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Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, U.K.
1Correspondence: Biochemistry Department, University of Western Australia, Nedlands, W.A. 6907, Australia. E-mail: mguppy{at}cyllene.uwa.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: oxygen consumption • metabolic hysteresis • pH • oxygen conformance • estivation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. It
occurs in virtually all major animal phyla in response to environmental
stresses such as temperature, desiccation, anoxia, hypersalinity, and
food deprivation. The extent of metabolic depression can vary
remarkably (2)
, from a minor lowering to ~80% of
control (in hibernating mammals when the temperature effect is
subtracted) to a more common 5–20% of control (estivating frogs and
snails), to extreme depression to 1% or less of control (anoxic
hydrated brine shrimp), to complete absence of measurable metabolism
(anhydrobiotic brine shrimp). There are five general characteristics of
cells that have depressed metabolic rates (2)
: decreased
pH (3)
; the presence of latent mRNA (4)
; an
altered phosphorylation state of many proteins (5)
; the
maintenance of one energy-utilizing process, ion pumping
(6)
; and the down-regulation of another, protein synthesis
(7)
. However, despite some progress associated with the
above themes, the fundamental phenomenon of metabolic depression
remains biochemically obscure, and to date no unequivocal molecular
mechanism or process associated with the control of metabolic
depression has been delineated.
In animals such as land snails and desert frogs, which estivate in dry
conditions, metabolic depression occurs in anticipation of
physiological stress; i.e., the animals strongly reduce their metabolic
rate before significant desiccation can occur. For example, when food
and water are withdrawn, without changes in atmospheric oxygen or
ambient temperature, the land snail (Helix aspersa)
depresses its metabolic rate to 16% of the control pre-estivation
value (8)
. Concomitantly, the pH of the hemolymph
decreases from 7.8 to 7.3 and its pO2 decreases
from 64 to 44 torr (8)
. Unraveling the mechanisms involved
in metabolic depression should be easier in these animals than with
other model systems (e.g., hibernation, anhydrobiosis, osmobiosis)
because there are no confounding effects of changes in the water
content of the animal, or in ambient temperature or
pO2.
Tissues isolated from these estivating animals, such as mantle from
snails (8)
and liver slices from frogs (9)
,
show a stable intrinsic metabolic depression and offer preparations
that are much more suitable than whole organisms for characterizing the
cellular and molecular processes involved. These preparations have
already been used to quantify the contributions of various effectors to
metabolic depression in snails (8)
and the role of protein
synthesis in metabolic depression in frogs (9)
.
However, such tissue preparations still present some problems, such as
the viability of cells on the borders of a slice and the access of
substrates and oxygen to the cells in the middle of an unperfused
tissue. Isolated cell preparations overcome these problems and provide
a more suitable system for biochemical analysis because, unlike an
isolated tissue, a cell population can be repeatedly sampled under
different conditions. For these reasons, investigations into the
biochemistry of metabolic depression have followed in the footsteps of
mainstream biochemistry, and isolated cell preparations have been
developed for turtles and goldfish, animals that depress metabolic rate
in response to anoxia (10
, 11)
. These preparations have
made it possible to ask sophisticated questions about topics such as
the sensitivities of various energy-consuming pathways to
pO2 and the identity of oxygen-sensing proteins
in metabolically depressing systems. However, until now, no such
preparation existed for estivating animals. We describe here an
isolated cell preparation from the hepatopancreas of the snail H.
aspersa in which we have measured the effect of
pO2, pH, and the estivating state on metabolic
rate.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
Garden snails (H. aspersa), fresh mass 8–10 g, were
collected locally in Cambridge and washed and given water, lettuce,
carrot mix, and cuttlefish bone three times per week. To prepare carrot
mix, 300 g carrots were homogenized in 300 ml water containing 85
mg CaCO3, 5 g bran, and 5 g milk
powder, then mixed with 300 ml of 1.67% (w/v) agar and frozen until
use. Snails were kept in glass tanks at 25°C, under 8 W fluorescent
light on a 14L:10D light:dark cycle starting at 9:00 h. After 2 wk,
some snails were removed and kept for 17 to 71 days in similar
conditions without food or water; control snails were maintained as
above. Full estivation is achieved within 11 days and can be sustained
for 6 months (8)
. In this paper we distinguish two
metabolic ‘states’ of the snails: either maintained awake and active
as above or estivating and inactive for at least 17 days.
