Circulation Research. 1997;81:1034-1044
(Circulation Research. 1997;81:1034-1044.)
© 1997 American Heart Association, Inc.
Low Efficiency of Ca2+ Entry Through the Na+-Ca2+ Exchanger as Trigger for Ca2+ Release From the Sarcoplasmic Reticulum
A Comparison Between L-Type Ca2+ Current and Reverse-Mode Na+-Ca2+ Exchange
Karin R. Sipido,
Micheline Maes,
Frans Van de Werf
From the Laboratory of Experimental Cardiology (K.R.S., F. Van de W.),
University of Leuven (Belgium), and the Laboratory of Electrobiology (M.M.),
University of Antwerp (Belgium).
Correspondence to Karin R. Sipido, MD, PhD, Laboratory of Experimental Cardiology, K.U.L., Campus Gasthuisberg O/N 7th Floor, Herestraat 49, B-3000 Leuven, Belgium. E-mail Karin.Sipido{at}med.kuleuven.ac.be.
 |
Abstract
|
|---|
Abstract It has been proposed that Ca
2+ entry
through the Na
+-Ca
2+ exchanger can contribute
significantly to the trigger for Ca
2+ release from the
sarcoplasmic reticulum (SR). We have compared
the characteristics of
Ca
2+ release triggered by reverse-mode
Na
+-Ca
2+ exchange and by L-type
Ca
2+ current (
ICaL) during
depolarizing
steps in single guinea pig ventricular
myocytes (whole-cell
voltage clamp, fluo 3 and fura-red as
[Ca
2+]
i indicators, 36±1°C,
K
+-based
pipette solution with 20 mmol/L
[Na
+]). Conditioning pulses
to +60 mV ensured comparable
Ca
2+ loading of the SR. In the
presence of
ICaL, [Ca
2+]
i
transients typically have an early
and rapid rising phase reflecting
Ca
2+ release, which has a
bell-shaped voltage dependence
with a peak at +10 mV. With Ca
2+ entry through
Na
+-Ca
2+ exchange only (20 µmol/L
nisoldipine),
Ca
2+ release flux from the SR is decreased
and directly related
to the amplitude of the depolarizing step.
Ca
2+ release is preceded
by a significant delay (81±21 ms
at +20 mV, 24±4
ms at +70 mV) related to Ca
2+ entry
through the exchanger. Triggered
release interrupts Ca
2+
entry, as evidenced by reversal of the
exchanger current. At potentials
positive to +40 mV, Ca
2+ influx
through
Na
+-Ca
2+ exchange, calculated from the outward
exchange
current, reaches magnitudes comparable to
ICaL, but Ca
2+ release
due to
reverse-mode Na
+-Ca
2+ exchange still has a
significant
delay. We calculated trigger efficiency as the ratio
between
the maximal rate of Ca
2+ release and the
Ca
2+ influx preceding
this release; efficiency of
reverse-mode Na
+-Ca
2+ exchange is
approximately
four times less than that of
ICaL. With both
ICaL and reverse-mode
Na
+-Ca
2+ exchange present, Ca
2+
release is
triggered by
ICaL, and a
contribution of reverse-mode Na
+-Ca
2+ exchange
to the trigger could not be detected at potentials
below +60 mV. These
characteristics of reverse-mode Na
+-Ca
2+
exchange
predict that its role as a trigger for Ca
2+
release during the
action potential is likely to be negligible.
Key Words: Na+-Ca2+ exchange sarcoplasmic reticulum Ca2+ channel cardiomyocyte
 |
Introduction
|
|---|
The
Ca
2+ release channel of the cardiac SR opens in response
to
an increase in [Ca
2+] at the cytoplasmic side of the
channel.
1 In cells from which the sarcolemma had been
removed, it was
shown that the rate of Ca
2+ release was
dependent on the rate
of change in [Ca
2+] near the SR,
being larger for rapid increases
in [Ca
2+] near the
SR,
2 and that Ca
2+ entry through the L-type
Ca
2+ channel would have all the characteristics necessary
for optimal
triggering of the Ca
2+ release
channel.
