|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online August 3, 2006
Journal of Experimental Biology 209, 3101-3113 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02357
Signalling pathways involved in hypertonicity- and acidification-induced activation of Na+/H+ exchange in trout hepatocytes
Institut für Zoologie and Center of Molecular Biosciences, Leopold Franzens Universität Innsbruck, Technikerstraße 25, A-6020 Innsbruck, Austria
* Author for correspondence (e-mail: Gerhard.Krumschnabel{at}uibk.ac.at)
Accepted 31 May 2006
 |
Summary |
|---|
 TOP Summary IntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Key words: trout hepatocyte, Na+/H+ exchange, intracellular pH, proton secretion, calcium, PKC, PKA, calmodulin
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
)
and in many cells NHE-1, the most widely distributed, so-called house-keeping
isoform, is stimulated by cell shrinkage, intracellular acidification and also
by growth factors (Dunham et al.,
2004). Various signalling pathways have been described that are
involved in activation of this important exchange protein, including protein
kinase C (PKC) (Wakabayashi et al.,
1997b; Maly et al.,
2002), protein kinase A (PKA)
(Kandasamy et al., 1995), and
Ca2+/calmodulin (Ca2+/CaM)
(Zavoico et al., 1986;
Wakabayashi et al., 1994). The
long cytoplasmic C terminus of NHE has been shown to contain various
subdomains that can be phosphorylated or bind to various regulatory proteins.
By creating truncated mutants (Bianchini et
al., 1995) these features could be confirmed at the molecular
level (for reviews, see Ritter et al.,
2001; Orlowski and Grinstein,
2004). In addition, one of the two proton-binding sites identified
on the intracellular side of the NHE-1 protein appears not to be involved in
the transport of protons, but, by allosterical modification of the protein,
changes the transport properties of the exchanger
(Zavoico et al., 1986;
Putney et al., 2002).
The receptor-mediated pathways activating NHE have been relatively
extensively studied, whereas the `receptor independent' activation of NHE by
osmotic stress or by intracellular acidification is less well characterized
(Putney et al., 2002). In
neutrophils, a stimulation of NHE activity was observed in response to an
isosmotic decrease in cell volume, suggesting that changes in cell size may be
recorded (Krump et al., 1997).
Direct phosphorylation of NHE as well as MAP kinase signalling have also been
reported to be involved in the activation of NHE
(Grinstein et al., 1992;
Moor and Fliegel, 1999), and
contradicting results exist concerning the involvement of Ca2+ ions
(Grinstein et al., 1985b;
Mitsuhashi and Ives, 1988;
Murao et al., 2005). In
Ehrlich Ascites tumour cells PKC appears to be involved in the hyperosmotic
activation of NHE (Pedersen et al.,
2002). Overall, however, intracellular signalling pathways
involved in the regulatory volume increase (RVI) response following
hyperosmotic exposure remain enigmatic. To make things more complicated, in
many fish red cells (Brauner et al., 2002) as well as hepatocytes
(Tuominen et al., 2003)
activation of NHE during hyperosmotic stress (red cells) or following
acidification (hepatocytes) is inhibited by oxygen, i.e. the NHE is more
strongly activated under hypoxic conditions. In addition, in fish red cells a
catecholamine-responsive NHE isoform has been described. This isoform,
ß-NHE, shows some similarity to NHE-1, but it was found to differ
considerably from mammalian NHE-1 isoform in many aspects
(Malapert et al., 1997;
Pedersen and Cala, 2004).
Despite these apparent peculiarities of fish cell NHE, most of the previous
investigations focused on red cells, and almost nothing is known regarding the
regulation of NHE in other cells from fish. An interesting model system to
investigate this are trout hepatocytes, since in these cells both RVI
following cell shrinkage (Ebner et al.,
2005) and the associated intracellular alkalinization
(Fossat et al., 1997;
Krumschnabel et al., 2003), as
well as pHi recovery after acidification
(Walsh, 1986;
Tuominen et al., 2003) are to
a large extent dependent on this ion exchanger, suggesting an important role
for NHE in cell function. Furthermore, changes in cell volume as well as pHi
may exert strong effects on hepatocyte metabolism
(Walsh and Mommsen, 1992;
Haussinger and Schliess, 1995)
and therefore control of this important transporter is clearly crucial for
these cells.
Thus, in order to shed light on the signalling pathways leading to NHE activation in trout hepatocytes, specifically focusing on NHE-1, the present study aimed to elucidate the possible role of altered extra- and intracellular pH, Ca2+, PKC, PKA and CaM in cell signalling upon induction of cell shrinkage with hypertonic medium or intracellular acidification by the weak acid, sodium propionate. Our data showed that NHE-1 is the main exchanger responsible for pHi changes during hypertonic challenge and, together with SITS-sensitive sodium-dependent Cl-/HCO -3 exchange it is responsible for pHi recovery after acidification of the cells. Absence of Ca2+ and changing the extracellular pH to levels above or below the normal values significantly reduced the hypertonicity-induced increase of pHi. By contrast, pHi recovery from sodium propionate loading increased with increasing medium pH. Both hypertonicity- and acidification-induced NHE-1 activity were reduced in the presence of inhibitors of PKA and CaM. Chelerythrine, a PKC inhibitor, also inhibited the acidification-, as well as the hypertonicity-induced NHE activity.
|
Material and methods |
|---|
|
TOPSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Experimental animal and hepatocytes isolation
Rainbow trout (Oncorhynchus mykiss Walbaum) were obtained from a
local hatchery and were acclimated in 200 l aquaria with running water at
15°C. Fish were fed daily with trout pellets (EWOS Aquaculture
International, Austria) ad libitum. Hepatocytes were isolated using
the collagenase digestion procedure described previously
(Krumschnabel et al., 1996).
Briefly, fish were killed by a blow on the head, the liver was exposed, and
the portal vein was cannulated. The liver was then perfused with a
Hepes-buffered saline to remove the blood, followed by perfusion with a
collagenase-containing saline (0.05% collagenase) until the tissue appeared
soft and swollen. Subsequently, the liver was excised, cut into fine fragments
with scissors, and further incubated with collagenase-containing saline for a
few minutes. The cells were finally filtered through two nylon screens (pore
diameter 250 and 150 µm) and washed three times (60 g, 4 min).
After isolation, hepatocytes were left to recover in standard saline (see
below) containing 1% BSA for 1 h in a shaking water bath thermostated to
19°C, which was also the temperature used during the experiments. Cells
viability, as determined from Trypan Blue exclusion, was always >85%.
Hepatocyte preparation
For the determination of intracellular free Ca2+ concentration
([Ca2+]i) and intracellular pH (pHi), hepatocytes
(1.5x106 to 2x106 cells ml-1)
were suspended in Leibovitz L15 medium (0.95 mmol l-1
CaCl2, 5.33 mmol l-1 KCl, 0.44 mmol l-1
KH2PO4, 0.46 mmol l-1 MgCl2, 0.40
mmol l-1 MgSO4, 137.9 mmol l-1 NaCl, 1.07
mmol l-1 Na2HPO4, 4.99 mmol l-1
galactose, 5 mmol l-1 sodium pyruvate, and amino acids and vitamins
according to the manufacturer's formulation) modified by addition of 10 mmol
l-1 Hepes, 5 mmol l-1 NaHCO3, 50 µg
ml-1 gentamycin and 100 µg ml-1 kanamycin, pH
titrated to 7.6. These cells were then plated on poly-L-lysine (5 µg
ml-1)-coated glass coverslips and maintained in an incubator
(19°C, 0.5% CO2) overnight. Before loading the cells with the
specific dye for the determination of [Ca2+]i or pHi, the cultures
were washed several times with fresh standard saline in order to remove
non-adherent cells and debris.
