INTRACELLULAR PH REGULATION BY THE PLASMA-MEMBRANE V-ATPASE IN MALPIGHIAN TUBULES OF DROSOPHILA LARVAE

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pH(i)) and luminal pH (pH(1u)) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pH(o) 6.9). In proximal segments both pH(i) (7.43 +/- 0.20) and pH(1u) (7.10 +/- 0.24)) were significantly lower than in distal segments (pH(i) 7.70+/-0.29, pH(1u) 8.09 +/- 0.15). Steady-state pH(i) of proximal segments was much less sensitive to changes in pH(o) than pH of the luminal fluid (Delta pH(1u)/Delta pK(o) was 0.49 while Delta pH(i)/Delta pH(o) was 0.18; pH(o) 6.50-7.20). Re-alkaliniziation from an NH4Cl-induced intracellular acid load (initial pH(i) recovery rate 0.55+/-0.34 pH.min(-1)) was nearly totally inhibited by 1 mmol.1(-1) KCN (96% inhibition) and to a large degree (79%) by 1 mu mol.1(-1) bafilomycin A(1). In contrast, both vanadate (1 mmol.1(-1)) and amiloride (1 mmol.1(-1)) inhibited pH(i) recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na+ from the bathing saline had no effect on pH(i) recovery, indicating that a Na+/H+ exchange is not significantly involved in pH(i) regulation. Instead pH(i) regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.