A vacuolar-type H+-ATPase and a Na+/H+exchanger contribute to intracellular pH regulation in cockroach salivary ducts

Abstract

SUMMARYCells of the dopaminergically innervated salivary ducts in the cockroach Periplaneta americana have a vacuolar-type H+-ATPase(V-ATPase) of unknown function in their apical membrane. We have studied whether dopamine affects intracellular pH (pHi) in duct cells and whether and to what extent the apical V-ATPase contributes to pHiregulation. pHi measurements with double-barrelled pH-sensitive microelectrodes and the fluorescent dye BCECF have revealed: (1) the steady-state pHi is 7.3±0.1; (2) dopamine induces a dose-dependent acidification up to pH 6.9±0.1 at 1 μmol l–1 dopamine, EC50 at 30 nmol l–1dopamine; (3) V-ATPase inhibition with concanamycin A or Na+-free physiological saline (PS) does not affect the steady-state pHi; (4)concanamycin A, Na+ -free PS and Na+/H+exchange inhibition with 5-(N-ethyl-N-isopropyl)-amiloride(EIPA) each reduce the rate of pHi recovery from a dopamine-induced acidification or an acidification induced by an NH4Cl pulse; (5)pHi recovery after NH4Cl-induced acidification is almost completely blocked by concanamycin A in Na+-free PS or by concanamycin A applied together with EIPA; (6) pHi recovery after dopamine-induced acidification is also completely blocked by concanamycin A in Na+-free PS but only partially blocked by concanamycin A applied together with EIPA. We therefore conclude that the apical V-ATPase and a basolateral Na+/H+ exchange play a minor role in steady-state pHi regulation but contribute both to H+extrusion after an acute dopamine- or NH4Cl-induced acid load.