Experimental media
The standard isosmotic saline used for measuring [Ca2+]i and pHi
consisted of (in mmol l-1) 10 Hepes, 136.9 NaCl, 5.4 KCl, 1
MgSO4, 0.33 NaH2PO4, 0.44
KH2PO4, 5 NaHCO3, 1.5 CaCl2, 5
glucose, pH 7.6 at 19°C, and had an osmolarity of 284 mosmol
l-1. To create hyperosmotic conditions, a mixture of one volume of
standard saline with an equal volume of the same medium containing an
additional 200 mmol l-1 NaCl was used, yielding an osmolarity of
465 mosmol l-1 (1.6x isosmolarity).
The standard isosmotic medium (low buffer capacity medium) used for measuring the H+ release with the Cytosensor microphysiometer consisted of (in mmol l-1) 138 NaCl, 5 KCl, 0.81 K2HPO4, 0.5 MgCl2, 0.11 KH2PO4, 1.3 CaCl2, 5 glucose, titrated to pH 7.6. For hyperosmotic conditions, an additional 100 mmol l-1 NaCl was added to the same medium.
Intracellular free Ca2+ measurement
Intracellular free Ca2+ ([Ca2+]i) of hepatocytes was
measured in individual attached cells using the membrane-permeable
Ca2+-sensitive fluorescence dye Fura 2-AM. Cells, cultured as
described above, were loaded with the dye for 1 h followed by two careful
washes with standard saline, then the coverslips were mounted in a measuring
chamber containing 1 ml saline and the chamber was fixed on the stage of an
inverted Axiovert 100 epifluorescence microscope (Zeiss, Vienna, Austria)
equipped with a 40x ultraviolet objective. By means of a slow scan CCD
video camera, fluorescence images were captured every 60 s, with excitation
set to 340 nm and 380 nm, and emission was detected above 510 nm. The images
were stored on a computer using the tillVISion software package (T.I.L.L.
Photonics). Basal levels of [Ca2+]i in standard saline were
measured for at least 5 min before half of the saline covering the cells was
carefully exchanged for an equal volume of hyperosmotic stock and measurements
were continued for at least another 30 min. In control experiments, in which
half of the bathing medium was exchanged for the same volume of isotonic
standard medium, no effect on [Ca2+]i (or pHi, see below) was
noted, ruling out the possibility that detected changes were due to mechanical
stress created by fluid exchange. At the end of each experiment, a calibration
was performed by determination of a maximum fluorescence ratio, obtained after
addition of 4.5 mmol l-1 CaCl2, and a minimum ratio,
obtained after adding 20 mmol l-1 EGTA, both in the presence of 7.2
µmol l-1 of the calcium ionophore ionomycin. Applying these
values and a dissociation constant (KD) of 680 nmol
l-1 previously determined for our experimental setup by the use of
a commercial calibration kit (Molecular Probes, Leiden, The Netherlands),
absolute levels of [Ca2+]i could be calculated using the formula
given by (Grynkiewicz et al.,
1985).
To assess the effect of hyperosmotic challenge on [Ca2+]i in the absence of extracellular Ca2+ (Ca2+e), cells, loaded with Fura 2-AM in standard saline, were incubated with Ca2+-free saline and, after determination of a baseline during the initial 5 min the cells were exposed to Ca2+-free hyperosmotic medium. In order to obtain Ca2+-depleted cells, hepatocytes were incubated in standard saline containing 25 µmol l-1 BAPTA-AM during Fura 2-loading and these cells were then exposed to Ca2+-free saline during measurements.
Intracellular pH measurement
Intracellular pH of individual hepatocytes was measured in cells loaded
with the pH-sensitive fluorescent dye BCPCF-AM, applying the same microscope
setup 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 l-1) and valinomycin (5 µmol
l-1) with a pH adjusted to 6.80, 7.20 or 7.60
(Pocock and Richards, 1992;
Seo et al., 1994). When
experimental media of pH values higher than 7.6 were used, calibration media
were adjusted to cover the range of pHi values determined.
To assess the effect the extracellular pH (pHe) on pHi during hyperosmotic challenge, cultured cells were incubated with BCPCF-AM for 30 min using standard saline titrated to the desired pH. All other media used during the course of the experiment were also titrated to the same pH.
Recovery of pHi from an acid load was assessed by adding 30 mmol l-1 of the weak acid sodium propionate and following changes of pHi for 15 min before performing calibration.
To determine the effect of the presence of different inhibitors on both pHi and H+ secretion in response to hyperosmotic challenge or to an acid load, the cells -after stabilisation -were exposed to the individual or to the combined inhibitors (concentrations are given below) and were then left for several minutes before exchanging for hyperosmotic medium or addition of sodium propionate.
The rate of alkalinization after hypertonic exposure or acid loading was
calculated from the linear portion of the increase of pHi after addition of
hypertonic saline or sodium propionate (pH units min-1),
respectively, and multiplication of this slope by the total apparent
intracellular buffering capacity (ß) determined from the extent of
acidification induced after addition of a known amount sodium propionate,
following the equation:
 |
where [propionate] is 30 mmol l-1, the pKa
of propionate at room temperature is 4.87, pHmin is minimal pHcyto following
acid load, pHinput is the pH of the medium (7.6) and pHcyto is baseline pHi
prior to acid load (Furimsky et al.,
2000).
Inhibitors were made up in concentrated stock solutions dissolved in distilled water or DMSO and were applied at the following final concentrations: cariporide mesilate 10 µmol l-1 (2 mmol l-1 stock in H2 O), SITS 0.5 mmol l-1 (100 mmol l-1 stock in DMSO), chelerythrine 1 and 5 µmol l-1 (2.5 mmol l-1 stock in H2O), calmidazolium 5 µmol l-1 (2.5 mmol l-1 stock in DMSO) and H-89 1 µmol l-1 (1 mmol l-1 stock in DMSO). The final concentration of DMSO was always kept below 0.5%, a concentration that did not interfere with the measurements.
Measurement of proton release
Proton secretion of the hepatocytes was estimated from the rate of
acidification of external medium measured with a cytosensor microphysiometer
(Molecular Devices, Munich, Germany) as previously described
(Pelster, 1995;
Krumschnabel et al., 2001a).
Hepatocytes (0.45x106 cells) were embedded in low-melting
point agarose gel (1.5%) on polycarbonate capsules, loaded into the cytosensor
chamber, and superfused with low buffer capacity medium (given above). By the
use of an electromagnetic valve, perfusion conditions could be rapidly
switched from a control to a test solution. The perfusion cycle was set to 3
min, with 130 s of constant perfusion followed by a 40 s flow-off period.
During the latter period, protons released by the hepatocytes acidify the
measuring chamber and this signal is recorded via a light-addressable
potentiometric sensor. From the slope of a line fitted to the sensor data the
rate of acidification was calculated. The following experimental protocol was
used with all measurements: first the cells were allowed to recover from
embedding for at least 1 h, then a baseline of acid secretion was determined
in freshly titrated saline and this was followed by switch to identical saline
for the control cells and to the different test salines for treated cells.
