Cadmium induces both pyruvate kinase and Na+/H+ exchanger activity through protein kinase C mediated signal transduction, in isolated digestive gland cells of Mytilus galloprovincialis (L.)
Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki 54124, Greece
* Author for correspondence (e-mail: Kaloyian{at}bio.auth.gr)
Received for publication 5 February 2004.
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Summary |
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Key words: mussel, Mytilus galloprovincialis, digestive gland, Na+/H+ exchanger, cadmium, pyruvate kinase, signal transduction
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Introduction |
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Although cellular metabolism has been investigated in marine species
(Almeida et al., 2001), the
consequences of cadmium effects have been little studied
(Watjen et al., 2001
;
Fabbri et al., 2003
). There
are a few data concerning the role of Hg, Cu, Zn and Cd on the regulation of
metabolic rates in the cell and specifically on key glycolytic enzymes
(Lai and Blass, 1984
; Canesi
et al., 1998
,
2001
;
Huang and Tao, 2001
;
Viselina and Lukyanova, 2000
).
Nikinmaa (1983
), Borgese et
al. (1987
) and Kaloyianni et
al. (2000
) reported that the
activation of pyruvate kinase (PK), a key glycolytic enzyme, observed after
adrenergic stimulation was related to Na+/H+ exchanger
activity in vertebrate red cells. It was therefore of interest to investigate
the effect of cadmium on Na+/H+ exchanger and PK
activities in mussel cells.
The Na+/H+ exchanger is a membrane system involved in
the coupled exchange of Na+ with H+ in a variety of
eukaryotic cells. This system seems to be a major regulating element of
intracellular pH (pHi) (Moolenaar et al.,
1983; Bianchini and Pouyssegur,
1994
) and cell volume control (Cala,
1983a
,b
).
The activity of this cation exchanger is regulated by growth factors, hormones
(Ceolotto et al., 1997
;
Rutherford et al., 1997
;
Kaloyianni et al., 1997
,
2000
,
2001
;
Sauvage et al., 2000
;
Konstantinou-Tegou et al.,
2001
; Bourikas et al.,
2003
), second messengers (Tse
et al., 1993
), neurotransmitters
(Zange et al., 1990
) and
osmotic stress (Grinstein et al.,
1992
). This antiport is involved in signal transduction since
intracellular pH changes have been closely related to signaling
(Incerpi etal., 1996
).
Furthermore, other transporters such as the
Na+-K+-2Cl cotransporter are related
to cell signaling (Flatman,
2002
). In mussel digestive gland cells no
Na+/H+ exchanger function has been reported. The effect
of heavy metals on Na+/H+ exchanger function has only
recently been mentioned (Villela et al.,
1999
). To our knowledge, in invertebrate cells,
Na+/H+ exchanger function has been reported
(Zange et al., 1990
;
Willoughby et al., 1999
;
Giannakou and Dow, 2001
) but
not in relation to heavy metals.
Pyruvate kinase (PK) is one of the key enzymes of the glycolytic pathway
(Storey and Storey, 1990) and
catalyzes the conversion of phosphoenolpyruvate to pyruvate in one of the two
ATP-producing steps in glycolysis. In mollusc tissues, pyruvate kinase
activity is controlled by FDP, ATP and L-alanine, as well as ATP,
cAMP and Ca2+ (Holwerda et al.,
1989
). In addition, molluscan pyruvate kinase is regulated by the
phosphorylation/dephosphorylation process
(Marie et al., 1979
;
Siebenaller 1979
;
Kiener and Westhead, 1980
;
Nakashima et al., 1982
;
Hakim et al., 1984
;
Holwerda et al., 1989
; Carillo
et al., 2001). Moreover, there are many physiological factors such as
temperature, anaerobic conditions, tidal cycle, seasonal and ion concentration
variations that can alter pyruvate kinase activity in molluscs
(Cortesi and Carpene, 1981
;
Carpene et al., 1984
;
Hakim et al., 1984
;
Cortesi et al., 1985
;
Ibarguren et al., 1990
;
Simpfendorfer et al.,
1997
).
Recent data indicate that zinc and cadmium are involved in modulating
signal transduction pathways (Hansson,
1996; Risso-de Faverney et
al., 2001
; Watjen et al.,
2001
). In mussels, there are reports on the effect of heavy metals
on cell signaling (Canesi et al.,
1998
,
2000a
,
2001
;
Viarengo et al., 2000
), so we
investigated the role of the toxic metal cadmium in isolated digestive gland
cells of Mytilus galloprovincialis.
