FIGURE 5.

Block of calpain-mediated ezrin hydrolysis rescues VacA-induced inhibition of acid secretion. A, aliquots of cultured parietal cells transiently transfected to express wild type GFP-ezrin and GFP-ezrin^V466G mutant. Thirty-six hours after the transfection, the parietal cells were incubated with activated VacA (5 μg/ml) and boiled VacA in the presence of 1.8 mm Ca²⁺ for 20 min prior to being solubilized in SDS-PAGE sample buffer. Equivalent amounts of proteins from these samples were applied to SDS-PAGE and subsequently transblotted onto nitrocellulose membrane. The blot was probed with an anti-GFP antibody using an ECL kit (Pierce). Note that wild type GFP-ezrin but not GFP-ezrin^V466G is hydrolyzed in VacA-treated glandular cells. B, cultured parietal cells transiently transfected to express wild type GFP-ezrin and GFP-ezrin^V466G mutant. Thirty-six hours after the transfection, the parietal cells were incubated with activated VacA (10 μg/ml) and 100 mm histamine plus 50 mm IBMX in the presence of 1.8 mm Ca²⁺ for 20 min. Treated cells were fixed and stained for ezrin, F-actin, and H,K-ATPase. Note that the apical vacuoles are dilated in the GFP-ezrin^V466G-expressing cells but not wild type GFP-ezrin-expressing cells. Bar, 10 μm. C, 35 parietal cells positively expressing GFP-ezrin (wild type and ezrin^V466G mutant) and stimulated parietal cells were surveyed for apical vacuole diameters in the same focal plane as described in Fig. 4D. Data are expressed as means ± S.E.