Since both the geometry of the cytosensor chamber and the embedding procedure of the cells make it very difficult to determine the number of cells actually releasing acid equivalents into the measuring chamber, acidification rates were not given as H+ s-1, but the signal (µV s-1) was converted to the percentage of the basal rate of proton secretion, measured under control conditions prior to the treatment.
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.
|
Results |
|---|
|
TOPSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
|
In the presence of the specific NHE-1 inhibitor cariporide, basal pHi decreased from 7.42±0.03 to 7.13±0.05 within 15 min. This decrease in pHi continued even after exposing the cells to hypertonic medium, and reached a value of 7.03±0.07 within the next 10 min where it stabilized (Fig. 1A). On measuring the rate of proton secretion, we observed that cariporide exerted a rather minute effect on the basal value, which transiently declined by about 3%. Upon exposing the cells to hypertonic medium in the presence of the inhibitor, proton secretion increased to only 130% of the basal rate, which was 21% of the hypertonicity-induced rate change in the absence of the inhibitor. Subsequently the rate started to decrease slowly until the end of the experiment (Fig. 1B).
pH-dependence of the hypertonic alkalinization
Media with pH adjusted to 6.8, 7.6 and 8.2 were used to study the effect of
pHe on changes of pHi during hypertonic challenge. After 30 min of incubation
in these media, hepatocytes had a basal pHi of 6.77±0.05 (pHe 6.8),
7.36±0.03 (pHe 7.6) and 7.56±0.03 (pHe 8.2). As shown in
Fig. 2, in spite of these
different baseline levels, hypertonicity induced an increase in pHi regardless
of the altered pHe, with the highest points being attained after about 10 min
in all experiments, when pHi remained more or less stable until the end of the
measuring period. In order to compare the rates of alkalinization following
hypertonic exposure, calculated from the linear part of each curve, we
determined the intracellular buffering capacities of the hepatocytes
maintained at different pHe values. These buffering capacities, which were
derived from experiments similar to those shown in
Fig. 9, amounted to
28.06±1.23 (pHe 6.8), 14.13±1.03 (pHe 7.6) and 14.14±1.74
(pHe 8.2) mmol H+ pH unit-1 l-l (25-97 cells
from four to six preparations). Using these values, we obtained rates of
alkalinization, which were significantly reduced at low pH (0.99±0.07
mmol H+ l-1 min-1, n=17 cells) and
in the high pH saline (1.14±0.09 mmol H+ l-1
min-1, n=35 cells) when compared to standard medium
(1.51±0.07 mmol H+ l-1 min-1,
n=42 cells).
|
|
Intracellular Ca2+ during hypertonic exposure
As shown in Fig. 3, exposing
cells to hypertonicity induced an increase in [Ca2+]i from
131±16 nmol l-1 to a maximum of 268±39 nmol
l-1 after approximately 6 min, a level where it remained until the
end of the experiment. In the absence of Ca2+e, the cells responded
with a similar initial increase in [Ca2+]i, from 114±8 nmol
l-1 to a maximum of 199±19 nmol l-1 within 5 min,
but subsequently [Ca2+]i started to decline to reach the baseline
value at the end of the experiment. Chelating intracellular free
Ca2+ using 25 µmol l-1 BAPTA-AM along with
Ca2+-free medium lowered basal [Ca2+]i to 23±4.3
nmol l-1 and completely abolished any alteration in
[Ca2+]i upon hypertonic exposure.
|
Effect of manipulating [Ca2+]i on pHi changes during hypertonic exposure
In order to elucidate whether there is a link between the
hypertonicity-induced changes in [Ca2+]i and pHi, alterations in
pHi were measured during hypertonic challenge in control saline, in
Ca2+-free medium and in Ca2+-free medium after
pretreating cells with BAPTA-AM. Fig.
4 shows that, in the absence of Ca2+e, pHi increased in
a similar, but slightly delayed fashion when compared to controls. Expressed
as initial rate of proton secretion, the alkalinization in control medium and
Ca2+-free medium amounted to 1.55±0.08 and 0.98±0.07
mmol H+ l-1 min-1, respectively, which were
significantly different (P<0.05). Chelating [Ca2+]i
with BAPTA-AM caused a dramatic decrease in pHi under isotonic conditions
which was not affected further by subsequent addition of Ca2+-free
medium (Fig. 4, inset). When
cells, pretreated in this manner, were exposed to hypertonicity, pHi increased
with the corresponding rate of proton secretion amounting to 1.14±0.12
mmol H+ l-1 min-1 (P<0.05
compared to controls), but did not attain a new steady state over the period
investigated.
|
|
Inhibiting PKA, using the inhibitor H-89, induced a slight and transient decrease of basal pHi, which then increased on exposing cells to hypertonicity (Fig. 6A). This increase, however, was significantly slower than in control hepatocytes, and the apparent rate of proton secretion amounted to 1.9±0.13 and 0.73±0.09 mmol H+ l-1 min-1 in the absence and presence of H-89, respectively (P<0.05). Measurement of proton secretion indicated that H-89 had no effect on the basal rate of proton secretion, whereas upon challenging the cells with hypertonic medium the change in proton secretion rate was significantly (P<0.05) reduced by 41% compared to the change in rate seen in controls (Fig. 6B).
|
|
In Fig. 8, a summary of the rates of proton secretion following hypertonic stimulation under the various conditions examined is given, allowing direct comparison of all treatments.
|
Exposing hepatocytes to SITS induced a gradual decrease in pHi and a final steady-state was attained after 15 min (not shown). During this time basal pHi was reduced to 6.96±0.02 and upon addition of sodium propionate it dropped to 6.5±0.02 (Fig. 9B). The recovery of pHi then occurred at a rate of 0.60±0.02 mmol H+ l-1 min-1, which was significantly lower than in controls (P<0.05). In the concurrent presence of both cariporide and SITS, basal pHi showed a continuous decrease during pre-incubation and upon addition of sodium propionate pHi decreased from 6.66±0.03 to 6.43±0.04 within 3 min. Recovery of pHi determined under these conditions was virtually abolished with proton secretion rate amounting to 0.07±0.04 mmol H+ l-1 min-1.
pH-dependence of pHi recovery following acidification
To determine the effect of pHe, after adjustment of pHi, on the pHi
recovery after acid loading, cells were incubated for 30 min in media with the
pH adjusted to 6.8, 7.6 and 8.2 before sodium propionate exposure. Basal pHi
values under these conditions were 6.78±0.03 (pH 6.8), 7.38±0.03
(pH 7.6) and 7.73±0.04 (pH 8.2). Upon exposing the cells to sodium
propionate, pHi decreased within 2 min to 6.26±0.03 (pH 6.8),
6.84±0.03 (pH 7.6) and 7.1±0.05 (pH 8.2). The rate of proton
secretion during pHi recovery for the three conditions amounted to
0.87±0.11 (6.8; P<0.05 versus controls),
0.88±0.04 (pH 7.6), and to 1.53±0.12 (8.2; P<0.05)
mmol H+ l-1 min-1.
