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+/Ca2+ 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 [Ca2+]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.
Kraut
R P
RP
Manitoba Institute of Cell Biology, Winnipeg, Canada.
Bose
R
R
Cragoe
E J
EJ
Greenberg
A H
AH
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000502:physiology*; D003602:Cytotoxicity, Immunologic; D003603:Cytotoxins / Q000494:pharmacology*; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000494:pharmacology*; D051379:Mice; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D003603:Cytotoxins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; D019831:Sodium-Calcium Exchanger; D054353:Perforin; C053494:2',4'-dimethylbenzamil; D000584:Amiloride; D002118:Calcium