Since the Na+/H+ exchanger is affected by hormones
(Borgese et al., 1987) and PK
is also hormonally regulated in fish cells
(Nikinmaa, 1983
) it was of
interest to investigate if the two proteins are influenced by common signal
transduction pathways. The present study investigates the possible effects of
cadmium on the Na+/H+ exchanger, as well as on pyruvate
kinase (PK) activity in digestive gland cells of the mussel Mytilus
galloprovincialis. Both PK and Na+/H+ exchanger
were studied in relation to ß1, ß2 and
1-adrenergic receptor stimulation and protein kinase C (PKC)
activation, in order to clarify the transduction pathway induced by cadmium in
the isolated digestive gland cells of Mytilus galloprovincialis.
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Materials and methods |
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Mussels, 56 cm long, were transferred from Kalamaria to the
laboratory and maintained in static tanks containing filtered natural seawater
(3540% salinity) for 7 days at 15°C. The seawater was changed every
2 days. During the adaptation period, the animals were not fed. The
concentrations of cadmium used in the present study ranged from 0.05 to 500
µmol l1, the lower concentration being close to that
found in Thermaikos Gulf (EKTHE
Oceanographic Institute, 1997).
Isolation of digestive cells
Digestive glands from 35 animals were cut into pieces and washed
with buffer without added Ca and Mg (CMFS; 1100 mOsm, pH 7.3, containing 20
mmol l1 Hepes buffer, 500 mmol l1 NaCl,
12.5 mmol l1 KCl, 5 mmol l1 EDTA). Tissue
samples were cut with scissors into small pieces and transferred to a flask
containing 15 ml dissociating solution (0.01% collagenase type CLS IV, 175 U
mg1, Biochrom AG, Berlin, Germany) in CMFS, followed by
gentle stirring for 60 min at 15°C. After filtration of the cell
suspension through 250 µm and 60 µm diameter nylon filters, the
procedure as described by Canesi et al.
(1997) was followed. Cell
suspensions, maintained in Leibovitz L-15 medium (supplemented with 350 mmol
l1 NaCl, 7 mmol l1 KCl, 4 mmol
l1 CaCl2, 8 mmol l1
MgSO4 and 40 mmol l1 MgCl2), were kept
at 15°C for at least 3 h before being used for experiments. After the
tissue treatment, cell viability was tested using Eosin exclusion and was
about 83%. Subsequently, cell viability was tested after incubation with
various concentrations of cadmium (0.05500 µmol
l1) and 98% of the cells remained intact. Concentrations of
cadmium higher than 500 µmol l1 resulted in a 50% cell
death.
Isolated cells were incubated with 0.05500 µmol l1 of cadmium for 30 min in the presence or absence of inhibitors or activators as mentioned in the Results. Preliminary experiments showed that adrenaline at the range of 5x109 mol l1 caused maximum stimulation of pHi.
Pyruvate kinase activity determination
1 ml of cell suspension (approx. 106 cells
ml1) was exposed to different concentrations of cadmium
chloride (CdCl2) and/or other substrates for 30 min. Samples were
removed at 0 and 30 min. The cell suspension was centrifuged at 160
g for 5 min, washed with L-15 medium, and the precipitate
representing the packed cells was lysed by the addition of 4 volumes of 20
mmol l1 imidazole/HCl buffer, pH 7.2 containing 10 mmol
l1 EGTA, 10 mmol l1 EDTA, 0.1 mmol
l1 phenyl-methyl-sulfonyl-fluoride (PMSF) and 15 mmol
l1 ß-mercaptoethanol. The suspension was subsequently
sonicated for 6x10 s in an MS E Sonicator (Soniprep 150 MSE, TCP Inc.,
NJ, USA), followed by centrifugation at 2400 g for 30 min at
4°C. The supernatant (200400 µl) was passed through a 5 ml
column of Sephadex G-25, equilibrated in 40 mmol l1
imidazole-HCl buffer (pH 7.1) containing 5 mmol l1 EDTA, 15
mmol l1 ß-mercaptoethanol and 20% (v/v) glycerol, in
order to remove low-molecular-mass metabolites
(Helmerhost and Stokes, 1980).