Effect of chelerythrine, H-89 and calmidazolium on the acidification-induced pHi changes
As seen in Fig. 10A,
exposure to 5 µmol l-1 chelerythrine induced a decrease in pHi
from a basal value of 7.33±0.04 to a value of 6.86±0.04 within
15 min and upon addition of sodium propionate, pHi decreased to a value of
6.63±0.03 within 4 min, after which pHi remained constant with the
proton secretion rate amounting to 0.15±0.04 mmol H+
l-1 min-1. In the corresponding controls, which were
also controls for H-89 and calmidazolium treatments, pHi decreased, upon acid
loading, from a basal value of 7.19±0.02 to 6.5±0.06 within 2
min and thereafter, pHi started to increase with a rate of proton secretion
amounting to 1.31±0.1 mmol H+ l-1
min-1.
|
Finally, as shown in Fig. 10C, a slight transient decrease in basal pHi was noted during exposure to calmidazolium, and upon exposing the cells to sodium propionate, pHi decreased from 7.36±0.05 to 6.6±0.06 within 2 min. Thereafter pHi recovery took place at a reduced rate of 0.96±0.05 mmol H+ l-1 min-1, which, however, was not significantly different from that of controls.
A summary of the rates of proton secretion following sodium-propionate-induced acidification under the various conditions examined is given in Fig. 11.
|
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
;
Kapus et al., 1994;
Pedersen et al., 2002), this
NHE-isoform is the main exchanger responsible for the hypertonicity-induced
change in pHi. Furthermore, the acidification of pHi in the presence of
cariporide (Fig. 1A) suggested
that the amiloride-sensitive proton secretion reported to be tonically active
under steady-state conditions in trout hepatocytes
(Furimsky et al., 1999;
Krumschnabel et al., 2001b)
also involves NHE-1. Looking at the acidification of the extracellular space,
however, inhibition of the NHE-1 with cariporide reduced steady-state proton
secretion only by about 3%, and NHE-1 activity accounted for only 80% of the
hypertonicity-induced proton secretion rate
(Fig. 1B). These findings
indicate that there are other mechanisms working in parallel with NHE-1 under
iso- and hyperosmotic conditions. In fact, it could be that other NHE isoforms
are involved, since amiloride has been shown to reduce steady-state proton
secretion by 20% (Krumschnabel et al.,
2001b) and to completely block hypertonicity-induced proton
secretion (Ebner et al., 2005).
Surprisingly, although these isoforms appear to be activated by hypertonicity,
there was no corresponding increase of pHi detectable, because cariporide
completely eliminated the increase in pHi during hypertonic exposure. A
speculative answer to this apparent paradox would be to postulate the presence
of intracellular cariporide-sensitive mechanisms, normally operating to remove
H+ from the cytosol into intracellular compartments. Thus, upon
inhibition with cariporide these protons would remain in the cytoplasm and
thus could counteract activities of the cariporide-insensitive NHE isoforms in
the cell membrane. In support for this, the non-isoform-specific inhibitor
amiloride also acidifies the cells but concurrently prevents enhanced acid
secretion (Krumschnabel et al.,
2003; Ebner et al.,
2005). The presence of intracellular NHE isoforms acidifying
Golgi- and post-Golgi compartments has been documented in human cells
(Nakamura et al., 2005), and
in cardiomyocytes cariporide was reported to inhibit the mitochondrial NHE,
thereby affecting mitochondrial matrix pH
(Ruiz-Meana et al., 2003).
The possible involvement of [Ca2+]i
A hypertonicity-induced increase in intracellular Ca2+ has been
observed in trout hepatocytes in a previous study
(Krumschnabel et al., 2003),
and our data indicated that this was due to Ca2+ mobilization from
intracellular stores accompanied by a sustained Ca2+ influx from
extracellular space (Fig. 3).
Removal of intracellular Ca2+ caused a significant reduction in
pHi, emphasizing a connection between steady-state pHi and Ca2+
movements. Similar observations have been reported for rat hepatocytes
(Martin-Requero et al., 1997)
and rat alveolar cells (Murao et al.,
2005). Although neither removal of extracellular Ca2+
nor a chelation of intra- and extracellular Ca2+ could abolish the
hypertonicity-induced alkalinization, the kinetics and magnitude of this
alkalinization were significantly affected by the absence of Ca2+
(Fig. 4). This contradicts
previous findings in other cell types, where shrinkage-induced activation of
NHE was found to be independent of changes in [Ca2+]i
(Grinstein et al., 1985b;
Mitsuhashi and Ives, 1988;
Dascalu et al., 1992;
Shrode et al., 1995;
Pedersen et al., 1996). On the
other hand, Ca2+-dependent activation was reported for other
conditions, including mitogenic stimulation
(Mitsuhashi and Ives, 1988;
Garnovskaya et al., 2003a) and
elevation of [Ca2+]i by a calcium ionophore or inhibition of the
endoplasmic reticulum Ca2+-ATPase
(Maly et al., 2002). In human
alveolar type II cells a decrease of [Ca2+]i has been shown to
elicit cell shrinkage (Murao et al.,
2005).
The conclusion that Ca2+ may be involved in the activation of
NHE is also supported by the observation that calmidazolium reduces the
hypertonicity-induced alkalinization. Ca2+-calmodulin (CaM) has
been shown to be involved in the activation process of NHE-1 in response to a
variety of stimuli including mitogenic factors, ionomycin and serotonin
(Wakabayashi et al., 1994;
Bertrand et al., 1994;
Garnovskaya et al., 2003a),
and, importantly, osmotic shrinkage
(Dascalu et al., 1992;
Shrode et al., 1995;
Shrode et al., 1997;
Garnovskaya et al., 2003b).
Consistent with the latter studies, our data confirmed an important role of a
CaM-dependent pathway in the hypertonicity-induced NHE-1 activation in trout
hepatocytes, as the inhibition of CaM by calmidazolium reduced the
hypertonicity-induced pHi increase by 54%
(Fig. 7A). A similar degree of
inhibition of hypertonicity-induced pHi increase, ranging from 40-50% has been
found in other cells with different CaM inhibitors
(Dascalu et al., 1992;
Pedersen et al., 1996).
Deletion of the CaM binding subdomain in the C-terminal of the human NHE-1
reduced its activation by shrinkage by about 80%
(Bertrand et al., 1994). In
parallel with its reduction in the intracellular alkalinization, calmidazolium
reduced the hypertonicity-induced proton secretion rate by 15% in the trout
cells (Fig. 7B). This is
considerably less than the 55 to 94% inhibition of hypertonicity-induced
proton secretion seen in CHO-K1 cells exposed to various CaM inhibitors
(Garnovskaya et al., 2003b).
Compared with the severe reduction in alkalinization, indicating that a
significant amount of protons remained in the cytoplasm during hypertonic
stress in the presence of calmidazolium, the reduction in the rate of proton
secretion was smaller than might be expected. This could indicate that again a
proton movement between intracellular compartments and the cytoplasm is
involved under these conditions (see above). A point that needs to be
clarified regarding the action of CaM in trout hepatocytes is, whether it
directly acts on NHE-1 via CaM binding sites at the cytoplasmic tail
of the protein (Wakabayashi et al.,
1997a; Garnovskaya et al.,
2003b; Orlowski and Grinstein,
2004), or if it affects NHE-1 via stimulation of
CaM-dependent kinases, as shown in an astrocyte cell model
(Shrode et al., 1997).
Interestingly, the CaM-dependent myosin light chain kinase involved in NHE-1
stimulation in these cells provides a link between antiporter activity and the
cytoskeleton. A similar interaction might thus underlie the pronounced
inhibition of NHE-1 we have recently observed in trout hepatocytes upon
disruption of the cytoskeleton (Ebner et
al., 2005).
pH-dependence of the NHE under hyperosmotic conditions
Intracellularly two proton binding sites have been identified, one involved
in the transfer of protons, whereas the other binding site appears to be an
allosteric binding site modifying the transport characteristics of NHE-1
(Wakabayashi et al., 1992).
