Abstract
Brevetoxins (designated PbTx-1 to -10) are potent lipid-soluble polyether compounds that are known to bind to and modulate voltage-gated sodium channel activity. To investigate whether brevetoxins produce direct central nervous system neurotoxic effects, cultured rat cerebellar granule neurons were exposed to brevetoxins in Locke’s buffer for 2 h at 22°C. Neuronal injury was quantified by assaying lactate dehydrogenase activity in the exposure buffer and in conditioned growth media collected at 22 h after brevetoxin exposure. Brevetoxins produced acute neuronal injury and death in neurons with a rank order potency of PbTx-1 (EC50 = 9.31 ± 0.45 nM) > PbTx-3 (EC50 = 53.9 ± 2.8 nM) > PbTx-2 (EC50 = 80.5 ± 5.9 nM) > PbTx-6 (EC50 = 1417 ± 32 nM), which is similar to their previously determined rank order potency for brevetoxin-induced icthyotoxicity and binding to [3H]PbTx-3-labeled sodium channels on synaptosomes. The neurotoxic response could be prevented by coapplication of the sodium channel antagonist tetrodotoxin or by the competitive or noncompetitiveN-methyl-d-aspartate (NMDA) receptor antagonists D-AP5 and MK-801, ketamine, dextromethorphan, and dextrorphan, respectively. NMDA receptor antagonists afforded neuroprotection with rank order potencies comparable to those measured previously for protection against glutamate-induced excitotoxic responses. Further analysis revealed that brevetoxins induced a concentration-dependent release of l-glutamate andl-aspartate into the exposure buffer. These data indicate that brevetoxin-induced injury in cultured rat cerebellar granule neurons is mediated by NMDA receptors that are activated indirectly as a consequence of PbTx-induced sodium channel activation and attendant excitatory amino acid release.
Footnotes
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Send reprint requests to: Dr. Thomas F. Murray, Department of Physiology and Pharmacology, Room 2223, College of Veterinary Medicine, The University of Georgia, Athens, GA 30601. E-mail:tmurray{at}calc.vet.uga.edu
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↵1 This work was supported in part through a pilot project grant from the Marine/Freshwater Biomedical Science Center of Oregon State University (ES03850).
- Abbreviations:
- CGN
- cerebellar granule neuron
- CNS
- central nervous system
- DIC
- days in culture
- LDH
- lactate dehydrogenase
- AMPA
- α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
- MK-801
- (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate
- D-AP5
- d-(−)-2-amino-5-phosphonopentanoic acid
- EAA
- excitatory amino acid
- OPD
- o-phthaldialdehyde
- NMDA
- N-methyl-d-aspartate
- Received January 11, 1999.
- Accepted March 3, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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