title: Effect of the ostreolysin A/pleurotolysin B pore-forming complex on intracellular Ca2+ activity in the vascular smooth muscle cell line A10.
abstract: Ostreolysin A/pleurotolysin B (OlyA/PlyB) is a binary pore-forming protein complex that produces a rapid cardiorespiratory arrest. Increased tonus of the coronary vascular wall produced by OlyA/PlyB may lead to ischemia, arrhythmias, the hypoxic injury of cardiomyocytes and cardiotoxicity. We evaluated the effects of OlyA/PlyB in cultured vascular smooth muscle A10 cells. Fluorometric measurements using the Ca(2+) indicator Fluo-4 AM and Fura-2 AM revealed that nanomolar concentrations of OlyA/PlyB increased the intracellular Ca(2+) activity [Ca(2+)]i in A10 cells. This effect was absent in a Ca(2+)-free medium, indicating that OlyA/PlyB-induced [Ca(2+)]i increase was dependent on Ca(2+) influx into cells. The increase in [Ca(2+)]i by OlyA/PlyB was partially prevented by: i) the calcium channel blockers verapamil and La(3+), ii) the inhibitor of the sodium-calcium exchanger (NCX) benzamil, and iii) the iso-osmotic replacement of NaCl by sucrose. The pre-treatment of cells with the Ca(2+)-ATPase inhibitor thapsigargin reduced the [Ca(2+)]i increase evoked by OlyA/PlyB, whereas the plasma membrane depolarization with high K(+) in the medium did not prevent OlyA/PlyB-induced [Ca(2+)]i. In summary, our data could suggest that the OlyA/PlyB-induced increase in [Ca(2+)]i is due to an influx of Ca(2+) through a variety of co-existing plasma membrane Ca(2+)-permeable channels, Ca(2+) entry through non-selective ion permeable pores formed de novo by OlyA/PlyB in the plasma membrane and calcium-induced intracellular Ca(2+) release, altogether leading to disturbed Ca(2+) homeostasis in A10 cells.
authors: Veterinary Faculty, Institute of Anatomy, Histology and Embryology, Gerbičeva 60, SI-1000 Ljubljana, Slovenia.
authors: Milka
authors: M
authors: Vrecl
authors: Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia.
authors: Monika
authors: M
authors: Babnik
authors: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia.
authors: Kristina
authors: K
authors: Sepčić
authors: Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia.
authors: Monika C
authors: MC
authors: Žužek
authors: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia.
authors: Peter
authors: P
authors: Maček
authors: Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia.
authors: Uroš
authors: U
authors: Diacci
authors: Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia. Electronic address: robert.frangez@vf.uni-lj.si.
authors: Robert
authors: R
authors: Frangež
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005656:Fungal Proteins / Q000633:toxicity*; D006706:Homeostasis / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects*; D051381:Rats
chemical_list: D005656:Fungal Proteins; D002118:Calcium
keywords: Fluo-4/AM; Fura-2/AM; OlyA/PlyB; Ostreolysin; Smooth muscle cells; [Ca(2+)](i)