Characterization of the voltage-activated currents in cultured atrial myocytes isolated from the heart of the common oyster Crassostrea gigas
EA 3879, Unité de Physiologie Comparée et Intégrative, Institut de Synergie des Sciences et de la Santé, 22 avenue Camille Desmoulins, CS93837, 29238, Brest-cedex 3, France
* Author for correspondence (e-mail: jpennec{at}univ-brest.fr)
Accepted 2 August 2004
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Summary |
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Key words: bivalve, oyster, Crassostrea gigas, heart, ionic current, cell culture, patch clamp, bioassay, cryopreservation
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Introduction |
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Some studies have shown that it is possible to isolate and cultivate cells
from bivalves, especially from bivalve heart
(Curtis et al., 1999;
Le Marrec et al., 1999
;
Pennec et al., 2002
). There
are few reports concerning fundamental electrophysiological studies in such
cells, however, and the characterization of the ionic currents in the heart
cells of the oyster remains to be done. In a previous study
(Pennec et al., 2002
) we
showed that it is possible to isolate viable cardiomyocytes from the heart of
the oyster and to culture them for longer periods (several days to a few
weeks), and to perform electrophysiological studies in these cells using a
macro-patch clamp technique. These cells were spontaneously beating after a
few days of culture and formed contractile networks in the culture dishes. The
beating rhythm could be decreased by acetylcholine and increased by
epinephrine. These effects could be blocked by atropine and propranolol; this
shows that the cells have functional cholinergic and adrenergic receptors. In
addition, we showed that a significant reduction of beating rate was induced
by tributyltin (TBT) and cadmium (Cd). These effects were attributed to a
reduction of the inward current that is thought to be mainly a calcium
current. However, there was no precise characterization of the different ionic
currents and, hence, the identification and determination of the
characteristics of the main ionic currents in these cells remained to be
carried out. Similarly, the role of the different channels in the
determination of the spontaneous beating rate of the isolated cells was
unknown. Besides such fundamental aspects, a better knowledge of these
channels could be helpful for determining the level of toxicity of sublethal,
low concentrations of marine pollutants. The aims of the present paper were
(1) to characterize the main ion channels in cultured cells isolated from the
atrium of the oyster heart and to compare these channels to those already
described in other mollusc cells; (2) to investigate the role of these
channels in the modulation of spontaneous contractions observed in the
cultured cells; and (3) to demonstrate that freezing and thawing does not
significantly change the electrophysiological properties of the cells, so
enabling the use of thawed cells as a useful bioassay for various
pharmacological or ecotoxicological studies.
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Materials and methods |
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Cryopreservation of the cells
Cells were frozen in 12% dimethylsulfoxide (DMSO) containing Leibovitz
medium at a cooling rate of approximately 23°C
min1, down to 80°C. After 2 h, the cells were
placed in liquid nitrogen where they were stored until thawing. This technique
has been extensively described by Le Marrec-Croq et al.
(1998). After thawing, DMSO
containing medium was removed and the cells were resuspended in normal culture
medium, then seeded in 35 mm Petri dishes and cultured at 18°C as
described above. The percentage of viable thawed cells was evaluated by a
Trypan Blue exclusion test.
Electrophysiological technique
A macro-patch clamp technique (see
Malecot and Duval, 1992) was
used to record the membrane currents of the cultured cardiomyocytes under
voltage-clamped conditions in the cell-attached configuration. Pipettes were
pulled from borosilicate glass capillaries (GC150 TF10; Clark Electromedical,
Phymep, Paris, France) using a microprocessor-controlled puller (DMZ,
München, Germany). They were automatically heat polished. The diameter of
the opening (3.3±0.02 µm) was checked by electron microscopy. The
average resistance of pipettes filled with the standard medium (modified
seawater) was 0.8 M
. Junction potential was corrected before
realization of a seal. The tip of the pipette was positioned in contact with
the cell membrane using a hydraulic micromanipulator (Narishige, Tokyo,
Japan), and a moderate suction was applied to induce the formation of a seal
better than 1 G
(gigaseal); thereafter the depression was released.
