Volumetric and ionic responses of goldfish hepatocytes to anisotonic exposure and energetic limitation
1 Instituto de Química y Fisicoquímica Biológicas
(Facultad de Farmacia y Bioquímica), Universidad de Buenos Aires,
C1113AAD Buenos Aires, Argentina
2 Laboratorio de Biomembranas (Facultad de Medicina), Universidad de Buenos
Aires, C1121ABG Buenos Aires, Argentina
3 Institut für Zoologie, Abteilung für Ökophysiologie,
Universität Innsbruck, A-6020, Austria
* Author for correspondence (e-mail: pablos{at}qb.ffyb.uba.ar)
Accepted 21 October 2002
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Summary |
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In trout and rat hepatocytes, hyposmotic (180 mosmoll-1) exposure at pH 7.45 caused cell swelling followed by a regulatory volume decrease (RVD), a response reported to be mediated by net efflux of KCl and osmotically obliged water. By contrast, goldfish hepatocytes swelled but showed no RVD under these conditions. Although in goldfish hepatocytes a net (86Rb+)K+ efflux could be activated by N-ethylmaleimide, this flux was not, or only partially, activated by hyposmotic swelling (120-180 mosmoll-1).
Blockage of glycolysis by iodoacetic acid (IAA) did not alter cell volume in goldfish hepatocytes, whereas in the presence of cyanide (CN-), an inhibitor of oxidative phosphorylation, or CN- plus IAA (CN-+IAA), cell volume decreased by 3-7%. Although in goldfish hepatocytes, energetic limitation had no effect on (86Rb+)K+ efflux, (86Rb+)K+ influx decreased by 57-66% in the presence of CN- and CN-+IAA but was not significantly altered by IAA alone. Intracellular K+ loss after 20 min of exposure to CN- and CN-+IAA amounted to only 3% of the total intracellular K+.
Collectively, these observations suggest that goldfish hepatocytes, unlike hepatocytes of anoxia-intolerant species, avoid a decoupling of transmembrane K+ fluxes in response to an osmotic challenge. This may underlie both the inability of swollen cells to undergo RVD but also the capability of anoxic cells to maintain intracellular K+ concentrations that are almost unaltered, thereby prolonging cell survival.
Key words: cell volume, goldfish, Carassius auratus, hepatocyte, trout, Oncorhynchus mykiss, rat, anoxia, K+ flux, water transport
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Introduction |
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In hepatocytes of the goldfish, a facultative anaerobe vertebrate, the
above changes are slow to develop or do not occur at all. Specifically, during
anoxia, goldfish hepatocytes display a set of dynamic metabolic responses that
prolong cell survival, including: (1) reallocating metabolic energy to
essential functions (Krumschnabel and
Wieser, 1994); (2) avoiding the rise of cytosolic Ca2+
by a net extrusion of the cation
(Krumschnabel et al., 1997
)
and (3) secreting protons that, together with a high cytosolic buffer
capacity, allow cytosolic pH to be maintained
(Krumschnabel et al.,
2001a
).
Although the principal mechanisms that allow goldfish hepatocytes to
tolerate metabolic inhibition have thus been characterized, the potential
osmotic disturbance associated with these conditions has not been studied so
far. However, investigation of the osmotic responses is important because, in
hepatocytes from anoxia-intolerant species, impairment of cell volume
regulation associated with severe metabolic inhibition contributes
significantly to the events leading to cell necrosis
(Carini et al., 1999).
Previous studies on volume regulation using mammalian hepatocytes
(Häussinger and Lang,
1991
; Wehner and Tinel,
2000
) and fish hepatocytes (Bianchini et al.,
1988
,
1991
) provided some insight
into how vertebrate hepatocytes respond to an osmotic gradient in the absence
or presence of metabolic inhibitors. Under non-inhibited conditions,
vertebrate hepatocytes challenged by either hyposmotic media or sodium-coupled
amino acid uptake increase their volume, which is followed by a loss of
K+, Cl- and water, resulting in a reduced cell volume
[i.e. a regulatory volume decrease (RVD)].
A mismatch between intracellular and extracellular osmolarity may also
develop during metabolic inhibition. For example, in rat hepatocytes, blockage
of oxidative phosphorylation promotes an increase of intracellular
Na+, causing cell swelling
(Carini et al., 1995).