Accordingly, NHE-1 has been shown to be sensitive to changes in pHi, and a
reduced pHi activates the protein
(Grinstein et al., 1985a;
Takaichi et al., 1993;
Bianchini et al., 1995). The pH
dependence of NHE in trout hepatocytes has only been investigated in isotonic
conditions, showing, at acutely altered pHi or pHe, a decrease in NHE activity
when pHi was increased, and an increase when pHe was elevated
(Fossat et al., 1997). This
reflects that NHE activity of trout cells, similar to that of other species
(Grinstein et al., 1985b), is
independently affected by extra- and intracellular protons. In our
experiments, hepatocytes were allowed to adjust their pHi to altered pHe
conditions before hyperosmotic challenge, which, as expected, resulted in an
increase and decrease of basal pHi at elevated and diminished pHe,
respectively. Under these conditions, hypertonic NHE activity was reduced both
at higher and lower pHe when compared to that seen in our standard pH 7.6
saline. Stimulation and inhibition by intracellular and extracellular protons,
respectively, thus results in a rather complex activation pattern of NHE by
hypertonicity, which we believe would be best explained by assuming a pH
optimum for the antiporter under these conditions. In rat astrocytes
(Shrode et al., 1997) and rat
mesangial cells (Bevensee et al.,
1999) a shift of the pHi dependence of NHE to more alkaline values
under hyperosmotic conditions was observed, and according to these authors
this shift may explain why cell shrinkage causes steady-state pHi to increase.
Nevertheless, at alkaline pH the activity of the NHE typically was reduced,
and a saturation of NHE activity in both iso- and hyperosmotic saline at pHe
above 7.8 has previously been documented for rat lymphocytes
(Grinstein et al., 1985b).
PKC- and PKA-dependent pathways are involved in the hypertonicity-induced activation of NHE
Evidence about the involvement of PKC in hypertonicity-induced activation
of NHE is contradictory in different cell types. In a study on Ehrlich ascites
tumour cells, the PKC inhibitor chelerythrine inhibited hypertonicity-induced
amiloride-sensitive pHi increase (Pedersen
et al., 1996; Pedersen et al.,
2002), whereas in rat bone cells
(Dascalu et al., 1992), rat
astrocytes (Shrode et al.,
1995), rat hepatocytes
(Heinzinger et al., 2001) and
CHO-K1 cells (Garnovskaya et al.,
2003b) no PKC-dependent hypertonicity-induced activation of NHE
was noted. In our study, the concentration-dependent decrease in basal pHi
upon addition of 1 or 5 µmol l-1 (and 10 or 30 µmol
l-1, not shown) chelerythrine
(Fig. 5A), indicates a
potential role for PKC in the steady-state pHi regulation in trout
hepatocytes. Importantly, the decrease of pHi was accompanied by an increase
in proton secretion, indicating that not only NHE activity but also other pHi
regulatory mechanisms are influenced by the activity of PKC, some of which
appear to be stimulated upon PKC inhibition. This conclusion was supported by
the observation that the decrease in pHi after addition of chelerythrine
exceeded the intracellular acidification observed after complete inhibition of
NHE by EIPA, and by the fact that even after blockade of NHE chelerythrine was
still able to reduce pHi (Gende,
1996). In addition, we observed that following the addition of
chelerythrine, oxygen consumption of trout hepatocytes is increased by up to a
factor of two within 15 min and this stimulation persists for at least 30 min
(unpublished). Although the underlying mechanism is not clear at present, this
enhanced metabolic activity could have contributed to elevated proton
production. Under hypertonic conditions chelerythrine completely inhibited the
intracellular alkalinization in a dosedependent manner, but at the same time
it again increased the rate of proton secretion. As stated before, this can be
explained by assuming a stimulation of proton production within the cell due
to the inhibition of PKC. Secretion of these protons would result in a
constant intracellular pH and a lowering of extracellular pH. This again
suggests that besides possible membrane effects inhibition of PKC affected
intracellular mechanisms involved in the acid-base homeostasis. Pedersen et
al. (Pedersen et al., 1996;
Pedersen et al., 2002),
however, interpreted the reduction in the hypertonicity-induced increase in
pHi by chelerythrine as an attenuation of NHE activity in Ehrlich ascites
tumour cells. At higher concentrations (30 µmol l-1)
chelerythrine completely blocked the hypertonicity-induced proton secretion
rate, but intracellular pH measurements indicated a reduced viability of the
cells, and therefore these data were not included in this study. Negative
effects of higher concentrations of chelerythrine on the viability of cells
have also been reported (Zhang et al.,
2005). However, the specificity of chelerythrine has been
questioned (Davies et al.,
2000). The dose-dependent effect observed in the lower
concentration range in our study suggests, however, that unspecific side
effects did not affect our results.
Upon pharmacological stimulation, the activation of PKA by increased cAMP
levels has been shown to decrease activity of NHE-3
(Kandasamy et al., 1995;
Moe et al., 1995) and NHE-5
(Attaphitaya et al., 2001),
while the activity of NHE-1 and NHE-2 was enhanced
(Kandasamy et al., 1995). In
line with the first observation inhibition of PKA by decreasing cAMP
production has been shown to increase NHE activity
(Saccomani et al., 1990). In
contrast, in hyperosmotic conditions PKA is apparently not involved in the
activation of NHE, as cell shrinkage often, although not universally
(Orlic et al., 2002), fails to
raise cAMP concentrations (Kregenow et
al., 1976; Shrode et al.,
1995) and even pharmacological elevation of cAMP was found to be
without effect in several mammalian cell types
(Dascalu et al., 1992;
Shrode et al., 1995). However,
our present data indicate that, in trout hepatocytes, PKA appears to
contribute to the activation of NHE-1, both under steady-state conditions as
well as in response to hyperosmotic stress. Thus, the addition of the PKA
inhibitor H-89 decreased steady-state pHi, reduced the hypertonicity-induced
pHi increase by 58% (Fig. 6A),
and attenuated the hypertonicity-induced proton secretion rate by 41%
(Fig. 6B). This different
responsiveness of trout and mammalian NHE-1 is consistent with molecular
features of the antiporter. The cytoplasmic domain of the mammalian NHE-1
homologues, which is the main site of NHE-1 regulation, lacks PKA consensus
sites, whereas in teleosts these domains appear to be consistently present
(Pedersen and Cala, 2004).
Whether the osmotic activation in trout hepatocytes involves a direct
phosphorylation of NHE-1 remains to be determined. In winter flounder,
cAMP-mediated stimulation of NHE-1 caused PKA-sensitive phosphorylation of the
antiporter (Pedersen and Cala,
2004), whereas direct phosphorylation appeared to play no role in
hyperosmotic activation (Pedersen et al.,
2003).
Involvement of PKC-, PKA- and CaM-dependent pathways in the acid loading-induced pH regulation mechanism
Although the dependence of NHE activity on pHi or pHe during cell recovery
from acidification has been studied in great detail
(Grinstein et al., 1985a;
Takaichi et al., 1993;
Bianchini et al., 1995;
Bevensee et al., 1999),
comparatively little is known regarding the role of other factors possibly
contributing to NHE regulation under this condition. In general, the
stimulation of NHE upon acidification is believed to reflect its activation by
cytoplasmic H+, which interact with a H+ sensor site at
the N-terminal transmembranous region of the antiporter
(Wakabayashi et al., 1992).
