Leukotoxin activity from culture supernatants of Pasteurella haemolytica serotype 1 in logarithmic growth phase caused rapid (less than 5 min) release of intracellular K+, uptake of extracellular Ca2+, and swelling of cultured
bovine lymphoma cells (BL3 cells). Release of 51CrO4(2-) and
lactate dehydrogenase (LDH) from BL3 cells began after 15 minutes of incubation with
leukotoxin at 37 C and was completed between 60 and 120 minutes of incubation. In addition,
leukotoxin exposure of BL3 cells resulted in cell aggregation and adherence to glass surfaces. Scanning electron microscopy indicated that after 10 minutes of
leukotoxin exposure, BL3 cells increased in size, and large membrane defects developed between 20 and 60 minutes of exposure. The rate of release of LDH from
leukotoxin-exposed BL3 cells was proportional to the amount of
leukotoxin added. At high cell concentrations, the activity of LDH released at completion was directly proportional to the amount of
leukotoxin added.
Leukotoxin-induced release of LDH required a divalent
cation, whereas K+ release and cell swelling did not. The addition of Ca2+, Mn2+, and Ba2+ resulted in increased
leukotoxin-induced release of LDH. Divalent
cation concentrations of 0.5 to 2.5 mM resulted in 50% of maximal stimulation.
Ethylene glycol-bis(beta-aminoethyl
ether) N,N,N',N'-tetraacetic
acid blocked increased release of LDH caused by Ca2+ addition, but had no effect on K+ release or cell swelling.
Leukotoxin action on BL3 cells (K+ release, cell swelling, Ca2+ uptake, and release of LDH) was prevented by incubation at 4 C.