:brevetoxin; D000095702:Polyether Toxins