Preparation of cells
The isolation of hepatopancreas cells was based on previous
methods for mammalian and mollusk tissues (12
13
14
15
16
17)
. All
steps were at 20–25°C. The hepatopancreas (wet mass 250 mg) was
taken from four control or four estivating snails, and the surrounding
membrane and any adhering gut were carefully removed. Tissue from two
snails per vial was dissociated by stirring for 1 h in 5 ml of
dissociation buffer [70 mM sodium gluconate, 5 mM galactose, 2 mM
trehalose, 10 mM HEPES, 5 mM KCl, 5 mM
NaH2PO4, 2 mM
MgCl2, 1 mM CaCl2, 5 mM
glucose, 1 mM acetate (the latter two for fuel), 20 µg gentamicin/ml,
and 0.4 mg collagenase/ml, pH 7.5]. Every 15 min stirring was stopped,
the supernatant was removed and pooled, and fresh buffer was added.
Stirring conditions [27 mm diameter glass vial, Teflon-covered
magnetic stirrer prism (13 mmx6 mm, 5 mm high) at 8 Hz] were
optimized for cell yield and quality. Pooled supernatants were
centrifuged for 5 min at 78 g (Beckman JA20); the pellets
were resuspended in 10 ml of standing buffer (dissociation buffer
without collagenase, but with 15 mM NaCl and 10 mg defatted bovine
serum albumin/ml) and left to settle for 1 h. The supernatant (9
ml) was removed, and the loosely sedimented cells were mixed with 9 ml
standing buffer, then layered over two preformed Percoll gradients (3
ml Percoll plus 2 ml 210 mM NaCl centrifuged at 30,000 g for
15 min), and centrifuged for 5 min at 300 g in a swing-out
head (MSE bench centrifuge). The cell layer was removed, mixed with 15
ml incubation buffer (10 mM HEPES, 90 mM NaCl, 5 mM KCl, 5 mM
NaH2PO4, 2 mM
MgCl2, 1 mM CaCl2, 5 mM
glucose, 1 mM acetate, 10 mg bovine serum albumin/ml, and 20 µg
gentamicin/ml, pH 7.8 or 7.3), centrifuged twice at 33 g for
5 min, and resuspended in incubation buffer at the appropriate pH at
106cells/ml. Yield was ~10 mg wet mass of cells
per snail.
Measurement of oxygen consumption
Cells were diluted with the appropriate incubation medium to
0.5 x 106 cells/ml in 0.5 ml, and
pO2 was measured using a Clark oxygen electrode
at 25°C. Initial oxygen concentration at air saturation was assumed
to be 479 nmol O/ml (18)
. Diffusion of oxygen into the
electrode was minimal. Oxygen consumption rates (nmoles
O/min/106 cells) were measured at least twice on
each preparation in each condition, then averaged to give
n=1.
Viability, cell types, and bacterial contamination
Viability of the cells in 0.4% trypan blue averaged 93%. The
cell count decreased by less than 10% over 3 h with no change in
viability. We arbitrarily divided the cells observed by light
microscopy using 0.1% neutral red into three types (Fig. 1
). Forty-five percent of the cells were small (mean diameter, 13 µm);
8% were vacuolated (32 µm) with lobed vacuoles in clear cytoplasm,
and 47% were granular (25 µm) with many neutral red-staining
granules. These cells may be smaller digestive, excretory, and larger
digestive plus calcium cells, respectively (19
, 20)
. The
preparation was optimized to remove contaminating small vesicles and
granules; these were rare in the final cell suspension, and fractions
enriched in them did not respire. Bacterial contamination was small:
respiration of the filtrate after passing the preparation through a 5
µm pore nitrocellulose filter was less than 10% of the rate of the
cell preparation at 80% air saturation. None of these parameters was
affected by the state of the animals from which the cells were derived,
or the pH of the incubations. Changes in the abundance of different
cell types could cause differences between respiration rates of cell
preparations isolated from control or estivating snails. However,
changes in the ratio of cell types in situ are small before
3 months of estivation (19
, 20)
, and there were no
significant differences in composition of the control and estivating
cell preparations used here.
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Statistics
The respiration rates in Fig. 2
were compared by ANOVA using a polynomial contrast (SPSSv8.0). The
respiration rates in Fig. 3
were compared using a 3-factor ANOVA (SPSSv8.0), the three factors
being state (awake/estivating), pH (7.8/7.3), and oxygen tension (64
torr/44 torr).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The cells are
stable for several hours, and sufficient cells can be isolated from
four control or four estivating snails for several determinations of
oxygen consumption or other biochemical parameters. The cell
preparation can easily be scaled-up if greater yields are needed. The
population is heterogeneous, with cells of at least three different
types identifiable by size or appearance. However, the composition of
the preparations is reproducible and the same in preparations from
control and estivating animals. It should be possible to separate the
different cell types in the preparation by conventional methods, but we
have not done so.