The concentration of intracellular H+ thus appears to be the
primary determinant of NHE activity. In addition, however, some studies have
documented alterations of amiloride-dependent pHi recovery in the presence of
a CaM antagonist, of PKC stimulatory agents, or of elevated cAMP, all of which
appear to act by altering the pH sensitivity of NHE
(Moule and McGivan, 1990;
Takaichi et al., 1993;
Kandasamy et al., 1995).
Our present data showed that, in line with previous data
(Walsh, 1986;
Furimsky et al., 1999;
Krumschnabel et al., 2001b;
Tuominen et al., 2003), in
trout hepatocytes pHi recovery following sodium propionate addition is
governed by NHE and a SITS-sensitive mechanism, presumably a
Na+-dependent Cl-/HCO -3
exchanger. Specifically, we observed that using cariporide and SITS alone
reduced pHi recovery by 61% and 33%, respectively, and upon using both
inhibitors simultaneously, the reduction in proton secretion rate (93%) was
almost equal to the sum of their individual inhibition
(Fig. 9A,B). Thus, the main
share of alkalinization was due to NHE-1 activity, and the impact on pHi
recovery of the various agents applied will therefore largely reflect their
effect on NHE-1 activity. In principle, all measurements could have been
conducted with SITS present, but since SITS significantly reduced the resting
pHi of the cells and could thereby have affected the action of other
inhibitors tested it was omitted from the subsequent experiments.
In our experiments examining the interactive effect of, on the one hand,
pHe and spontaneously adjusted pHi levels and, on the other hand, pHi recovery
after an acidic load, we obtained results indicating a positive correlation
between these parameters (Fig.
11), similar to findings on rat lymphocytes
(Grinstein et al., 1985b). As
outlined above, regarding the impact of pHi, this differs from previous
findings, where, at constant pHe, NHE activity decreased with an increase of
pHi (Fossat et al., 1997). By
contrast, the increase of NHE activity with increasing pHe would be in line
with the study of Fossat et al. (Fossat et
al., 1997). Together, this seems to suggest that when pHi is
allowed to adjust to the prevailing pHe, the latter is a quite strong
determinant of acidification-induced NHE activity. Although at first glance
this may seem to contradict the well known pivotal importance of intracellular
protons as a major determinant of NHE activity, it may also reflect an
additive effect of stimulation by intracellular protons and the favourable
proton gradient prevailing at high pHe.
Besides the impact of pHe, we observed that inhibition of PKA, and CaM to a
some extent (though not significant), affected NHE activity by reducing pHi
recovery (Fig. 11). The same
may be true for inhibition of PKC by chelerythrine, with the possibility that
PKC inhibition might have activated other hydrogen-producing mechanisms
acidifying the cell, as discussed in the previous section. These findings were
surprising in the light of previous reports showing that addition of agents
stimulating these pathways produced an acceleration of NHE activity, which was
additive to, and thus presumably independent from, the stimulation of NHE
activity by acidification (Moule and
McGivan, 1990; Kandasamy et
al., 1995). By contrast, our data suggest that these pathways
contribute to pHi recovery without prior stimulation of PKA, CaM or PKC. This
would imply that these pathways are, at least to some extent, already
stimulated during acidification. To our knowledge, this has not yet been
tested explicitly, but previous studies found enhanced PKC stimulation of
NHE-1 in acidified outer hair cells (Ikeda
et al., 1996), but also reduced cAMP responses to isoproterenol
and ionomycin in rat pinealocytes at low pHi
(Ho et al., 1992).
In summary our study provides evidence that, in trout hepatocytes, the NHE-1 isoform of the Na+/H+ exchanger is the main mechanism mediating pH changes under hypertonic stress and following acidification. Similar to other cells, NHE-1 activity is sensitive to alterations of pHe and pHi, with somewhat different activation profiles detectable under both conditions. Furthermore, hypertonic NHE-1 activation is partly dependent on Ca2+, which differs from most mammalian cells, where Ca2+ is mainly important in response to mitotic stimuli. Other signalling pathways contributing to activation of trout hepatocyte NHE-1 involve PKA, PKC and, at least in hypertonic conditions, CaM. The role of these pathways in hypertonic NHE-1 activation agrees with findings on many, although not all, mammalian cells. In contrast, their contribution to NHE-1-mediated acid recovery has, to our knowledge, not yet been documented in mammalian cells.
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Attaphitaya, S., Nehrke, K. and Melvin, J. E. (2001). Acute inhibition of brain-specific Na(+)/H(+) exchanger isoform 5 by protein kinases A and C and cell shrinkage. Am. J. Physiol. 281,C1146 -C1157.
Bertrand, B., Wakabayashi, S., Ikeda, T., Pouyssegur, J. and
Shigekawa, M. (1994). The Na+/H+ exchanger isoform 1 (NHE1)
is a novel member of the calmodulin-binding proteins. Identification and
characterization of calmodulin-binding sites. J. Biol.
Chem. 269,13703
-13709.
Bevensee, M. O., Bashi, E., Schlue, W. R., Boyarsky, G. and Boron, W. F. (1999). Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells. Am. J. Physiol. 276,C674 -C683.
Bianchini, L., Kapus, A., Lukacs, G., Wasan, S., Wakabayashi, S., Pouyssegur, J., Yu, F. H., Orlowski, J. and Grinstein, S. (1995). Responsiveness of mutants of NHE1 isoform of Na+/H+ antiport to osmotic stress. Am. J. Physiol. 269,C998 -C1007.
Brainer, J. C., Wang, T. and Jensen, F. B. (2002). Influence of hyperosmotic shrinkage and beta-adrenergic stimulation on red blood cell volume regulation and oxygen binding properties in rainbow trout and carp. J. Comp. Physiol. 172,251 -262.
Dascalu, A., Nevo, Z. and Korenstein, R.
(1992). Hyperosmotic activation of the
Na(+)-H+ exchanger in a rat bone cell line: temperature
dependence and activation pathways. J. Physiol.
456,503
-518.
Davies, S. P., Reddy, H., Caivano, M. and Cohen, P. (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351,95 -105.[CrossRef][Medline]
Dunham, P. B., Kelley, S. J. and Logue, P. J.
(2004). Extracellular Na+ inhibits
Na+/H+ exchange: cell shrinkage reduces the inhibition.
Am. J. Physiol. Cell Physiol.
287,C336
-C344.
Ebner, H. L., Cordas, A., Pafundo, D. E., Schwarzbaum, P. J., Pelster, B. and Krumschnabel, G. (2005). Importance of cytoskeletal elements in volume regulatory responses of trout hepatocytes. Am. J. Physiol. 289,R877 -R890.
