Fig. 5
a All inhibitors exert influence on the d-v pHi-gradient in the FCE during S10b (SIM-experiment; CFDA). Especially glibenclamide and furosemide, but also verapamil and 9-anthroic acid led to alkalisation in both FC types. Verapamil, glibenclamide, and furosemide resulted in a steeper d-v gradient (angle × 2, × 5, and × 100, respectively). The increase in fluorescence intensity as well as in the inclination of the d-v gradient (× 3) caused by bafilomycin was due to the alkalisation of vesicles (see b), leading to increasing cytoplasmic acidification as well as to a shallower d-v gradient. Amiloride and 9-anthroic acid led to a stronger acidification of vFC, thus reducing the angle of the d-v gradient (× 0.5 and × 0.1, respectively) as well (cf. Additional file 2: Table S5). For statistics, see legend to Fig. 4. b Alkalisation of vesicles in the FCE by bafilomycin. SIM-fluorescence images of S10b-follicles incubated in R-14 medium with bafilomycin or with DMSO (control) for 20 min. Inhibition of V-ATPases led to a more granular staining (arrowhead) compared to the controls, indicating alkalisation of vesicles. In addition, the cytoplasm of the FC appeared darker than in the controls, indicating cytoplasmic acidification