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The Journal of Experimental Biology 206, 2211-2219 (2003)
doi: 10.1242/jeb.00385

The V-type H⁺-ATPase in Malpighian tubules of Aedes aegypti: localization and activity

Xing-He Weng¹, Markus Huss², Helmut Wieczorek² and Klaus W. Beyenbach^1,*

¹ Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA
² Department of Biology, University of Osnabrück, D-49019 Osnabrück, Germany

^* Author for correspondence (e-mail: kwb1{at}cornell.edu)

Accepted 17 March 2003

The V-type H⁺-ATPase is thought to provide the driving force for transepithelial electrolyte and fluid secretion in Malpighian tubules. To confirm the presence of this proton pump in Malpighian tubules of the yellow fever mosquito Aedes aegypti, we used several antibodies raised against the V-type H⁺-ATPase of Manduca sexta. Western blot analysis confirmed the presence of the V-type H⁺-ATPase in Malpighian tubules of Aedes aegypti. In situ immunostaining identified the V-type H⁺-ATPase at the apical membrane of the mitochondrion-rich brush border of principal cells. The V-type H⁺-ATPase was not found in stellate cells. Measurements of ATPase activity revealed that bafilomycin-sensitive and NO₃^--sensitive ATPase activity accounted for 50–60% of total ATPase activity in crude extracts of Malpighian tubules. No significant ouabain- or vanadate-sensitive Na⁺/K⁺-ATPase activity was detected. These results support the conclusion reached previously in electrophysiological studies that the mechanisms for transepithelial electrolyte secretion in the Aedes Malpighian tubules rely on the V-type H⁺-ATPase as the principal energizer of epithelial transport. Measures of transepithelial Na⁺ and K⁺ secretion and estimates of the H⁺ flux mediated by the V-type H⁺-ATPase suggest a 1:1 stoichiometry for Na⁺/H⁺ and K⁺/H⁺ exchange transport across the apical membrane.

Key words: V-type H⁺-ATPase, Na⁺/K⁺-ATPase, Malpighian tubule, electrolyte secretion, Na⁺/H⁺ exchange, polyclonal antibody, immunohistochemistry, western blot

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