The columns were centrifuged for 1 min and the filtrates (100150 µl)
were used as a source of enzyme. PK activity was determined
spectrophotometrically, as described by Ward et al.
(1969
). Optimal assays
conditions for PK were 20 mmol l1 imidazole-HCl (pH 7.2), 2
mmol l1 ADP, 5 mmol l1 MgCl, 20 mmol
l1 KCl, 0.15 mmol l1 NADH, 1.5 mmol
l1 phosphoenol pyruvate (PEP) and 1 U ml1
LDH. Protein content was determined using bicinchoninic acid (BCA) protein
assay reagent protocol using bovine serum albumin (BSA) as standard
(Sorensen and Brodbeck,
1986
).
Enzyme activities are means ± S.D. for 7
experiments and are expressed as percentage (%) of control value. Enzyme
activity of control cells was 5.23±1.04 nmol min mg1
protein, which is in the same range as measured in a previous study
(Canesi et al., 1999).
Determination of intracellular pH
The intracellular pH was measured using a modification of the experimental
procedure as reported by Incerpi et al.
(1996). Digestive gland cells
suspended in physiological saline buffer (PS; 1100 mOsm, pH 7.3, containing 20
mmol l1 Hepes buffer, 436 mmol l1 NaCl, 10
mmol l1 KCl, 10 mmol l1 CaCl2,
53 mmol l1 MgSO4 and 10 mmol l1
glucose), were loaded with the fluorescent indicator 2', 7'-bis
(carboxyethyl)-5(6)-carboxyfluorescein tetra-acetoxymethylester (BCECF/AM;
AppliChem, Inc., Darmstadt, Germany), which is highly lipid soluble, membrane
permeable and readily cleaved by intracellular esterases.
Appropriate amounts of cells were incubated in PS buffer with BCECF/AM
(12 µg 106 cells), (the volume of BCECF was taken
from a stock solution and adjusted to the number of cells each time) at
15°C for 45 min in the dark. The isolated cells were then washed three
times or more with the same medium to remove the fluorescent indicator and
then resuspended in the same medium (23x106 cells/3
ml). When appropriate, various concentrations of cadmium chloride (0.05, 5, 50
and 500 µmol l1), 10 nmol l1
phorbol-12-myristate-13-acetate (PMA; an activator of protein kinase C), 5.5
nmol l1 adrenaline (L-epinephrine; an -
and ß-adrenergic agonist), 1 µmol l1
isoprenaline-HCl (a ß-adrenergic agonist), 1 µmol l1
phenylephrine-HCl (an
1-adrenergic agonist), 20 nmol
l1 calphostin C (from Cladosporium cladosporioides;
a specific inhibitor of protein kinase C), 1 µmol l1
prazosin-HCl (a peripheral
1-adrenergic antagonist), 1
µmol l1 metoprolol-tartrate (a
ß1-adrenergic antagonist), 20 nmol l1
ethyl-N-isopropyl-amiloride (EIPA; a selective inhibitor of
Na+/H+ exchanger) and 10 µmol l1
propranolol-HCl (an antagonist of ß-adrenergic receptors), were
added.
Fluorescence was measured while the suspension was continuously stirred using a magnetic stirrer at 20°C in a Perkin-Elmer (Connecticut, USA) LS 50B Fluorescence thermostatic spectrometer equipped with a thermostatic holder. Data were obtained as the ratio of the pH-sensitive excitation wavelength (495 nm) to the pH-insensitive excitation signal wavelength (440 nm), with the emission wavelength set at 530 nm.
Routinely, for each experiment, and also for the cell sample preparations
used to evaluate pHi values, fluorescence was calibrated against pH. Cells
were diluted in a series of standard-buffer solutions containing MES 30 mmol
l1 (pH 6), Hepes 30 mmol l1 (pH 6.5) Mops
30 mmol l1 (pH 7) and Tris 30 mmol l1 (pH
8). Each buffer contained 440 mmol l1 KCl and 1 mmol
l1 MgCl2. Fluorescence was calibrated against pH
as described by Thomas et al.
(1979), using the polyether
ionophore nigericin (6.7 mmol l1), which couples
K+ and H+ gradients across the plasma membrane. High
[K+] solution in combination with the K+/H+
exchange ionophore nigericin leads to equilibration of extracellular
K+ with intracellular K+ and pHi at the clamped
extracellular pH. The calibration curve, done at the end of each experiment,
was linear in the pH range 68 (r2=0.99; typically
y=3.13x16.5). A standard curve was also conducted in
the presence of cadmium and was the same as that in the absence of the metal.
