Effect of ß-adrenergic stimulation on the relationship between membrane potential, intracellular [Ca2+] and sarcoplasmic reticulum Ca2+ uptake in rainbow trout atrial myocytes
1 Unitat de Fisiologia Animal, Departamento de Biologia Celular, Fisiologia
i Immunología, Facultat de Ciencies, Universitat Autònoma de
Barcelona, 08193, Cerdanyola, Barcelona, España
2 Cardiac Membrane Research Laboratory, Department of Kinesiology, Simon
Fraser University, Burnaby, BC, Canada, V5A 1S6
Author for correspondence (e-mail: lhove{at}hsp.santpau.es)
Accepted 19 January 2004
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Summary |
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Key words: trout, excitation-contraction coupling, Na-Ca exchange, membrane current, teleost heart, caffeine
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Introduction |
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In the teleost heart, the effect of ß-adrenergic stimulation on
ICa has recently been characterized
(Hove-Madsen and Tort, 1998;
Vornanen, 1998
), with a two-
to threefold stimulation of ICa. This is similar to values obtained
in mammals, but much smaller that the seven- to tenfold stimulation reported
in frog (Fischmeister and Shrier,
1989
). Based on the stimulatory effect of ß-adrenergic
stimulation of ICa, it has been suggested that sarcolemmal
Ca2+ influx is sufficient to activate contraction under these
conditions (Vornanen, 1998
).
ß-adrenergic stimulation does, however, also affect other important
factors such as myofilament sensitivity and SR function, which critically
determine the amplitude and kinetics of cell shortening
(Freestone et al., 2000
;
Li et al., 2000
).
With respect to the SR function, the short duration of the Ca2+
transient in the mammalian cardiac myocyte and the simultaneous regulation of
the intercellular [Ca2+] by several mechanisms have made it
difficult to characterize the relationship between
[Ca2+]i and the rate of Ca2+ uptake in the SR
in intact mammalian cardiac myocytes. In contrast to this, long
depolarizations in lower vertebrate myocytes lead to a tonic cell shortening,
a maintained outward current (Hove-Madsen et al.,
1998,
2000
) and presumably an
intracellular [Ca2+] that is maintained elevated. Furthermore, a
voltage dependent Ca2+ uptake by the SR takes place during long
depolarizations in these cells and can be used to estimate the average SR
Ca2+ uptake rate (Hove-Madsen et al.,
1998
,
2000
). Using simultaneous
measurements of intracellular [Ca2+] and whole membrane current,
the present study has taken advantage of these properties of trout cardiac
myocytes in order to (1) characterize the relationship between membrane
potential, [Ca2+]i and the SR Ca2+ uptake
rate under basal and phosphorylating conditions, (2) use this relationship
between cytosolic [Ca2+] and SR Ca2+ uptake to calculate
the SR Ca2+ uptake during a Ca2+ transient induced by a
200 ms depolarization, and (3) determine the relative contribution of SR
Ca2+ uptake to total Ca2+ transport in the absence and
presence of ß-adrenergic stimulation.
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Materials and methods |
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Electrophysiological measurements
Whole membrane current was measured using a software driven patch-clamp
amplifier (EPC-9, Heka, Germany) or an Axopatch 200, (Axon, California, USA).
After seal formation, the cell was placed in front of one of eight capillaries
containing the desired extracellular solution. Standard internal and external
solutions were used to eliminate Na+ and K+ currents.
The pipette solution contained (in mmol l1): CsCl 100, MgATP
3.1; MgCl2 1; sodium phosphocreatine 5; Li2GTP 0.42;
EGTA 0.025 (no EGTA was included when using fluo-3 loaded cells); Hepes 10;
tetraethyl ammonium (TEA) 20. The pH was adjusted to 7.2 with CsOH. The
external medium contained (in mmol l1): NaCl 107; CsCl 20;
MgCl2 1.8; NaHCO3 4; NaH2PO4 0.8;
CaCl2 1.8; Hepes 10; glucose 5; pyruvate 5. The pH was adjusted to
7.4 with NaOH. The Na+ current was eliminated with 1 µmol
l1 tetrodotoxin (TTX). Whole membrane current was measured
in the ruptured or the perforated patch configuration with 200250 µg
ml1 amphotericin B (Figs
4,
5,
6,
7) at room temperature
(20°C). The pipette resistance was 25 M, seal resistance was
220 G
, and the access resistance was 6.5±1.2 M
for
ruptured patches. When using amphotericin B, equilibration of the preparation
by continuous stimulation at 0.2 Hz was started when access resistance had
decreased to
20 M
, and the access resistance was 11.6±3.1
M
at the end of the experiments. Cells showing a sudden drop in access
resistance were discarded.
