QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online April 18, 2006
Journal of Experimental Biology 209, 1716-1724 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02187

This Article Figures Only Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rein, J. Articles by Baumann, O. Search for Related Content PubMed PubMed Citation Articles by Rein, J. Articles by Baumann, O.

Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly Calliphora vicina

Julia Rein¹, Bernhard Zimmermann^1,2, Carsten Hille¹, Ingo Lang^1,*, Bernd Walz¹ and Otto Baumann^1,

¹ Institut für Biochemie und Biologie, Universität Potsdam, D-14415 Potsdam, Germany
² Carl Zeiss Jena GmbH, Advanced Imaging Microscopy, D-07745 Jena, Germany

Author for correspondence (e-mail: obaumann{at}rz.uni-potsdam.de)

Accepted 28 February 2006

Secretion in blowfly salivary glands is induced by the neurohormone serotonin and powered by a vacuolar-type H⁺-ATPase (V-ATPase) located in the apical membrane of the secretory cells. We have established a microfluorometric method for analysing pH changes at the luminal surface of the secretory epithelial cells by using the fluorescent dye 5-N-hexadecanoyl-aminofluorescein (HAF). After injection of HAF into the lumen of the tubular salivary gland, the fatty acyl chain of the dye molecule partitions into the outer leaflet of the plasma membrane and its pH-sensitive fluorescent moiety is exposed at the cell surface. Confocal imaging has confirmed that HAF distributes over the entire apical membrane of the secretory cells and remains restricted to this membrane domain. Ratiometric analysis of HAF fluorescence demonstrates that serotonin leads to a reversible dose-dependent acidification at the luminal surface. Inhibition by concanamycin A confirms that the serotonin-induced acidification at the luminal surface is due to H⁺ transport across the apical membrane via V-ATPase. Measurements with pH-sensitive microelectrodes corroborate a serotonin-induced luminal acidification and demonstrate that luminal pH decreases by about 0.4 pH units at saturating serotonin concentrations. We conclude that ratiometric measurements of HAF fluorescence provide an elegant method for monitoring V-ATPase-dependent H⁺ transport in the blowfly salivary gland in vivo and for analysing the spatiotemporal pattern of pH changes at the luminal surface.

Key words: vacuolar ATPase, V-ATPase, 5-N-hexadecanoyl-aminofluorescein, pH, concanamycin A, serotonin, 5-hydroxytryptamine, insect, blowfly, Calliphora vicina

This article has been cited by other articles:

				B. Schewe, E. Schmalzlin, and B. Walz Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase J. Exp. Biol., March 1, 2008; 211(5): 805 - 815. [Abstract] [Full Text] [PDF]

				J. Rein, M. Voss, W. Blenau, B. Walz, and O. Baumann Hormone-induced assembly and activation of V-ATPase in blowfly salivary glands is mediated by protein kinase A Am J Physiol Cell Physiol, January 1, 2008; 294(1): C56 - C65. [Abstract] [Full Text] [PDF]

				M. Voss, O. Vitavska, B. Walz, H. Wieczorek, and O. Baumann Stimulus-induced Phosphorylation of Vacuolar H+-ATPase by Protein Kinase A J. Biol. Chem., November 16, 2007; 282(46): 33735 - 33742. [Abstract] [Full Text] [PDF]

				C. Hille and B. Walz A vacuolar-type H+-ATPase and a Na+/H+ exchanger contribute to intracellular pH regulation in cockroach salivary ducts J. Exp. Biol., April 15, 2007; 210(8): 1463 - 1471. [Abstract] [Full Text] [PDF]