Metabolic and ionic responses of trout hepatocytes to anisosmotic exposure
1 Institut für Zoologie und Limnologie, Abteilung für
Ökophysiologie, Universität Innsbruck, Technikerstraße 25,
A-6020 Innsbruck, Austria
2 Instituto de Química y Fisicoquímica Biológicas
(Facultad de Farmacia y Bioquímica), Universidad de Buenos
Aires, C1113AAD Buenos Aires, Argentina
* Author for correspondence (e-mail: gerhard.krumschnabel{at}uibk.ac.at)
Accepted 12 March 2003
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Summary |
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Key words: rainbow trout, Oncorhynchus mykiss, hepatocyte, cell volume, oxygen consumption, intracellular free calcium, intracellular pH.
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Introduction |
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Given the dependence of basal metabolic processes on the existence of a
more or less tightly regulated intracellular milieu, changes of cell volume
may also affect cellular metabolism. The latter aspect is best illustrated by
the fact that, in liver cells, the action of several hormones known to affect
both metabolism and cell volume may to a large extent be mimicked by volume
changes evoked by anisosmotic exposure in the absence of the hormones. This
led Häussinger and Lang
(1991) to propose that cell
volume changes associated with the effect of hormones act as a second
messenger in metabolism. Although interactive effects of cell volume and
metabolism have also been documented in other cell types
(Lang et al., 1998
;
Smets et al., 2002
), most of
these studies addressed cells of mammalian origin.
In fish, such studies have almost exclusively been conducted with red
cells, revealing complex interactions between haemoglobin oxygen binding,
acidbase regulation and ion transport
(Nikinmaa, 1992;
Ferguson and Boutilier, 1989
;
Roig et al., 1997
). However,
in these cells, metabolism is largely governed by their role in gas transfer
and it therefore appears likely that a rather different relationship between
metabolism and volume control will prevail in hepatocytes.
Given the role of the liver as a central metabolic organ and the various
differences in liver metabolism between teleosts and mammals that have already
been documented (Walsh and Mommsen,
1992), we considered a study of the impact of anisosmotic
conditions on metabolic and ionic aspects in teleost hepatocytes of great
interest, both in a general physiological context as well as from a
comparative point of view.
Since, over the past decade, hepatocytes from the rainbow trout
Oncorhynchus mykiss have been established as a model system for the
investigation of teleostean cellular physiology
(Canals et al., 1992;
Furimsky et al., 2000
;
Krumschnabel et al., 1998
;
Mommsen et al., 1988
;
Schwarzbaum et al., 1996
;
Walsh, 1986
), including the
study of volume regulatory processes
(Bianchini et al., 1988
;
Fossat et al., 1997
;
Michel et al., 1994
), we chose
to use these cells for our purpose. In the present study, we first examined
how anisosmotic exposure of trout hepatocytes affects their cell volume. We
then investigated the impact of the volume changes associated with these
conditions on both aerobic (oxygen consumption) and anaerobic (lactate
production) metabolism and on the production of glucose, which is considered
an important function of hepatic metabolism with regard to the maintenance of
plasma glucose level.
In addition, we studied the consequences of anisosmotic conditions on [Ca2+]i and on pHi. As already noted above, various ion transporters involved in volume regulation are also of importance for the control of pHi. Hence, in order to gain some insight into the nature of these mechanisms, the impact of anisosmotic conditions on pHi was further investigated both in complete and in ion-substituted media and in the presence of inhibitors of specific ion transporters.
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Materials and methods |
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Preparation of hepatocytes and cell culture
Hepatocytes were isolated from rainbow trout (Oncorhynchus mykiss
Walbaum) acclimated to 15°C as previously described
(Krumschnabel et al., 1996).
Following isolation, hepatocytes were suspended in standard saline (for
composition, see below) and were left to recover for one hour in a shaking
water bath maintained at 19°C, which was also the temperature used in the
experiments. Measurements of metabolic parameters (oxygen consumption, lactate
accumulation and glucose production) were then conducted with these freshly
isolated cells. For the determination of intracellular free calcium and
intracellular pH, cells (1x106 cells ml1)
were suspended in Leibovitz L15 medium (0.95 mmol l1
CaCl2, 5.33 mmol l1 KCl, 0.44 mmol
l1 KH2PO4, 0.46 mmol
l1 MgCl2, 0.40 mmol l1
MgSO4, 137.9 mmol l1 NaCl, 1.07 mmol
l1 Na2HPO4, 4.99 mmol
l1 galactose, 5 mmol l1 sodium pyruvate,
and amino acids and vitamins according to the manufacturer's formulation)
modified by addition of 10 mmol l1 Hepes, 5 mmol
l1 NaHCO3, 50 µg ml1
gentamycin and 100 µg ml1 kanamycin, pH titrated to 7.6.
