Cytolysins increase intracellular calcium and induce eicosanoids release by pheochromocytoma PC12 cell cultures

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Abstract
Cytolysins are the most commonly occurring toxins among bacteria, plants, and animals. By disturbing cell membrane, they impair ionic permeability, leading to cell death. In an attempt to investigate cytolysin action on catecholaminergic neurons, we have treated pheochromocytoma cell cultures with Strep-tolysin S, Staphylococcus aureus α and δ, Stoichatus, Parcelsin, and cobra direct lytic factor. To measure neurotoxicity, PC12 cultures were loaded with 51Cr and exposed for 1 hr at 37°C to different concentrations of cytolysins. Cytotoxic dose-response curves have been generated resulting in CD50 (cytotoxic dose 50%) in the range of 1–50 μg toxin/culture. Using subcytotoxic concentrations of cytolysins (which are of clinical relevance), changes on intracellular calcium were measured by Fura-2 fluorescence technique. Addition of either Stoichatus toxin and tetanolysin or streptococcus and staphylococcus cytolysins to PC12 cells caused rapidly or gradually a progressive increase in [Ca2+]i, respectively. Under similar conditions, samples of PC12 culture medium were assayed for 3H-arachidonic acid released and by radioimmunoassay for the content of PGE2 (prostaglandin), TXB2 (stable metabolite of thromboxane), and 5-HETE (hydroxy acid lipoxygenase product). PLA2 was activated 4.5–6.0-fold and the levels of all three eicosanoids were increased by 2.5–9-fold (PGE2), 4–6-fold (TXB2), and over 100-fold (5-HETE) by Stoichatus and Parcelsin cytolysins. Upon treatment with Streptolysin S and staphylococcus α toxins PLA2 (phospholipase A2) was slightly activated (1.5-fold) and the levels of PGE2 and TXB2 increased 1.3–2.0-fold and that of 5-HETE up to 30-fold. These results indicate that the increased intracellular calcium followed by PLA2 activation and eicosanoid production and release might be involved in neurotoxic action of cytolysins.