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Experimental protocols
The cells were stimulated continuously with a 200 ms depolarization from
80 mV to 0 mV every 5 s. This protocol allowed evaluation of
ICa and Caf induced Na+Ca2+ exchange
(NCX) current (INCX) under steady state conditions in the absence
and presence of ß-adrenergic stimulation. ß-adrenergic stimulation
was produced by exposing cell to 1 µmol l1 isoproterenol
(ISO). The Caf induced INCX allows determination of the SR
Ca2+ content in the absence and presence of ISO as described
previously (Hove-Madsen et al.,
1998; Negretti et al.,
1995
; Varro et al.,
1993
). The basic experimental protocol outlined in
Fig. 1 was used to determine
the effect of ß-adrenergic stimulation on Ca2+ uptake in the
SR. First the SR Ca2+ content was cleared with a brief exposure to
10 mmol l1 caffeine (Caf). Then Ca2+ uptake was
induced by depolarizing the cell to different membrane potentials for 3 or 10
s (labeled Load in Fig. 1). The
amount of Ca2+ accumulated in the SR was determined from the
INCX elicited by exposing the cell to 10 mmol l1
Caf after repolarizing the cell to 80 mV (labeled Caf in
Fig. 1). The SR Ca2+
uptake rate was calculated by dividing the Ca2+ accumulated by the
SR with the duration of the depolarization. For conversion of the time
integrals of Ca2+ carrying currents to Ca2+
concentrations, a conversion factor of 15.4 pF pl1 was used
(Hove-Madsen and Tort, 1998
)
and the Ca2+ accessible cell volume was considered to equal the
non-mitochondrial cell volume, which in trout corresponds to 55% of the total
cell volume (Vornanen,
1998
).
|
Measurement of intracellular [Ca2+]
The intracellular Ca2+ concentration
[Ca2+]i was measured with the Ca2+ indicator
fluo-3. Cells were incubated with 5 µmol l1 fluo-3 AM for
1015 min at room temperature. After incubation cells were washed and
left for 30 min at room temperature before starting experiments. Fluo-3 was
excited at 488±1 nm and fluorescence emission was measured using a 530
nm bandpass filter. The bandpass width was ±20 nm. A sampling interval
of 10 ms was used to achieve a reasonable signal-to-noise ratio. This sampling
interval was necessary as the cell volume of trout atrial myocytes is
24 pl, which is about ten times less than the cell volume of mammalian
cardiac myocytes. Calibration of the fluo-3 signal in each cell was done by
subtraction of background fluorescence and measurement of the maximal
fluorescence at the end of each experiment. As long depolarizations lead to a
tonic elevation of [Ca2+]i and contraction in trout
cardiac myocytes (Hove-Madsen et al.,
1998,
2000
), maximal fluorescence
was obtained by depolarizing the cell for 3 s to increasingly more positive
potentials until an irreversible cell contracture was achieved. The fluo-3
signal measured at irreversible cell contracture was taken as the maximal
fluorescence since it also coincided with a large and irreversible increase in
leak, and it was similar to the fluorescence obtained when leak was induced by
squeezing the cell with the patch pipette. [Ca2+]i was
then calculated from
[Ca2+]i=FxKd/(FmaxF)
where F is the measured net fluorescence, Fmax is
the maximal net fluorescence and Kd is the dissociation
constant for Ca2+ for fluo-3
(Trafford et al., 1999
). A
Kd for fluo-3 in cells of 1100 nmol l1
(Harkins et al., 1993
) was used
in the present study.
Data analysis and statistics
Unless otherwise stated, cells served as their own control when the effects
of ß-adrenergic stimulation were examined. For statistical analysis of
the data, Student's t-test for paired samples was used to compare two
experimental conditions. Values are given as means ±
S.E.M. Analysis of variance (ANOVA) was used
to evaluate the effect of ß-adrenergic stimulation on the relationships
between membrane potential, [Ca2+]i and SR
Ca2+ uptake (Figs 3,
6 and
9). While SR Ca2+
uptake does not depend directly on the membrane potential, it does depend on
the [Ca2+]i concentration, and since the
[Ca2+]i concentration depends on the membrane potential,
the Boltzmann equation was used to empirically fit the relationship between
[Ca2+]i or SR Ca2+ uptake and membrane
potential (Figs 3 and
9). Similarly, the relationship
between SR Ca2+ uptake and [Ca2+]i has been
described by a Hill equation (Hove-Madsen
and Bers, 1993) and this equation was therefore used to fit the
data in Fig. 6C.