These cells were then plated on Cell-Tak (2 µg cm2
surface area)-coated glass cover slips and maintained in an incubator
(19°C, 0.5% CO2) overnight. Before use of the hepatocytes in
ion measurements, the cultures were washed several times with fresh standard
saline in order to remove non-adherent cells and debris. Both in freshly
isolated cells and in cell cultures, viability, as determined from Trypan blue
exclusion, was maintained at >95% throughout the experiments described.
Experimental media
The standard isosmotic incubation saline consisted of 10 mmol
l1 Hepes, 136.9 mmol l1 NaCl, 5.4 mmol
l1 KCl, 1 mmol l1 MgSO4, 0.33
mmol l1 NaH2PO4, 0.44 mmol
l1 KH2PO4, 5 mmol l1
NaHCO3, 1.5 mmol l1 CaCl2, 5 mmol
l1 glucose, pH 7.6 at 19°C, and had an osmolarity of 284
mosmol l1. Hyposmotic conditions were created by exposing
cells to a mixture of one volume of standard saline with an equal volume of
the same medium lacking NaCl, yielding an osmolarity of 166 mosmol
l1 and corresponding to 0.58 x isosmolarity. A mixture
of equal volumes of standard saline with and without 400 mmol
l1 sucrose served to establish hyperosmotic conditions. This
medium had an osmolarity of 465 mosmol l1, equivalent to 1.6
x isosmolarity. Osmolarity of the media was measured by freezing point
depression (Knaur Semi-Micro Osmometer, Berlin, Germany). For the measurement
of the production of glucose and lactate, glucose was omitted from all media.
Furthermore, as preliminary experiments showed that sucrose may be partially
hydrolysed by the cells, resulting in a disturbing background signal in
glucose measurements, sucrose was substituted by trehalose in these
experiments. In ion substitution experiments, either Na+ salts or
Cl salts were substituted by equimolar amounts of
tetramethylammonium or gluconate, respectively.
Cell volume
Cell water volume was assessed by epifluorescence microscopy as described
previously (Espelt et al.,
2003). Briefly, hepatocytes were plated on glass cover slips that
had been previously coated with 0.1% w/v poly-L-lysine. Each cover slip with
attached cells was mounted in a chamber filled with isosmotic medium and
placed on the stage of a Nikon TE-200 epifluorescence inverted microscope.
Hepatocytes were then loaded with 2 µmol l1 of calcein-AM
for 4560 min, and the loading solution was washed out with isosmotic
medium for at least 1 h before starting the experimental data acquisition.
Experimental solutions were superfused at a rate of 2 ml
min1. Changes in cell water volume were inferred from
readings of the fluorescence intensity recorded by exciting calcein through a
470 CWL excitation filter and imaged with a 500 nm LP dichroic mirror and a
515 LP barrier filter. Normalised values of cell water volume
(Vr) were computed from monitored changes in relative
fluorescence (Ft/Fo) according to the
following equation:
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A detailed description of the technique, its validation and corresponding
computations can be found elsewhere
(Alvarez-Leefmans et al., 1995;
Altamirano et al., 1998
).
Oxygen consumption
Rates of oxygen consumption
(O2) were
determined with a dual-chamber Cyclobios Oxygraph
(Haller et al., 1994
) as
described previously (Krumschnabel et al.,
1994
). Before each measurement, two groups of cells from one
preparation, at a density of 30x106 cells
ml1, were incubated under either isosmotic or anisosmotic
(hypo- or hyperosmotic) conditions for 30 min. Hepatocytes
(5x106 cells ml1) were then injected into
one of two measuring chambers containing the equivalent incubation medium, and
oxygen consumption was followed over approximately 10 min. At the end of the
experiment, cells were re-counted with a hemocytometer (Bürker-Türk,
Friedrichsdorf, Germany).
