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
Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase
Bettina Schewe1,2,
Elmar Schmälzlin3 and
Bernd Walz1,2,*
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
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Fig. 1. (A–E) Distribution of BCECF and TMRE fluorescence in a
double-labelled isolated salivary gland. (A) Tangential optical section of the
gland tubule under differential interference contrast optics. (B–D)
Confocal optical sections of the gland excited to display BCECF fluorescence
(B) and TMRE fluorescence (C). BCECF-AM loading results in a punctate staining
pattern on a diffuse background. (D) Overlaid of images of B and C in which
the yellow colour indicates colocalization of BCECF-fluorescent spots and
TMRE-stained mitochondria. (E) Punctate BCECF fluorescence in a permeabilized
gland stained with BCECF-free acid; confocal optical section. Scale bars, 10
µm. (F,G) Drop in BCECF fluorescence excited at 490 nm and 439 nm after
bath application of β-escin indicates loss of unbound dye from the
cytoplasm resulting from permeabilization. (H,I) Traces showing BCECF
fluorescence excited at 490 nm and 439 nm; β-escin permeabilization leads
to loss of cytoplasmic dye because fluorescence emission drops at both
excitation wavelengths (indicated by red arrows). A subsequent decrease in
bath pH induces antiparallel changes in BCECF fluorescence (a drop in
fluorescence excited at 490 nm; an increase in fluorescence excited at 439 nm,
indicated by blue arrows) suggesting that the BCECF that remains after
permeabilization records cytoplasmic pH changes.
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Fig. 3. (A–E) pHi changes induced by bath application of 10 mmol
l–1 cAMP (A), 10 µmol l–1 8-CPT-cAMP (B),
100 µmol l–1 of the adenylyl cyclase inhibitor forskolin
(C) and 500 µmol l–1 of the phosphodiesterase inhibitor
IBMX (D). These experiments show that increases in intracellular cAMP
concentration mimic the acidifying effect of 5-HT stimuli. (E) Quantitative
analysis of the experiments. Data are means ± s.e.m.; the number of
experiments is given in parentheses.
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Fig. 4. Microfluorometric measurements of oxygen content in the lumen of isolated
salivary gland tubules injected with polystyrene beads containing the
O2-sensitive luminescent dye PtPFPP. 100% O2
concentration in A–C corresponds to the O2 content in the
bath PS, which is in equilibrium with ambient air. (A) 10 nmol
l–1 5-HT stimulates cellular respiration and a drop in
luminal O2 concentration that is not significantly reduced in the
presence of concanamycin A (A,D). The 8-CPT-cAMP-induced drop in luminal
O2 concentration is significantly reduced by concanamycin A (B,D).
Application of 1 µmol l–1 thapsigargin in
Ca2+-free PS causes an increase in luminal O2
concentration, and the 5-HT-induced drop in luminal O2
concentration is significantly reduced under these conditions (C,D). Data in D
are means ± s.e.m.; the number of experiments is given in parentheses;
*P<0.05.
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Fig. 5. (A–C) Location of carbonic anhydrase (CA) in cryostat sections of
salivary gland tubules. The dark reaction product indicating CA activity is
localized at the basal pole of the secretory cells as shown in a cross section
(B) and a longitudinal section (C). Formation of the reaction product is
completely inhibited by 10–5 mol l–1 of the
CA inhibitor acetazolamide (A). Scale bars, 20 µm.
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Fig. 6. Effects of acetazolamide and concanamycin A on 5-HT-induced pHi
changes. (A) Bath application of 500 µmol l–1
acetazolamide causes a cytoplasmic acidification, whereas 10 nmol
l–1 5-HT, applied in the presence of acetazolamide, produces
an alkalinization. (B) In the presence of acetazolamide, 10 µmol
l–1 8-CPT-cAMP induces an intracellular alkalinization. (C,D)
Inhibition of the apical V-ATPase by 1 µmol l–1
concanamycin A causes a small acidification. In preparations in which 10 nmol
l–1 5-HT induces a monophasic acidification (C), this pH
change is almost unaffected by concanamycin A. When 10 nmol
l–1 5-HT induces a multiphasic pH change, concanamycin A
blocks the alkalizing response component (D). (E) Acetazolamide and
concanamycin A applied together cause additive acidifications, and 10 nmol
l–1 5-HT causes an alkalinization in the presence of these
two inhibitors (E). The alkalinization produced by 10 nmol
l–1 5-HT in the presence of acetazolamide is significantly
(P<0.05) smaller when the V-ATPase is simultaneously inhibited by
concanamycin A (F). Data in F are means ± s.e.m., N=9.
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Fig. 7. Removal of Na+ from the bath causes an alkalinization (A). (A,E)
Under Na+-free conditions, 10 nmol l–1 5-HT
induces a small intracellular alkalinization (*P<0.05).
(B,E) In the continuous presence of 50 µmol l–1 EIPA, 10
nmol l–1 5-HT induces a significantly smaller acidification
than under control conditions (**P<0.01). (C,E) In the
absence of extracellular Cl–, 10 nmol l–1
5-HT induces a small alkalinization, which is significantly different from the
control stimulation (**P<0.01). (D,E) Application of
500 µmol l–1 DIDS causes a strong acidification but does
not significantly influence the 5-HT-induced acidification
(*P>0.05). Data in E are means ± s.e.m. The
number of replicates is given in parentheses.
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Fig. 8. (A) Removal of extracellular Na+ leads to an intracellular
alkalinization (0.22±0.03 pH units; N=29). (B) Removal of
extracellular glutamate induces an intracellular alkalinization
(0.13±0.02, N=6) that is not significantly different from that
observed under Na+-free conditions (P>0.05) (C). Data
in C are means ± s.e.m.
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Fig. 9. Schematic representation of transporters that we discuss in conjunction
with steady-state pHi regulation (A) and the 5-HT- and cAMP-induced
acidification (B). Note: 5-HT causes an acidification, a cAMP-mediated
increase in the number of H+-pumping V-ATPase holoenzymes in the
apical membrane of the gland cells and stimulates cellular respiration (B).
For details, see Discussion.
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© The Company of Biologists Ltd 2008
10 nmol l–1 induce monophasic
drops in pHi. Only concentrations 
10 nmol l–1
produce bi- or multiphasic pH changes. (G) Summary data for acidifications
induced by 0.1–10 nmol l–1 5-HT (total pHi
changes). Summary data for 10 nmol l–1 5-HT represent only
monophasic acidification. Data in G are means ± s.e.m.; the number of
experiments is given in parentheses.