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Results |
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ß-adrenergic stimulation of steady state ICa and Ca2+ transients
These results show that ISO stimulates Ca2+ accumulation, so it
was important to determine whether the stimulation was a result of an
increased sarcolemmal Ca2+ entry or a direct stimulation of SR
Ca2+ uptake. Therefore, the free [Ca2+]i and
the whole membrane current resulting from a long depolarization were measured
simultaneously. First, the steady state effect of ISO on ionic current and
[Ca2+]i was established using continuous stimulation
with 200 ms depolarizations from 80 to 0 mV at a frequency of 0.2 Hz.
As shown in Fig. 4, ISO
increased both ICa (Fig.
4A) and Ca2+ transient
(Fig. 4B) significantly by
91±29 (P<0,01, N=7) and 41±10%,
respectively (P<0.01, N=7). Furthermore, when fitting the
decaying phase of the Ca2+ transient with a single exponential, ISO
decreased the half life of the Ca2+ transient significantly from
151±12 to 111±6 ms (P<0.001, N=7).
Effect of ß-adrenergic stimulation on the relationship between [Ca2+]i and SR Ca2+ uptake
Fig. 5 shows superimposed
fluo-3 traces (Fig. 5A) and
ionic currents (Fig. 5B)
elicited by exposure to 10 mmol l1 Caf (right panels) after
a 3 s depolarization to +10 mV (left panels), before (black traces) and after
stimulation with 1 µmol l1 ISO (gray traces). The
membrane potential is indicated in the panel above the traces and the period
of Caf application is shown. Notice that both the
[Ca2+]i and the whole membrane currents are permanently
elevated throughout the 3 s depolarization to +10 mV. Also notice that except
from the first few hundred ms, ISO reduced the [Ca2+]i
while it stimulated the outward current during depolarization. This
observation was consistent in all seven cells examined.
Fig. 5C shows that the effect
of ISO on the Caf induced ionic current and Ca2+ transient was
reversible. This was also true for the effects of ISO on the whole membrane
current and the Ca2+ transient elicited with long depolarizations
or with repetitive 200 ms depolarizations (data not shown).
Fig. 6A shows recordings of the
[Ca2+]i during depolarization to different membrane
potentials and caffeine applications in the absence (left panels) and the
presence of 1 µmol l1 ISO (right panels). The membrane
potential is given in the top of the two bars above the recordings, and the
absence () or presence of 10 mmol l1 Caf (+) in the
bath solution in the bottom bar. Fig.
6B compares the average Ca2+ transient during
depolarization (black bars) and the peak Caf induced Ca2+ transient
(white bars) after depolarization in the absence (left panel) or the presence
of 1 µmol l1 ISO (right panel). The membrane potential
during the depolarization is given below the bars. Notice that there was no
significant difference between the average Ca2+ transient during
depolarization and the peak Ca2+ transient elicited by Caf after
depolarization in control conditions, while the Caf induced Ca2+
transient was significantly bigger than the average Ca2+ transient
during the preceding depolarization in the presence of ISO
(P<0.001, N=7). This suggest that ISO directly stimulates
SR Ca2+ uptake, and Fig.
6C shows the relationship between SR Ca2+ uptake
(dCa/dt) and the average [Ca2+]i during depolarization
without (filled circles) and with 1 µmol l1 ISO (open
circles). The lines represent fits of the data points with a Hill equation.
This gave Hill slopes of 1.74 and 2.24 without and with ISO, respectively,
while the max values were
unchanged at 418 and 435 µmol l1 s1,
respectively. K0.5 was shifted from 1.27 µmol
l1 in the control to 0.80 µmol l1 with
1 µmol l1 ISO. Fitting of data points from individual
experiments showed that there was a significant difference between the average
K0.5 in the absence and the presence of ISO
(P<0.05), while ISO did not significantly affect the Hill slope or
the
max.