Patches showing either bleb formation or unstable seal values were discarded.
The formation of the seal and the capacitance compensation were monitored on
an oscilloscope (TDS 340A; Tektronics, Beaverton, OR, USA). Recordings were
made at room temperature (averaging 20°C). The resting membrane potential
of the cells was previously measured using an intracellular microelectrode
filled with 2.7 mol l1 KCl (diameter <1 µm, average
resistance 40 M
). The microelectrodes were connected to the amplifier
(Axon; Geneclamp 500 B, Foster City, CA, USA) via a headstage
designed for voltage measurement (HS-2A; Axon). Current measurements were made
using a patch-clamp amplifier (Geneclamp 500 B) equipped with a
current-to-voltage converter headstage (CV5 series; Axon). The outputs
(voltage and current) were connected to the scope and to a micro-computer (PC
compatible) via an analog-to-digital converter (CED 1401 plus;
Cambridge, UK) running at 125 kHz. Aprogram (WCP v. 3.06 from Strathclyde
University, Scotland, UK) was used to record the currents and to deliver
sequences of programmed voltage pulses via the analog output of the
CED to the Geneclamp and then to the membrane patch. A classical P/4 protocol
of pulses was used to remove residual leak current, if any, and residual
capacitance artifact (Almers et al.,
1983
). The currents were further analysed off-line using WCP to
calculate the ionic conductances and other parameters such as time constants.
The currents and conductances were standardized assuming that the surface of
patched membrane was equal to the opening of the tip of the pipette. Some
experiments were carried out in parallel to observe the effect of some of the
compounds on the spontaneous beating rhythm of the cultured cells. In these
cases the beating frequency was recorded using an automated system based on a
specially designed system connected to a computer via the serial
port. Visual observation was used to check the method.
Patch clamp solutions
As the stability and the resistance of the seals were lower in the culture
medium, the patch experiments were performed after replacing the culture
medium with a modified sterilized and filtered (0.22µm) seawater containing
the following major ions: NaCl, 456 mmol l1; KCl, 9.7 mmol
l1; CaCl2, 11 mmol l1;
MgCl2, 55.6 mmol l1. This medium contained
neither proteins nor antibiotics, but the ions were present at the same
concentration as in the culture medium. It was equilibrated with air; the pH
was 7.3 at room temperature. The medium was changed more than 3 h before the
experiments in order to allow stabilization of the characteristics of the
cells. If used, channel blockers were added to the medium bathing the cells
and to the medium filling the pipette: tetraethyl ammonium chloride (TEA),
tetrodotoxin (TTX), charybdotoxin (CHTX) and verapamil at the concentrations
stated in the text or in the figure legends. Effects of TTX, CHTX and
verapamil were rapid, so recordings were made 310 min following their
addition. For TEA, recordings were only made after 30 min, which was the
incubation time required to achieve a maximum and steady inhibition. All the
chemicals were purchased from Sigma (L'Isle-d'Abeau, France).
Statistical analysis
Results are given as means ± S.E.M. The normality of the
distributions was verified using the ShapiroWilk test; mean values were
compared by using parametric (Student's t-test or AspinWelch
test) or non-parametric (MannWhitney) tests. Values were considered to
be significantly different when P<0.05.