3 Studies that found
a close relationship between
the voltage dependence of the L-type
Ca
2+ current and the
voltage dependence of contraction and of
Ca
2+ release in
intact isolated single cardiac cells have supported
this
hypothesis.
48 The colocalization of L-type
Ca
2+ channels
and Ca
2+ release channels in the
junctional complex of T tubules
would facilitate direct
interaction.
9,10 Theoretical modeling
has indicated that
control of Ca
2+ release by local interactions
between
individual L-type Ca
2+ channels and Ca
2+
release channels
of the SR could indeed reproduce the characteristics
of Ca
2+ release in the intact cell.
11,12
Recently, [Ca
2+]
i transients
resulting from
the activation of small release units have been
observed during
confocal microscopy as Ca
2+ sparks or local
Ca
2+ transients. These results indicate that
Ca
2+ entry through one
L-type Ca
2+ channel
controls activation of a small cluster of
Ca
2+ release
channels.
13,14
Although the important role of the L-type Ca2+ current is
well established, it has been proposed that it may not be the only
trigger for Ca2+ release (reviewed in Reference 1515 ).
Studies of force development in multicellular preparations and of cell
shortening in single cells have shown significant force and/or
shortening at positive potentials, where Ca2+ entry through
the L-type Ca2+ channel was unlikely. Evidence was
presented supporting the hypothesis that at these positive
potentials Ca2+ entry through the
Na+-Ca2+ exchanger (reverse-mode
Na+-Ca2+ exchange) provided the trigger for
Ca2+ release from the SR.1618 It was also
proposed that Na+ accumulation associated with the
Na+ current induced reverse-mode
Na+-Ca2+ exchange and sufficient
Ca2+ entry to trigger Ca2+
release,19,20 but conflicting results have been
reported.2124 A different line of evidence for a role of
reverse-mode Na+-Ca2+ exchange as a trigger for
release comes from experiments showing that a considerable amount of
cell shortening or a significant [Ca2+]i
transient still remains in the presence of Ca2+ channel
blockers applied shortly before the test pulse by means of a rapid
solution switcher.2528 In these studies, it was proposed
that Na+-Ca2+ exchange would contribute
significantly to the triggering of Ca2+ release in the
voltage range around 0 mV (where typically Ca2+ current is
maximally activated) and during the action potential.
The extent of the contribution of reverse-mode
Na+-Ca2+ exchange to the triggering of
Ca2+ release during the action potential remains
controversial. In contrast to numerous studies on the Ca2+
currentrelated release, many characteristics of
Na+-Ca2+ exchange as a trigger for release are
unknown. Although in the data presented by Bridge, Levi, and
colleagues27,28 the time course of Ca2+ release
triggered by the exchanger is the same as the one triggered by the
Ca2+ current, differences in the time course of shortening
at positive potentials suggest that there are important kinetic
differences between the two trigger mechanisms.16,17
Thermodynamics of the exchanger predict a decrease and/or reversal of
the exchange current during Ca2+ release. However, the
outward Na+-Ca2+ exchange current and its
predicted decrease or reversal during Ca2+ release have not
yet been not documented, except in one study involving Ca2+
influx during action potential recording.29 In
contrast to ICaL-related Ca2+
release, the efficiency of reverse-mode
Na+-Ca2+ exchange, ie, the relation between the
amplitude of Ca2+ entry through the exchanger and the
amplitude of Ca2+ release, has not been studied. Therefore,
in the present study we have investigated the characteristics of
Ca2+ entry through the exchanger as a trigger for
Ca2+ release and compared those characteristics with those
of Ca2+ entry through the L-type Ca2+ channel.