Using this relationship between cytosolic [Ca2+] and SR Ca2+ uptake rate, the total SR Ca2+ uptake during the Ca2+ transients shown in Fig. 4B can be calculated by first calculating the SR Ca2+ uptake rate during the Ca2+ transient, and then integrating this signal to obtain the total SR Ca2+ uptake during the Ca2+ transient. Similarly the total sarcolemmal Ca2+ extrusion can be calculated from the time integral of the tail currents in Fig. 4A. Fig. 7A shows the total SR Ca2+ uptake (labeled SR), total sarcolemmal Ca2+ extrusion (labeled SL) and total Ca2+ transport (labeled TOT) without (black bars) and with 1 µmol l1 ISO (gray bars). Notice that ISO significantly increased all three parameters. When normalizing the SR Ca2+ uptake to the total Ca2+ transport, it constituted 35±6% and 41±4% of the total Ca2+ eliminated from the cytosol without and with 1 µmol l1 ISO, respectively.
Effect of ß-adrenergic stimulation and low extracellular [Ca2+] on SR Ca2+ uptake
To examine whether the stimulatory effect of ISO was able to compensate for
a lowering of the extracellular [Ca2+], the effect of ISO applied
in the presence of a low extracellular [Ca2+] (0.3 or 0.5 mmol
l1) was compared with control values obtained in the absence
of ISO with 1.8 mmol l1 extracellular Ca2+.
Fig. 8A shows representative
current traces elicited by a 200 ms depolarization in control conditions and
with ISO + low Ca2+. On average, ISO increased the ICa
amplitude by 67±40% (N=5).
Fig. 8B shows the corresponding
current traces elicited by a rapid Caf application after a 10 s
depolarization. Notice that SR Ca2+ uptake was strongly reduced in
the presence of ISO + low Ca2+.
Fig. 9 compares the
relationship between the membrane potential during a 10 s depolarization and
the amount of Ca2+ taken up by the SR in control conditions and
with ISO + low Ca2+. The relationship between SR Ca2+
uptake and membrane potential was significantly affected by application of ISO
+ low Ca2+ (P<0.001, N=5) resulting in a
reduced uptake at membrane potentials above 30 mV. Thus while ISO could
overcome the effect of lowering extracellular Ca2+ on
ICa it was unable to prevent a strong reduction in SR
Ca2+ uptake at physiologically relevant membrane potentials.
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Discussion |
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This experimental approach is based on the following assumptions: (1) SR
Ca2+ uptake can be quantified as the amount of Ca2+
released from the SR by Caf, (2) SR Ca2+ uptake takes place
throughout a long depolarization, (3) the average SR Ca2+ uptake
rate during a depolarization can be calculated because uptake is uniform
throughout the depolarization. With respect to the first assumption, it has
been shown that Ca2+ released from the SR by Caf can be used as a
measure of the SR Ca2+ content in mammalian and trout
cardiomyocytes (Hove-Madsen et al.,
1998,
2000
;
Trafford et al., 1999
). Since
K is substituted with Cs and 15 mmol l1 extracellular NaCl
is replaced with CsCl in the present experiments, SR Ca2+ uptake
could on the one hand be underestimated, because Cs has been shown to decrease
SR Ca2+ uptake in mammalian cardiomyocytes
(Kawai et al., 1998
), and on
the other hand be overestimated, because of the 10% reduction in the
extracellular [Na+]. The effect of Cs on SR Ca2+ uptake
is, however, not expected to differ in the absence and presence of ISO.
Regarding the second assumption, Ca2+ uptake has been shown to
increase proportionally to the length of the depolarization as long as the SR
Ca2+ content is not near saturation
(Hove-Madsen et al., 1998
),
suggesting that SR Ca2+ uptake takes place throughout the
depolarizations used in the present study. Finally,
Fig. 5 shows that both the
Ca2+ transient and the outward current remain elevated throughout a
3 s depolarization so that the average SR Ca2+ uptake rate can be
calculated as the amount of Ca2+ released by Caf divided by the
duration of the preceding depolarization.
Direct stimulation of SR Ca2+ uptake by ß-adrenergic stimulation
When measuring SR Ca2+ uptake as a function of membrane
potential, ß-adrenergic stimulation affects neither the maximal SR
Ca2+ uptake nor the SR Ca2+ uptake at rest. It does,
however, significantly shift the relationship between SR Ca2+
uptake and membrane potential by 26 mV, so that the SR can more
efficiently remove Ca2+ from the cytosol during the repolarization
of the action potential and during diastole. This in turn may contribute to
the faster decay of the Ca2+ transient elicited by a standard 200
ms depolarization observed in Fig.