Furthermore, exposure of hepatocytes from rat
(Anundi and de Groot, 1989
),
trout and goldfish (Krumschnabel et al.,
1996
) to hypoxia and anoxia enhances the glycolytic flux (a
process termed the `Pasteur effect'), thereby increasing the concentration of
glycolytic intermediates. The degradation of glycogen to glucose phosphate and
the anaerobic increase of glycolytic intermediates have been postulated, but
not proven, to increase osmotically active substances in the cytosol, causing
hepatocyte swelling (Wehner et al.,
1992
; Corassanti et al.,
1990
; Häussinger and
Lang, 1991
; Lang et al.,
1998
). At the same time, enhanced anaerobic metabolism is
accompanied by an increased rate of export of organic osmolytes. For example,
as reported for hepatocytes of trout and goldfish, chemical anoxia leads to an
elevated transport of glucose and lactate out of the cell
(Krumschnabel et al., 2001a
),
a response that might counteract the potential increases of intracellular
osmolarity of energetically compromised cells.
In the present study, we examined how osmotic gradients affect cell volume and (86Rb+)K+ transmembrane fluxes of goldfish hepatocytes, with particular focus on the osmotic effects of metabolic inhibition. The major aims of this investigation were to characterize the volumetric responses of these anoxia-tolerant cells under conditions of energetic steady state and energetic limitation and to compare these responses with those of anoxia-intolerant hepatocytes. Furthermore, as goldfish hepatocytes have an exceptional capability of maintaining ion homeostasis, we wanted to elucidate whether K+ flux balance is also preserved in response to an osmotic challenge or whether, under these conditions, K+ homeostasis is temporarily suspended in order to restore cell volume.
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Materials and methods |
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Animals
Goldfish Carassius auratus L. (10-30 g) were obtained commercially
from local dealers in Buenos Aires. They were kept in 2001 tanks at 20°C.
Rainbow trout Oncorhynchus mykiss Walbaum (150-250 g) were obtained
from the Center of Aquaculture from the Universidad del Comahue (Bariloche,
Argentina) and were maintained in 2001 tanks at 15°C. Fish were acclimated
to the above-specified temperatures for at least two weeks before being used.
Male Wistar rats Rattus rattus (200-300 g) were fasted for 12 h
before being used.
Isolation of hepatocytes
Fish were killed by a blow to the head and transection of the spinal cord,
whereas rats were anesthetized with pentobarbital (150 mg kg-1,
intraperitoneal). Hepatocytes of trout and rat were isolated by collagenase
digestion methods, which required perfusion of the portal vein as described
previously (Berry, 1974;
Krumschnabel et al., 1996
).
The small size of goldfish, together with the diffuse distribution of hepatic
tissues in cyprinids, prevented the use of the perfusion techniques for the
isolation of hepatocytes. Therefore, goldfish hepatocytes were isolated by
incubating fragments of liver tissue with a collagenase medium as described
previously (Schwarzbaum et al.,
1992
; Krumschnabel et al.,
1994
).
Alternatively, in a few experiments shown in
Fig. 2, goldfish hepatocytes
were isolated by a similar procedure, except that collagenase medium was
replaced by a collagenase-free medium containing EDTA, as described by Seddon
and Prosser (1999).
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|
Conversion factors in goldfish hepatocytes
The ratio of total protein content to cell number was 0.7±0.1 (mg
protein 10-6 cells-1) (N=13). Cell diameter
assayed by videomicroscopy was 12.77±0.24 µm (147 cells from 10
independent preparations) and the percentage of dry mass was 25±1%
(N=19). The yield of cells was 62.2±9.7x106
cells g fresh mass-1 (N=15). For each independent
experiment, livers from 1-3 fish individuals were pooled.
Incubation protocols
Except where otherwise stated, cells from goldfish, rat and trout were
incubated in media at 20°C (pH 7.45) with the following composition.
Isotonic control media:
Hypotonic media were prepared by mixing different amounts of isotonic media with medium D, which consisted of 10 mmol l-1 Hepes, 5 mmol l-1 KCl. Medium E (osmolarity, 503 mosmol l-1) had the following composition: 10 mmol l-1 Hepes, 135.2 mmol l-1 NaCl, 3.8 mmol l-1, KCl, 1.3 mmol l-1 CaCl2, 1.2 mmol l-1 KH2PO4, 1.2 mmol l-1 MgSO4, 10 mmol l-1 NaHCO3, 200 mmol l-1 sucrose.
