Electrogenic H+ transport and pH gradients generated by a V-H+ -ATPase in the isolated perfused larval Drosophila midgut

Abstract

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by approximately 17-fold and approximately 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (V(t)) of 38 to 45 mV and high resistance (R(t)) of 800 to 1,400 Omega x cm(2) measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10(-4) M, ouabain 10(-4) M, bumetanide 10(-4) M), K(+) -free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect V(t) or R(t). Bafilomycin-A(1) (3 to 5 microM) decreased V(t) by approximately 40% and short-circuit current (I(sc)) by approximately 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H(+) -ATPase located in the basal membrane. Gradients of H(+) were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO(3)(-)), driven by the basal V-H(+) -ATPase.