Determinants of intracellular pH in gas gland cells of the swimbladder of the European eel Anguilla anguilla
Institut für Zoologie und Limnologie, Universität Innsbruck, A-6020 Innsbruck, Austria
* Author for correspondence (e-mail: Bernd.Pelster{at}uibk.ac.at )
Accepted 30 January 2002
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Summary |
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Key words: swimbladder, gas gland cell, V-ATPase, Na+/H+ exchange, anion exchange, European eel, Anguilla anguilla, pHi
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Introduction |
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The reduction in the effective gas-transport capacity of the blood is
achieved by the production and secretion of acidic metabolites by swimbladder
gas gland cells. Gas gland cells are highly specialized for the production of
lactic acid and CO2 via anaerobic metabolic pathways, such
as glycolysis or the pentose phosphate shunt
(Pelster, 1995b). This acid is
released into the bloodstream to reduce the physical solubility of gases
(salting out effect) and the haemoglobin oxygen-carrying capacity (Root
effect) and, thus, to increase gas partial pressures in the blood. Although
the acid secretion of gas gland cells has been characterized in cultured cells
(Pelster 1995a
;
Pelster and Niederstätter,
1997
), nothing is known about the intracellular pH of these cells
or of how it is regulated.
Three major mechanisms are known to regulate intracellular pH: the
Na+/H+ exchanger, which uses the Na+
concentration gradient as a driving force to remove protons from the cell
(Roos and Boron, 1981;
Doppler et al., 1986
;
Harvey and Ehrenfeld, 1988
;
Kramhoft et al., 1988
); anion
exchangers which, in conjunction with the carbonic-anhydrase-catalyzed
equilibrium of the CO2/HCO3- reaction,
transfer protons from one side of the membrane to the other; and
H+-ATPases, such as V-ATPase
(Deitmer and Rose, 1996
).
Different types of bicarbonate exchange have been described, the most
important here being the
Na++HCO3-/H++Cl-
exchanger, also called the Na+-dependent bicarbonate exchanger, and
the Na+-independent HCO3-/Cl-
exchanger, which normally promotes an efflux of HCO3- to
decrease pHi after alkalization
(Reinertsen et al., 1988
;
Kramhoft et al., 1994
). All
these mechanisms have been shown to play a role in proton release by gas gland
cells in primary cell culture (Pelster,
1995a
; Pelster and
Niederstätter, 1997
). The question unanswered so far is how
intracellular pH (pHi) is influenced by these mechanisms, or even by
extracellular pH (pHe), which can be as low as pH 6.6-6.8 during periods of
gas deposition (Kobayashi et al.,
1990
). The aim of the present study was therefore to analyze how
the various mechanisms of proton secretion contribute to the control of pHi in
swimbladder gas gland cells.
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Materials and methods |
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DMEM F12 (Dulbecco's modified Eagle's medium mixture F-12) and foetal calf serum (FCS) were obtained from Gibco-BRL; Ala-Gln (alanine-glutamine), albumin, bafilomycin A1, 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), desoxyribonuclease I (DNAse I), epidermal growth factor (EGF), gentamycin, Hepes, kanamycin, 5-(N-methyl-N-isobutyl)-amiloride (MIA), NH4Cl, pituitary extract, progesterone, protease, putrescine and valinomycin were obtained from Sigma (Deisenhofen, Germany); 2',7'-bis-(2-carboxypropyl)-5-(6-)-carboxyfluorescein (BCPCF) and nigericin were obtained from Molecular Probes Europe (Leiden, Netherlands); collagen S (Type I, from calf skin), collagenase and insulin-tranferrin-sodium-selenite supplement (ITS) were obtained from Roche M.B. (formerly Boehringer, Mannheim, Germany).
Cell preparation and cell culture
The eels were quickly killed by decerebration and spinal pithing. The body
wall was opened ventrally, and the swimbladder was carefully exposed. A
catheter was inserted into the swimbladder artery to perfuse the organ with
saline solution (in mmoll-1: 140 NaCl, 5.4 KCl, 1 MgCl2,
10 Hepes, pH 7.4). The swimbladder epithelium was then removed, cut into small
pieces and incubated in saline solution containing (in g l-1) 0.50
albumin, 0.22 collagenase, 0.165 protease, 0.15 DNAse and 3-4 ml
l-1 elastase for 10 min. The solution was filtered through a 70
µm cell strainer into a stop solution (DMEM F12 with 10 % FCS). For the
strained tissue, the enzyme incubation and filtration procedure was repeated
twice. Cells were separated from the solution by centrifugation (10 min at 700
revs min-1, 4 °C) and resuspension (see
Pelster, 1995a;
Pelster and Niederstätter,
1997
). Sedimentation and resuspension of the cells were repeated
twice to remove all traces of the solution used for the digestion of the
tissue. Gas gland cells were cultured on collagen-S-coated coverslips in DMEM
F12 (cell culture medium enriched with, in mg l-1, 5 ITS, 0.001
EGF, 0.00629 progesterone, 0.5 pituitary extract, 1000 albumin and 2172
Ala-Glu, and, in ml l-1, 5 FCS, 5 gentamycin (1 mol l-1)
and 10 kanamycin (0.5 mol l-1). Cells were incubated at 18-20
°C and 0.6 % CO2 for 2-5 days until they formed a broad
monolayer of cell patches, but not yet a confluent layer. Cell culture medium
was renewed as soon as a change in colour indicated low pH.