The background ratio of unlabelled cells was 6 times less than that of the
labelled ones. The intracellular pH (pHi) was 7.4±0.01. The results are
means ± S.D. from at least 7 experiments.
Measurement of 22Na influx
Influx of 22Na by the digestive gland cells was measured by a
modification of a previously described method
(Sauvage et al., 2000;
Bourikas et al., 2003
). After
isolating the digestive gland cells, the suspension was centrifuged at 160
g for 5 min at 4°C. The packed cells were washed four
times in a buffer free of Na+ containing 10 mmol
l1 Tris/MES buffer (pH 6), 446 mmol l1
KCl, 53 mmol l1 MgSO4, 35 mmol
l1 sucrose, 10 mmol l1 glucose, 0.1 mmol
l1 ouabain, and finally the packed cells were suspended
either in 10 mmol l1 Tris/MES buffer (pH 6) or in 10 mmol
l1 Tris/Mops buffer (pH 8), both of which also contained
4,4-diisothiocyanatostilbene-2.2-disulfonic acid (DIDS; an inhibitor of anion
exchanger) and 6-ethoxyzolamide (a carbonic anhydrase inhibitor), in order to
avoid pHi disturbances, and bumetanide (a Na-K-2Cl
cotransport inhibitor), in order to avoid Na+ transients (at final
concentrations 0.125 mmol l1, 0.4 mmol l1
and 0.01 mmol l1, respectively). The cells were allowed to
resuspend in the above-mentioned buffer for 20 min.
Afterwards, the cells were centrifuged at 160 g for 5 min
and the packed cells were finally suspended either in 10 mmol
l1 Tris/Mops buffer (pH 8) or in 20 mmol
l1 Tris/MES buffer (pH 6). Both these buffers contained 436
mmol l1 NaCl, 53 mmol l1 MgSO4,
35 mmol l1 sucrose, 10 mmol l1 glucose,
0.1 mmol l1 ouabain (an inhibitor of sodium pump, in order
to prevent Na+ movement), 1 mmol l1 iodoacetic
acid (an inhibitor of glycolysis at the step catalysed by
glyceraldehyde-P-dehydrogenase), 0.125 mmol l1 DIDS (an
anion exchanger inhibitor), freshly prepared antimycin A (final concentration
6 mg ml1), 0.4 mmol l1 6-ethoxyzolamide,
0.01 mmol l1 bumetanide and labelled 22Na (3.7
kBq/sample). When appropriate, cadmium chloride at final concentration of 50
µmol l1, calphostin C at 20 nmol l1,
propranolol at 10 µmol l1 and EIPA at 20 nmol
l1 were added immediately after the addition of the above
buffer, and the cells incubated for 30 min. Then, the cell suspensions were
transferred in plastic tubes containing 10 mmol l1 Tris/Mops
buffer (pH 8) or 20 mmol l1 Tris/MES buffer (pH 6), plus 30%
sucrose and gently layered over 0.2 ml of dibutyl-phthalate. Centrifugation of
the cell suspension followed in order to collect the packed cells at the
bottom of the tubes without the incubation buffer. A volume of 10 µl of the
supernatant, without disturbing the dibutyl-phthalate liquid phase, was
transferred in plastic vials for 22Na counting (extracellular Na),
while the rest was removed gently. Finally 100 µl of 30% perchloric acid
(PCA) was added to the packed cells for cell lysis to occur and after
centrifugation at 1600 g for 5 min the supernatant was
transferred into plastic vials for 22Na counting. Radioactivity was
measured for 10 min per sample in a beta scintillation counter (LKB 1209
Rackbeta). Sodium influx, expressed as µmol Na+
h1 106 cells, was calculated from the
percentage of total radioactivity incorporated into the cells and the total
concentration in the incubation buffer. Data were corrected for backflux of
22Na. The intracellular sodium concentration measured in cells
suspended in solution at pH 8 represents the total sodium influx, whereas
intracellular sodium concentration measured in cells suspended in a solution
at pH 6 represents the influx due to passive permeability alone. The
difference between these two influxes represents the sodium influx stimulated
by the pH gradient, and is expressed as the influx of Na+ due to
maximal activity of the Na+/H+ exchanger
(Vmax) (Delva et al.,
1993; Sauvage et al.,
2000
).