Following previously published reports that have emphasized the
importance of experimental conditions, our experiments were done at
36°C and with K+-containing pipette solutions. To
increase Ca2+ entry through the exchanger, we included
20 mmol/L Na+ in the pipette solution. Our
findings indicate that Na+-Ca2+ exchange can
trigger Ca2+ release from the SR but that this trigger is
less efficient than L-type Ca2+ current and that its
contribution during the action potential is likely to be negligible.
 |
Materials and Methods
|
|---|
Cell Isolation
Single guinea pig ventricular myocytes were isolated
enzymatically
as previously described.
23 Cell isolation
routinely yielded
60% to 70% of viable rod-shaped cells.
Voltage-Clamp and [Ca2+]i
Measurements
We used the whole-cell ruptured patch-clamp
technique.30 Membrane currents were recorded with an
Axopatch 1D amplifier, filtered at 1 kHz, and sampled and digitized at
4 kHz (Fastlb45, Indec Systems).
[Ca2+]i was monitored with fluo 3 (60
µmol/L) or a combination of fluo 3 (30 µmol/L)
and fura-red (70 µmol/L). We chose to use fluo 3 because
of the brightness of the signal and fast response time, which made it
easy to study the rising phase of the [Ca2+]i
transient. In combination with fura-red, motion artifacts could be
minimized, and [Ca2+]i could be estimated
after calibration of the ratio signal. Excitation wavelength was 485±8
nm. The dichroic mirror under the objective was centered at 510 nm. The
emission light was split by a second dichroic mirror centered at 585
nm. Fluo 3 fluorescence was sampled at 535±15 nm; the
fura-reddependent emission was recorded by a second red-sensitive
photomultiplier with a bandpass filter of 615±25 nm in front of it.
The microscope was also equipped with a transmitted light source at 700
nm; a CCD camera made it possible to follow cell shortening visually on
a TV monitor.
With fluo 3 alone, fluorescence values are normalized for
baseline fluorescence. For the combination of fluo 3 and
fura-red, we used calibration parameters that were obtained
partly during in vitro calibration (ßxKd) and
partly during in vivo calibration (maximum and minimum
fluorescence ratios).31,32 Such calibration can
yield a best estimate of [Ca2+]i but is less
reliable than calibration of the ratiometric dyes, such as fura 2 or
indo 1, because of possible inhomogeneous equilibration of
the two dyes. Despite these limitations, calibrated
[Ca2+]i values between cells were well
reproducible for a given experimental protocol.
Ca2+ release from the SR is indicated by a rapid increase
of [Ca2+]i, and it has been shown previously
that the Ca2+ release flux is proportional to the rate of
rise or derivative of
[Ca2+]i.12,33 Quantitative
calculations of the release flux were not possible in the presence of
K+ currents and Na+-Ca2+ exchange
currents. We have therefore used an indirect approach: we have measured
and compared the amplitude of the derivative of
[Ca2+]i in different conditions as an
indirect estimate of the amplitude of the SR Ca2+ release
flux.34 To analyze the rate of rise of
[Ca2+]i, we have used the derivative of the
fluo 3 signal. With the derivative of the calibrated
[Ca2+]i signal, results were qualitatively
comparable, but the signal-to-noise ratio was less favorable.
Solutions and Experimental Protocols
The pipette solution contained (mmol/L) potassium
aspartate 120, KCl 20, potassium HEPES 10, MgATP 5, MgCl2
0.5, NaCl 20, and fluo 3 0.06 (or fluo 3 0.03 and fura-red 0.07), pH
7.20. Alternatively, potassium aspartate and KCl were replaced with
CsCl (130 mmol/L). For some experiments, a pipette solution
without NaCl was used. The presence of high
[Na+]i with 20 mmol/L
[Na+]pip compared with 0 mmol/L
[Na+]pip was confirmed by the presence of
4-fold larger dihydro-ouabainsensitive Na+-K+
pump currents (K.R. Sipido and F. Verdonck, unpublished data, 1997).
