title: Effect of the ostreolysin A/pleurotolysin B pore-forming complex on neuroblastoma cell morphology and intracellular Ca²⁺ activity.
abstract: Ostreolysin A (OlyA) and pleurotolysin B (PlyB), isolated from edible oyster mushrooms, form a cytolytic complex (OlyA/PlyB) in membrane cells that causes respiratory arrest. This study evaluated the mechanisms underlying cytotoxic OlyA/PlyB activity in neuroblastoma NG108-15 cells. Confocal microscopy with morphometric analysis revealed that OlyA/PlyB increased the 3-dimensional projected area of differentiated cells. Iso-osmotic replacement of NaCl by sucrose or Na-isethionate prevented the cellular swelling. This suggests that formation of cellular edema requires the presence of Na(+) and/or Cl(-) in the extracellular space and may be related to an influx of Na(+) and/or a shift in Cl(-), which induce a marked influx of water that is ultimately responsible for cellular swelling. In addition, extracellular Ca(2+) moderately contributed to the swelling because benzamil (10 µM), a 3Na(+)/Ca(2+) exchange (NCX) inhibitor, and Ca(2+)-free medium partially prevented this response. Fluorometric measurements revealed that OlyA/PlyB, at approximately 15-fold higher concentrations, increased the intracellular Ca(2+) activity [Ca(2+)]i. This increase was dependent on the presence of Na(+) and Ca(2+) in the external medium and was sensitive to benzamil. It is thus likely that a switch in the NCX mode, associated with the de novo formation of non-selective ion pores by OlyA/PlyB in cellular plasma membranes, plays an important role in this effect. Overall, OlyA/PlyB affects neuroblastoma cell morphology and Ca(2+) homeostasis to influence the toxin-induced respiratory arrest.
authors.affiliation: *Veterinary Faculty, Institute of Anatomy, Histology and Embryology, Veterinary Faculty, Institute of Physiology, Pharmacology and Toxicology, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia and CNRS, Institut de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie et Développement Bât. 32-33, 91198 Gif sur Yvette cedex, France.
authors.forename: Milka
authors.initials: M
authors.lastname: Vrecl
authors.affiliation: *Veterinary Faculty, Institute of Anatomy, Histology and Embryology, Veterinary Faculty, Institute of Physiology, Pharmacology and Toxicology, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia and CNRS, Institut de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie et Développement Bât. 32-33, 91198 Gif sur Yvette cedex, France.
authors.forename: Monika
authors.initials: M
authors.lastname: Babnik
authors.affiliation: *Veterinary Faculty, Institute of Anatomy, Histology and Embryology, Veterinary Faculty, Institute of Physiology, Pharmacology and Toxicology, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia and CNRS, Institut de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie et Développement Bât. 32-33, 91198 Gif sur Yvette cedex, France.
authors.forename: Uroš
authors.initials: U
authors.lastname: Diacci
authors.affiliation: *Veterinary Faculty, Institute of Anatomy, Histology and Embryology, Veterinary Faculty, Institute of Physiology, Pharmacology and Toxicology, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia and CNRS, Institut de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie et Développement Bât. 32-33, 91198 Gif sur Yvette cedex, France.
authors.forename: Evelyne
authors.initials: E
authors.lastname: Benoit
authors.affiliation: *Veterinary Faculty, Institute of Anatomy, Histology and Embryology, Veterinary Faculty, Institute of Physiology, Pharmacology and Toxicology, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia and CNRS, Institut de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie et Développement Bât. 32-33, 91198 Gif sur Yvette cedex, France robert.frangez@vf.uni-lj.si.
authors.forename: Robert
authors.initials: R
authors.lastname: Frangež
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D005656:Fungal Proteins / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000494:pharmacology*; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D051381:Rats
chemical_list: D005656:Fungal Proteins; D006460:Hemolysin Proteins; C461280:ostreolysin; C586632:pleurotolysin B, oyster mushroom; D002118:Calcium
keywords: MACPF domain; [Ca2+]i; cellular swelling; neuroblastoma NG108-15 cells; ostreolysin A; pleurotolysin B