V-ATPase inhibition prevents recovery from anoxia in Artemia franciscana embryos: quiescence signaling through dissipation of proton gradients
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
* Author for correspondence (e-mail: jcovi1{at}lsu.edu)
Accepted 11 May 2005
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Summary |
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Key words: vacuolar-type ATPase, CCCP, bafilomycin A1, quiescence, gradient dissipation, anoxia, acidification, intracellular pH, 31P-NMR
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Introduction |
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Descriptive observations of Artemia development can be traced to
the early 1900s (for review, see Clegg and
Conte, 1980), and extensive investigation of the mechanisms
involved in the extreme anoxia tolerance displayed by the encysted embryonic
stage continues on many fronts (e.g. Clegg
et al., 2000
; Eads and Hand,
2003
; Menze et al.,
2005
; Warner et al.,
2002
; Willsie and Clegg,
2002
). However, until now, functional examination of these
mechanisms in vivo has been hampered by the presence of a cyst shell
that encapsulates these embryos. This shell can be divided into two main
sections: tertiary envelope (chorion) and embryonic cuticle
(Drinkwater and Clegg, 1991
),
which together present a physical barrier conferring impermeability to all but
low-molecular-mass gasses and water
(Trotman, 1991
). It even
appears to prevent the passage of protons
(Busa et al., 1982
) and
volatile solvents such as DMSO (Price,
1967
). In our companion paper, however, we demonstrated that
removal of the chorion confers permeability to macrolide antibiotics such as
bafilomycin A1, a highly specific and lipid-soluble inhibitor of
the V-ATPase (Covi and Hand,
2005
). This method of permeabilization is used advantageously in
the present study and promises to expand the experimental repertoire available
for other in vivo experiments in the future with these encysted
embryos.
The V-ATPase is a proton pump that is well known for its nearly ubiquitous
role in the acidification of intracellular compartments
(Forgac, 2000;
Futai et al., 2000
). These
compartments, diagrammed in Fig.
1, include early and late endosomes, Golgi complex, exocytotic and
protein trafficking vesicles, lysosomes and yolk platelets
(Fagotto, 1995
;
Nishi and Forgac, 2002
). Not
surprisingly, a large number of physiological processes depend on V-ATPase
activity, including endocytosis, exocytosis, protein targeting, receptor
recycling and lysosomal function (Nishi
and Forgac, 2002
). Of particular relevance when considering A.
franciscana embryos is the finding that the acidification of yolk
platelets for degradation requires an ATP-dependent proton pump in insects
(Abreu et al., 2004
) and is
inhibited by bafilomycin in Xenopus oocytes
(Fagotto and Maxfield, 1994
)
and Rhodnius prolixus eggs (Motta
et al., 2004
). The only form of regulation of V-ATPase activity
known to have physiological relevance is the dissociation of the peripheral
(V1) sector from the membrane-spanning (V0) domain
(Wieczorek et al., 2003
), and
this disassembly may be a mechanism for inactivating the V-ATPase when energy
availability is limited (Kane and Smardon,
2003
).
|
The ability to survive prolonged bouts of anoxia is directly linked to the
depression of cellular energy turnover
(Hand, 1998). This correlate
is exemplified in A. franciscana embryos, for which it has reasonably
been argued that long-term survivorship under anoxia is accomplished in part
through the establishment of a nearly ametabolic state
(Clegg, 1997
;
Warner and Clegg, 2001
). We
previously proposed that the drop in cellular ATP:ADP ratio and increase in
free inorganic phosphate (Pi) are likely to inactivate the V-ATPase
within minutes of exposing these embryos to anoxia and that subsequent
dissipation of proton chemical gradients could result in a substantial
cytoplasmic acidification (Covi and Hand,
2005
). Here, we use the technique of phosphorous nuclear magnetic
resonance (31P-NMR) to directly test this hypothesis by observing
the effects of V-ATPase inhibition and proton gradient dissipation on the
pHi status of intact brine shrimp embryos. The protonophore CCCP is
also used to examine the effects of complete dissipation of cellular proton
gradients. Specifically, this lipid-soluble weak acid facilitates a comparison
of intracellular acidification under anoxia with the acidification induced by
combining the uncoupling of oxidative phosphorylation with the dissipation of
proton gradients.