4.
The effect of ß-adrenergic stimulation on the relationship between SR
Ca2+ uptake and membrane potential could either be due to a direct
effect on SR Ca2+ uptake or a consequence of its stimulatory effect
on ICa and the intracellular Ca2+ transient (see
Fig. 4). In this respect, the
voltage dependency of ICa is also shifted towards more negative
membrane potentials by ß-adrenergic stimulation
(Brum et al., 1984) and this
could potentially account for the observed shift in the relationship between
membrane potential and SR Ca2+ uptake. ß-adrenergic
stimulation does, however, also stimulate SR Ca2+ uptake through
phospholamban phosphorylation (Bassani et
al., 1995
; Li et al.,
2000
; Mattiazzi et al.,
1994
), and PKA-dependent phosporylation of phospholamban has been
reported to shift the Kd for SR Ca2+ uptake
towards lower [Ca2+] (Mattiazzi
et al., 1994
). The fact that ß-adrenergic stimulation lowers
the average [Ca2+]i during long depolarizations and
shifts the K0.5 for the relationship between cytosolic
[Ca2+] and SR Ca2+ uptake from 1.27 to 0.8 µmol
l1 does therefore suggest that its stimulatory effect on SR
Ca2+ loading is due to a stimulation of the SR Ca2+
uptake rather than being a consequence of ICa stimulation.
The SR Ca2+ uptake in trout atrial myocytes is comparable to
values previously reported in trout ventricular myocytes
(Hove-Madsen et al., 1998),
and the uptake rate is within the range reported in intact or permeabilized
mammalian cardiac myocytes at room temperature
(Balke et al., 1994
;
Wimsatt et al., 1990
). Also, a
40% reduction of the K0.5 for SR Ca2+ uptake by
ISO is similar to values reported in mammals
(Mattiazzi et al., 1994
). The
absolute value for K0.5 in control is similar to a value
previously reported for permeabilized trout ventricular myocytes
(Hove-Madsen et al., 1998
),
while values for both control and ISO are 23 times higher than values
reported in several mammalian studies
(Balke et al., 1994
;
Hove-Madsen and Bers, 1993
;
Mattiazzi et al., 1994
). These
differences may reflect species dependent differences in the SR
Ca2+ ATPase, but could also be related to the calibration of the
fluorescent Ca2+ indicator. The present work uses a
Kd for fluo-3 of 1.1 µmol l1 obtained
from in situ calibration in frog skeletal muscle fibers
(Harkins et al., 1993
), while
other studies have often used in vitro calibration of the fluorescent
dyes (Balke et al., 1994
;
Trafford et al., 1999
). Using
a Kd for fluo-3 of 400 nmol l1 from
in vitro calibration would give K0.5 values of
0.46 µmol l1 in controls and 0.29 µmol
l1 with ISO. These values are similar to those reported in
mammals (Balke et al., 1994
;
Trafford et al., 1999
),
suggesting that differences in dye calibration could at least partially
account for the differences in K0.5 in the present study
and values measured in mammals.
Indirect effects of ß-adrenergic stimulation on SR Ca2+ uptake
While the present results do show that ß-adrenergic stimulation causes
a direct stimulation of the SR Ca2+ uptake, they do not exclude an
indirect effect of ß-adrenergic stimulation on SR Ca2+ uptake
with more physiological stimulation pulses. Thus, the contribution of
ICa to the elevation of the [Ca2+]i during a
3 s or a 10 s depolarization is expected to be significantly smaller than its
contribution during a 200 ms depolarization. In agreement with this,
ICa is not reduced when the extracellular solution is switched from
control to ISO plus low extracellular [Ca2+] while the SR
Ca2+ uptake during a 10 s depolarization is drastically reduced at
all membrane potentials examined (see Figs
8 and
9). This agrees with previous
results, which suggested that the maintained outward current and tonic
contraction during long depolarizations in trout cardiac myocytes
(Hove-Madsen et al., 2000;
Hove-Madsen and Tort, 2001
)
are due to reverse mode Na+Ca2+ exchange.