Production of lactate and glucose
For the determination of the rates of lactate accumulation and glucose
production, cells (10x106 cells ml1) were
washed and subsequently incubated in glucose-free iso- or anisosmotic medium
over a period of 60 min. At 0 min, 15 min, 30 min and 60 min, duplicate 50
µl samples were removed, immediately precipitated with 10% ice-cold
metaphosphoric acid and frozen for later measurement of glucose or lactate,
using standard enzymatic NAD(P)H-coupled assays
(Bergmeyer, 1984).
Intracellular free calcium
The effect of anisosmotic exposure on intracellular free calcium
concentration ([Ca2+]i) was assessed in individual
attached cells, cultured as described above. Hepatocytes were loaded with the
Ca2+-sensitive fluorescence dye Fura 2-AM and were mounted on the
stage of an inverted fluorescence microscope as described in detail before
(Krumschnabel et al., 2001a).
Fluorescence images were captured every 30 s, with excitation set to 340 nm
and 380 nm, and emission was measured above 510 nm. Basal levels of
[Ca2+]i in standard saline were measured for at least 5
min before half of the saline covering the cells was exchanged for an equal
volume of hypo- or hyperosmotic stock and measurements were continued for at
least another 30 min. At the end of each experiment, a calibration was
performed by determination of a maximum fluorescence ratio, obtained after
addition of 4.5 mmol l1 CaCl2, and a minimum
ratio, obtained after adding 20 mmol l1 EGTA, both in the
presence of 7.2 µmol l1 of the calcium ionophore
ionomycin. Applying these values and a dissociation constant
(KD) value of 680 nmol l1, determined
for our experimental set-up by use of a commercial calibration kit (Molecular
Probes), absolute levels of [Ca2+]i could be calculated
using the formula given by Grynkiewicz et al.
(1985
).
Intracellular pH
Intracellular pH (pHi) of individual hepatocytes was determined in cells
loaded with the pH-sensitive fluorescence dye BCPCF-AM, applying the same
microscopic set-up and experimental protocol as above. Excitation was set to
490 nm and 440 nm, and emission was again recorded above 510 nm. Calibrations
were performed by replacing the experimental medium with high K+
saline, where the concentrations of NaCl and KCl were reversed, containing the
cation ionophores nigericin (10 µmol l1) and valinomycin
(5 µmol l1) with a pH adjusted to 6.80, 7.20 or 7.60
(Pocock and Richards, 1992;
Seo et al., 1994
).
Statistics
Data are presented as means ± S.E.M. of N independent
preparations. In experiments on cell cultures, data are shown as means
± S.E.M. of n individual cells. In this case, at least three
independent cultures from three different preparations were used. Differences
between treatments were evaluated with Student's t-test or analysis
of variance (ANOVA) followed by Tukey's post-hoc test, with a
P value of <0.05 being considered as significant.
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Results |
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Effects of anisosmotic exposure on cellular metabolism
As the two Oxygraph chambers allowed only comparisons of two treatments at
a time, independent controls were measured for both hypo- and hyperosmotically
treated cells. Controls and hyposmotically exposed cells respired at a rate of
0.88±0.06 nmol 106 cells min1 and
0.71±0.05 nmol 106 cells min1,
respectively, which was significantly different (P<0.05).
Hepatocytes serving as controls for hyperosmotic conditions had a
O2 of
0.76±0.05 nmol 106 cells min1,
which was not significantly different from the rate of 0.78±0.04 nmol
106 cells min1 observed in
hyperosmotically treated cells. In Fig.
2, these data are summarised in a normalised form so as to allow a
better comparison between treatments.
|
Rates of lactate accumulation were rather variable, both in isosmotic and anisosmotic conditions (Fig. 3). Hence, despite large differences in the means, hyposmotic exposure did not result in a significant change in lactate production as compared with controls. Similarly, although under hyperosmotic conditions lactate production appeared to be reduced throughout the entire experiment, this decrease was not significant.
|
Compared with isosmotic controls, the rate of glucose production was significantly diminished by approximately 38% between 0 min and 15 min of hyposmotic incubation but was no longer different from the control values during the rest of the experiment (Fig. 4). In hyperosmotic medium, a significant reduction of glucose production was seen throughout the entire period investigated, the reduction amounting to 4055% of control values.
|
Effects of anisosmotic exposure on intracellular free calcium
Both hypo- and hyperosmotic conditions elicited significant changes in
[Ca2+]i in most hepatocytes, which, however, showed high
variability between different cells. As demonstrated by the examples depicted
in Fig. 5, individual responses
to hyposmotic exposure ranged from an early drop in
[Ca2+]i, which was absent in some cells, followed by a
sustained increase (Fig. 5A;
approximately 36% of the cells), through a series of oscillatory increases
(Fig. 5B; 26%), to
comparatively slight fluctuations around the baseline value of
[Ca2+]i (Fig.