Other test media for goldfish hepatocytes had the following composition:
The osmolarity of all media was measured with a vapor pressure osmometer (5100 B; Wescor Inc., Logan, UT, USA).
Experiments on metabolic inhibition were performed by incubating hepatocytes in isotonic media A (goldfish) or C (rat) in the absence of inhibitors (control condition) or in the presence of 2 mmol l-1 sodium cyanide (CN-), 0.5 mmol l-1 iodoacetic acid (IAA) or both (CN-+IAA). In order to evaluate the nature of the K+ channels potentially activated under chemical anoxia, goldfish hepatocytes were incubated in the presence of either 5 mmol l-1 BaCl2, 1 mmol l-1 tetraethylammonium (both blockers of voltage-sensitive K+ channels) or 1 mmol l-1 quinine (a blocker of Ca2+-sensitive K+ channels). Blockers were added 4 min before CN- addition to block the specific K+ channels before inhibition of mitochondrial activity.
Rates of (86Rb+)K+ influx and
efflux
Influx and efflux of K+ were estimated using the radioactive
isotope of Rb+ (86Rb+), which acts as a
K+ analog (Krumschnabel et al.,
1996). Pulse experiments were used to determine the initial rate
of ion uptake at various times. Cells were incubated in iso- (medium A) and
hypotonic (180 mosmol l-1) media.
(86Rb+)K+ influx
Cells (12x106 cells ml-1) were diluted 1:2 in
medium A (isotonic condition) or hypotonic medium (180 mosmol l-1),
as required for each experiment. At 10 min, 20 min and 40 min, 3.5 ml of the
cell suspension was transferred to 7 ml conical tubes containing
4.67x104 Bq mg-1 of 86Rb+
(the dilution was negligible) and was gently agitated for 60 s. After 1 min, 4
min and 7 min of incubation, 500 µl duplicate samples of the cell
suspension were transferred to 1.5 ml Eppendorf reaction tubes containing 500
µl of ice-cold 100 mmol l-1 MgCl2 medium to stop the
uptake. Cells were centrifuged for 4 s at 6700 g; the
supernatant was sucked out of the reaction tube, and external medium adhering
to the cell pellet and the walls of the reaction tube was then diluted twice
by carefully layering 1 ml of ice-cold MgCl2 medium on top of the
cells and removing it by aspiration. This procedure ensured a small background
signal. The final cell pellet was vortexed with 1 ml of scintillation
cocktail, and radioactivity was assessed by scintillation counting. 5 µl
duplicate samples were used to determine specific radioactivity. Results were
expressed as nmol 86Rb+ 10-6
cells-1 min-1.
(86Rb+)K+ efflux
Hepatocytes were incubated in the presence of 6.47x104
86Bq Rb+ ml-1 for 2.5 h. Subsequently, cells
were diluted 1:2 in medium A or in hyposmotic medium, both containing
6.47x104 86Bq Rb+ ml-1.
After 10 min, 20 min and 40 min of incubation, 1 ml of the cell suspension was
transferred to 1.5 ml Eppendorf reaction tubes and centrifuged for 4 s at 6700
g. The supernatant was sucked out of the reaction tube, and
external medium adhering to the cell pellet and the walls of the reaction tube
was then diluted twice by carefully layering 1 ml of either iso- or hypotonic
medium on top of the cells and removing it by aspiration.
Following this, the cell pellet was resuspended in 1 ml of unlabeled medium A or hypotonic medium. From this cell suspension, 200 µl duplicate samples were withdrawn after 4 min and 8 min, and the pellets were separated from medium by rapid centrifugation as described for 86Rb+ influx. An aliquot of the supernatant was removed, and radioactivity was determined by scintillation counting. Results were expressed as c.p.m. 10-6 cells-1 min-1.
Measurement of intracellular Na+
200 µl aliquots of cell suspension (40x106 cells
ml-1) were layered on the top of 1 ml of phtalic/phtalate solution
(40% phtalic acid plus 60% dibutyl-phthalate) and spun for 1 min at 6700
g. After centrifugation, the phtalic/phtalate solution was
removed by aspiration and the cell pellet diluted in 500 µl distilled water
and sonicated for 30 min. In some experiments, aliquots of cell suspensions
were preincubated in 100 mmol l-1 MgCl2 (an
Na+-free medium) for 30 min. Cells were then spun for 4 s at 6700
g and resuspended in 100 mmol l-1 MgCl2
before the onset of the experiment. The level of intracellular Na+
was measured by flame photometry using an EEL Flame photometer (Evans
Electroselenium Ltd, Halstead, UK).