The external solution contained (mmol/L) NaCl 130, KCl 5.4,
sodium HEPES 11.8, MgCl2 0.5, CaCl2 1.8, and
glucose 6, pH 7.35. For some experiments, KCl was omitted and replaced
with 10 mmol/L CsCl. All experiments were done at
36±1°C. External solution exchange was done by a rapid perfusion
system positioned close to the cell. From the block of the inward
rectifier K+ current at a holding potential of -50 mV by
application of CsCl (10 mmol/L) or BaCl2
(0.5 mmol/L), we measured a solution exchange time of
2
s. To block L-type Ca2+ channels, we used 20
µmol/L nisoldipine (Bayer), prepared as a 20
mmol/L stock in dimethyl sulfoxide. Ca2+ release
from the SR was disabled with 10 µmol/L ryanodine (Sigma
Chemical Co) prepared as a 10 mmol/L stock in water. In the
presence of nisoldipine, NiCl2 (2 or 5 mmol/L)
was used to block Na+-Ca2+ exchange.
The characteristics of Ca2+ release were studied during
225-ms test depolarizations from -40 or -45 mV to -30 up to +70 mV.
These depolarizations were preceded by two conditioning steps (300 ms)
to +60 mV at 1-s intervals; the test depolarization followed the last
conditioning step with an interval of 2 s. The pulse protocol was
repeated every 20 or 30 s, and the holding voltage between pulse
trains was -70 mV.
With 20 mmol/L NaCl in the pipette, the SR was not depleted
at rest.35 With two conditioning depolarizing steps to +60
mV, large and reproducible loading of the SR could be obtained. We
tested and confirmed that with this loading protocol, steady-state
block of the L-type Ca2+ channel did not significantly
affect the extent of loading. This is illustrated in Fig 1
. After the two conditioning steps to
+60 mV, the Ca2+ content of the SR was estimated from the
amplitude of Ca2+ release induced by a brief application of
caffeine.36,37 The protocol was then repeated in the
presence of 20 µmol/L nisoldipine (n=7). Caffeine
application induced more than one [Ca2+]i
transient in five of seven cells, probably related to the high loading
state of the SR, since this was not observed with lower
[Na+]pip. Therefore, we compared both the
peak fluorescence of the first release signal and the
integrated transient inward current during Ca2+ release,
which mainly results from Ca2+ extrusion through
Na+-Ca2+ exchange. Neither
parameter was significantly affected by Ca2+
channel block, indicating that loading of the SR in the present
conditions occurs primarily through Ca2+ entry through
Na+-Ca2+ exchange.

View larger version (26K):
[in this window]
[in a new window]
|
Figure 1. Ca2+ content of the SR after two
conditioning pulses to +60 mV is estimated from Ca2+
release induced by rapid application of caffeine, as shown in the
insert at the top. A, Membrane current (I) and fluorescence (F)
records of the last conditioning pulse and during caffeine (10
mmol/L) application in control conditions (left) and after
Ca2+ channel block (right). a.u. indicates arbitrary units.
B, Pooled data of seven cells (mean±SEM). On the left is the
integrated transient inward current (C) during caffeine application; on
the right, peak value of first fluorescence transient (F)
during caffeine application. Experiments were performed in potassium
aspartatebased pipette solution; [Na+]pip
was 20 mmol/L.
|
|
 |
Results
|
|---|
Voltage Dependence and Time Course of
[Ca2+]i Transients With Both
ICaL and
Na+-Ca2+ Exchange
In Fig 2

, membrane currents (top
traces) and [Ca
2+]
i transients
(bottom
traces) during test depolarizations between -30 and
+70 mV are
illustrated by a representative example. The early
inward
current during the depolarizing step is
ICaL (nisoldipine sensitive;
see below). On
repolarization, a time-dependent inward current
is seen, reflecting
Ca
2+ efflux through the Na
+-Ca
2+
exchanger
([Ca
2+]
i dependent and blocked by
NiCl
2). At -30 mV, there is
only a very small increase in
[Ca
2+]
i, but as the test potential
becomes
more positive, the amplitude and rate of rise of
[Ca
2+]
i increase. At +10 mV, the rate of rise
of [Ca
2+]
i is maximal,
and peak
[Ca
2+]
i is reached 20 ms after the onset of
the depolarizing
step. At potentials negative to +30,
[Ca
2+]
i declines after
this early peak and
before the membrane is repolarized. As the
depolarizing pulse becomes
more positive, a second component
of slow increase of
[Ca
2+]
i can be seen after the fast initial
rise.