Statistical analysis
For PK activity, pHi and Na+ influx determination, statistical
analysis was carried out using Instat 2 Software (Graphpad Instat, San Diego,
CA, USA), Dunnet's test. The minimal level of significance chosen was
P<0.01. The analyses were carried out using the STATISTICA
statistical package (Microsoft Co., Thessalonika, Greece).
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Results |
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The influence of cadmium on pHi and 22Na influx was inhibited by
EIPA (20 nmol l1), (Fig.
2), a known selective inhibitor of the
Na+/H+ exchanger. The transport system can also engage
in Na+ exchanges with a very low Na+ transport rate,
which is 6% of the rate of Na+ transport in the
Na+/K+ exchange reaction
(Skou, 1988). Since most other
sodium transporters are inhibited (see Materials and methods) the observed
effect can be attributed to Na+/H+ exchanger inhibition.
The increase of pHi under the influence of cadmium was not affected by DIDS, a
antiport inhibitor (results not shown).
Studies on vertebrate cells have shown that Na+/H+
activation is mediated by protein kinase C (PKC) activity
(Kaloyianni et al., 2001;
Bourikas et al., 2003
). To
investigate whether the effect of cadmium on pHi elevation of mussel cells was
related to any metal-induced effect on protein kinase C mediated signal
transduction, we tested the effects of: (1) the phorbol ester PMA, an
activator of PKC and (2) calphostin C, a specific inhibitor of protein kinase
C. PMA (10 nmol l1) treatment caused a significant rise in
pHi after 20 min, reaching a plateau after 30 min of incubation
(Fig. 3), suggesting that the
antiport is influenced by PKC. In addition, the presence of calphostin C (20
nmol l1) together with cadmium resulted in a significant
decrease in pHi value compared to that observed after treatment with cadmium
alone (Fig. 3). Calphostin C
together with cadmium also inhibited the increased influx of Na+ in
the digestive gland cells, relative to the Na+ influx after
treatment with cadmium alone (Fig.
2). The influence of cadmium on pHi as well as on Na+
influx was inhibited by propranolol, an antagonist of ß-adrenergic
receptors, thus indicating that signal transduction induced by cadmium occurs
via interaction with ß-adrenergic receptors
(Fig.
2,Table 1). ß1- and
1-adrenergic receptor antagonists
also counterbalanced the heavy metal effect on pHi rise
(Table 1).
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Effects of cadmium on PK activity
Cadmium added at micromolar concentrations to isolated digestive gland
cells of M. galloprovincialis resulted in a time-dependent response
in PK activity (Table 2). The
dose-dependent response of cadmium showed that PK activity was maximally
increased (11.69±2.08 nmol min1 mg1
protein) at 50 µmol l1 CdCl2, followed by a
decrease at 500 µmol l1 CdCl2 (8±1.75
nmol min1 mg1 protein), while 0.05 µmol
l1 of CdCl2 showed only a small increase in PK
activity (6.07±1.23 nmol min1 mg1
protein), in relation to control (5.23±1.04 nmol min1
mg1 protein), which represents PK activity in the absence of
CdCl2 (not shown). During the exposure period of 30 min, no
significant changes in enzymatic activity were observed in control cells. To
determine whether the effect of cadmium treatment on PK activity of mussel
cells was related to a metal-induced effect on protein kinase C mediated
signal transduction, we tested the effects of inhibitors in experiments
similar to those employed for the Na+/H+ exchanger: (1)
the phorbol ester PMA (an activator of PKC) and (2) staurosporine (antibiotic
AM-2282, from Streptomyces sp.; an inhibitor of PKC) and calphostin C
(a specific inhibitor of protein kinase C). Treatment of isolated digestive
gland cells with 10 nmol l1 PMA resulted in a significant
increase of enzymatic activity, almost at the same range as observed after
treatment with cadmium alone (Table
3). Additionally, staurosporine or calphostin C inhibited the
cadmium effect on digestive gland cells
(Table 4). The presence of
propranolol (10 µmol l1) or prazosin (1 µmol
l1) or metoprolol (1 µmol l1) together
with cadmium caused an inhibition of PK increase
(Table 4).
