Calcium ion involvement in the action of Pasteurella haemolytica leukotoxin

The influence of Ca2+ ions on the cytotoxic activity of Pasteurella haemolytica leukotoxin was investigated. The divalent cation influenced the cytotoxic effect of the leukotoxin for sensitive BL-3 target cells, but its absence did not eliminate cytotoxicity. In short-term 1-h assays using neutral red uptake as a measure of cell viability, depletion of Ca2+ either by exhaustive dialysis or by addition of the Ca2+ chelators EDTA and EGTA eliminated the cytolytic effect of low doses of the toxin. Addition of Ca2+ to target cell cultures depleted of the divalent cation restored the cytolytic effect of the leukotoxin. Prolonged exposure of the BL-3 cells to the toxin abrogated the protective effect of EDTA and EGTA. Cell death measured by uptake of neutral red, exclusion of trypan blue and 51Cr release indicated that protection observed in the absence of free Ca2+ was temporary. Toxin-induced cytolysis equivalent to that observed in the presence of Ca2+ occurred following the initial 2-h exposure. In addition, verapamil, a Ca2+ channel blocker, prevented cell death during 1-h cytotoxicity assays. The protection afforded by verapamil was dose-dependent and was influenced by the concentration of Ca2+ in the buffer medium. The results suggest that Ca2+ positively influences the rapid initial phase of cell death resulting from exposure to the toxin, but is not required for the entirety of the cytolytic process.