5C; 38%). Upon hyperosmotic exposure, cells responded with an
early pronounced increase in [Ca2+]i followed by smaller
(Fig. 5D; 49%) or larger
(Fig. 5E; 16%) oscillatory
increases, or by a single and sustained increase of
[Ca2+]i (Fig.
5F; 35%). The mean response of all cells examined yielded a slow
progressive increase of [Ca2+]i under hyposmotic
conditions, whereas in hyperosmotic medium a faster increase was observed,
which peaked at 10 min of exposure and persisted thereafter
(Fig. 6).
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Effects of anisosmotic exposure on intracellular pH
The impact of hypo- and hyperosmotic conditions on pHi of trout hepatocytes
is depicted in Fig. 7.
Hyposmotic medium caused a decrease in pHi of 0.2 units within 510 min
to a lower steady level, and this decrease was fully reversible within 5 min
after re-exposure to isosmotic conditions. In hyperosmotic medium, pHi showed
an increase of 0.4 units, which was completed after approximately 15 min, and
re-exposure to isosmotic medium caused a decrease of pHi within 15 min to a
value not significantly different from the initial pHi.
|
In order to gain some insight into the mechanisms involved in these pHi responses, the effects of anisosmotic conditions on pHi were then repeated in both complete and ion-substituted media and in the presence of ion transport inhibitors. Examples of these experiments are shown in Fig. 8; a summary of the data is given in Table 1.
|
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As shown in Table 1, omission of Na+ from the incubation medium resulted in a significant decrease of pHi by 0.10 units. Subsequent exposure to hyposmotic Na+-free medium caused a further significant decrease by 0.08 pH units. Although this value tended to be lower than the decrease of 0.19 pH units evoked by hyposmotic medium in the presence of Na+, this difference was not significant. A similar picture was seen with EIPA, the inhibitor of Na+/H+ exchange, which produced a decrease of pHi by 0.22 units under isosmotic conditions and a further acidification by 0.20 units in hyposmotic medium. Exposure to Cl-free conditions resulted in an alkalinization of pHi by 0.37 units, but subsequently imposed hyposmotic conditions caused no further significant change of pHi. SITS, the inhibitor of both Na+-dependent and Na+-independent Cl/HCO3 exchange, caused a significant decrease of pHi by 0.27 units; hyposmotic exposure with SITS significantly decreased pHi further by 0.10 units.
In the experimental series investigating the effect of hyperosmotic medium, Na+-free conditions elicited a decrease in pHi of 0.24 units. Subsequent exposure to hyperosmotic conditions caused virtually no change in pHi, whereas an increase of 0.42 pH units was observed in the presence of Na+. Similar to the effect of Na+-free medium, EIPA caused a significant intracellular acidification, whereas no further change of pHi was seen under hyperosmotic conditions in the presence of EIPA. By contrast, the increase of pHi evoked in Cl-free medium was followed by further significant alkalinization of 0.25 pH units in hyperosmotic medium. This increase, however, was significantly smaller than the one measured in Cl-containing medium. SITS decreased pHi by 0.10 units. Exposure to hyperosmotic medium in the presence of SITS elicited an increase of pHi by 0.18 units, which was significantly different from that seen in its absence.
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Discussion |
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Almost a mirror image to that described above was observed regarding volume
changes of trout hepatocytes in hyperosmotic medium. That is, cells first
shrank and then exerted a regulatory volume increase (RVI), which almost,
although not completely, restored cell volume within the time investigated
(Fig. 1B). This is remarkable,
since in many cells, including rat hepatocytes, RVI is rather slow
(Wehner and Tinel, 2000) or
virtually absent (Corasanti et al.,
1990
) unless the cells are subjected to pseudo-hypertonic
conditions as mentioned above. In contrast to hyposmotic medium,
hyperosmolarity left
O2 of trout
hepatocytes unaltered, despite the fact that the cells were still in a
shrunken state at the time respiratory rate was assessed
(Fig. 2). Similar observations
have been reported for kidney cortex
(Gyory et al., 1981
) and L-929
cells (Clegg and Gordon,
1985
), in which hyperosmotic conditions were also without effect
on
O2. It has
been hypothesised by Clegg and Gordon
(1985
) that this reflects the
highly structured organisation of cellular metabolism, which may render
endogenous respiration of the cells largely independent of metabolite
concentration in the aqueous cell compartments.
