Ca2+ 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 thr ee-dimensional projected area of differentiated neuroblastoma (NG108-15) ce lls provided Ca2+ was present in the medium. No swelling was detected when external NaCl was replaced by sucrose, but replacement of NaCl by Na-isethi onate did not prevent the swelling, as revealed by confocal laser scanning microscopy. In addition, microspectrofluorometric measurements in cells pre loaded with the Ca2+ indicator fura-2/AM revealed that EqTx-II (100 nM) mar kedly increased the fluorescence (F-340/F-380) ratio indicating a rise of i ntracellular Ca2+ concentration ([Ca2+](i)). The elevation of [Ca2+](i) exh ibited two components that seem to be related to the kinetics of EqTx-II-in duced Ca2+ entry since pretreatment of cells with Ca2+-ATPase inhibitors (t hapsigargin), Ca2+ channel blockers (nifedipine and Gd3+) or prolonged expo sure to a high K+ (75 mM) medium did not alter EqTx-II-induced Ca2+ signals . As far as we know, this is the first demonstration that EqTx-II causes sw elling of neuroblastoma cells and that this effect is correlated both with an increase of [Ca2+](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 t he cells. (C) 2000 Elsevier Science Ltd. All rights reserved.