Furthermore, the fact that ISO has a stimulatory effect on ICa even when the extracellular Ca2+ is lowered while SR Ca2+ uptake during long depolarizations is strongly reduced under these conditions (see Figs 8, 9), suggests that ISO do not have any stimulatory effect on INCX.
In this respect the average Ca2+ transient is smaller in the
presence of ISO, while the corresponding outward current is larger in
Fig. 5. Assuming that the
outward current is reverse mode INCX, this suggests that under
these conditions the amplitude of reverse mode INCX during long
depolarizations is being determined by the [Ca2+]i
rather than being the amplitude of reverse mode INCX that
determines the [Ca2+]i. Therefore, ISO not only
stimulates SR Ca2+ uptake as shown in Figs
2,
3,
5,
6,
7, but the SR Ca2+
uptake relative to reverse mode NCX does in fact become faster in the presence
of ISO. The relatively faster SR Ca2+ uptake in turn leads to a
reduction in [Ca2+]i, an increase in the
transsarcolemmal Ca2+ gradient and as a consequence a larger
reverse mode INCX as observed in
Fig. 5. Thus, the apparent
stimulation of reverse mode INCX in
Fig. 5 is more likely to be an
indirect effect of ISO on SR Ca2+ uptake. The absence of
ß-adrenergic stimulation of the INCX agrees with data from the
toad heart (Ju and Allen,
1999). It would also be in agreement with the absence, in both the
trout and mammalian NCX protein (Nicoll et
al., 1990
; Xue et al.,
1999
), of the putative PKA-dependent phosporylation site described
in frog (Fan et al., 1996
). It
is, however, contrary to results from the frog heart, where a putative
PKA-dependent phosporylation site on the NCX has been described
(Fan et al., 1996
) and a
PKA-dependent inhibition of NCX activity has been reported in both frog and
shark cardiac myocytes (Fan et al.,
1996
; Woo and Morad,
2001
). The present results also show that the importance of SR
Ca2+ uptake relative to sarcolemmal Ca2+ extrusion by
the NCX is unchanged with ß-adrenergic stimulation in trout atrial
myocytes. This is contrary to conclusions reached from measurements of SR
function in tissue preparations (Shiels
and Farrell, 1997
) and the difference is most likely related to
the indirect nature of the measurements in the tissue preparation. Also, the
stimulation of L-type Ca2+ current by ISO in isolated
trout myocytes has been taken as evidence for a larger contribution of
sarcolemmal Ca2+ entry to the activation of contraction in the
presence of ß-adrenergic stimulation
(Vornanen, 1998
). This
conclusion is not easily compatible with an unchanged relative contribution of
forward mode INCX to relaxation with ß-adrenergic stimulation
found in the present study, since it indirectly implies a similar contribution
of sarcolemmal Ca2+ entry at steady state.
Finally, it should be noticed that the present results were obtained at
room temperature, while the acclimation temperature was 16°C and the
preferred temperature for rainbow trout is around 14°C. We cannot rule out
that this may have consequences for the measured effects of ß-adrenergic
stimulation on SR Ca2+ content and uptake rates, but we recently
found that neither SR Ca2+ uptake nor SR Ca2+ release
were significantly changed when the experimental temperature was lowered from
21 to 7°C (Hove-Madsen et al.,
2001). Furthermore, it is not clear that a difference between
acclimation and experimental temperature of 4°C would selectively alter
the SR Ca2+ uptake under basal or phosporylating conditions. An
unequivocal answer to this question will, however, have to await further
experimentation that directly addresses it.
In conclusion, the present study has characterized the relationship between the membrane potential, cytosolic [Ca2+] and SR Ca2+ uptake rate in trout atrial myocytes in the absence and presence of ß-adrenergic stimulation. The results show that ß-adrenergic stimulation shifts the relationship between membrane potential and SR Ca2+ uptake by 26 mV and decreases the K0.5 for SR Ca2+ uptake from 1.27 to 0.8 µmol l1, while it has no effect on resting or maximal SR Ca2+ loading. This may contribute to the faster decay of the Ca2+ transient with ß-adrenergic stimulation in these cells. Furthermore, ß-adrenergic stimulation does not alter the importance of SR Ca2+ uptake relative to Ca2+ extrusion by the INCX, and the characterization of the dependency of SR Ca2+ uptake on membrane potential and [Ca2+]i should be useful for quantitative comparisons of SR Ca2+ uptake and Ca2+ extrusion by the INCX under specific experimental conditions.