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To investigate whether the metal induced effect on PK activity was related to Na+/H+ exchanger, the inhibitor of Na+/H+ exchanger and other sodium transporters, amiloride was used. It is noteworthy that treatment of isolated digestive cells with 10 µmol l1 amiloride, together with cadmium, resulted in a significant decrease of the cadmium effect on enzymatic activity (Fig. 4). The same results were observed after treatment of cells with EIPA (20 nmol l1), which is a selective amiloride-type inhibitor of the Na+/H+ exchanger (Fig. 4). Treatment with each inhibitor alone, without cadmium, did not show any significant difference from enzymatic activity measured in control cells.
|
Effect of ß- and -agonists on pHi and PK activity
The effect of adrenaline on pHi change as well as on PK activity was
tested. A significant rise in pHi was caused after adrenaline (5.5 nmol
l1) treatment (Table
5). It is noteworthy that after adrenaline treatment, pHi reached
even higher levels than those observed after cadmium treatment. After exposure
of the cells to cadmium chloride together with adrenaline, the increased pHi
observed was significantly higher than the value in control cells but
significantly lower than the pHi rise obtained after incubation either with
cadmium or with the agonist alone (Table
5). Therefore, the response of the cells in pHi increase after
exposure to cadmium together with adrenaline seems to be synergistic and not
additive.
|
Furthermore, PK activity was increased after adrenaline treatment of
digestive gland cells (Table
3). The ß- and 1 agonists isoprenaline and
phenylephrine, respectively, caused a significant rise of pHi and PK activity
in isolated digestive gland cells (Tables
3,
5). Isoprenaline (1 µmol
l1) caused an elevation of pHi, which was maximum after 15
min oftreatment and decreased after 30 min, but was still significantly higher
than the control. The same kinetics for PK activity were also observed after
treatment with isoprenaline (results not shown). Similar to the pHi increase
after adrenaline plus cadmium treatment, the response of the cells both in PK
activity and on pHi after co-exposure to cadmium together with various
agonists or PMA was synergistic (Tables
3,
5).
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Discussion |
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Further support for this interpretation is provided by the experiments
using blockers of the exchanger. EIPA is a selective inhibitor of the
Na+/H+ exchanger, which competes with sodium for binding
to the exchanger (Clark and Limbird,
1991). At a concentration of 20 nmol l1 it
significantly reduces the effect of cadmium on both Na+ influx and
pHi increase, compared to the increase observed after treatment with cadmium
alone. Therefore, the results show the existence of an amiloride-sensitive
Na+/H+ exchanger in digestive gland cells of mussel
Mytilus galloprovincialis, which is activated by cadmium.
A known stimulator of PKC, treatment by phorbol-esters produced similar pHi
changes to those of cadmium, regarding pHi change in digestive gland cells. In
accordance with the latter, the inhibitor of PKC calphostin C together with
cadmium, diminished the stimulatory effects of cadmium on pHi increase as well
as on Na+ influx. From the results we could suggest that cadmium
activates the exchanger by affecting the signaling pathway via
activating protein kinase C. Our data is in accordance with other studies,
where the stimulation of the Na+/H+ exchanger by
hormones via protein kinase C was reported
(Grinstein et al., 1985;
Incerpi et al., 1994
;
Kaloyianni et al., 2001
;
Bourikas et al., 2003
).
There are few data concerning the effect of heavy metals on the activity of
key glycolytic enzymes (Canesi et al.,
1998,
2000a
,
2001
). The present results
demonstrate that 50 µmol l1 of cadmium significantly
activates the glycolytic enzyme PK after 30 min of treatment of digestive
gland cells of Mytilus galloprovincialis. These data are consistent
with the effect of zinc, insulin and growth factors on PK activity in
digestive gland cells of mussels (Canesi et al.,
1997
,
1999
,
2000b
,
2001
). Furthermore, after
treatment of the cells with amiloride or EIPA, together with cadmium, a
significant decrease of PK activity was observed
(Fig. 4). It is known that
amiloride inhibits most plasma membrane Na+ transport systems, as
epithelial Na+ channel, the Na+/H+ exchanger
and the Na+/Ca2+ exchanger
(Frelin et al., 1988
;
Kleyman and Cragoe, 1988
).
These data show that the stimulus from heavy metals passes to PK via
Na+/H+ exchanger, or that PK activation depends on
intracellular pH changes, caused by the Na+/H+ exchanger
activity. Thus, the signal transduction pathway induced by cadmium leads to
the activation of PK together or via Na+/H+
exchanger activation.