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Materials and methods |
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Animals
Encysted embryos of the brine shrimp Artemia franciscana Kellog
(Great Salt Lake population) were obtained in the dehydrated state
(post-diapause) from Sanders Brine Shrimp Co. (Ogden, UT, USA) in 2001. These
post-diapause embryos were stored dry at 20°C and hydrated
overnight at 0°C in 0.25 mol l1 NaCl prior to
dechorionation or aseptic washing. Dechorionation was performed as previously
described (Kwast and Hand,
1993). Aseptic washing was performed using the same protocol, but
the antiformin (NaOH/hypochlorite solution) wash was kept at 0°C to
prevent complete removal of the chorion. Aseptically treated embryos were used
in the 16 h anoxia study only. Dechorionated embryos were used in all other
experiments. Embryos were stored for no more than 16 h in ice cold 0.125 mol
l1 NaCl prior to experimentation. All solutions were
prepared less than 24 h prior to use, and the length of incubation time for
embryos in antiformin was strictly maintained at 20±1 min at room
temperature. Developmental staging of embryos was conducted using the same
criteria described by Conte et al.
(1977
).
NMR spectroscopy
Proton-decoupled 31P-NMR spectra were collected at a frequency
of 202.458 MHz at a temperature of 298±0.1 Kusing a Brucker (Bellerica,
MA, USA) AMX-500 spectrometer. 2048 data points were collected over a spectral
width of 82 p.p.m., and datasets were zero-filled to 32 K. Except during the
first hour of anoxia and recovery from anoxia, 3072 transients were recorded
over a period of 10 min in each experiment. During these transitions between
aerobic and anoxic conditions, 1536 transients were recorded over 5 min in
order to provide better time resolution of pH shifts. Data were exponentially
multiplied by 10 Hz line-broadening prior to Fourier transformation. Zero
reference was established using a 85% H3PO4 standard.
Field drift was assessed by the acquisition of 85% H3PO4
spectra before and after each experiment. A deuterium lock was not required,
as field drift in our system (0.057 Hz h1) was much lower
than resolution (0.4 Hz data point1).
Embryos were packed by gravity into a 10 mm glass NMR tube, and movement
during perfusion was prevented by placing a foam plug at the top of the embryo
column. For bafilomycin treatments and controls, embryos were superfused from
the bottom of the tube with 0.2 mol l1 sucrose. For CCCP
treatment and 16 h anoxia study, embryos were superfused with 0.25 mol
l1 NaCl. It should be noted that control spectra obtained
using sucrose superfusion were indiscernible from those obtained with NaCl
superfusion (data not shown). Both sucrose and NaCl solutions were sterile
filtered before use, and the final concentration of ethanol during treatments
was 0.2%. Maintenance of aerobic conditions and induction of anoxia during
in vivo spectroscopy were conducted as described by Kwast et al.
(1995). In brief, embryos were
superfused with oxygen saturated medium at a rate of 2 ml
min1, and anoxia was induced by stopping perfusion. As
discussed by Kwast et al.
(1995
), the use of
oxygen-saturated medium has no noticeable affect on embryonic development, and
stopping superfusion provides the most rapid production of anoxic conditions.
Peak identification was accomplished by comparison with published spectra
(Busa et al., 1982
;
Kwast et al., 1995
).
Intracellular pH was estimated from the chemical shift (resonance frequency)
of Pi using a titration curve generated in our facility
(Fig. 2C). This standard curve
was produced using a solution of 10 mmol l1 Pi,
110 mmol l1 KCl and 20 mmol l1 NaCl, and
pH was set by the combination of mono- and dibasic phosphate.
|
Hatching assays
Hatching success was observed for embryos used in 31P-NMR
experiments. To remove inhibitors and ethanol from the incubation medium after
NMR measurements were made, embryos were gently resuspended in the NMR tube
using 35 artificial seawater at room temperature, and care was taken
to use only the embryos that were contained within the spectrometer detection
window. These embryos were transferred to a Durawipe (Johnson and Johnson
Advanced Materials Co., New Brunswick, NJ, USA) filter funnel, rinsed with
artificial seawater and resuspended in fresh 35
artificial seawater
for 1530 min prior to transferring to cell culture plates for hatching
tests. Embryos were allowed to develop in the dark for 6669 h before
counting. This incubation period was sufficient for completion of hatching, as
no further development was observed in individuals that did not reach the
naupliar stage after 66 h. It is relevant to note that recovery of pHi after
removal of bafilomycin was not tested, as it is extremely difficult to remove
bafilomycin from A. franciscana embryos. For example, the depressed
hatching rates caused by exposure to 4 µmol l1
bafilomycin for 24 h at 0°C (10% hatch) are only slightly reversed by 5.5
days of washing in inhibitor-free medium at 0°C (21% hatch). Given the low
KD (108 mol l1) and
lipophilic nature of bafilomycin (Drose
and Altendorf, 1997
), this could be the result of strong binding
between inhibitor and enzyme, or a product of slow diffusion out of the
lipid-rich cell mass of these embryos.