Peak [Ca
2+]
i coincides with the end of
the depolarization; decline
of [Ca
2+]
i starts
on repolarization.

View larger version (19K):
[in this window]
[in a new window]
|
Figure 2. Membrane currents (I) (top traces) and
[Ca2+]i transients (bottom traces) during
225-ms depolarizing steps to the indicated potentials from a holding
potential of -45 mV. Test depolarization was preceded by two
conditioning steps to +60 mV, as illustrated in the insert at the top.
Experiments were performed in potassium aspartatebased pipette
solution; [Na+]pip was 20 mmol/L.
|
|
Because of this second slow component, a plot of the maximal values of
[Ca2+]i reached during the depolarizing pulse
is directly related to the test membrane potential, as shown in Fig 3B
(solid circles, mean±SEM of nine
cells). The presence of a second slow component at positive potentials
is also evident from the plot of the amplitude of the time-dependent
inward current on repolarization, reflecting Ca2+ efflux
via the Na+-Ca2+ exchanger (Fig 3B
, solid
squares). This plot illustrates the pronounced increase in this current
at positive potentials. Although the plot of peak
[Ca2+]i is a monotonic function of membrane
voltage, there are clear differences in the time course of the
[Ca2+]i transient at different potentials.
These are illustrated by the superimposed traces obtained at +10 and at
+70 mV (Fig 3A
). The rate of rise of the
[Ca2+]i transient at +70 mV appears to be
delayed compared with the [Ca2+]i transient
at +10 mV. Therefore, if we plot [Ca2+]i at
20 ms after the depolarizing step as a function of the test membrane
potential, a different voltage dependence is seen (Fig 3B
, open
circles). The bell-shaped voltage dependence of the early increase in
[Ca2+]i suggests a close relation to
ICaL; however, the voltage dependence of the
peak value of [Ca2+]i suggests that a second
process with more linear voltage dependence also contributes to the
[Ca2+]i transient. This process was absent in
cells studied with a pipette solution without NaCl, as illustrated in
Fig 3C
and 3D
. Taken together, these data indicate the presence of a
contribution of reverse-mode Na+-Ca2+ exchange
to the [Ca2+]i transients of cells studied
with 20 mmol/L [Na+]pip.
Therefore, we further examined the [Ca2+]i
transient after block of ICaL in cells with
20 mmol/L [Na+]pip.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 3. A, Membrane currents (I) and
[Ca2+]i transients at +10 mV and at +70 mV on
expanded time scale (same records as in Fig 1 ). Note the delay
before the rapid upstroke of the [Ca2+]i
transient at +70 mV and the presence of a second slow component,
resulting in a high [Ca2+]i at the end of the
depolarizing step and a large inward current on repolarization. B,
Pooled data of eight cells studied with 20 mmol/L
[Na+]pip (mean±SEM) for maximal
[Ca2+]i during the depolarizing step ( ),
[Ca2+]i at 20 ms after the depolarizing step
( ), and the inward current 10 ms after repolarization ( ). C,
Membrane currents and [Ca2+]i transients at
+10 mV and at +70 mV in a cell studied with 0 mmol/L
[Na+]pip. At +70 mV, the
[Ca2+]i transient is very small. D, Pooled
data from six cells studied with 0 mmol/L
[Na+]pip (mean±SEM) for maximal
[Ca2+]i during the depolarizing step ( ),
[Ca2+]i at 20 ms after the depolarizing step
( ), and the inward current 10 ms after repolarization
(Iinw on rep, ). All parameters have a
similar bell-shaped voltage dependence.