Somewhat different responses to hypo- and hyperosmotic exposure were also seen in the experiments examining the rates of lactate accumulation and glucose production. In hyposmotic medium, lactate production was not significantly altered, although the lack of significance may in part be due to the large variability observed (Fig. 3). At the same time, glucose production was initially significantly diminished but returned to control levels thereafter (Fig. 4). By contrast, while hyperosmotic conditions also had no effect on the rate of lactate production, glucose production was significantly decreased throughout the entire experiment.
In rat hepatocytes, alterations in glycogen synthesis and glycolytic
activity were found to be related to the decrease in pHi associated with
hyposmotic conditions (Peak et al.,
1992). However, although intracellular acidification was also seen
in hyposmotically induced trout hepatocytes
(Fig. 7), we recently observed
that both
O2 and
lactate production were largely unaffected by a decrease in extracellular pH
by as much as one pH unit (Krumschnabel et
al., 2001b
). As such a decrease in extracellular pH is accompanied
by a pronounced acidification of pHi in trout hepatocytes
(Walsh, 1986
;
Krumschnabel et al., 2001b
),
the change in pHi does not appear to be directly involved in the modification
of glucose metabolism in these cells.
As noted before, the lack of an effect of hyperosmolarity on
O2 has been
previously described in other cells, but the unaltered rate of lactate
production and the decreased cellular output of glucose under these conditions
contradict earlier observations on perfused rat liver
(Lang et al., 1989
) and L-929
cells (Clegg et al., 1990
),
where glycolytic flux was enhanced. Furthermore, in rat hepatocytes,
alkalinization per se stimulated glycolysis
(Peak et al., 1992
), which was
apparently not the case in the present study. Thus, in summary, both hypo- and
hyperosmotic conditions tended to cause a decrease of metabolic functions in
trout hepatocytes, which is in contrast to the generally opposing effects
observed in mammalian cells
(Häussinger et al.,
1994
).
Cell volume and [Ca2+]i
Although alterations of [Ca2+]i have often been found
to be associated with cell volume changes in mammalian cells
(Hoffmann and Dunham, 1995),
and recently also in teleost cells (Leguen
and Prunet, 2001
), the role of [Ca2+]i in
the volume regulatory responses of cells appears to be rather variable. In
fish, a strict dependence of RVD on [Ca2+]i has been
reported for goldfish renal proximal tubule cells
(Terreros and Kanli, 1992
),
whereas no requirement for Ca2+ has been documented in trout tubule
cells (Kanli and Norderhus,
1998
). In the present study, the importance of
[Ca2+]i changes for RVD and RVI has not been
specifically addressed, but our results clearly showed that anisosmotic
conditions evoke alterations of [Ca2+]i in trout
hepatocytes (Figs 5,
6). In hyposmotic medium, these
[Ca2+]i responses were not reflected in dramatic changes
of the mean [Ca2+]i levels but were clearly visible at
the individual cell level. The reason underlying the heterogeneity of cellular
responses is not clear, but a remarkable variability at the cell level has
also been reported for other cells
(Jorgensen et al., 1997
). As
in this previous study, the alterations of [Ca2+]i did
not seem to be closely related to the changes of cell volume in the trout
hepatocytes. Furthermore, in previous investigations it has been shown that
the increase of K+ efflux induced by hyposmotic conditions was not
altered by the Ca2+ ionophore A23187 or inhibitors of
Ca2+-dependent K+ channels
(Bianchini et al., 1988
),
whereas a significant reduction of RVD and of taurine release was seen in the
absence of extracellular Ca2+
(Michel et al., 1994
). The
role of [Ca2+]i in the RVD response of trout hepatocytes
therefore remains ambiguous at present.
