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The Journal of Membrane Biology

, Volume 206, Issue 1, pp 61–72 | Cite as

Electrogenic H+ Transport and pH Gradients Generated by a V-H+-ATPase in the Isolated Perfused Larval Drosophila Midgut

  • S. Shanbhag
  • S. Tripathi
Article

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 ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (Vt) of 38 to 45 mV and high resistance (Rt) of 800 to 1400 Ω • cm2 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 Vt or Rt. Bafilomycin-A1 (3 to 5 μM) decreased Vt by ≈ 40% and short-circuit current (Isc) by ≈ 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 HCO3), driven by the basal V-H+-ATPase.

Key words

Bafilomycin-A1 Ultrastructural morphometry Cable analysis Ion-selective microelectrodes Microperfusion 

Abbreviations

BEL,

basal extracellular labyrinth;

DMSO,

dimethyl-sulfoxide;

HEPES,

N-2-hydroxyethylpiperazine-N’-2-ethaesulfonic acid;

Isc,

short-circuit current;

LIS,

lateral intracellular space;

Rt,

transepithelial resistance;

Va,

apical cell membrane potential;

Vb,

basal cell membrane potential;

V-H+-ATPase,

vacuolar-proton-adenosinetriphosphatase;

Vt,

transepithelial potential, perfusion end;

Vc,

transepithelial potential, collection end.

Notes

Acknowledgement

We record our gratitude to the late Peter D’Souza and to J. N. Parmar for fabrication of the microperfusion apparatus and excellent machining support. We thank T. V. Abraham for electronics and data acquisition software. Supported by Interdisciplinary Programme 10P-809.

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Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  1. 1.Tata Institute of Fundamental ResearchMumbaiIndia

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