Respirometry
Respiration measurements were conducted in a solution of 0.2 mol
l1 sucrose. CCCP was added 2.75 h after embryos were placed
in respiration medium at 25°C, and the final ethanol concentration after
addition was 0.5%. Respiration medium was sterile filtered prior to use, and
oxygen consumption due to any bacterial contamination was limited by the
inclusion of 50 µg ml1 gentamycin sulfate. Gentamycin
sulfate has no noticeable effect on survivorship of A. franciscana
embryos (data not shown).
Dechorionated embryos were blotted dry, and 23 mg(approximately
230350 embryos) were weighed directly in a 1 ml Eppendorf tube. Embryos
were resuspended in respiration medium within 46 min of blotting dry
and immediately transferred to a water-jacketed respiration chamber (model
RC350; Strathkelvin Instruments, Glasgow, Scotland, UK) that was maintained at
25°C. Oxygen tension was measured in 1.5 ml of medium using a
polarographic oxygen electrode (Strathkelvin model 1302) under room lighting,
and data were collected with DataCan V acquisition software from Sable Systems
(Las Vegas, NV, USA). Raw data were analyzed with DatGraf software from
Cyclobios (Innsbruck, Austria). Oxygen consumption by the electrodes, back
diffusion into the system, and electrode time constants were corrected for as
previously described (Kwast et al.,
1995). Oxygen tension was recorded continuously for 6.5 h with
gentle stirring, and the electrode was raised at regular predetermined
intervals to introduce air for gas exchange. In this way, CO2
accumulation was limited, and oxygen tension was prevented from dropping below
65% air saturation. It is important to note that, while we observed no lysis
or homogenization of embryos during respirometry experiments, continuous
stirring does appear to increase permeability of the cuticle and decrease
subsequent hatching success in controls. Thus, respiration effects observed
with stirring cannot be directly compared with 31P-NMR, which has
no detrimental effect on hatching rates for control treatments.
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Results |
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Aerobic superfusion of encysted embryos produced a single Pi resonance during the first 1.5 h of development, after which a second resonance appeared slightly upfield (to the right) of the first (Fig. 3A,B). The pH indicated by both of these chemical shifts was relatively stable in control embryos until exposure to anoxia (Fig. 3A), upon which they merged and moved progressively upfield in accordance with the predicted intracellular acidification. Relative to the control, exposure to 4 µmol l1 bafilomycin beginning at 1.5 h of development had no noticeable effect on steady-state pHi, acidification induced by anoxia or alkalinization during aerobic recovery (Fig. 3A). By contrast, when the embryos were incubated with bafilomycin for 24 h at 0°C (metabolism and development blocked by cold) prior to 31P-NMR, steady-state aerobic pHi was noticeably acidified, and realkalinization of pHi after anoxia was severely blunted (Fig. 4A). After 20 min of aerobic incubation, a single broad Pi resonance indicated a slight acidification in embryos pretreated for 24 h with bafilomycin (Fig. 4A,B). Recovery from this acidification appears to be asynchronous, given that the breadth of the Pi peak lessens during aerobic incubation (Fig. 4B). Importantly, the three phosphate resonances produced by di- and trinucleotide phosphates (NDP and NTP; primarily ATP, ADP, GTP and GDP) appear to be unaffected by bafilomycin treatment (compare Fig. 4B to Fig. 2A), which indicates that the embryos are indeed viable and metabolically active after the 24 htreatment with bafilomycin.
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The disparity in response observed between acute and prolonged treatments with bafilomycin suggests a time dependency for loading effective quantities of the inhibitor. This possibility is supported by the observation that pre-incubation of dechorionated embryos with 1 µmol l1 bafilomycin for 24 h on ice did not impair recovery from anoxia (data not shown), while 4 µmol l1 bafilomycin clearly did. Movement of the inhibitor across the embryonic cuticle may be too slow to effect a change in pH status during acute treatment.
