THE NA+/CA2+ EXCHANGER REGULATES CYTOLYSIN/PERFORIN-INDUCED INCREASES IN INTRACELLULAR CA2+ AND SUSCEPTIBILITY TO CYTOLYSIS

The Ca2+ dependency of NK cell-mediated and cytolysin-mediated cytolysis may be related to increases in target cell intracellular Ca2+. In a previous study we hypothesized that the Na+/Ca2+ exchanger can act as a counter-lytic mechanism by regulating the damaging increases in intracellular free calcium ([Ca2+]i) produced by cytolysin. We found that conditions said to inhibit Ca2+ extrusion by Na+/Ca2+ exchange, namely low extracellular Na+ or the presence of certain amiloride analogs which block Na+/Ca+ exchange, enhanced the cytolysin-mediated cytolysis of YAC-1 lymphoma cells. In the present work we have confirmed the above hypothesis by measuring the [Ca2+]i of fura-2- or aequorin-labeled YAC-1 cells treated with cytolysin and low Na+ medium or amiloride analogs. YAC-1 cells appear to have a Na+/Ca2+ exchange system: low Na+ medium caused gradual increases in [Ca2+]i, and this effect was reversed in Na+-replete medium. Cytolysin purified from NK cell granules caused rapid dose-dependent increases in [Ca2+]i, and low Na+ medium enhanced these cytolysin-mediated increases. The Na+/Ca2+ exchange system appeared to be more active in cytolysin-challenged cells: amiloride analogs, which inhibit Na+/Ca2+ exchange in other systems, acted synergistically with cytolysin to cause large increases in [Ca2+]i, but had little effect, if any, on their own. 5-(N-4-Chlorobenzyl)-2',4'-dimethylbenzamil, the amiloride analog which has the greatest specificity for the Na+/Ca2+ exchanger and which previously was found to be the most potent enhancer of cytolysin-mediated cytolysis, was the most potent enhancer of cytolysin-mediated increases in [Ca+]i. The above results suggest that Na+/Ca2+ exchange may be one of the target cell mechanisms of resistance to cytolysin and NK cell-mediated cytolysis.