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Results |
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Potassium currents
Delayed potassium current
In fibres treated with tetrodotoxin (TTX) and verapamil to block
Na+ and Ca++ inward currents, respectively, outward
currents were observed in all cells when depolarizing pulses were applied to
the patch (Fig. 1B). These
currents appeared to be composed of at least two different currents involving
two different types of channels. A fast-activated, rapidly inactivated outward
current was observed in most cells, superimposed by a slow-activated,
sustained current. By using 105 mol l1 of
4-aminopyridine (4-AP), the first current that showed fast activation and
inactivation could be suppressed. Only the slow-activating, non-inactivating
current remained. This current was blocked by 40 mmol l1 of
tetraethyl ammonium (TEA), a well known potassium channel blocker, and was
identified as a classical delayed outward rectified potassium current
(IK), which is observed in most excitable cells
(Fig. 1C). The
currentvoltage relationship (IV curve) showed the usual
pattern (Fig. 1D) with a strong
rectification. The maximum conductance was calculated from the slope of the
linear part of the IV relationship and normalized in mS
cm2, as described in Materials and methods. Under normal
conditions, the mean value of the maximum conductance was 11.47±0.90 mS
cm2 (N=8). With 30 mmol l1 of TEA
in the bath, the conductance was reduced to 2.54±0.84 mS
cm2 (N=6), which gives a strong inhibition (78%).
An order of magnitude of the time constant of the activation of the
IK current in control conditions can be determined by fitting the
current obtained with the 150 mV depolarizing pulse onto a single exponential
curve. In these conditions, the computed value was 53.01±0.40 ms
(N=8).
Fast inactivating current
In well-polarized cells, or when the depolarizing test pulse was preceded
by a hyperpolarizing pre-pulse (40 mV, 120 ms), a transient outward
current could be observed (see Fig.
1A,B). Characterization of the transient current was carried out
in medium containing 30 mmol l1 TEA, which strongly
inhibited the delayed potassium current (see above) but only slightly reduced
the amplitude of the transient current
(Fig. 2A). It should be noticed
that higher concentrations of TEA in the medium (40 mmol l1;
30 min incubation) had a significant inhibitory effect on the transient
current, but this current was more selectively blocked by 4-AP at a
concentration of 105 mol l1 in the pipette
(Fig. 2B), and was also
inactivated by 120 ms depolarizing pulses applied immediately before the test
pulse (Fig. 2C). Application of
depolarizing steps regularly spaced from 30 to +110 mVallowed the
plotting of the inactivation curve showed in
Fig. 2D. From this curve it can
be determined that a depolarizing voltage averaging +53 mV above the resting
potential induced a 50% inhibition of the normalized current. This voltage
corresponds to a membrane potential of about +7 mV,according to the mean value
of the resting membrane potential.
|
The IV curve corresponding to the fast IK
current is depicted in Fig. 3.
It also shows a strong rectification; the maximum conductance was
30.33±3.73 mS cm2 (N=7). By performing a
regression according to HodgkinHuxley type equations, the time constant
of activation (m; 0.484±0.027 ms) and the time constant
of inactivation (
h; 2.160±0.051 ms) could be determined
for a 150 mV depolarizing pulse.
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Maxi potassium current
In most cells, but not in all the cells, a large outward current was
observed with strong depolarizing pulses
(Fig. 4A). The
IV curve gave a maximum conductance of 387.13±31.42 mS
cm2 (N=6). This current was reduced in
verapamil-treated cells and suppressed by 105 mol
l1 of charybdotoxin (CHTX); it was then attributed to a
calcium activated potassium channel (KCa). This current was mainly
elicited by strong depolarisations, but its large conductance suggested that,
even with moderate depolarisations (see
Fig. 4A), its intensity was not
negligible. Hence, this current could play a role in the stabilization of
membrane potential and in the reduction of the spontaneous beating rate. This
hypothesis was in agreement with the fact that, in spontaneously beating
cultured cells, inhibition of this current by CHTX increases the beating rate
in a dose-dependent way, as shown in Fig.
4B.