BCPCF incubation and pHi measurement.
Cell patches were incubated for 30 min with 1 µmol l-1 BCPCF,
a derivative of the pH-sensitive fluorescent dye
biscarboxyethyl-carboxyfluorescein (BCECF)
(Graber et al., 1986;
Wood and Pärt, 2000
).
Excess dye was removed by washing the cells twice with phosphate-buffered
saline (PBS; containing, in mmol l-1, 137 NaCl, 2.68 KCl, 1.47
KH2PO4, 8.06 Na2HPO4.
7H2O, 10 glucose, 1 CaCl2 and 1 MgSO4; pH
7.4). Measurements were started approximately 10 min later so that pHi was not
influenced by BCPCF incubation itself, which could lower pHi
(Negulescu and Machen, 1990
).
Both cell patches and media were tested before incubation with BCPCF to
exclude other disturbing influences such as autofluorescence of the cells or
of the chemicals used for measurements
(Buckler and Vaughan-Jones,
1990
; Kramhoft et al.,
1988
; Wong and Huang,
1989
; Pocock and Richards,
1992
).
A common phenomenon was leakage of BCPCF out of the cell
(Allen et al., 1990;
Muallem et al., 1992
), which
could not be avoided despite different loading methods; data from experiments
with a significant rate of leakage were rejected.
The fluorescence intensity of BCPCF is independent of proton concentration
at an excitation wavelength of 440 nm, but changes according to pH when
excited at 490nm. Intracellular pH was therefore quantified by calculating the
ratio of fluorescence recorded at these two excitation wavelengths
(Graber et al., 1986;
Negulescu and Machen, 1990
).
Measurements were supported by Tillvision 3.3 software from T.I.L.L. Photonics
(Martinsried, Germany). Measurements were acquired once every minute, or once
every 15 s, immediately after the incubation medium had been changed.
The excitation ratio was calibrated for pH by constructing a calibration
curve. This was achieved by equilibrating pHi and pHe. Cells were
permeabilized by treatment with the cation ionophores nigericin
(10-5 mol l-1) and valinomycin (5x10-6
mol l-1) and exposed to a modified PBS medium containing a high
concentration of K+ (NaCl replaced by KCl)
(Pocock and Richards, 1992;
Seo et al., 1994
). A
time-dependent bleaching of the BCPCF
(Borzak et al., 1990
;
Weinlich et al., 1993
) was
occasionally observed.
Experimental protocol
In the first series of experiments, cells were exposed to PBS titrated to
different pH values, and the fluorescence ratio was recorded without any
further treatment to analyze the relationship between pHe and pHi. In the
second set of experiments, the effects of specific inhibitors of the various
proton-translocating mechanisms (MIA, DIDS and bafilomycin A1) were
analyzed at a constant pHe (7.4). In the third series of experiments, the
influence of these inhibitors on pHi was examined during artificial lowering
of pHi using the NH4+ pulse technique, i.e. by
incubating the cells with 10 mmol l-1 NH4Cl in PBS (pH
7.4) for 10 min (Negulescu and Machen,
1990). Addition of NH4Cl solution, in which an
equilibrium is established between gaseous NH3 and
NH4+, results in the diffusion of NH3 into
the cell. Protonation of NH3 results in alkalization of pHi. On
removal of NH4Cl, the opposite takes place: NH3 rapidly
leaves the cell by diffusion and the proton stays behind, causing an
acidification of the cell, which is then compensated by active or secondary
active removal of protons out of the cell.
Statistical analyses
Values are presented as means ± S.E.M. (N is the number of
cell patches examined). For each set of experiments, cells from at least four
different cell preparations were used. Differences from control values were
tested by analysis of variance (ANOVA). Significance was accepted at
P<0.05. Values of P<0.01 are reported as highly
significant.
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Results |
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Influence of MIA
Incubation of cells with 10-5 mol l-1 MIA caused a
more-or-less continuous decrease in pHi during the time of MIA exposure
(Fig. 2A). After 10 min of
exposure, pHi had decreased by approximately 1 unit. After removal of MIA,
recovery of pHi was very slow. pHi still was significantly lower than control
values 15 min after a return to control medium. Even after 1 h, some cells had
not returned to control pHi (results not shown).