Changes of [Ca2+]i under hyperosmotic conditions have
received comparatively little attention, but an increase (frog skeletal
muscle; Chawla et al., 2001),
no change (Ehrlich ascites tumour cells;
Pedersen et al., 1998
) and
even a decrease of [Ca2+]i have been reported (rabbit
proximal tubular cells; Raat et al.,
1995
). In trout hepatocytes, hyperosmolarity unequivocally caused
an increase of [Ca2+]i that was apparent both at the
population and the single cell level (Figs
5,
6). Importantly, this elevation
was seen in all cells studied, and the occurrence of the initial
Ca2+ peak coincided with the time of maximum cell shrinkage.
Furthermore, the absolute increase to nearly twice the resting level clearly
exceeded a mere concentration effect due to volume reduction, which would have
accounted for an increase of about 25%. Although the importance of these
changes for RVI remains to be elucidated, we believe that the sum of these
findings is suggestive of their physiological significance.
Volume regulatory mechanisms and their impact on pHi
The pHi regulatory mechanisms of trout hepatocytes have been repeatedly
studied before (Walsh, 1986;
Furimsky et al., 2000
;
Krumschnabel et al., 2001b
).
Our current data, collected as a corollary during the ion substitution and
transport inhibitor experiments, largely corroborate these studies, and
therefore the interested reader should refer to these reports for a detailed
analysis on this topic.
Available reports on the transport mechanisms involved in RVD of trout
hepatocytes indicate that, upon hyposmotic swelling, KCl cotransport and
taurine release are the main routes mediating the loss of osmolytes (Bianchini
et al., 1988,
1991
;
Michel et al., 1994
). In
principle, neither of these transport pathways should directly modify pHi, but
our observations here show that a significant and reversible acidification was
induced by hyposmolarity (Fig.
7). Although similar observations have been reported for many
other cells, the reasons underlying this decrease in pHi appear to be largely
unresolved (Jakab et al.,
2002
). Our present data indicate that, in the trout cells,
Na+/H+ exchange was not responsible for the
acidification, as neither Na+-free conditions nor EIPA altered the
decrease of pHi (Table 1).
Similarly, Cl/HCO3 exchange was
apparently not involved, since SITS did not affect the hyposmotic pHi change.
Interestingly, however, the omission of Cl from the
incubation medium prevented the hyposmotic acidification. A tentative
explanation for this is that, under these conditions, RVD, and in turn also
the pHi changes associated with it, might be blocked, since it was shown that
both the increases in K+ permeability
(Bianchini et al., 1988
) and in
taurine release (Michel et al.,
1994
) were diminished in Cl-free media. A
partial inhibition of RVD has also been reported for trout renal tubules in
the presence of either Cl channel blockers or an inhibitor
of KCl cotransport (Kanli and Norderhus,
1998
).
The RVI response of many cells includes activation of
Na+/H+ exchange and parallel
Cl/HCO3 exchange, causing the
net uptake of NaCl followed by water
(Hoffmann and Dunham, 1995).
The same is obviously true for trout hepatocytes, since in the present and a
previous study (Fossat et al.,
1997
) hyperosmotic shrinkage induced an increase in pHi that was
sensitive to inhibitors of Na+/H+ exchange and
Na+-free conditions, as well as to an inhibitor of
Cl/HCO3 exchange or
Cl-free conditions. Furthermore, Fossat et al.
(1997
) directly demonstrated
an amiloride- and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic
acid (DIDS)-sensitive increase in Na+ and Cl
uptake, respectively, in hyperosmotic medium. But while inhibition of
Na+/H+ exchange completely abolished the pHi changes,
this was not the case with inhibition of
Cl/HCO3 exchange, which only
diminished the extent of alkalinization. This suggests that, as in other cells
(Bevensee et al., 1999
;
Cabado et al., 2000
),
Na+/H+ exchange is the main mechanism involved in
alkalinization in hyperosmotic medium. In addition, despite significantly
different effects on baseline pHi of the removal of extracellular
Cl and the presence of SITS, the alkalinization induced by
hyperosmolarity was similar in magnitude. Thus, although the activity of the
Na+/H+ exchanger is known to be dependent on pHi in
isosmotic conditions (Fossat et al.,
1997
), this may be less pronounced in hyperosmotic conditions.
Furthermore, in several cell types, e.g. rat lymphocytes
(Grinstein et al., 1985
) and
renal mesangial cells (Bevensee et al.,
1999
), the pHi dependence of Na+/H+ exchange
was found to be alkali shifted in hyperosmotic medium, allowing sustained acid
extrusion at increased pHi.
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Acknowledgments |
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