Assessment of cell volume
Videomicroscopy
The size of hepatocytes was assessed by quantitative phase-contrast
microscopy. Cells were loaded onto a 500 µl glass chamber (no adhesion
medium was necessary because cells remained attached during measurements) and
allowed to equilibrate for 20 min in a continuously superfused medium A at 0.5
ml min-1. At specific times following the start of the experiment,
different media were superfused into the chamber at the same flow, according
to the treatment.
Hepatocytes were viewed through phase-contrast optics (total magnification, 300x) on an inverted microscope (Olympus IMT-2) equipped with a 20x, N.A. 0.40 objective. Images were captured by means of a CCD camera (EDC-1000, Electrim Corp., Princeton, USA) operating at fixed gain. Images were recorded on a computer through data translation hardware boards and were processed by means of the Optimet program (Bioscan, Inc., Edmonds, USA). Cell volumes were estimated from diameters, assuming that the cells had spherical shape and that their volume changed by the same magnitude in all radial directions. In all cases, the diameters of the same cells were measured throughout the experiment and cell volumes were only computed for cells that remained alive during the whole experiment.
Results are presented as means ± S.E.M. of four independent experiments, using 13-15 cells per experiment. Volume data were expressed as total volume (V; in cm3) or as relative cell volume (Vr), where Vr=Vt/V0; Vt is the value of V at time t, and V0 is the value of V at time 0. Under the experimental conditions applied in this study, this technique allowed changes in V of within 8% to be detected.
Epifluorescence microscopy
Hepatocytes were plated on 25 mm-diameter glass coverslips (Fischer
Scientific, Pittsburg, PA, USA) that had been previously coated with 0.1% w/v
poly-L-lysine. Each coverslip with attached cells was mounted in a chamber
filled with isotonic medium and placed on the stage of a Nikon TE-200
epifluorescence inverted microscope. Hepatocytes were then loaded with 2
µmol l-1 of calcein-AM. Dye loading was monitored
fluorometrically by sampling the signal of single cells every 180 s until
fluorescence of the cells reached 5-10 times the autofluorescence level. The
loading time was 45-60 min. The loading solution was then washed out with
isotonic medium for at least 1 h before starting the experimental data
acquisition. Experimental solutions were superfused at a rate of 2 ml
min-1.
Changes in cell water volume were inferred from readings of the
fluorescence intensity recorded by exciting calcein through a 470 CWL
excitation filter (Nikon Inc., Melville, USA) and were imaged with a 500 nm LP
dichroic mirror (Nikon Inc.) and a 515 LP barrier filter (Nikon Inc.). Changes
in fluorescence intensity due to changes in intracellular fluorophore
concentration were recorded from a small region of dye-loaded cells using a
customized microspectrophotometry system described in detail elsewhere
(Alvarez-Leefmans et al.,
1997). Values of Vr were computed from
monitored changes in relative fluorescence
(Ft/Fo), where Fo
is the fluorescence from a pinhole region of the cell equilibrated with
isotonic medium, and Ft is the fluorescence of the same
region of the cell exposed to an anisotonic medium. This technique allows for
continuous measurements of Vr changes to within 1%
(Alvarez-Leefmans et al.,
1995
). A detailed description of the technique, its validation and
corresponding computations can be found elsewhere
(Alvarez-Leefmans et al., 1995
;
Altamirano et al., 1998
).
Statistics
The effect of the different treatments on 86Rb+
transmembrane fluxes was evaluated by one-way analysis of variance (ANOVA)
followed by a TukeyKramer multiple comparisons test. P0.05
was considered significant. Exponential fits for the calculation of
Vm (maximal V) ± S.E. were calculated by
non-linear regression.
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Results |
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The absence of RVD in hyposmotic medium led us to perform a series of experiments to check the validity of this result as follows.
(1) Goldfish hepatocytes that had been isolated in collagenase-free medium were exposed to 180 mosmol l-1 medium (Fig. 2A), showing a volumetric response similar to that of hepatocytes isolated with collagenase and, importantly, no RVD response.