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A similar result was observed after artificial acidification of the cells using the NH4+ pulse technique. In the presence of MIA, the decrease in pHi following the removal of ammonium was significantly more pronounced than in the absence of MIA. After removal of ammonia, the pHi of control cells remained only slightly below the prepulse value, but in the presence of MIA the cells were acidified by more than 1 pH unit, and recovery from this severe acidification was very slow (Fig. 2B).
It is also noteworthy that, during the ammonium pulse, pHi in MIA-incubated
cells became more alkaline than in control cells. At pH values higher than
7.8, however, the relationship between fluorescence intensity and pHi changes
(Graber et al., 1986), so pHi
values calculated at this point may represent an overestimate.
Influence of DIDS
Addition of DIDS (10-4 mol l-1) to the incubation
medium resulted in a rapid decrease in pHi, but this decrease was not as
pronounced as in the presence of MIA. Intracellular pH decreased by
approximately 0.3 unit, and after 5 min started to return towards control
levels, even in the presence of DIDS (Fig.
3A).
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Compared with controls, acidification of cells after the NH4+ pulse was more pronounced in the presence of DIDS (Fig. 3B). While pHi stabilised at 7.2 in control cells after the NH4+ pulse, in the presence of DIDS it stabilised at approximately 6.8. After removal of DIDS, pHi returned to control values. Towards the end of the NH4+ pulse, pHi was less alkaline in DIDS-incubated cells.
Influence of bafilomycin A1
Addition of bafilomycin A1 to the incubation medium at pHe=7.4
had no effect on steady-state pHi (data not shown). After acidification of the
cells by the NH4+ pulse, however, a severe acidification
was observed in the presence of 10-5 mol l-1 bafilomycin
(Fig. 4). In the presence of
bafilomycin, pHi decreased to 6.2, compared with 7.1 in control cells. After
removal of NH4+, only a minor increase in pHi was
observed in the presence of bafilomycin. Following the removal of bafilomycin,
pHi increased significantly, but it did not return to control levels within
the first 10 min.
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Discussion |
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At low extracellular pH values (near 7.0), pHi in many cells approaches
pHe, but in gas gland cells pHi remained significantly lower than pHe. During
subsequent alkalization, pHi recovered to alkaline values, which clearly shows
that the low pHi does not indicate any loss of control or cell damage.
However, the danger of an excessive acidification of the cells is probably
alleviated by a reduction in glycolytic activity at low pH
(Pelster, 1995b), and the rate
of acid secretion indeed decreases with decreasing pHe
(Pelster, 1995a
).
The role of Na+/H+ exchange in gas gland
cells
Previous studies have demonstrated the existence of a variety of
proton-translocating mechanisms in gas gland cells, such as
Na+/H+ exchange, anion exchange and V-ATPase, but their
importance for the homeostasis of pHi remains unclear
(Pelster, 1995a). The results
of the present study reveal that Na+/H+ exchange is
essential for the stability of pHi in resting cells, but also for the recovery
of pHi from an artificial acid load. The Na+/H+
exchanger is present in almost all cells and contributes significantly to the
removal of protons from the cytoplasm
(Frelin et al., 1988
;
Fliegel and Dibrov, 1996
). Even
in parietal cells, which during stimulation secrete protons via the
K+/H+-ATPase, Na+/H+ exchange
appears to be the main route of proton release after an artificial acid load
(Rossmann et al., 2001
).
Na+/H+ exchange is typically activated at low pHi and
inactivated at alkaline pH (Frelin et al.,
1988
). As shown previously, the relative contribution of
Na+/H+ exchange to acid secretion by gas gland cells is
enhanced at low pHe (Pelster and
Niederstätter, 1997
), and this is supported by the results of
the present study. The use of Na+-dependent mechanisms for ion
transport requires Na+/K+-ATPase activity, and the
importance of this ATPase for acid secretion by gas gland cells has been
demonstrated previously (Pelster and
Niederstätter, 1997
). Incubation of gas gland cells with 1
mmol l-1 ouabain induced a slow decrease in the rate of proton
secretion, and this effect was particularly pronounced at low pH, which is in
line with our results on the pH-dependence of Na+/H+
exchange. The rapid decrease in the rate of acid secretion on removal of
extracellular Na+ in comparison with the slow response to removal
of K+ or application of ouabain suggested that the Na+
gradient was critical for acid secretion and that the membrane potential was
not of primary importance (Pelster and
Niederstätter, 1997
).