In addition to macrolide antibiotics, dechorionated embryos of A. franciscana are also permeable to the protonophore CCCP. Incubation with 50 µmol l1 CCCP at 25°C produced a rapid rise in oxygen consumption by whole embryos (Fig. 5), as would be predicted if the protonophore reached the mitochondrial inner membrane and uncoupled oxidative phosphorylation. This initial increase was followed by prolonged decrease in oxygen consumption consistent with increasing mortality, which is expected to accompany complete uncoupling oxidative phosphorylation (Fig. 5). Pre-incubation with 20 µmol l1 CCCP for 30 h at 0°C (metabolism and development blocked by cold) has a large effect on the metabolic status and pHi of dechorionated embryos when rewarmed to 25°C. In these CCCP-treated embryos, the predominant Pi resonance, even though quite broad, indicates an acidified pHi of 6.77 after a brief 20 min of aerobic incubation (Fig. 6A). Also, as expected from exposure to an uncoupler of oxidative phosphorylation, NTP and NDP resonance peaks are greatly reduced relative to control embryos (Fig. 6A versus Fig. 2A). The broadness of the main Pi resonance, and presence of a small additional alkaline peak, may indicate differential permeability to CCCP among embryos. Upon exposure to anoxia, very little shift was observed for the main (acidic) Pi resonance (Fig. 6B), although the broadness of the peak decreased. Concurrently, the remaining NTP and NDP signals disappeared. Based on the nature of this main Pi resonance, a key point to be made is that exposure to anoxia induced little additional acidification beyond that already caused by CCCP.
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Discussion |
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The 31P-NMR data presented here suggest that the sequestration
of protons in compartment(s) with low Pi content is the major
contributor to intracellular alkalinization during aerobic recovery from
anoxia. Following 1 h of anoxia in the presence of 4 µmol
l1 bafilomycin, NTP chemical shifts reappear less than 30
min after the restoration of oxygenated conditions. This is consistent with
the restoration of oxidative phosphorylation. During the same period, however,
only a 0.3 unit alkalinization of pHi (6.66.9) is observed in embryos
sufficiently loaded with bafilomycin (Fig.
4). Using these data, and assuming that buffering capacity is
relatively constant between pH 6.7 and 7.7, one can estimate that oxidative
phosphorylation and the resynthesis of ATP combined could only account for
30% of the protons required for the full recovery of pHi after 1 h of
anoxia. This value is at the lower end of the range estimated by Kwast et al.
(1995
). Their calculations
suggest that oxidative phosphorylation, under acidic conditions known to
inhibit carbohydrate metabolism in A. franciscana embryos, could
account for 1345% of the proton consumption required to shift pHi from
the anoxic value of 6.7 to the aerobic value of 7.7, while the resynthesis of
NTP could contribute another 1327% over the same pH range. Our data
contribute to this work by demonstrating that the V-ATPase is active at a pH
of
6.9 and that proton pumping by this enzyme is responsible for a large
fraction of the alkalinization occurring during aerobic recovery following 1 h
of anoxia.
Recovery from intracellular acidification in A. franciscana
embryos is more likely to involve sequestration of protons in intracellular
compartments than secretion of protons across the plasma membrane. While the
V-ATPase has been observed to function in proton secretion in the gas gland
cells of eel swimbladder when pHi is low
(Sotz et al., 2002), this
recovery mechanism would be less effective in A. franciscana embryos
because the extracellular compartment is spatially constrained by a cuticle
and likely to have a buffering capacity equal to or less than the cytosol.
Thus, although it is possible that protons are extruded to the extracellular
space during recovery from the intracellular acidification induced by anoxia,
it seems more probable that alkalinization of pHi would involve the
sequestration of protons within intracellular compartments. These compartments
would require a Pi content low enough relative to the cytosol to
make their visualization with 31P-NMR difficult. In this way, the
protons used to acidify the main intracellular compartment under anoxia would
effectively be masked during recovery and aerobic development. As mentioned
earlier, compartments involved in this event could include lysosomes,
transport vesicles, Golgi apparatus and yolk platelets. It seems unlikely that
such organelles would `hyper acidify' at a time when metabolic processes are
being restarted, because their functional capacity could be adversely
affected. It is more probable that they serve as proton reservoirs that are
emptied upon exposure to anoxia and refilled during aerobic recovery. This
would suggest that dissipation of proton gradients under anoxia contributes to
the observed intracellular acidification.
While incubation with bafilomycin did produce a slight acidification of the
intracellular space under aerobic conditions, the transient nature of this
acidification suggests that V-ATPase inhibition alone is not enough to induce
a rapid dissipation of proton gradients. One might speculate that inactivation
of proton pumping would be coordinated with the opening/activation of
dissipative paths if proton gradients collapse under anoxia. Opening of
dissipative channels might be triggered directly by oxygen removal; mechanisms
of oxygen sensing is an exciting area of current research in many organisms
including A. franciscana embryos
(Kwast and Hand, 1996).
Even if the aerobic embryos are able to maintain pHi in the
absence of V-ATPase activity, inhibition of this enzyme is still likely to
affect development given its broad functional repertoire. Such is the case for
Xenopus, in which bafilomycin treatment appears to block both yolk
platelet degradation and cellular differentiation
(Fagotto and Maxfield, 1994).
