Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase.
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.
Schewe
Bettina
B
University of Potsdam, Institute of Biochemistry and Biology, University Campus Golm, Karl-Liebknecht-Strasse 24-25, Potsdam-Golm, Germany.
Schmälzlin
Elmar
E
Walz
Bernd
B
D000818:Animals; D002256:Carbonic Anhydrases / Q000378:metabolism*; D003592:Cytophotometry; D004175:Diptera / Q000378:metabolism*; D005452:Fluoresceins; D006706:Homeostasis / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D018942:Macrolides / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D012469:Salivary Glands / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
D005452:Fluoresceins; D018942:Macrolides; D012701:Serotonin; C036978:concanamycin A; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D025262:Vacuolar Proton-Translocating ATPases; D002256:Carbonic Anhydrases; D010100:Oxygen