The role of anion exchange
DIDS, as an inhibitor of bicarbonate exchangers, also caused a decrease in
pHi, but this decrease was not as pronounced as that observed after addition
of MIA. It can therefore be concluded that an anion exchanger is involved in
maintaining steady-state pHi. The Cl-/HCO3-
exchanger typically extrudes base, and an inhibition would therefore result in
an accumulation of base inside the cell, which is equivalent to an
alkalization. The Na+-dependent anion exchanger, however, uses the
Na+ gradient and transports HCO3- into the
cell. In this case, inhibition of the anion exchanger would result in a lower
HCO3- concentration inside the cell and, thus, in a
reduced buffering capacity. In consequence, pHi will become more acidic. The
observed decrease in pHi in the presence of DIDS is therefore in line with the
conclusion that a Na+-dependent anion exchanger is present in
swimbladder gas gland cells (Pelster,
1995a). The influence of DIDS was reduced after the ammonium
pulse, suggesting that the importance of bicarbonate-exchanging mechanisms is
reduced at low pHi. Similar results have been reported for muscle and in
Ehrlich ascites tumour cells, in which bicarbonate exchange appears to be
mainly responsible for the regulation of steadystate pHi, but
Na+/H+ exchange is activated at low pHi
(Kramhoft et al., 1994
). This
conclusion is also supported by the observation that, during the
NH4Cl incubation phase, the alkalization of control cells was not
as pronounced as in the presence of DIDS. HCO3-
extrusion by the Na+-independent
HCO3-/Cl- exchanger would have resulted in
the downregulation of pHi in this situation
(Reinertsen et al., 1988
;
Wood and Pärt, 2000
).
The role of V-ATPase
A reduction in the rate of acid secretion in the presence of bafilomycin
A1 suggests that a V-type ATPase is present in gas gland cells
(Pelster, 1995a), and two
isoforms of the B-subunit of V-ATPase have recently been cloned and sequenced
(Niederstätter and Pelster,
2000
). The presence of two different isoforms may, of course, be
related to different properties and/or functions of the protein. Gas gland
cells are responsible not only for the production and secretion of acidic
metabolites but also for the production and secretion of surfactant at their
luminal surface (Prem et al.,
2000
). Before exocytosis, surfactant is stored in multilamellar
bodies, which can be stained with the fluorescent dye LysoTracker Green
(Mair et al., 1999
), a weak
base that accumulates in acidic organelles. This staining can be prevented by
preincubation of the gas gland cells with submicromolar concentrations of
bafilomycin A1 (H. Niederstätter and B. Pelster, unpublished
observations), which demonstrates that the low pH established in these
vesicles requires the presence of V-ATPase. The present study revealed that
inhibition of V-ATPase with bafilomycin A1 significantly impairs
proton extrusion after an artificial acid load. Therefore, at low pHi,
V-ATPase is activated and required for the regulation of cell pH. At alkaline
pH values, bafilomycin A1 had no effect on pHi in the present
study, but in an earlier study a 15-20 % reduction in the rate of acid
secretion was observed (Pelster,
1995a
). It appears quite possible that these different results may
be related to a possible shuttling of V-ATPase in relation to the activity
status of the cell, as observed in kidney cells for example
(Gluck et al., 1998
). Like
parietal cells, in which K+/H+-ATPase is mainly involved
in acid secretion during periods of stimulation and
Na+/H+ exchange is mainly responsible for homeostasis of
intracellular pH (Muallem et al.,
1988
; Yanaka et al.,
1991
; Rossmann et al.,
2001
), in gas gland cells, V-ATPase would be responsible for the
secretion of acid during periods of gas deposition into the swimbladder and
pHi would be controlled mainly by Na+/H+ exchange and in
part by anion exchange at alkaline pHe values. Unfortunately, attempts to
stimulate gas gland cells and thus to enhance acid secretion have not been
successful (Pelster and Pott,
1996
). Verification of this hypothesis must await further
experimentation.
Concluding remarks
Our results suggest that Na+/H+ exchange and anion
exchange are important for the regulation of pHi at alkaline pHe values.
Recovery from an intracellular acidification depends mainly on the activity of
the Na+/H+ exchanger, and a V-ATPase contributes
significantly to the removal of protons from the cytoplasm. The activity of
bicarbonate-exchanging mechanisms, however, is greatly reduced at low pHi. A
model of a gas gland cell and the mechanisms of acid transport developed on
the basis of the present study and the results of previous studies
(Pelster, 1995a;
Pelster and Niederstätter,
1997
) is shown in Fig.
5. The presence of various mechanisms for the secretion of protons
therefore appears to be attributable to the fact that gas gland cells need to
secrete protons at a wide range of pHe values. The mechanisms that control the
activity status of gas gland cells are still unknown, but this promises to be
a fascinating area of research.
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Acknowledgments |
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