These results are consistent with our observations, which demonstrate that the
development of brine shrimp embryos is arrested by incubation in low
concentrations of bafilomycin (Covi and
Hand, 2005
), despite an ability to maintain pHi and NTP
status for at least 5 h during exposure to this inhibitor. One potential
explanation would be an inability to initiate the first round of
lysosome-mediated yolk degradation, which peaks at the start of emergence
(Perona et al., 1988
;
Perona and Vallejo, 1989
).
This possibility is supported by the observation that both yolk degradation
and development can be reversibly inhibited in Artemia embryos by
incubation with lysosomotrophic agents
(Perona et al., 1987
).
Importantly, 31P-NMR experiments using CCCP support previous
theoretical calculations, which suggested that the dissipation of proton
concentration gradients will have a large impact on pHi of encysted
embryos. We estimated that the dissipation of proton gradients across the
membranes of Golgi complex, endosomal and exocytotic vesicles, lysosomes and
yolk platelets in A. franciscana embryos could release enough protons
to account for 50% of the acidification occurring during the first hour of
exposure to anoxia (Covi and Hand,
2005). Here, we directly tested the effect of proton gradient
dissipation with the protonophore CCCP. If a global dissipation of proton
gradients contributes to pHi acidification under anoxia, then
exposure of aerobic embryos to CCCP should induce a pHi similar to
that observed in anoxic embryos. This acidification would be produced by both
a net hydrolysis of ATP due to an uncoupling of oxidative
phosphorylation and a dissipation of proton concentration gradients.
In addition, subsequent exposure of CCCP-treated embryos to anoxia should not
cause further acidification of the already acidic pHi produced by
CCCP exposure. These responses are in fact exactly what we observed in whole
embryos incubated with this protonophore
(Fig. 6). Thus, it appears that
global proton gradient dissipation occurs under anoxia, and this event
contributes to the acidification observed in quiescent embryos.
There is a mismatch between the kinetics of acidification under anoxia and
alkalinization upon reoxygenation, which is consistent with the involvement of
proton gradient dissipation in the acidification event. Under anoxia,
pHi declines approximately 0.8 units during the first 22.5 min of
exposure (Busa et al., 1982;
Kwast et al., 1995
). This
rapid drop is followed by a slower phase of acidification during which
pHi decreases another 0.2 units over a period of 30 min (57.5 min
total anoxia exposure). Busa et al.
(1982
) observed that this slow
phase of acidification continues overnight, eventually reaching a pH of 6.3
(
1.4 unit total acidification). Our results support this finding, as we
observed a pH of 6.36 in embryos incubated for 16.5 h under anoxia, and 6.25
after 30 h of exposure. However, unlike the acidification occurring under
anoxia, alkalinization of pHi during recovery is remarkably fast.
Kwast et al. (1995
)
demonstrated that pHi is completely restored within the first 22.5
min of reoxygenation following 2 h of anoxia. Our work extends this finding by
demonstrating a 1.35 unit alkalinization during only 25 min of aerobic
recovery following 16.5 h of anoxia (Fig.
7). One potential explanation for the disparity in kinetics
between the two transitions could be that proton gradient dissipation under
anoxia is slow relative to the resequestration of protons by active transport
during recovery.
In conclusion, the data presented here demonstrate that the dissipation of
proton gradients under anoxia helps to produce an intracellular acidification
that is critical to the induction of quiescence in encysted embryos of the
brine shrimp, Artemia franciscana. Taking into account changes in the
metabolic state of the embryo, we constructed a schematic model of the role
played by proton chemical gradients during the induction of, and recovery
from, anoxia-induced quiescence (Fig.
8). In this model, exposure to anoxia induces a drop in cellular
ATP as oxidative phosphorylation is arrested. Within the first 5 min of
exposure to anoxia, this leads to a cessation of proton transport by pumps
such as the V-ATPase and an activation of proton dissipative paths. The
combined result is a 1 unit drop in intracellular pH over approximately 1 h.
The return of oxidative phosphorylation upon reoxygenation of the embryos
causes ATP levels to rise, which in turn activates the V-ATPase. Subsequent
proton pumping into the intracellular compartment causes further
alkalinization of pHi, which in turn helps to facilitate the
resumption of development and metabolic processes inhibited by low pH.
Importantly, this novel mechanism for regulated acidification/alkalinization
of the intracellular space may represent a critical adaptive trait conferring
extreme anoxia tolerance to this species. Such an adaptive trait could answer
the question of what sets the anoxia tolerance of Artemia embryos
apart from that of animals such as goldfish and turtles, which was recently
posed by Guppy and Withers
(1999).
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Acknowledgments |
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