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Acknowledgments |
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Footnotes |
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References |
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Balke, C. W., Egan, T. M. and Wier, W. G. (1994). Processes that remove calcium from the cytoplasm during excitation-contraction coupling in intact rat heart cells. J. Physiol. 474,447 -462.[Abstract]
Bassani, R. A., Mattiazzi, A. and Bers, D. M. (1995). CaMKII is responsible for activity-dependent acceleration of relaxation in rat ventricular myocytes. Am. J. Physiol. 268,H703 -H712.[Medline]
Brum, G., Osterrieder, W. and Trautwein, W. (1984). Beta-adrenergic increase in the calcium conductance of cardiac myocytes studied with the patch clamp. Pflugers Arch. 401,111 -118.[Medline]
Chu, G., Lester, J. W., Young, K. B., Luo, W., Zhai, J. and
Kranias, E. G. (2000). A single site (Ser16)
phosphorylation in phospholamban is sufficient in mediating its maximal
cardiac responses to beta-agonists. J. Biol. Chem.
275,38938
-38943.
Fan, J., Shuba, Y. M. and Morad, M. (1996).
Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.
Proc. Natl. Acad. Sci. USA
93,5527
-5532.
Fischmeister, R. and Shrier, A. (1989). Interactive effects of isoprenaline, forskolin and acetylcholine on Ca2+ current in frog ventricular myocytes. J. Physiol. 417,213 -239.[Abstract]
Freestone, N. S., Ribaric, S., Scheuermann, M., Mauser, U., Paul, M. and Vetter, R. (2000). Differential lusitropic responsiveness to beta-adrenergic stimulation in rat atrial and ventricular cardiac myocytes. Pflugers Arch. 441, 78-87.[CrossRef][Medline]
Gomez, A. M., Cheng, H., Lederer, W. J. and Bers, D. M. (1996). Ca2+ diffusion and sarcoplasmic reticulum transport both contribute to [Ca2+]i decline during Ca2+ sparks in rat ventricular myocytes. J. Physiol. 496,575 -581.[Abstract]
Harkins, A. B., Kurebayashi, N. and Baylor, S. M. (1993). Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3. Biophys. J. 65,865 -881.[Abstract]
Hartzell, H. C., Mery, P. F., Fischmeister, R. and Szabo, G. (1991). Sympathetic regulation of cardiac calcium current is due exclusively to cAMP-dependent phosphorylation. Nature 351,573 -576.[CrossRef][Medline]
Hove-Madsen, L. and Bers, D. M. (1993). Sarcoplasmic reticulum Ca2+ uptake and thapsigargin sensitivity in permeabilized rabbit and rat ventricular myocytes. Circ. Res. 73,820 -828.[Abstract]
Hove-Madsen, L., Llach, A. and Tort, L. (1998). Quantification of Ca2+ uptake in the sarcoplasmic reticulum of trout ventricular myocytes. Am. J. Physiol. 275,R2070 -R2080.[Medline]
Hove-Madsen, L., Llach, A. and Tort, L. (1999). Quantification of calcium release from the sarcoplasmic reticulum in rainbow trout atrial myocytes. Pflugers Arch. 438,545 -552.[CrossRef][Medline]
Hove-Madsen, L., Llach, A. and Tort, L. (2000).
Na(+)/Ca(2+)-exchange activity regulates contraction and
SR Ca(2+) content in rainbow trout atrial myocytes. Am.
J. Physiol. Regul. Integr. Comp. Physiol.
279,R1856
-R1864.
Hove-Madsen, L., Llach, A. and Tort, L. (2001).
The function of the sarcoplasmic reticulum is not inhibited by low
temperatures in trout atrial myocytes. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 281,R1902
-R1906.
Hove-Madsen, L., Mery, P. F., Jurevicius, J., Skeberdis, A. V. and Fischmeister, R. (1996). Regulation of myocardial calcium channels by cyclic AMP metabolism. Basic Res. Cardiol. 91 Suppl 2,1 -8.[Medline]
Hove-Madsen, L. and Tort, L. (1998). L-type Ca2+ current and excitation-contraction coupling in single atrial myocytes from rainbow trout. Am. J. Physiol. 275,R2061 -R2069.[Medline]
Hove-Madsen, L. and Tort, L. (2001). Characterization of the relationship between Na+-Ca2+ exchange rate and cytosolic calcium in trout cardiac myocytes. Pflugers Arch. 441,701 -708.[CrossRef][Medline]
Ju, Y. K. and Allen, D. G. (1999). Does adrenaline modulate the Na+-Ca2+ exchanger in isolated toad pacemaker cells? Pflugers Arch. 438,338 -343.[CrossRef][Medline]
Jurevicius, J. and Fischmeister, R. (1996).
cAMP compartmentation is responsible for a local activation of cardiac
Ca2+ channels by beta-adrenergic agonists. Proc. Natl.