(2) As a comparison, rat and trout hepatocytes were exposed to iso- and hypotonic media under conditions identical to those used for goldfish hepatocytes. In the cells from both species, exposure to 180 mosmol l-1 media induced an increase of Vr to a maximum, followed by a volume decrease towards initial values (Fig. 2B). An exponential function of the form Vr=Vr0+Ate-nt was fitted to the experimental data, yielding maximal Vr values of 1.34 (rat) and 1.18 (trout) when t=1/n (Vr0 is the value of Vr at time 0, whereas A and n denote constants).
(3) To evaluate the possibility that the occurrence of RVD in goldfish hepatocytes is activated only at a more alkaline extracellular pH, cells were exposed to iso- and hypotonic (180 mosmol l-1) media at pH 7.8. Under this condition, exposure to 180 mosmol l-1 induced an increase of Vr to a maximum at 1.58±0.05, followed by a volume decrease of approximately 13% (Vr=1.38±0.06) of isosmotic values (Fig. 2B).
Volume changes under conditions of energetic limitation
As preliminary experiments indicated that changes in cell volume during
metabolic inhibition may be very small and thus lie within the error of the
videomicroscopy technique, we decided to measure the effect of metabolic
inhibition on Vr by using quantitative epifluorescence
microscopy. Results of these experiments are shown in Figs
3,
4.
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|
In goldfish hepatocytes, addition of IAA had no significant effect on Vr (Fig. 3A). In the presence of CN-, Vr decreased by 3.2±0.51% (t=34 min; P<0.01, N=4), with subsequent recovery towards initial values (Fig. 4A). A similar pattern of shrinking followed by an increase in Vr, and even cell swelling, was observed in the presence of both CN- and IAA, although initial Vr shrinkage was more pronounced (6.6±1.6% compared with controls at 42 min; Fig. 3B; P<0.01, N=4).
Comparative experiments with rat hepatocytes incubated with CN- showed that these cells increased in Vr by 5.1±1.1% over control values without any regulation being detected (Fig. 4B; P<0.01, N=4).
Unidirectional (86Rb+)K+ fluxes in
goldfish hepatocytes
Using 86Rb+ as a K+ analog, we determined
the impact of the different inhibitory treatments on the transmembrane influx
and efflux of K+ in goldfish hepatocytes.
Anisotonic media
Because in many cell types K+ efflux is an early event mediating
the RVD (Häussinger et al.,
1994; Fugelli et al.,
1995
), we tested whether
(86Rb+)K+ transmembrane fluxes decouple
during the first 40 min of incubation under hyposmotic conditions. Results in
Figs 5,
6 show that the magnitude of
(86Rb+)K+ efflux, as well as that of the
influx, remained constant in isosmotic as well as in hyposmotic (i.e. 180
mosmol l-1) media. In separate experiments, swelling of hepatocytes
was induced by exposure to 180 mosmol l-1 medium for 30 min and
then 1 mmol l-1 NEM, an activator of Cl--dependent
K+ transport in fish cells was added
(Bianchini et al., 1988
;
Jensen, 1994
;
Bogdanova and Nikinmaa, 2001
).
In this condition, (86Rb+)K+ efflux increased
approximately 3.9-fold (386±44%, N=4) with respect to control
values (Fig. 7).
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As a slight RVD had been observed in hyposmotic medium at pH 7.8 (Fig. 2B), we performed another series of (86Rb+)K+ flux experiments as described above but with media adjusted to this higher pH value. Again, (86Rb+)K+ efflux and influx remained constant in isosmotic media. In 180 mosmol l-1 medium, however, although (86Rb+)K+ influx remained constant (0.30±0.04 nmol 10-6 cells-1 min-1 in controls versus 0.36±0.03 nmol 10-6 cells-1 min-1 in hypotonically treated cells, N=4), efflux increased significantly to 152±13% (N=4) of the isosmotic control values (Fig. 7).
Metabolic inhibition
Incubation in the presence of IAA did not significantly affect
86Rb+ influx. In the presence of both CN- and
CN-+IAA, however, 86Rb+ influx decreased
acutely, with flux values reaching 47±11% (CN-) and
34±5% (CN-+IAA) of control values
(Fig. 5) after 4 min of
incubation.
