Estimation of pH and the number of lytic granules in a CD8+ CTL clone treated with an inhibitor of vacuolar type H(+)-ATPase concanamycin A.

Abstract

A vacuolar type H(+)-ATPase inhibitor, concanamycin A (CMA), raised the pH and induced morphologic changes such as vacuolation in lytic granules in the CD8+ cytotoxic T lymphocyte clone OE4. Here we measured the pH in lytic granules present in OE4 by immunoelectron microscopic observation using (3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) as a pH probe. In control OE4, only the peripheral region, but not cores, were heavily stained with DAMP. Exposure to 100 nM CMA for 60 min raised the pH in the lytic granules from 5.7 +/- 0.2 to 6.8 +/- 0.2. Furthermore, the number of lytic granules in OE4 was estimated under phase contrast microscopy after Wright staining. About 20 lytic granules in average were detected in control cells, while CMA induced vacuolation and decreased their number by 20 to 30%.