Acad. Sci. USA 93,295
-299.
Kawai, M., Hussain, M. and Orchard, C. H. (1998). Cs+ inhibits spontaneous Ca2+ release from sarcoplasmic reticulum of skinned cardiac myocytes. Am. J. Physiol. 275,H422 -H430.[Medline]
Li, L., Chu, G., Kranias, E. G. and Bers, D. M. (1998). Cardiac myocyte calcium transport in phospholamban knockout mouse, relaxation and endogenous CaMKII effects. Am. J. Physiol. 274,H1335 -H1347.[Medline]
Li, L., DeSantiago, J., Chu, G., Kranias, E. G. and Bers, D.
M. (2000). Phosphorylation of phospholamban and troponin I in
beta-adrenergic-induced acceleration of cardiac relaxation. Am. J.
Physiol. Heart Circ. Physiol. 278,H769
-H779.
Mattiazzi, A., Hove-Madsen, L. and Bers, D. M. (1994). Protein kinase inhibitors reduce SR Ca transport in permeabilized cardiac myocytes. Am. J. Physiol. 267,H812 -H820.[Medline]
Negretti, N., Varro, A. and Eisner, D. A. (1995). Estimate of net calcium fluxes and sarcoplasmic reticulum calcium content during systole in rat ventricular myocytes. J. Physiol. 486,581 -591.[Abstract]
Nicoll, D. A., Longoni, S. and Philipson, K. D. (1990). Molecular cloning and functional expression of the cardiac sarcolemmal Na(+)-Ca2+ exchanger. Science 250,562 -565.[Medline]
Shiels, H. and Farrell, A. (1997). The effect
of temperature and adrenaline on the relative importance of the sarcoplasmic
reticulum in contributing Ca2+ to force development in isolated
ventricular trabeculae from rainbow trout. J. Exp.
Biol. 200,1607
-1621.
Trafford, A. W., Diaz, M. E. and Eisner, D. A. (1999). A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes. Pflugers Arch. 437,501 -503.[CrossRef][Medline]
Varro, A., Negretti, N., Hester, S. B. and Eisner, D. A. (1993). An estimate of the calcium content of the sarcoplasmic reticulum in rat ventricular myocytes. Pflugers Arch. 423,158 -160.[Medline]
Vornanen, M. (1998). L-type Ca2+
current in fish cardiac myocytes, effects of thermal acclimation and
beta-adrenergic stimulation. J. Exp. Biol.
201,533
-547.
Wimsatt, D. K., Hohl, C. M., Brierley, G. P. and Altschuld, R.
A. (1990). Calcium accumulation and release by the
sarcoplasmic reticulum of digitonin-lysed adult mammalian ventricular
cardiomyocytes. J. Biol. Chem.
265,14849
-14857.
Woo, S. H. and Morad, M. (2001). Bimodal
regulation of Na(+)Ca(2+) exchanger by
beta-adrenergic signaling pathway in shark ventricular myocytes.
Proc. Natl. Acad. Sci. USA
98,2023
-2028.
Xue, X. H., Hryshko, L. V., Nicoll, D. A., Philipson, K. D. and Tibbits, G. F. (1999). Cloning, expression, and characterization of the trout cardiac Na(+)/Ca(2+) exchanger. Am. J. Physiol. 277,C693 -C700.[Medline]
Zhou, Y. Y., Song, L. S., Lakatta, E. G., Xiao, R. P. and Cheng,
H. (1999). Constitutive beta2-adrenergic signalling enhances
sarcoplasmic reticulum Ca2+ cycling to augment contraction in mouse
heart. J. Physiol. 521,351
-361.
Ziolo, M. T., Katoh, H. and Bers, D. M. (2001).
Positive and negative effects of nitric oxide on Ca(2+) sparks,
influence of beta-adrenergic stimulation. Am. J. Physiol. Heart
Circ. Physiol. 281,H2295
-H2303.