Maitotoxin-induced intracellular calcium rise in PC12 cells: involvement of dihydropyridine-sensitive and omega-conotoxin-sensitive calcium channels and phosphoinositide breakdown.
The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.
Meucci
O
O
Department of Human Communicative Sciences, II School of Medicine, University of Naples, Italy.
Grimaldi
M
M
Scorziello
A
A
Govoni
S
S
Bergamaschi
S
S
Yasumoto
T
T
Schettini
G
G
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects*; D003600:Cytosol / Q000737:chemistry; D004095:Dihydropyridines / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000502:physiology; D008387:Marine Toxins / Q000494:pharmacology*; D009529:Nicardipine / Q000494:pharmacology; D035964:Oxocins; D016716:PC12 Cells / Q000737:chemistry / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000494:pharmacology*; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D020960:omega-Conotoxins; D000095702:Polyether Toxins
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D002118:Calcium; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D019204:GTP-Binding Proteins; D008387:Marine Toxins; D009529:Nicardipine; D035964:Oxocins; D010456:Peptides, Cyclic; D037342:Pertussis Toxin; D010716:Phosphatidylinositols; D012964:Sodium; D010566:Virulence Factors, Bordetella; D020960:omega-Conotoxins; C053423:Conus magus toxin; C020394:maitotoxin; D000095702:Polyether Toxins