|
|
Voltage Dependence and Time Course of
[Ca2+]i Transients After Block of
ICaL
Nisoldipine (20 µmol/L) was applied and
Ca2+ release was examined after full solution exchange,
during steady-state perfusion. With conditioning pulses to +60 mV and
with 20 mmol/L [Na+]pip, the
block of ICaL had no significant effect on the
loading of the SR (see "Materials and Methods" and Fig 1
). Fig 4
shows the
[Ca2+]i transients before and after block of
ICaL. The rapidly rising
[Ca2+]i transients at potentials below +30 mV
are blocked; however, the [Ca2+]i transient
at the most positive potential (+70 mV) is only slightly changed. This
is illustrated in more detail in Fig 5
, where membrane currents and [Ca2+]i
transients at +10, +40 and +70 mV are shown on an expanded time scale.
The membrane current traces clearly show the block of
ICaL by nisoldipine. The rapid rise of the
[Ca2+]i transient at +10 mV is also blocked,
and instead, a delayed, more slowly rising
[Ca2+]i transient is seen. At +70 mV the
[Ca2+]i transient has changed very little,
whereas at +40 mV an intermediate picture is seen. The changes in the
[Ca2+]i transients at +10 and at +40 mV can
be described by two parameters: (1) a decrease in the rate
of rise of [Ca2+]i, corresponding to a
decrease in the Ca2+ release flux,12,33,34 and
(2) a delay between the onset of depolarization and the onset of a more
rapid rise in [Ca2+]i. These
parameters were quantified in a total of six cells by
measuring the maximal value of the derivative of the fluo 3 signal and
of the time to this maximal value in control conditions and after the
block of ICaL by nisoldipine, illustrated in Fig 6
. The plot of the maximal rate of rise
in control conditions (Fig 6A
, solid squares) is bell-shaped, with a
maximum at +10 mV. With nisoldipine (open squares), the maximal rate of
rise is decreased significantly at potentials between -30 and +40 mV.
At more positive potentials, little change or even a slight increase is
seen. However, even at these potentials the rate of rise is still less
than the maximal rate of rise observed with ICaL
at +10 mV. The delay to the onset of the rapid increase in
[Ca2+]i is shown in Fig 6B
as the time to the
maximal dF/dt. In control conditions, the maximal rate of rise is
reached within 10 to 15 ms after the onset of the depolarizing pulse,
with the minimal delay seen around +10 mV. After block of
ICaL, this delay is increased significantly and
exceeds 30 ms even at the most positive voltages; the time to peak
dF/dt is now a monotonic decreasing function of the amplitude of the
depolarizing step.

View larger version (17K):
[in this window]
[in a new window]
|
Figure 5. Membrane currents (I) (top traces) and
[Ca2+]i transients (bottom traces) during
depolarization to +10, +40, and +70 mV before and after block of
ICaL by 20 µmol/L nisoldipine (+nis).
This is the same cell as in Fig 4 .
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Figure 6. A, The rate of rise of
[Ca2+]i was quantified as the maximal value
of the first derivative of the fluo 3 signal; for each cell, these
values were normalized to the maximal value of that cell. Values are
mean±SEM (pooled data from six cells, potassium aspartatebased
pipette solution, [Na+]pip=20 mmol/L). V
indicates voltage. B, The delay to the rapid rise of
[Ca2+]i was quantified as the time to the
maximal value of the first derivative.
|
|
The monotonic and nearly linear relation between membrane voltage and
the amplitude and rate of rise of the [Ca2+]i
transients after block of ICaL strongly suggest
that these [Ca2+]i transients are related to
reverse-mode Na+-Ca2+ exchange. The presence of
a phase of rapid increase in [Ca2+]i also
suggests that these [Ca2+]i transients do not
merely result from Ca2+ entry through the exchanger but
that Ca2+ is released from the SR. We hypothesized that the
presence of a delay to the more rapid rise in Ca,2+ a delay
that is inversely related to the membrane potential, reflected the time
between Ca2+ entry and Ca2+ release. To
investigate this hypothesis, we compared the
[Ca2+]i transients in the presence of
nisoldipine with the [Ca2+]i transients after
block of Ca2+ release by ryanodine.