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Sodium current
A fast transient inward current was observed in some cells. This current
was inhibited by 106 mol l1 of TTX
(tetrodotoxin) and was identified as the classical voltage-gated sodium
current. An example of this current is reported in
Fig. 5. Note that it was not
observed in all the cells. The sodium current was only triggered if a
hyperpolarizing pulse (amplitude 60 mV, duration 120 ms) was applied
just before the test pulse, and suggests that this current should be
inactivated at the normal membrane potential of the cultured cells and cannot
be involved in the triggering of the spontaneous beatings. Calculated from the
IV curve, the maximum conductance was 20.54±0.56 mS
cm2 (N=8). m and
h
of the sodium current were calculated according to the HodgkinHuxley
equation using the curve-fitting option available in the WCP software; the
values obtained were 0.330±0.011 ms and 1.110±0.31 ms,
respectively.
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Calcium currents
In cells treated with 40 mmol l1 TEA, an inward current,
which was inhibited by 105 mol l1
verapamil or 5103 mol l1 cobalt
ions, was identified as a calcium inward current
(Fig. 6A). The activation of
this current was rather slow, with a time constant averaging 44.01±0.17
ms; noinactivation was seen with relatively short duration pulses
(100120 ms). This slow inward current was increased by serotonin (5HT)
as shown in Fig. 6B. The
corresponding conductance was similarly increased; control conductance,
6.82±1.44 mS cm2 (N=16), conductance with
106 mol l1 of 5HT, 14.09±0.25 mS
cm2 (N=10). The dose-dependant increase induced by
5HT was reversed by verapamil (106 mol
l1); the conductance was reduced to a value not differing
from control (6.05±0.84 mS cm2; N=6).
m was also decreased. In addition it was noticed that 5HT
could induce a spontaneous activity in some quiescent cells. Experiments in
cultured, spontaneously beating cells (46 days old cultures) showed
that 5HT induced a dose-dependent increase in the beating rate of the cells,
as shown in Fig. 7; 50% of the
maximum effect was obtained with 109 mol
l1. Concentrations of 5HT higher than 106
mol l1 induced very strong contractions, which ruptured the
cellular networks and caused detaching of the cells from the bottom of the
dishes. The effect of 5HT on the beating rate was reversed by verapamil
(106 mol l1), which induced a return to a
frequency not different from control (16.00±0.97 beats
min1 vs 16.11±1.63 beats
min1; N=16 and N=8, respectively). Higher
concentrations of verapamil (105 mol l1 or
higher) largely impaired the spontaneous contractile activity. The slow inward
current was also inhibited by cobalt ions, as shown in
Fig. 6C. In some cells, when
delivering a 80 mV hyperpolarizing pulse to the membrane before the test
pulse, a transient inward current was observed, with
h
41.50±2.50 ms (Fig.
8).
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Cryopreserved cells
In order to standardize the culture and then to facilitate the use of the
cultured cells, e.g. for carrying out toxicological experiments, attempts were
made to cryopreserve the cells. The technique modified from Le Marrec et al.
(1998) and briefly described
in the Materials and methods, was used to freeze isolated cells. The frozen
cells could be stored for long periods in liquid nitrogen and then thawed and
cultured just like freshly isolated cells. The percentage of viable cells,
evaluated after thawing by a Trypan Blue exclusion test, was better than 75%.
This is lower than the percentage of viable unfrozen cells (95%) but higher
than viable thawed cells from Pecten maximus (P. Fritayre,
unpublished), which represented only 30%. The cells, cultured after thawing,
were adherent to the bottom of the plastic dishes in 56 days; they were
spontaneously beating at 12 days and they formed contractile networks after
1015 days, instead of, respectively, 2 days, 5 days and 8 days for the
unfrozen cells. It can be assumed that the first stages of culture are slower
for thawed cells than for unfrozen cells. However after the first 2 weeks, the
cultures appeared very similar and the spontaneous beating rate was not
significantly different; 29.4±1.2 beats min1 in the
normally cultured cells vs 27±0.82 beats
min1 in the thawed cells (N=8). At this stage,
measurements were made to determine if freezing/thawing induced significant
and durable modifications in the major ionic currents. Only slight
modifications were recorded; the delayed potassium conductance was
9.44±0.02 mS cm2 and the calcium conductance was
6.30±0.71 mS cm2. These values were slightly lower
but not significantly (P>0.05) different from the values found in
normally cultured cells. Similarly, the fast potassium conductance was not
modified. Only the sodium current could not be clearly identified in thawed
cells, but it was not present in all the normally cultured cells in any event.