Fossat, B., PortheNibelle, J., Pedersen, S. and Lahlou, B. (1997). Na+/H+ exchange and osmotic shrinkage in isolated trout hepatocytes. J. Exp. Biol. 200,2369 -2376.[Abstract]
Furimsky, M., Moon, T. W. and Perry, S. F. (1999). Intracellular pH regulation in hepatocytes isolated from three teleost species. J. Exp. Zool. 284,361 -367.[CrossRef][Medline]
Furimsky, M., Moon, T. W. and Perry, S. F. (2000). Evidence for the role of a Na(+)/HCO(3)(-) cotransporter in trout hepatocyte pHi regulation. J. Exp. Biol. 203,2201 -2208.[Abstract]
Garnovskaya, M. N., Mukhin, Y. V., Turner, J. H., Vlasova, T. M., Ullian, M. E. and Raymond, J. R. (2003a). Mitogen-induced activation of Na+/H+ exchange in vascular smooth muscle cells involves janus kinase 2 and Ca2+/calmodulin. Biochemistry 42,7178 -7187.[CrossRef][Medline]
Garnovskaya, M. N., Mukhin, Y. V., Vlasova, T. M. and Raymond,
J. R. (2003b). Hypertonicity activates
Na+/H+ exchange through Janus kinase 2 and calmodulin.
J. Biol. Chem. 278,16908
-16915.
Gende, O. A. (1996). Chelerythrine inhibits
Na(+)-H+ exchange in platelets from spontaneously
hypertensive rats. Hypertension
28,1013
-1017.
Grinstein, S., Cohen, S., Goetz, J. D., Rothstein, A. and
Gelfand, E. W. (1985a). Characterization of the activation of
Na+/H+ exchange in lymphocytes by phorbol esters: change
in cytoplasmic pH dependence of the antiport. Proc. Natl. Acad.
Sci. USA 82,1429
-1433.
Grinstein, S., Rothstein, A. and Cohen, S.
(1985b). Mechanism of osmotic activation of
Na+/H+ exchange in rat thymic lymphocytes. J.
Gen. Physiol. 85,765
-787.
Grinstein, S., Woodside, M., Sardet, C., Pouyssegur, J. and
Rotin, D. (1992). Activation of the
Na+/H+ antiporter during cell volume regulation.
Evidence for a phosphorylation-independent mechanism. J. Biol.
Chem. 267,23823
-23828.
Grynkiewicz, G., Poenie, M. and Tsien, R. Y.
(1985). A new generation of Ca2+ indicators with greatly improved
fluorescence properties. J. Biol. Chem.
260,3440
-3450.
Haussinger, D. and Schliess, F. (1995). Cell volume and hepatocellular function. J. Hepatol. 22, 94-100.[CrossRef][Medline]
Heinzinger, H., van den Boom, F., Tinel, H. and Wehner, F.
(2001). In rat hepatocytes, the hypertonic activation of
Na(+) conductance and
Na(+)-K(+)-2Cl(-) symport -but not
Na(+)-H(+) antiport -is mediated by protein kinase C.
J. Physiol. 536,703
-715.
Ho, A. K., O'Brien, L., Girard, M. and Chik, C. L. (1992). Intracellular pH on protein kinase C and ionomycin potentiation of isoproterenol-stimulated cyclic AMP and cyclic GMP production in rat pinealocytes. J. Neurochem. 59,2304 -2310.[Medline]
Ikeda, K., Sunose, H. and Takasaka, T. (1996). Effects of protein kinase C on the Na(+)-H+ exchange in the cochlear outer hair cell. Acta Otolaryngol. 116,828 -832.[Medline]
Kandasamy, R. A., Yu, F. H., Harris, R., Boucher, A., Hanrahan,
J. W. and Orlowski, J. (1995). Plasma membrane
Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are
differentially responsive to second messenger agonists of the protein kinase A
and C pathways. J. Biol. Chem.
270,29209
-29216.
Kapus, A., Grinstein, S., Wasan, S., Kandasamy, R. and Orlowski,
J. (1994). Functional characterization of three isoforms of
the Na+/H+ exchanger stably expressed in Chinese hamster
ovary cells. ATP dependence, osmotic sensitivity, and role in cell
proliferation. J. Biol. Chem.
269,23544
-23552.
Kregenow, F. M., Robbie, D. E. and Orloff, J.
(1976). Effect of norepinephrine and hypertonicity on K influx
and cyclic AMP in duck erythrocytes. Am. J. Physiol.
231,306
-311.
Krump, E., Nikitas, K. and Grinstein, S.
(1997). Induction of tyrosine phosphorylation and
Na+/H+ exchanger activation during shrinkage of human
neutrophils. J. Biol. Chem.
272,17303
-17311.
Krumschnabel, G., Biasi, C., Schwarzbaum, P. J. and Wieser, W. (1996). Membrane-metabolic coupling and ion homeostasis in anoxia-tolerant and anoxia-intolerant hepatocytes. Am. J. Physiol. 270,R614 -R620.
Krumschnabel, G., Gstir, R., Manzl, C., Prem, C., Pafundo, D.
and Schwarzbaum, P. J. (2003). Metabolic and ionic responses
of trout hepatocytes to anisosmotic exposure. J. Exp.
Biol. 206,1799
-1808.
Krumschnabel, G., Manzl, C. and Schwarzbaum, P. J. (2001a). Metabolic responses to epinephrine stimulation in goldfish hepatocytes: Evidence for the presence of alpha-adrenoceptors. Gen. Comp. Endocrinol. 121,205 -213.[CrossRef][Medline]
Krumschnabel, G., Manzl, C. and Schwarzbaum, P. J. (2001b). Regulation of intracellular pH in anoxia-tolerant and anoxia-intolerant teleost hepatocytes. J. Exp. Biol. 204,3943 -3951.
Malapert, M., Guizouarn, H., Fievet, B., Jahns, R., GarciaRomeu, F., Motais, R. and Borgese, F. (1997). Regulation of Na+/H+ antiporter in trout red blood cells. J. Exp. Biol. 200,353 -360.[Abstract]
Maly, K., Strese, K., Kampfer, S., Ueberall, F., Baier, G., Ghaffari-Tabrizi, N., Grunicke, H. H. and Leitges, M. (2002). Critical role of protein kinase C alpha and calcium in growth factor induced activation of the Na(+)/H(+) exchanger NHE1. FEBS Lett. 521,205 -210.[CrossRef][Medline]
Martin-Requero, A., Daza, F. J., Hermida, O. G., Butta, N. and Parrilla, R. (1997). Role of Ca2+ and protein kinase C in the receptor-mediated activation of Na+/H+ exchange in isolated liver cells. Biochem. J. 325,631 -636.
Mitsuhashi, T. and Ives, H. E. (1988).
Intracellular Ca2+ requirement for activation of the
Na+/H+ exchanger in vascular smooth muscle cells.
J. Biol. Chem. 263,8790
-8795.
Moe, O. W., Amemiya, M. and Yamaji, Y. (1995). Activation of protein kinase A acutely inhibits and phosphorylates Na/H exchanger NHE-3. J. Clin. Invest. 96,2187 -2194.[Medline]
Moor, A. N. and Fliegel, L. (1999). Protein
kinase-mediated regulation of the Na(+)/H(+) exchanger
in the rat myocardium by mitogen-activated protein kinase-dependent pathways.
J. Biol. Chem. 274,22985
-22992.
Moule, S. K. and McGivan, J. D. (1990). Epidermal growth factor and cyclic AMP stimulate Na+/H+ exchange in isolated rat hepatocytes. Eur. J. Biochem. 187,677 -682.[Medline]
Murao, H., Shimizu, A., Hosoi, K., Iwagaki, A., Min, K. Y.,
Kishima, G., Hanafusa, T., Kubota, T., Kato, M., Yoshida, H. et al.
(2005). Cell shrinkage evoked by Ca2+-free solution in
rat alveolar type II cells: Ca2+ regulation of
Na+-H+ exchange. Exp. Physiol.
90,203
-213.
