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Pasteurella haemolytica A1 leukotoxin causes rapid (5 to 15 min) leakage of intracellular K+ and cell swelling and slower (15 to 60 min), Ca2+-dependent formation of large plasma membrane defects (congruent to 100 nm) and leakage of lactate dehydrogenase from bovine lymphoma cells (BL3 cells) (K. D. Clinkenbeard, D. A. Mosier, A. L. Timko, and A. W. Confer, Am. J. Vet. Res., in press). Incubation of BL3 cells in medium made hypertonic by inclusion of 75 mM sucrose blocked leukotoxin-induced cell swelling, formation of large plasma membrane defects, and leakage of lactate dehydrogenase but did not block leukotoxin-induced leakage of intracellular K+. Carbohydrates with molecular weights less than that of sucrose, e.g., mannitol, did not block leukotoxin-induced cell swelling of BL3 cells. Increasing the concentration of mannitol to twice that of sucrose still resulted in no protective effect. Assuming that leukotoxin acts as a transmembrane molecular sieve, then the functional transmembrane pore size formed by leukotoxin in BL3 cells is slightly less than the size of sucrose, i.e., 0.9 nm. Exposure of BL3 cells to leukotoxin for 15 or 45 min followed by the addition of hypertonic sucrose to the incubation medium reversed leukotoxin-induced cell swelling and prevented further leakage of lactate dehydrogenase. Leukotoxin-induced leakage of lactate dehydrogenase required both cell swelling and Ca2+-dependent processes. The Ca2+-dependent steps can occur before or concurrent with cell swelling.

Transmembrane pore size and role of cell swelling in cytotoxicity caused by Pasteurella haemolytica leukotoxin