In addition, the effects of blockers are not modified. After 2 weeks, no
significant difference was found between the two types of cultured cells.
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Discussion |
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It was necessary to characterize the ion channels in steady-state conditions and in fully functional cells. As it was technically not possible to perform patch-clamp experiments on cells in vivo, and as the cells were forming contractile clusters that were not suitable for patch-clamp studies after 4 weeks of culture, the present study focused on the characterization of voltage-dependent ion currents underlying the electrophysiological properties of cultured atrial cardiomyocytes of the oyster within the culture period giving steady electrophysiological properties (525 days after seeding).
The following currents were identified under these conditions:
(1) A non-inactivating or delayed outward rectifying potassium current,
very similar to that observed in many other cells, e.g. neurons or in
cardiomyocytes isolated from molluscs
(Curtis et al., 1999;
Yeoman and Benjamin, 1999
).
This current is TEA-sensitive but the sensitivity can vary according to the
tissue or species. In oyster atrial cells, the sensitivity appears to be
higher than in mussel cardiac cells: TEA (20x103 mol
l1) induced a significant inhibition and at
30x103 mol l1, a drastic (78%)
inhibition of the potassium current in the oyster cells, whereas
30x103 mol l1 of TEA induced only a
27% inhibition in the mussel cells (Curtis
et al., 1999
). The range is closer to that observed in mammals,
where a total inhibition is observed with TEA at a concentration of
50x103 mol l1. One explanation for
the greater sensitivity of oyster cells compared to mussel cells could be
that, as in the present study, the cells were incubated in the TEA-containing
medium for 30 min before recording, thus allowing the penetration of some
amount of TEA into the cells and increasing the efficiency of the blockage.
However, the possibility of a specific difference cannot be excluded.
(2) A fast activated, spontaneously inactivated potassium current, which
has been identified in many cardiac cells in invertebrates
(Curtis et al., 1999;
Yeoman and Benjamin, 1999
) and
vertebrates, including mammals (Coraboeuf
and Carmeliet, 1982
; Giles and
van Ginneken, 1985
). This current looks like the A-current
previously identified in mollusc neurons
(Connor and Stevens, 1971
) or
the Ito current identified in mammalian heart cells
(Greenstein et al., 2000
) and
involved in the rapid initial phase of action potential repolarization. More
recently, such a current was identified in mussel heart cells
(Curtis et al., 1999
). This
current is partly responsible for the initial repolarization process: the
`notch' in mammalian ventricular action potential. It was not present in
pacemaker cells from the sino-atrial node in mammals
(Irisawa et al., 1993
) but was
identified in isolated spontaneously beating cells of Lymnea
(Yeoman and Benjamin, 1999
),
where it was involved in the determination of the beating rate. According to
this hypothesis, our experiments demonstrate that 4-AP induced an increase in
the spontaneous beating rate of the cultured oyster cells by inhibiting this
fast inactivating K+ current. This current appears less sensitive
to TEA than the delayed rectifying K+ current, in contrast to the
observations in mussel (Curtis et al.,
1999
), where the sensitivity of both K+ currents is in
the same range. In oyster, it was not observed in medium containing high
concentrations of TEA (60 mmol l1) but as it was not
observed in all the cells, even in normal medium, it is difficult to be sure
if there was a total inhibition. The reason why it was lacking in some cells
could be either the tissue origin or, more certainly, a different
physiological state; all the cells were isolated from the atrium but they were
not all spontaneously beating. In addition, this spontaneous activity appeared
only after 45 days of culture, depending on the temperature, and in
parallel this current was identified neither in freshly isolated cells nor
during the first days of culture. In addition, this current was inactivated by
application of preliminary depolarizing pulses as shown by the inactivation
curve, so in cells showing a low membrane potential, this current may be
simply inactivated. Then it could be hypothesized that this current is
characteristic for well-polarized, differentiated cells with fully functional
channels. It could be involved in the modulation of the spontaneous activity
even if it was not responsible for the triggering of this activity because of
its repolarizing effect.
