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First published online February 15, 2008
Journal of Experimental Biology 211, 805-815 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.002667

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Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase

Bettina Schewe^1,2, Elmar Schmälzlin³ and Bernd Walz^1,2,*

¹ University of Potsdam, Institute of Biochemistry and Biology, University Campus Golm, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany
² University of Potsdam, Department of Animal Physiology, University Campus Golm, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany
³ University of Potsdam, Department of Chemistry, Physical Chemistry, University Campus Golm, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany

^* Author for correspondence (e-mail: walz{at}uni-potsdam.de)

Accepted 6 November 2007

Blowfly salivary gland cells have a vacuolar-type H⁺-ATPase (V-ATPase) in their apical membrane that energizes secretion of a KCl-rich saliva upon stimulation with serotonin (5-hydroxytryptamine, 5-HT). We have used BCECF to study microfluometrically whether V-ATPase and carbonic anhydrase (CA) are involved in intracellular pH (pH_i) regulation, and we have localized CA activity by histochemistry. We show: (1) mean pH_i in salivary gland cells is 7.5±0.3 pH units (N=96), higher than that expected from passive H⁺ distribution; (2) low 5-HT concentrations (0.3–3 nmol l^–1) induce a dose-dependent acidification of up to 0.2 pH units, with 5-HT concentrations >10 nmol l^–1, causing monophasic or multiphasic pH changes; (3) the acidifying effect of 5-HT is mimicked by bath application of cAMP, forskolin or IBMX; (4) salivary gland cells exhibit CA activity; (5) CA inhibition with acetazolamide and V-ATPase inhibition with concanamycin A lead to a slow acidification of steady-state pH_i; (6) 5-HT stimuli in the presence of acetazolamide induce an alkalinization that can be decreased by simultaneous application of the V-ATPase inhibitor concanamycin A; (7) concanamycin A removes alkali-going components from multiphasic 5-HT-induced pH changes; (8) NHE activity and a Cl^–-dependent process are involved in generating 5-HT-induced pH changes; (9) the salivary glands probably contain a Na⁺-driven amino acid transporter. We conclude that V-ATPase and CA contribute to steady-state pH_i regulation and 5-HT-induced outward H⁺ pumping does not cause an alkalinization of pH_i because of cytosolic H⁺ accumulation attributable to stimulated cellular respiration and AE activity, masking the alkalizing effect of V-ATPase-mediated acid extrusion.

Key words: intracellular pH, BCECF, salivary glands, blowfly, Calliphora vicina, serotonin, vacuolar H⁺-ATPase, V-ATPase, NHE, AE, carbonic anhydrase, oxygen consumption, Na⁺/amino acid cotransporter