First published online July 6, 2005
Journal of Experimental Biology 208, 2799-2808 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01681
V-ATPase inhibition prevents recovery from anoxia in Artemia franciscana embryos: quiescence signaling through dissipation of proton gradients
Joseph A. Covi1,*,
W. Dale Treleaven2 and
Steven C. Hand1
1 Division of Cellular, Developmental and Integrative Biology, Department of
Biological Science
2 NMR Facility, College of Basic Sciences, Louisiana State University, Baton
Rouge, LA 70803, USA
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Fig. 1. Diagram describing the potential acidic compartments within a developing
encysted gastrula of the brine shrimp, Artemia franciscana. Degree of
acidification is indicated by grey scale. Maintenance of steady-state pH
within acidic compartments is assumed to be regulated largely by ATP-dependent
proton pumping, ion leak rates and buffering capacity.
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Fig. 3. Intracellular pH and NTP/NDP status of dechorionated A.
franciscana embryos during acute treatment with bafilomycin as observed
with 31P-NMR. (A) Intracellular pH is plotted as a function of time
for two Pi chemical shifts identified during early development.
Circles represent the downfield (left) Pi resonance, while diamonds
represent the upfield (right) Pi resonance. Open symbols indicate 4
µmol l–1 bafilomycin treatment while filled symbols
indicate 0.2% ethanol control treatment. Period of bafilomycin or ethanol
treatment is indicated by a hashed bar, and anoxic period is indicated by a
solid bar. (B) Representative spectrum for aerobic embryos, demonstrating the
presence of two distinct Pi peaks, which appear as a part of normal
aerobic development. Two pH values are given to indicate that a range of pH is
evident from the observed Pi peaks.
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Fig. 4. Intracellular pH and NTP/NDP status of dechorionated A.
franciscana embryos for long-term treatment with bafilomycin as observed
with 31P-NMR. (A) Intracellular pH is plotted as a function of time
for two Pi chemical shifts identified during early development.
Open circles indicate pH estimated using the predominant Pi
chemical shifts for embryos pretreated with 4 µmol l–1
bafilomycin on ice for 24 h prior to acquisition of NMR spectra. Filled
circles indicate pH determined from main downfield Pi resonance for
control embryos given identical treatment but in the absence of bafilomycin.
Filled diamonds indicate the upfield (acidic) Pi resonance for
control embryos, which appears after 1.5 h of aerobic incubation as a part of
normal development. Period of ethanol (control) or bafilomycin treatment is
indicated by a hatched bar, and anoxic treatment is indicated by a solid bar.
Spectra used for plotting bafilomycin treatment are selectively displayed for
(B) 1 h of aerobic development, followed by (C) 1 h of anoxia-induced
quiescence and (D) 2 h of subsequent aerobic recovery. Control spectra are not
shown. All shaded boxes are identical, and serve to emphasize changes in
chemical shift and shape of the Pi peaks.
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Fig. 5. Effect of 50 µmol l–1 CCCP on oxygen consumption
( O2) by dechorionated
A. franciscana embryos. Embryos were allowed to develop for 2.75 h
before the addition of the protonophore CCCP. Period of incubation with CCCP
is indicated by a solid bar.
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Fig. 6. Representative 31P-NMR spectra for long-term treatment of
dechorionated embryos with CCCP. Embryos were incubated for 30 h on ice in a
sterile-filtered solution of 0.25 mol l–1 NaCl and 20 µmol
l–1 CCCP. 31P-NMR spectra were subsequently
acquired during 1 h of superfusion with CCCP under (A) aerobic conditions, (B)
followed by anoxia.
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Fig. 7. Intracellular pH status for dechorionated A. franciscana embryos
during exposure to, and recovery from, 16.5 h of anoxia. Intracellular pH is
plotted as a function of time for two Pi chemical shifts identified
during early development for aseptically treated embryos superfused with 0.25
mol l–1 NaCl. Solid circles indicate pH determined from main
downfield (alkaline) Pi resonance, and open diamonds indicate the
upfield (acidic) Pi resonance. Period of anoxic treatment is
indicated by a solid bar. Treatment conditions used mimic those employed in
the anoxia studies of Kwast et al.
(1995 ).
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Fig. 8. Schematic representation for the role of proton concentration gradients in
intracellular pH shifts induced by transitions between anoxic and aerobic
states in encysted embryos of the brine shrimp, Artemia franciscana.
Plots were modified from Kwast et al.
(1995), and the data were
derived from 31P-NMR estimates of intracellular pH. Broken lines
indicate time points corresponding to the embryo diagrams above and are
numbered accordingly.
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© The Company of Biologists Ltd 2005
-NTP and ß-NDP; d, 
-NTP and NDP; e,
ß-NTP. (C) A standard curve relating the chemical shift of Pi
(
) to solution pH was used to determine the average intracellular pH
during treatments. This curve is described by the equation
pH=6.76+log[(