(3) A maxi potassium current, mainly activated with large depolarizing
pulses, and largely sensitive to calcium influx, as shown by the inhibiting
effect of verapamil. This current should be a calcium-activated potassium
current (KCa), as it is blocked by CHTX. It was not observed in
mussel cells (Curtis et al.,
1999) but it is present in many species of vertebrates and in
molluscs like the squid (Ödblom et
al., 2000
). This channel could be activated by small
depolarisations and, because of its large conductance, could also be involved
in the control of the spontaneous activity of these cells by stabilizing the
membrane potential. The increase observed in spontaneous beating rate of the
cultured cells following the inhibition of this current by CHTX is in
agreement with such a hypothesis.
An inward rectifying potassium channel was not found in oyster atrial
cells. This parallels findings on cardiac cells of the mussel
(Curtis et al., 1999).
(4) A rapid sodium inward current. This current appears to be very similar
to the fast sodium current observed in many excitable cells, and especially
cardiac cells, in molluscs such as mussel
(Curtis et al., 1999), and
vertebrates. The difference could be a slower activation and a slower
inactivation when compared to the values reported in mammals
(Ruff, 1992
), which are about
the half of those determined here. In order to better evaluate the difference,
the fast sodium current was measured in isolated rat muscle fibres using the
same experimental setup and at the same temperature. Values of
0.168±0.046 ms and 0.207±0.019 ms (N=8) were obtained
for
m and
h, respectively, in rat muscle
fibres. These values were one half and one fifth, respectively, of the values
found in the oyster atrial cells. The maximum value of the conductance
measured in the rat fibres was 62.0±1.0 ms cm2
(N=8); this value is about three times higher than the value found in
the atrial cells. There are two possible explanations for this: either the
sodium channels are less abundant in these atrial cells than in the rat muscle
fibres or, but not exclusively, these channels are of a different subtype. The
differences in activation and inactivation time constants at similar
temperature and potentials favoured this second possibility.
(5) A calcium inward current. Such a current is well known in cardiac cells
that demonstrate contractility. Evidence that the calcium current indicated in
our experiments is flowing through L-type calcium channels includes: (i) it
showed no noticeable inactivation within the duration of our test pulses, and
was inhibited by verapamil and cobalt ions; (ii) it can also be strongly
reduced by cadmium (Pennec et al.,
2002). A more precise investigation should be carried out to
assess whether both types (L and T) of calcium channels are present in the
cells. However, when a strong hyperpolarization (60 mV to 70 mV)
was applied to the membrane before the test pulse a transient inward current
was observed in some cells, suggesting that transient calcium channels exist
in the membrane but are normally inactivated by the relatively low resting
potential of the cultured cells (45.8 mV). 5HT increased both the
calcium conductance and the beating rate of the cultured cells in
vitro. Moreover, 5HT triggered a spontaneous beating activity in
quiescent cells. These effects were reversed by verapamil, showing that
calcium channels are involved in the determination of the spontaneous beating
rate as reported by Yeoman et al.
(1999
) in the ventricular
muscle cells of the Lymnaea heart. Pennec et al.
