title: Cell volume decrease as a link between azaspiracid-induced cytotoxicity and c-Jun-N-terminal kinase activation in cultured neurons.
abstract: Azaspiracids (AZAs) are a group of marine toxins recently described that currently includes 20 members. Not much is known about their mechanism of action, although the predominant analog in nature, AZA-1 targets several organs in vivo, including the central nervous system, and exhibits high neurotoxicity in vitro. AZA distribution is increasing globally with mussels being most widely implicated in AZA-related food poisoning events, with human poisoning by AZAs emerging as an increasing worldwide problem in recent years. We used pharmacological tools to inhibit the cytotoxic effect of the toxin in primary cultured neurons. Several targets for AZA-induced neurotoxicity were evaluated. AZA-1 elicited a concentration-dependent hyperpolarization in cerebellar granule cells of 2-3 days in vitro; however, it did not modify membrane potential in mature neurons. Furthermore, in immature cells, AZA-1 decreased the membrane depolarization evoked by exposure of the neurons to 50mM K(+). Preincubation of the neurons with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), amiloride, or ouabain before addition of AZA-1 decreased the AZA-1-induced neurotoxicity and the increase in phosphorylated c-Jun-N-terminal kinase (JNK) caused by the toxin, indicating that disruption in ion fluxes was involved in the neurotoxic effect of AZA-1. Furthermore, short exposures of cultured neurons to AZA-1 caused a significant decrease in neuronal volume that was reverted by preincubation of the neurons with DIDS or amiloride before addition of the toxin. The results presented here indicate that the JNK activation induced by AZA-1 is secondary to the decrease in cellular volume elicited by the toxin.
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo 27002, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Vale
authors.forename: Kyriacos C
authors.initials: KC
authors.lastname: Nicolaou
authors.forename: Michael O
authors.initials: MO
authors.lastname: Frederick
authors.forename: Mercedes R
authors.initials: MR
authors.lastname: Vieytes
authors.forename: Luis M
authors.initials: LM
authors.lastname: Botana
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D000838:Anions; D048429:Cell Size / Q000187:drug effects*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008387:Marine Toxins / Q000633:toxicity*; D008564:Membrane Potentials; D049990:Membrane Transport Modulators / Q000494:pharmacology; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D009579:Nitrobenzoates / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000378:metabolism; D013141:Spiro Compounds / Q000633:toxicity*; D013997:Time Factors; D000095702:Polyether Toxins
chemical_list: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000584:Amiloride; D000838:Anions; D007473:Ion Channels; D048031:JNK Mitogen-Activated Protein Kinases; D008387:Marine Toxins; D049990:Membrane Transport Modulators; D009579:Nitrobenzoates; D010042:Ouabain; D011188:Potassium; D017923:Sodium-Hydrogen Exchangers; D013141:Spiro Compounds; C406592:azaspiracid; D000095702:Polyether Toxins; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid