First published online October 7, 2008
Journal of Experimental Biology 211, 3306-3314 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.020776
Ionic determinants of pH of acidic compartments under hypertonic conditions in trout hepatocytes
Khaled H. Ahmed and
Bernd Pelster*
Institut für Zoologie and Center of Molecular Biosciences, Leopold
Franzens Universität Innsbruck, Technikerstrasse 25, A-6020 Innsbruck,
Austria
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Fig. 1. Image of trout hepatocytes – using laser-scanning microscopy (LSM
510, Zeis) – pre-incubated with 5 µmol l–1 acridine
orange (AO) for 10 min showing two distinct emission spectra: (1) green
fluorescence corresponding to the monomeric AO emission, weak in the cytoplasm
and bright in the nucleus, and (2) red fluorescence from separated or
aggregated compartments in the cytoplasm, corresponding to aggregated AO
emission. The red fluorescence indicates an accumulation of AO in the form of
dimers and/or polymers due to the acidic pH inside the compartmental lumen.
The LSM is equipped with a x32 oil-immersion objective; 488 nm line of
an argon laser was used for excitation; fluorescence emissions were
simultaneously recorded in green (505–530 nm) and red (>650 nm)
channels.
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Fig. 2. Changes in AO fluorescence in response to (A) exposure of cells to
hypertonicity (1.6xisosmolarity) followed by restoration of isotonicity
and (B) exposure of cells to bafilomycin A1 (0.3 µmol l–1)
followed by the same exposure conditions as in A in the continuous presence of
bafilomycin A1. Data are means ± s.e.m. of 64–66 cells from three
to four independent preparations. Data were normalized to the mean calculated
from five points preceding the hypertonicity exposure. Dotted line corresponds
to normal quenching of AO (three independent preparations, 54 cells, data
normalized to the mean calculated from the first five points).
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Fig. 3. Changes in AO fluorescence following exposure of cells to valinomycin (10
µmol l–1) under steady-state conditions followed by
exposure to hypertonicity then restoration of isotonic conditions, in the
continuous presence of valinomycin. Data are means ± s.e.m. of 67 cells
from three independent preparations. Data were normalized to the mean
calculated from the first five points. For comparison, the control
(Fig. 2A) is shown as a dotted
line.
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Fig. 4. Changes in AO fluorescence following exposure of cells to
Cl–-free medium or SITS (0.5 mmol l–1) under
steady-state conditions followed by exposure to hypertonicity then restoration
of isotonic conditions, in the continuous absence of Cl– or
presence of SITS. Data are means ± s.e.m. of 34–38 cells from
three independent preparations. Data were normalized as in
Fig. 3. For comparison, the
control (Fig. 2A) is shown as a
dotted line.
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Fig. 5. Changes in AO fluorescence following exposure of cells to
Cl–-free medium in the presence or absence of
HCO3– under steady-state conditions followed by
exposure to hypertonicity then restoration of isotonic conditions, in the
continuous absence of Cl– or both Cl– and
HCO3–. Data are means ± s.e.m. of
38–55 cells from three independent preparations. Data were normalized as
in Fig. 3. For comparison, the
control (Fig. 2A) is shown as a
dotted line.
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Fig. 6. Changes in AO fluorescence following exposure of cells to
Na+-free medium or amiloride (100 µmol l–1)
under steady-state conditions followed by exposure to hypertonicity then
restoration of isotonic conditions, in the continuous absence of
Na+ or presence of amiloride. Data are means ± s.e.m. of
34–41 cells from three independent preparations. Data were normalized as
in Fig. 3. For comparison, the
control (Fig. 2A) is shown as a
dotted line.
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Fig. 7. Changes in AO fluorescence following exposure of cells to
Ca2+-free medium under steady-state conditions followed by exposure
to hypertonicity then restoration of isotonic conditions, in the continuous
absence of Ca2+. Data are means ± s.e.m. of 65 cells from
four independent preparations. Data were normalized as in
Fig. 3. For comparison, the
control (Fig. 2A) is shown as a
dotted line.
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Fig. 8. Changes in AO fluorescence following (A) exposure of cells to BAPTA-AM (25
µmol l–1) in the presence and absence of extracellular
Ca2+ and (B) exposure of cells (pre-incubated with 25 µmol
l–1 BAPTA-AM for 1 h) to hypertonicity followed by
restoration of isotonic conditions, in the continuous absence of extracellular
Ca2+. Data are means ± s.e.m. of 61–126 cells from
four to five independent preparations. Data were normalized as in
Fig. 3. For comparison, the
control (Fig. 2A) is shown as a
dotted line in B.
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Fig. 9. (A) Effect of sequential addition of bafilomycin A1 (0.3 µmol
l–1) and BAPTA-AM (25 µmol l–1) on the
hypertonicity-induced changes in AO fluorescence. Data are means ±
s.e.m. of 56–68 cells from three independent preparations. Data were
normalized as in Fig. 3. (B)
Changes in AO fluorescence following exposure of cells to A23187 (2 µmol
l–1) in Ca2+-free medium followed by BAPTA-AM (25
µmol l–1), in the continuous presence of A23187 and
absence of extracellular Ca2+. Data are means ± s.e.m. of 90
cells from five independent preparations. Data were normalized as in
Fig. 3.
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© The Company of Biologists Ltd 2008
