In NK cell-mediated cytolysis, the degranulation of the NK cell, and the binding and polymerization of its effector molecule cytolysin/perforin, are Ca2+ dependent. To determine if increases in the target cell free intracellular Ca2+ concentration ([Ca2+]i) are also important for cytotoxicity, we examined the effects of Ca2+ substitutes, promotors of Ca2+ influx; and inhibitors of Ca2+ efflux on the cytolysis mediated by purified cytolysin. Ca2+ promoted cytolysin-mediated cytolysis but could also inactivate cytolysin on preincubation in the absence of target cells. Ba2+ and Sr2+ could not alone promote cytolysin-mediated cytolysis but did enhance Ca2(+)-driven cytolysis. Preincubation with Ba2+ and Sr2+ did not inactivate cytolysin. One interpretation which these results suggested was that neither Ba2+ nor Sr2+ were involved in cytolysin polymerization and pore formation, but could act intracellularly as Ca2+ analogs once pore formation had occurred. The synergistic interaction of cytolysin and the Ca2+ ionophore A23187 further supported a role for increased [Ca2+]i in cytolysis. Inhibitors of Na+/Ca2+ exchange, namely low Na+ medium, ouabain, and the amiloride analogs 2',4'-dimethylbenzamil, 5-(N-4-chlorobenzyl)-2',4'dimethylbenzamil, and alpha-phenylbenzamil all markedly enhanced cytolysin-mediated cytolysis. These results support the hypothesis that the Ca2+ dependency of NK cell- and cytolysin-mediated cytolysis is related to increases in target cell [Ca2+]i. Furthermore, they indicate that Na+/Ca2+ exchange is an important counterlytic mechanism.

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