Nakamura, N., Tanaka, S., Teko, Y., Mitsui, K. and Kanazawa,
H. (2005). Four Na+/H+ exchanger isoforms are distributed to
Golgi and post-Golgi compartments and are involved in organelle pH regulation.
J. Biol. Chem. 280,1561
-1572.
Orlic, T., Loomis, W. H., Shreve, A., Namiki, S. and Junger, W. G. (2002). Hypertonicity increases cAMP in PMN and blocks oxidative burst by PKA-dependent and -independent mechanisms. Am. J. Physiol. 282,C1261 -C1269.
Orlowski, J. and Grinstein, S. (2004). Diversity of the mammalian sodium/proton exchanger SLC9 gene family. Pflugers Arch. 447,549 -565.[CrossRef][Medline]
Pedersen, S. F. and Cala, P. M. (2004). Comparative biology of the ubiquitous Na+/H+ exchanger, NHE1: lessons from erythrocytes. J. Exp. Zool. A 301,569 -578.
Pedersen, S. F., Kramhoft, B., Jorgensen, N. K. and Hoffmann, E. K. (1996). Shrinkage-induced activation of the Na+/H+ exchanger in Ehrlich ascites tumor cells: mechanisms involved in the activation and a role for the exchanger in cell volume regulation. J. Membr. Biol. 149,141 -159.[CrossRef][Medline]
Pedersen, S. F., Varming, C., Christensen, S. T. and Hoffmann, E. K. (2002). Mechanisms of activation of NHE by cell shrinkage and by calyculin A in ehrlich ascites tumor cells. J. Membr. Biol. 189,67 -81.[CrossRef][Medline]
Pedersen, S. F., King, S. A., Rigor, R. R., Zhuang, Z., Warren,
J. M. and Cala, P. M. (2003). Molecular cloning of NHE1 from
winter flounder RBCs: activation by osmotic shrinkage, cAMP, and calyculin A.
Am. J. Physiol. Cell Physiol.
284,C1561
-C1576.
Pelster, B. (1995). Mechanisms of acid release in isolated gas gland-cells of the European eel Anguilla anguilla.Am. J. Physiol. 38,R793 -R799.
Pocock, G. and Richards, C. D. (1992). Hydrogen ion regulation in rat cerebellar granule cells studied by single-cell fluorescence microscopy. Eur. J. Neurosci. 4, 136-143.[CrossRef][Medline]
Putney, L. K., Denker, S. P. and Barber, D. L. (2002). The changing face of the Na+/H+ exchanger, NHE1: structure, regulation, and cellular actions. Annu. Rev. Pharmacol. Toxicol. 42,527 -552.[CrossRef][Medline]
Ritter, M., Fuerst, J., Woll, E., Chwatal, S., Gschwentner, M., Lang, F., Deetjen, P. and Paulmichl, M. (2001). Na(+)/H(+) exchangers: linking osmotic dysequilibrium to modified cell function. Cell Physiol. Biochem. 11, 1-18.[Medline]
Ruiz-Meana, M., Garcia-Dorado, D., Pina, P., Inserte, J., Agullo, L. and Soler-Soler, J. (2003). Cariporide preserves mitochondrial proton gradient and delays ATP depletion in cardiomyocytes during ischemic conditions. Am. J. Physiol. 285,H999 -H1006.
Saccomani, G., Mitchell, K. D. and Navar, L. G. (1990). Angiotensin II stimulation of Na(+)-H+ exchange in proximal tubule cells. Am. J. Physiol. 258,F1188 -F1195.
Seo, J. T., Steward, M. C., Larcombe-McDouall, J. B., Cook, L. J. and Case, R. M. (1994). Continuous fluorometric measurement of intracellular pH and Ca2+ in perfused salivary gland and pancreas. Pflugers Arch. 426, 75-82.[CrossRef][Medline]
Shrode, L. D., Klein, J. D., O'Neill, W. C. and Putnam, R. W. (1995). Shrinkage-induced activation of Na+/H+ exchange in primary rat astrocytes: role of myosin light-chain kinase. Am. J. Physiol. 269,C257 -C266.
Shrode, L. D., Klein, J. D., Douglas, P. B., O'Neill, W. C. and Putnam, R. W. (1997). Shrinkage-induced activation of Na+/H+ exchange: role of cell density and myosin light chain phosphorylation. Am. J. Physiol. 272,C1968 -C1979.
Takaichi, K., Balkovetz, D. F., Van Meir, E. and Warnock, D. G. (1993). Cytosolic pH sensitivity of an expressed human NHE-1 Na(+)-H+ exchanger. Am. J. Physiol. 264,C944 -C950.
Tuominen, A., Rissanen, E., Bogdanova, A. and Nikinmaa, M. (2003). Intracellular pH regulation in rainbow trout (Oncorhynchus mykiss) hepatocytes: the activity of sodium/proton exchange is oxygen-dependent. J. Comp. Physiol. B 173,301 -308.[CrossRef][Medline]
Wakabayashi, S., Fafournoux, P., Sardet, C. and Pouyssegur,
J. (1992). The Na+/H+ antiporter
cytoplasmic domain mediates growth factor signals and controls
`H(+)-sensing'. Proc. Natl. Acad. Sci. USA
89,2424
-2428.
Wakabayashi, S., Bertrand, B., Ikeda, T., Pouyssegur, J. and
Shigekawa, M. (1994). Mutation of calmodulin-binding site
renders the Na+/H+ exchanger (NHE1) highly
H(+)-sensitive and Ca2+ regulation-defective.
J. Biol. Chem. 269,13710
-13715.
Wakabayashi, S., Ikeda, T., Iwamoto, T., Pouyssegur, J. and Shigekawa, M. (1997a). Calmodulin-binding autoinhibitory domain controls `pHsensing' in the Na+/H+ exchanger NHE1 through sequence-specific interaction. Biochemistry 36,12854 -12861.[CrossRef][Medline]
Wakabayashi, S., Shigekawa, M. and Pouyssegur, J.
(1997b). Molecular physiology of vertebrate
Na+/H+ exchangers. Physiol. Rev.
77, 51-74.
Walsh, P. J. (1986). Ionic requirements for intracellular pH regulation in rainbow-trout hepatocytes. Am. J. Physiol. 250,R24 -R29.
Walsh, P. J. and Mommsen, T. P. (1992). Intracellular pH regulation and metabolic interactions in hepatic tissues. J. Exp. Zool. 263,176 -185.[CrossRef][Medline]
Zavoico, G. B., Cragoe, E. J., Jr and Feinstein, M. B.
(1986). Regulation of intracellular pH in human platelets.
Effects of thrombin, A23187, and ionomycin and evidence for activation of
Na+/H+ exchange and its inhibition by amiloride analogs.
J. Biol. Chem. 261,13160
-13167.
Zhang, Y. H., Alvarez de la Rosa, D., Canessa, C. M. and Hayslett, J. P. (2005). Insulin-induced phosphorylation of ENaC correlates with increased sodium channel function in A6 cells. Am. J. Physiol. 288,C141 -C147.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  K. H. Ahmed and B. Pelster Ionic determinants of pH of acidic compartments under hypertonic conditions in trout hepatocytes J. Exp. Biol., October 15, 2008; 211(20): 3306 - 3314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. H. Ahmed and B. Pelster Interdependence of Ca2+ and proton movements in trout hepatocytes J. Exp. Biol., October 1, 2007; 210(19): 3473 - 3483. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
40 cells from 