Metabolic inhibition by either CN-, IAA or CN-+IAA did not significantly (P>0.05, N=4) affect 86Rb+ efflux (Fig. 6). In addition, (86Rb+)K+ efflux under chemical anoxia was not altered in goldfish hepatocytes preincubated in the presence of either 5 mmol l-1 BaCl2, 1 mmol l-1 tetraethylammonium (both blockers of voltage-sensitive K+ channels) or 1 mmol l-1 quinine (a blocker of Ca2+-sensitive K+ channels) (P>0.75, N=4; data not shown).
Intracellular Na+
Exposure of goldfish hepatocytes to CN- for 40 min produced no
significant change in the level of intracellular Na+
(Fig. 8), whereas in the
presence of 1 mmol l-1 ouabain a significant increase in
Na+ content was noted after this time of incubation
(Fig. 8). Furthermore, in cells
preincubated for 30 min in the presence of 100 mmol l-1
MgCl2 (an isosmotic Na+-free medium), Na+
content was approximately 16% of control values and showed no significant
change over 40 min.
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Discussion |
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In hyperosmotic medium (503 mosmol l-1), goldfish hepatocytes
shrank to a constant volume without showing regulatory volume increase, a
feature also observed in hepatocytes from other vertebrate species
(Corassanti et al., 1990). On
the other hand, in hypotonic media (including isosmotic medium with
L-alanine), cells increased their volume following an exponential time course
(Fig. 1). In hepatocytes, as
well as other cells of most vertebrates, swelling triggers a volume regulatory
response that is largely mediated by an increased plasma membrane conductance
for K+ and Cl-
(Bakker-Grunwald, 1983
;
Wehner et al., 1992
;
Wang et al., 1996
;
Roe et al., 2001
). However,
unlike hepatocytes of trout and rat, experiments using videomicroscopy (Figs
1,
2) and fluorescence microscopy
(not shown) showed that goldfish hepatocytes displayed no (pH 7.45) or only a
slight (pH 7.8) RVD. As hypotonic swelling was not accompanied by an increase
in K+ efflux (Fig.
6), the almost complete absence of RVD might have been the
consequence of transporters of K+ and/or Cl- being
inactivated. Incubation of hypotonically swollen cells in the presence of NEM
showed that (86Rb+)K+ efflux can in fact be
increased almost 4-fold in goldfish cells
(Fig. 7). Thus, although a
mechanism promoting efflux of water appears to be present, it was seemingly
not activated under the conditions applied. Because Jensen
(1994
) showed that in red
cells of carp (a species belonging to the same family as the goldfish)
(86Rb+)K+ efflux is activated as
extracellular pH is increased from 7.6 to 7.8, we tested whether the same is
true for goldfish hepatocytes. Our results showed that activation of both
(86Rb+)K+ efflux as well as of a minor RVD
indeed occurred, but the relative change of both parameters was much lower
than in cells of rat and trout.
Another situation prone to result in a net K+ flux out of the
cells can occur as a consequence of ATP depletion caused by metabolic
inhibition (Wang et al.,
1996). Accordingly, results of the present study showed that, in
goldfish hepatocytes, CN- as well as CN-+IAA lead to a
60-67% reduction in (86Rb+)K+ influx (95-99%
of which is driven by Na+/K+-ATPase activity; see
Krumschnabel et al., 1996
). As
no concurrent reduction of K+ efflux was observed in the present
study, a significant net K+ efflux developed as a consequence of
Na+/K+-ATPase inhibition.
We therefore evaluated how the ensuing K+ flux imbalance would
alter cell volume. Using a fluorometric technique to detect small changes in
cell volume, we verified that rat hepatocytes swelled by approximately 5.1% in
the presence of cyanide. This increase agrees with previous reports showing
swelling in anoxic hepatocytes of anoxia-intolerant species, including rat
(Carini et al., 1999) and
trout (Krumschnabel et al.,
1998
). Moreover, incubation of rat hepatocytes with IAA and
CN- caused Vr to increase by more than 20%
(Gores et al., 1989
).
By contrast, goldfish hepatocytes showed the opposite response, as CN- alone or in combination with IAA led to an acute decrease in Vr by 3% and 7%, respectively (Figs 3, 4), followed by a subsequent volume increase to (CN-) or even above (CN-+IAA) initial values. Thus, in goldfish hepatocytes, changes in cell volume paralleled changes in intracellular K+. However, although (86Rb+)K+ influx decreased to 30-40% of control values in less than 4 min, the magnitude of net K+ efflux was relatively small, as was the magnitude of volume decrease.
