Ca(2+) and Na(+) contribute to the swelling of differentiated neuroblastoma cells induced by equinatoxin-II.
Equinatoxin-II (EqTx-II), a cytotoxic protein (mol.wt 20 kDa) isolated from the sea anemone Actinia equina, was found to consistently increase the three-dimensional projected area of differentiated neuroblastoma (NG108-15) cells provided Ca(2+) was present in the medium. No swelling was detected when external NaCl was replaced by sucrose, but replacement of NaCl by Na-isethionate did not prevent the swelling, as revealed by confocal laser scanning microscopy. In addition, microspectrofluorometric measurements in cells preloaded with the Ca(2+) indicator fura-2/AM revealed that EqTx-II (100 nM) markedly increased the fluorescence (F(340)/F(380)) ratio indicating a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). The elevation of [Ca(2+)](i) exhibited two components that seem to be related to the kinetics of EqTx-II-induced Ca(2+) entry since pretreatment of cells with Ca(2+)-ATPase inhibitors (thapsigargin), Ca(2+) channel blockers (nifedipine and Gd(3+)) or prolonged exposure to a high K(+) (75 mM) medium did not alter EqTx-II-induced Ca(2+) signals. As far as we know, this is the first demonstration that EqTx-II causes swelling of neuroblastoma cells and that this effect is correlated both with an increase of [Ca(2+)](i) and needs the presence of extracellular Na(+). It is suggested that EqTx-II has the ability to insert into the plasma membrane of neuroblastoma cells and to form pores altering the membrane permeability and the intracellular osmolality, inducing a marked influx of water into the cells.
Meunier
F A
FA
Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS, Gif sur Yvette, France.
Frangez
R
R
Benoit
E
E
Ouanounou
G
G
Rouzaire-Dubois
B
B
Suput
D
D
Molgó
J
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002454:Cell Differentiation; D003064:Cnidarian Venoms / Q000633:toxicity*; D003603:Cytotoxins / Q000633:toxicity*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D009447:Neuroblastoma / Q000473:pathology*; D051381:Rats; D012615:Sea Anemones / Q000737:chemistry; D012964:Sodium / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D003064:Cnidarian Venoms; D003603:Cytotoxins; D004791:Enzyme Inhibitors; C012307:equinatoxin; D012964:Sodium; D000252:Calcium-Transporting ATPases; D002118:Calcium