Oxygen conformance
Cell respiration depended strongly on oxygen concentration,
regardless of the state of the snail from which the cells were derived
or the pH of the incubation (Fig. 2A
, 2B
). The
oxygen dependence of respiration was the same for both states and both
pH values: when the curves were expressed as percent of respiration
rate at 142 torr for that condition, they were indistinguishable (not
shown). The decrease in respiration rate from 142 torr to 9 torr was
between 76% and 83% for all preparations. Coupled or uncoupled
hepatopancreas mitochondria from H. aspersa did not
oxygen-conform (T. Bishop and M. D. Brand, unpublished
observations).
Metabolic depression
Cells from estivating snails respired more slowly than cells from
control awake animals under all conditions tested (Fig. 2A
, 2B
). Estivating cells consumed oxygen at 47% of the rate of
controls at pH 7.8 (Fig. 2A
) and 67% of the rate of
controls at pH 7.3 (Fig. 2B
). This persistent metabolic
depression was apparent at all oxygen concentrations (Fig. 2)
and was
stable for several hours after preparation. The intrinsic depression of
hepatopancreas cell respiration demonstrated here was maximal by 16
days and stable for at least 3 months. It took up to 7 days to reverse
fully on reawakening, even though the snails moved and fed within an
hour of receiving water and food.
Effect of pH and osmolarity
When the pH of the resuspension and incubation medium was
decreased from 7.8 (the hemolymph pH in control snails) to 7.3 (the
hemolymph pH in estivating snails) (8)
, there was a
significant effect on respiration of cells from control snails but none
on cells from estivators (Fig. 2A
, 2B
). There was
no significant effect of varying the osmolarity of the incubation
medium from 100 to 300 mOsm using NaCl (not shown).
Effect of pO2, pH, and snail state under physiological
conditions
Figure 3
shows a section of cell respiration rates from Fig. 2
under combinations of physiologically relevant conditions: varying from
a pO2 of 64 torr and pH of 7.8 for cells from
control animals to a pO2 of 44 torr and pH of 7.3
for cells from estivating animals (8)
. There were
significant effects of pO2, pH, and state on cell
respiration rate, resulting in a 70% depression of metabolic rate from
control cells of awake animals at their physiological
pO2 and pH (point A) to metabolically depressed
cells of estivating animals at their physiological
pO2 and pH (point D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 10
, 21)
. The snail
hepatocyte system is easy to prepare and convenient to use. It yields
relatively large amounts of stable cells and should provide a valuable
model system for elucidation of the molecular and cellular mechanisms
by which organisms turn down metabolism under normal physiological
stress. We describe below the basic physiological properties of this
in vitro system that make it suitable for biochemical study
of metabolic depression. We anticipate that further investigation of
this model system will allow previous observations in this (5
, 8
, 22)
and other groups of organisms (1
2
3)
to be more
easily integrated into a complete and coherent overview of the
mechanisms of metabolic depression.
As a land snail enters estivation there are major changes in
pO2, pH, and the state of the animal. The
sensitivity of metabolic rate to changes in pO2
shown by our preparation, characteristically termed oxygen conformance,
has been demonstrated previously for a variety of mammalian cells
(e.g., ref 21
) in intact H. aspersa and in a
mantle preparation of H. aspersa (8)
. The
effect of pO2 on respiration rate is consistent
for snail hepatopancreas cells under all combinations of pH and state;
respiration rate is decreased to ~0.75 of normal by estivational
hypoxia (Table 1
). This phenomenon may involve oxygen sensing, which has been
demonstrated in a wide variety of organisms, including those that
depress metabolism (23
24
25)
. Hand (25)
invokes a mitochondrial oxygen sensor in the brine shrimp, whereas our
data suggest an extra mitochondrial oxygen sensor because neither
coupled nor uncoupled hepatopancreas mitochondria were oxygen
conformers.
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The effect of pH in our preparation is typical of the role of pH in
metabolic depression. The effect of pH has been demonstrated in the
whole animal, in isolated tissues, and in isolated mitochondria
(1
, 2)
. For the snail hepatopancreas cells a physiological
change in pH has an effect on respiration, but the effect is larger for
awake versus estivating snails (Table 1)
. The mechanism by which pH
effects metabolic depression is still to be delineated, but
Reipschlager and Portner (26)
suggest that pH changes may
work through the efficiencies of H+ and
Na+ pumps, and Kwast and Hand (27)
show that the rate of protein synthesis is sensitive to pH in the
mitochondria of brine shrimp.