In trout hepatocytes, by comparison, a somewhat different situation has
been described. Firstly, during hypotonic swelling and RVD, K+
efflux was 6-7 times higher than the influx (see
Bianchini et al., 1988) (in
goldfish cells at pH 7.8, this ratio was only 1.5), allowing complete volume
recovery in approximately 40 min (Fig.
2B). In addition, during acute chemical anoxia, K+
efflux was more than three times higher than the corresponding influx and
could be blocked 55% by BaCl2, an inhibitor of voltage-sensitive
K+ channels (Krumschnabel et al.,
1996
,
1998
). This suggests that a
significant part of anoxic K+ leakage is due to membrane
depolarization.
In goldfish cells, on the other hand,
(86Rb+)K+ efflux was insensitive to either
BaCl2 or TEA, a result that, together with the lower K+
influx decay, could explain the lower degree of decoupling of K+
transmembrane fluxes. Similar to trout hepatocytes, quinine, a known inhibitor
of Ca2+-sensitive K+ channels, did not prevent the
CN--induced K+ loss. Thus, in contrast to the case of
rat hepatic and hepatoma cells (Wang et
al., 1996; Roe et al.,
2001
), cytosolic calcium is not implicated in the decoupling of
K+ transmembrane fluxes.
In Fig. 9, we used the results on (86Rb+)K+ unidirectional fluxes of this and previous studies to calculate the relative K+ loss under various conditions. It can be seen that, in trout hepatocytes, the relative loss of intracellular K+ after 20 min of incubation with NEM, hyposmotic medium and cyanide amounts to 30%, 20% and 7%, respectively, whereas in the goldfish the same treatments yield 12%, 4% and 2.5% K+ loss.
|
The goldfish cells therefore appear to have the capability to maintain intracellular K+ relatively constant, which might underlie both the inability of hypotonically swollen goldfish hepatocytes to undergo RVD and, at the same time, prevent the swelling of anoxic cells.
What factors other than K+ fluxes can affect cell volume during
metabolic inhibition in goldfish hepatocytes? One could argue that, as
chemical anoxia in goldfish hepatocytes leads to an enhanced production of
lactate immediately followed by lactate export
(Krumschnabel et al., 2001a),
the loss of this organic osmolyte may trigger the observed cell shrinkage.
However, this hypothesis has to be discarded as cell shrinkage is also
observed in the presence of both IAA and CN-, a condition where
lactate production is fully inhibited
(Krumschnabel et al.,
2001b
).
In hepatocytes of anoxia-intolerant species, excessive accumulation of
lactic acid is one of the main factors responsible for cytosolic
acidification. Accordingly, it has been demonstrated that in hepatocytes of
the rat (Carini et al., 1995),
as well as in those of the trout
(Krumschnabel et al., 2001a
),
chemical anoxia leads to impairment of Na+/K+-ATPase
activity and cytosolic acidosis followed by the activation of the
Na+/H+ exchanger. This results in net Na+
influx, cell swelling and progressive loss of cell viability. In line with
this, in rat hepatocytes, cytoprotection was achieved by substituting NaCl
with choline chloride or by preventing sodium accumulation with glycine
(Carini et al., 1999
).
In the anoxic goldfish cells, the situation is different. Firstly, the
magnitude of the anoxia-induced decrease of
Na+/K+-ATPase activity is lower than in trout
hepatocytes, with no increase in intracellular Na+
(Fig. 8). Secondly, anoxic
goldfish hepatocytes do not activate Na+/H+ exchange,
and intracellular pH does not decrease, which may be attributed to a high
buffering capacity together with proton secretion sensitive to SITS
(4-acetamido-4-isothio-cyanostilbene-2,2-disulfonic acid)
(Krumschnabel et al., 2001a).
Thus, in goldfish hepatocytes under metabolic inhibition, increases in cell
volume due to sodium overload are prevented.
Altogether, these findings point to an outstanding capability of goldfish cells to maintain ionic gradients under a variety of conditions that are considered a severe challenge to ion homeostasis. While this is of great advantage during anoxic periods, as it allows the prevention or reduction of the transitory loss of intracellular K+ under conditions of energetic limitation, at the same time it prevents K+ being used as an osmolyte for RVD, thereby limiting the capability to regulate cell volume under certain conditions.
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Acknowledgments |
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References |
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