Ca2+ Entry via Na+-Ca2+
Exchange Precedes Triggered Ca2+ Release
Fig 7
shows a
representative example of superimposed membrane
currents and [Ca2+]i transients before and
after block of Ca2+ release with 10 µmol/L
ryanodine (all in the presence of nisoldipine). Similar results were
obtained in five other cells. In the presence of ryanodine, the rapidly
rising part of the [Ca2+]i transient is
blocked, and [Ca2+]i transients are slow,
increasing throughout the depolarizing pulse, as reported
previously.38 The [Ca2+]i
transient before application of ryanodine can be superimposed on the
one in the presence of ryanodine up to the time when a sudden increase
in the rate of rise occurs. These findings support the idea that the
rapidly rising phase represents Ca2+ release,
occurring with a delay during which Ca2+ entry occurs. This
is also reflected in the time course of the membrane currents.
Ryanodine induced a small linear leak current, but the most important
change during the depolarizing steps is the disappearance of the inward
shift of the membrane current coinciding with Ca2+ release.
Comparing [Ca2+]i transients and the time
course of membrane currents in the presence and absence of ryanodine
(as shown in Fig 7
), we have measured the delay for Ca2+
release triggered by Na+-Ca2+ exchange as the
time from onset of depolarization to the time of rapid increase in
[Ca2+]i, with its associated inward shift of
the membrane current. At +10 mV, this delay was 93±20 ms; at +40 mV,
55±13 ms; and at +70 mV, 24±4 ms (mean±SEM, n=6, same group of cells
as in Fig 6
). These data reflect the delay to the onset of
Ca2+ release and confirm that this delay is related to
Ca2+ entry.
Our results so far indicate that after block of
ICaL, Na+-Ca2+ exchange
can trigger Ca2+ release from the SR, but this
Ca2+ release differs in two important aspects from
Ca2+ release triggered by ICaL.
First, the same maximal rate of rise of
[Ca2+]i and therefore the same
Ca2+ release flux are not attained in the range of membrane
potentials studied. Second, Ca2+ release is delayed
significantly. One possible explanation for these differences is that
the rate of Ca2+ entry through the exchanger is much less
than the rate of Ca2+ entry through the L-type
Ca2+ channel. We therefore have quantified this rate of
entry by measuring ICaL and the
Na+-Ca2+ exchange current.
Amplitude and Time Course of the Na+-Ca2+
Exchange Current During Ca2+ Entry and
Ca2+ Release
Because of previous reports that substitution of internal
K+ with Cs+ affects Ca2+ release
from the SR,18,39,40 the experiments shown so far were done
with a K+-containing pipette solution. However, the
membrane currents are then always mixed with time-dependent
K+ currents, complicating the interpretation. Therefore, to
measure the Na+-Ca2+ exchange current, we have
repeated our experiments with a Cs+-containing pipette
solution and with external KCl replaced with CsCl (n=12). An example of
such an experiment is shown in Fig 8
. We
recorded membrane currents and [Ca2+]i
transients first in control conditions, then in the presence of
nisoldipine to block ICaL, and last in a
solution with 2 mmol/L NiCl2 added to the
nisoldipine-containing solution to block
Na+-Ca2+ exchange. The
Na+-Ca2+ exchange current is the difference
between the membrane current in the presence of nisoldipine and the
current in the presence of NiCl241 and is shown
in the middle panels. It is clear that the initial
Na+-Ca2+ exchange current is outward at the
onset of the depolarizing pulse and decreases during the pulse as
[Ca2+]i increases to become a net inward
current during Ca2+ release. Furthermore, the
Na+-Ca2+ exchange outward current increases in
amplitude with increasing membrane potential. The amplitude of the
outward Na+-Ca2+ exchange current was measured