(2002
) previously showed that
the slow inward current could also be increased by epinephrine, and that this
effect was reversed by high doses of propranolol, thus demonstrating that the
heart rate could also be modulated by beta-adrenergic agonists. However, the
effects of epinephrine were not highly dose-dependent and were observed at
relatively high concentrations (106 mol
l1), suggesting that epinephrine is not the prominent
catecholamine in the modulation of the cardiac rhythm. Serotonin is another
possibility as it is released by neurons and could then play a role in the
control of the heart rate of the oyster in vivo (Lee, 1993;
Kioaki Kuwasawa and Hill,
1997
). The present results, showing that the concentration giving
50% of the maximum effect was as low as 109 mol
l1, are in agreement with such a hypothesis.
Besides of its normal regulation, this current is inhibited by some toxic
chemicals such as tributyltin (TBT) and cadmium, which are found in moderately
polluted seawater at concentrations ranging from 1012 to
1010 mol l1
(Shim et al., 1998;
Thornton, 1992
). These
chemicals induced a decrease in both inward and outward currents, depending on
the concentration and on the time of exposure, as previously reported
(Pennec et al., 2002
). New
measurements gave an inhibition of 46% for the sodium conductance, 47% for the
calcium conductance and only 33% for the outward rectified potassium
conductance with a concentration of 109 mol
l1 of cadmium in the medium (J.-P.P., unpublished results).
Significant inhibition was observed with concentrations lower than
109 mol l1 (i.e. 1012
mol l1 after 12 days of exposure), revealing the sensitivity
of this bioassay.
In addition to voltage-operated channels, acetylcholine (Ach) increases the
global potassium conductance (Pennec et
al., 2002), showing that these cells also have Ach-activated
potassium channels. As the effect of Ach was blocked by atropine, it was
assumed that the cultured cells had functional muscarinic M2-like
receptors.
Globally, our results agree well with those reported for freshly isolated
mussel cardiac cells (Curtis et al.,
1999), despite the fact that our experiments were performed on
long-term cultured cells that showed spontaneous contractile activity. It can
be suggested that in freshly isolated cells, ion channels could be more or
less altered by the enzymatic dissociation while they are fully functional
after a few days of culture. In agreement with this, cells were beating after
only a few days of culture and epinephrine or acetylcholine had a minor or
even no effect during these very first days of culture, which could also
explain the differences in the identified currents that were noticed: the
increased sensitivity of the delayed potassium current to TEA, the existence
of a calcium-activated potassium current and the existence in some cells of
T-type calcium currents, which were not seen in mussel. These currents were
described in Lymaea ventricular cells
(Yeoman et al., 1999
). We
suggest that both the maxi potassium current and the fast inactivating
potassium current are involved in the modulation of spontaneous beating rate
along with the antagonistic calcium current. The potassium currents reduce the
spontaneous beating rate while the calcium current increases it. The transient
inward calcium current could be related to the triggering of the spontaneous
activity, as suggested by Yeoman et al.
(1999
) but as it was not seen
in all the cells of the present study we hypothesize that it is limited to the
pacemaker cells.
The technique of cryopreservation could be very useful for bioassays: cells can be stored for a long time before use without any significant alteration in their electrophysiological properties. Associated with long-term culture, it provides the possibility of testing cardioactive compounds in acute or in chronic conditions.
To summarize, the present work shows that the atrial cells isolated from
oyster heart present ionic currents very similar to the currents described in
homologous cells of many molluscs or vertebrates. This gives fundamental
information about the electrophysiological properties of these cells. Despite
a slightly slower development of the contractile networks, the culture of
cryopreserved cells is possible without any major alteration of their
electrophysiological properties, thus increasing the versatility and the
interest of the model. Hence freshly isolated and cryopreserved cells can be
used as a model for the study of acute and chronic effects of natural or
exogenous marine molecules. Preliminary studies with freshly isolated cells on
the toxicity of marine pollutants such as cadmium and tributyltin showed that
the sensivity of this assay was several orders of magnitude higher than the
sensitivity of the usual in vivo experiments
(Pennec et al., 2002).
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Acknowledgments |
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References |
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