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 Ca2+ indicator Fluo-4 AM and Fura-2 AM revealed that nanomolar concentrations of OlyA/PlyB increased the intracellular Ca2+ activity [Ca2+]i in A10 cells. This effect was absent in a Ca2+-free medium, indicating that OlyA/PlyB-induced [Ca2+]i increase was dependent on Ca2+ influx into cells. The increase in [Ca2+]i by OlyA/PlyB was partially prevented by: i) the calcium channel blockers verapamil and La3+, 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 Ca2+-ATPase inhibitor thapsigargin reduced the [Ca2+]i increase evoked by OlyA/PlyB, whereas the plasma membrane depolarization with high K+ in the medium did not prevent OlyA/PlyB-induced [Ca2+]i. In summary, our data could suggest that the OlyA/PlyB-induced increase in [Ca2+]i is due to an influx of Ca2+ through a variety of co-existing plasma membrane Ca2+-permeable channels, Ca2+ entry through non-selective ion permeable pores formed de novo by OlyA/PlyB in the plasma membrane and calcium-induced intracellular Ca2+ release, altogether leading to disturbed Ca2+ homeostasis in A10 cells.