Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae

Abstract

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pHi) and luminal pH (pHlu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pHo 6.9). In proximal segments both pHi (7.43 +/- 0.20) and pHlu (7.10 +/- 0.24)) were significantly lower than in distal segments (pHi 7.70 +/- 0.29, pHlu 8.09 +/- 0.15). Steady-state pHi of proximal segments was much less sensitive to changes in pHo than pH of the luminal fluid (delta pHlu/delta pHo was 0.49 while delta pHi/delta pHo was 0.18; pHo 6.50-7.20). Re-alkaliniziation from an NH4Cl-induced intracellular acid load (initial pHi recovery rate 0.55 +/- 0.34 pH.min-1) was nearly totally inhibited by 1 mmol.l-1 KCN (96% inhibition) and to a large degree (79%) by 1 mumol.l-1 bafilomycin A1. In contrast, both vanadate (1 mmol.l-1) and amiloride (1 mmol.l-1) inhibited pHi recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na+ from the bathing saline had no effect on pHi recovery, indicating that a Na+/H+ exchange is not significantly involved in pHi regulation. Instead pHi regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.