An intrinsic metabolic depression, or a stable effect of state on
metabolic rate, has been demonstrated previously in liver slices from
an estivating frog, in a mantle preparation from H. aspersa,
and in cell-free preparations from brine shrimp (8
, 9
, 28)
. For snail hepatopancreas cells there is a large intrinsic
metabolic depression, but the effect of state is greater at a pH that
reflects the awake condition; i.e., pH 7.8 (Table 1)
. Again, the
processes involved in this effect have not been identified but could be
one of many such as protein synthesis and the organization of membrane
composition.
The snail cell preparation described here is sensitive to all of these
changes (pO2, pH, and physiological state); but
how well do these in vitro effects represent the more
complex situation in vivo? We have addressed this question
in Fig. 3
. Hepatopancreas cells in control animals would be at point A.
As snails enter estivation, the cells immediately decrease their
metabolic rate in response to a decrease in hemolymph oxygen tension
(from point A to b) and then in response to changes in pH (b to c),
giving a decrease (A to c) in oxygen consumption of control cells to
45% of the value under awake conditions of pH and
pO2. This fits well with studies demonstrating
that whole animal respiration in a related snail decreases 50% within
1 h of imposed hypercapnia and hypoxia (29)
.
Achievement of full estivation over 16 days causes a further intrinsic
depression (c to D), to 65% of the rate of awake cells at estivating
values of oxygen and pH (c). The combined extrinsic effects of oxygen
tension and pH, and intrinsic effects of estivation (A to D), give an
overall cellular metabolic depression to 30% of the original rate,
approaching the response of the whole animal (depressed to 16% of
control) (8)
and surpassing the response of isolated
mantle (depressed to 52% of control) (8)
.
How might these separate influences interact in vivo, and
might their order of occurrence during entry into estivation, or
arousal from estivation, influence their relative importance? Analysis
of the data in Fig. 3
by 3-way ANOVA indicates that
pO2, pH, and physiological state all have
significant effects on respiration rate. The effect of
pO2 is independent of the other factors, whereas
the effects of pH and physiological state have a significant
interaction. This can be clearly seen from Table 1
, which shows that
the effect of pO2 on respiration rate is similar,
regardless of pH or state, whereas the effects of pH and state are
interdependent. This suggests that the effect of
pO2 on respiration rate is fundamentally
different from the effects of pH and state; i.e., there are likely to
be different mechanisms for the effects of hypoxia and pH/state on
respiration rate.
The independence of pO2 effects and interaction
of pH and state have implications for our interpretation of their
relative roles in entry into estivation and arousal from estivation.
Consider the scenario that entry into estivation involves a rapid
decrease in pulmonary ventilation, which induces a respiratory hypoxia
and hypercapnia/acidosis, followed by a longer term metabolic acidosis
(30)
and an even longer term change in intrinsic
respiration. [Full metabolic depression by estivation requires over 14
days (see Results, Metabolic depression).] The fractions in Fig. 3
show the successive contributions of hypoxia, acidosis, and change in
state for this sequence. The relative contribution of hypoxia to
metabolic depression is independent of the order; i.e., its relative
effect is the same regardless of whether it occurs before or after
acidosis and state changes (see Table 1
and Fig. 3
). In contrast, the
effect of pH is greater than the effect of change in state, if it
occurs before the change in state. For arousal from estivation, the
order of change might be the same. The increase in
pO2 has the same relative effect regardless of
whether it occurs first or last. The effect of pH (if it occurs before
change in state) has a relatively small effect, whereas change in state
now has a relatively larger effect. Regardless of the actual sequence
of these changes, or whether they occur more or less simultaneously, it
appears that hypoxia is having an effect on metabolic depression in a
fundamentally different way (noninteracting) than acidosis/intrinsic
effects. One interpretation of these data concerning the interactive
effect of pH and state could be as follows. These two variables act
through an unknown switch mechanism called MD. MD can be in two forms,
that which causes depression (MDd) or that which causes arousal (MDa).
The change from MDa to MDd can be triggered by a change in pH or state,
but the MDd form cannot be changed back to the MDa form unless both pH
and state change to the awake value or conformation.
The interpretation of our data for isolated snail hepatopancreas cells is tentative at present, but our study represents real progress in the research of mechanisms for metabolic depression. First, we present a model cellular system for the study of metabolic depression, and our data unequivocally demonstrate, at the cellular level, a significant role for the intrinsic or stable changes that occur during the estivation/arousal cycle. Second, our study presents the useful observation that pO2 and pH/state work through different mechanisms and presumably affect different energy-utilizing processes. This observation behooves us to ask whether there really are two different mechanisms for metabolic depression, and how an energy-utilizing metabolism is partitioned between that affected by pO2 and that affected by pH/state.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Zoology Department, University of Western Australia, Nedlands, W.A. 6907, Australia.
Received for publication June 24, 1999. Revised for publication November 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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