Figure 4.

pH_p determinations in SLO-permeabilized cells. The experimental protocol is diagramatically illustrated at the top. In brief, intact cells that normally have acidic phagosomes (left) were permeabilized using 0.1 μg/ml SLO in media devoid of ATP/Mg²⁺, resulting in inhibition of H⁺ pumping due to depletion of substrate (middle). Readdition of ATP/ Mg²⁺ induces resumption of pumping (right). Phagosomal pH was measured by ratio imaging as described for Fig. 1 (see Materials and Methods) in peritoneal macrophages obtained from wild-type (Nramp ^+/+) mice and from Nramp1 mutant mice (Nramp ^−/−). (A) Measurements in Nramp1 ^+/+ cells. Where indicated, cells were treated with SLO, and ATP and Mg were reintroduced in the absence (filled symbols) or presence of 100 nM concanamycin (CCM; open symbols). (B) Nramp1 ^+/+ cells (open symbols) or Nramp1 ^−/− cells (filled symbols) were treated with SLO. ATP and Mg were reintroduced where indicated. A and B are each representative of five determinations. (C) Summary of results from five experiments like that shown in B. The initial rate (black bars) and extent (stippled bars) of ATP/ Mg-induced acidification were measured. Data are means ± SE of five separate experiments.