To characterize the signaling pathway involved in stimulation of PK in
mussel digestive gland cells by cadmium treatment, the effects of
staurosporine, calphostin C, and PMA on PK were investigated. We found a
relationship between PKC activity and PK stimulation, after treatment of the
isolated digestive gland cells with the heavy metal. In parallel with our
results showing that cadmium uptake is related to the signaling pathway
activating PKC as well as Na+/H+ exchanger or/and PK,
the study of Adams et al.
(2002) shows that cadmium
induces signal transduction cascades such as PKC, tyrosine kinase and casein
kinase II. Canesi et al.
(2000a
,
2001
), reported that other
heavy metals such as Zn can modulate the activity of the glycolytic enzymes
phosphofructokinase (PFK) and pyruvate kinase (PK) through a tyrosine-mediated
signal transduction pathway, while the heavy metals Hg and Cu interfere with
Ca2+ mediated signaling in isolated digestive gland cells of mussel
M. galloprovincialis. The involvement of Ca2+ in cadmium
mediated signaling in the digestive gland cells of M.
galloprovincialis is also probable since it is reported that cadmium
impairs influx of Ca2+ in gills of M. galloprovincialis
(Viarengo et al., 1993
).
The signaling pathway induced by cadmium led us to hypothesise that the
cell responses to cadmium treatment are similar to those observed after
hormone treatment. Thus, the activity of PK and the change in pHi after
adrenaline and isoprenaline treatment was investigated (Tables
3,
5). Specifically, adrenaline
treatment of the digestive gland cells resulted in a significant increase of
enzymatic activity as well an increase in pHi. The increases in pHi and PK
activity were similar to ß-adrenergic receptor activation in frog and
human erythrocytes (Kaloyianni et al.,
1997,
2000
;
Bourikas et al., 2003
). The
fact that cadmium, adrenaline and isoprenaline induce PK activation as well as
increased pHi led us to investigate if cadmium also binds to ß-adrenergic
receptors. Treatment with propranolol, an antagonist of ß-adrenergic
receptors, together with the heavy metal, caused a significant decrease of PK
activity and inhibition of the pHi and Na+ increase
(Fig. 2, Tables
1,
4). The inhibition of pHi and
PK caused by prazosin and metoprolol, indicates that activation of
1 and ß1 receptors after cadmium may also
occur. From the results we could suggest that cadmium may exert its activation
on digestive gland cells by interacting with ß, ß1- and
1-adrenergic receptors. Even though it is reported that
cadmium is taken up by an inorganic anion exchanger and that cadmium transport
across the apical membrane occurs not only via passive diffusion but
also via a H+ antiport system
(Endo et al., 2000
), in
addition to its uptake by endocytosis and pinocytosis
(Cossa, 1989
;
Endo et al., 1998
), the use of
adrenergic antagonists showed that ß, ß1- and
1-adrenergic receptors may also be functioning. In
accordance with the latter, in oysters, an adrenaline mediated stress response
has been recorded (Lacoste et al.,
2001
), which indicates the functioning of
- and
ß-adrenergic receptors. However, the mechanism of cadmium uptake in
digestive gland cells of Mytilus remains to be elucidated.
In conclusion, under stress, cells adapt strategies for maintaining their
intracellular homeostasis and function. Since stimulation or inhibition of PKC
stimulates or inhibits both the Na+/H+ exchanger and PK
activity, it seems reasonable to suggest that the heavy metal interacts with
ß, ß1- and 1-adrenergic receptors,
which then pass through PKC affecting or stimulating both
Na+/H+ exchanger and PK. As a consequence of stimulating
the activity of PK, glycolysis is accelerated in response to the increase
demands for energy under stress. Increased glycolysis results in increased
production of H+, whose exit is facilitated by the already
activated Na+/H+ exchanger. On the other hand, it is
noteworthy that the inhibition of Na+/H+ exchanger by
EIPA results in inhibition of PK as well. These data suggest that either the
heavy metal stimulus passes from PKC to PK via
Na+/H+ exchanger or that PK activation depends on
intracellular pH changes caused by Na+/H+ exchanger
activity. These results support previous evidence that
Na+/H+ exchanger is a regulator of intracellular signal
transduction. Since Na+/H+ exchanger activity is
regulated by a variety of stimuli, this antiport may contribute to the
fine-tuning of several intracellular signal transduction pathways in the
digestive gland cells of Mytilus. Further research is needed in order
to clarify these mechanisms.
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