Fig. 5. Investigation of Na⁺-independent pH increases in granulosa cells. Granulosa cells were acidified by NH₄Cl pulse in bicarbonate-free medium and Na⁺-independent recovery examined. (A) Typical examples of each treatment group. Note that in control experiments, acidosis is almost fully relieved despite the absence of Na⁺. Drugs or vehicles were added to the bath at t=10 minutes, and remained throughout the experiment. NEM (100 µM) and bafilomycin (20 nM or 200 nM) retarded the Na⁺-independent alkalinization that occurs following NH₄Cl removal in controls. A lower level of NEM (10 µM) partially inhibited recovery, whereas a higher level (1000 µM) did not further inhibit recovery (not shown). Bafilomycin was maximally effective at both 20 and 200 nM, and these data were combined (three replicates: one of 200 nM and two of 20 nM; due to the high cost of bafilomycin and the large volumes required for these experiments, the data for 20 and 200 nM were combined to yield three replicates). Concanamycin (10 nM) caused partial inhibition, which was not increased at 100 nM (not shown). (B) Summary of Na⁺-independent acidosis correction. Each bar represents mean±s.d. of the initial rate of pH recovery from three replicates. Different letters indicate P<0.01 (ANOVA).