10 ms into the depolarizing pulse; ICaL was
measured as the nisoldipine-sensitive peak inward current at 5 to 10 ms
after depolarization. The average values of membrane current density
for five cells are shown in Fig 10A
. These values are in the same order
of magnitude as reported by others in whole-cell
experiments.38,41,42

View larger version (26K):
[in this window]
[in a new window]
|
Figure 8. Membrane currents (I) (top) and
[Ca2+]i transients (normalized
fluorescence records of fluo 3 signal [F]) (bottom) at
the indicated potentials with internal potassium aspartate and KCl
replaced with 130 mmol/L CsCl
([Na+]pip=20 mmol/L) and with 10
mmol/L CsCl added to the external solution. a.u. indicates arbitrary
units; con, records in control conditions; nis, records in the
presence of 20 µmol/L nisoldipine; and Ni, membrane currents
recorded with both 20 µmol/L nisoldipine and 2 mmol/L
NiCl2 (dashed line). Note the absence of a transient inward
current on repolarization; [Ca2+]i transients
in the presence of NiCl2 are flat. The difference currents
between nis and Ni are the Na+-Ca2+ exchange
currents (middle). The increase in peak
[Ca2+]i after block of
ICaL is probably related to higher loading
of the SR during Ca2+ entry via the exchanger preceding
release.54
|
|
Experiments with Cs+-containing pipette solutions were
complicated by the fact that in the majority of cells (8 of 12) after
block of ICaL the remaining
[Ca2+]i transient no longer had a rapidly
rising phase indicative of triggered Ca2+ release (versus
in only 1 of 15 cells tested with K+-containing pipette
solution). This is in line with previous reports.18,39,40
One possible explanation is that the Cs+ pipette solution
somehow decreased Na+-Ca2+ exchange. We
therefore repeated the protocol shown in Fig 8
with
K+-containing solutions. To block K+ currents
as much as possible, we added 1 µmol/L almokalant (to
block the rapidly activating component of the delayed rectifier
K+ current) and 0.25 or 0.5 mmol/L
BaCl2. Records of such an experiment are shown in Fig 9
. As with CsCl in the pipette, the
Na+-Ca2+ exchange current is outward at the
onset of depolarization and shows a large inward shift during
Ca2+ release. In a manner similar to that for the
experiments performed with Cs+ in the pipette, we have
pooled the results of five cells and have plotted the membrane current
density of the Ni2+-sensitive initial outward current and
of the nisoldipine-sensitive peak inward current (Fig 10B
). The maximal
ICaL appears to be shifted by +10 mV compared
with the data obtained with CsCl, which is compatible with a 10-mV
offset potential with low Cl- pipette solutions. With this
taken into account, the values for Na+-Ca2+
exchange current or for ICaL are not
significantly different between cells studied with a
K+-containing or with a Cs+-containing pipette
solution.

View larger version (27K):
[in this window]
[in a new window]
|
Figure 9. Membrane currents (I) (top) and
[Ca2+]i transients (normalized
fluorescence records of fluo-3 signal [F]) (bottom) at
the indicated potentials with a K+-based pipette solution
([Na+]pip=20 mmol/L) and with 1
µmol/L almokalant and 0.25 mmol/L BaCl2 added to the
external solution. a.u. indicates arbitrary units; con, records in
control conditions; nis, records in the presence of 20
µmol/L nisoldipine; and Ni, membrane currents recorded with both
20 µmol/L nisoldipine and 2 mmol/L NiCl2
(dashed line). The difference currents between nis and Ni are the
Na+-Ca2+ exchange currents (middle). The
increase in peak [Ca2+]i after block of
ICaL is probably related to higher loading
of the SR during Ca2+ entry via the exchanger preceding
release.54
|
|
Relation Between Ca2+ Entry and Release: Trigger
Efficiency
The data shown in Fig 10
indicate that high rates of
Ca2+ entry can be achieved through reverse-mode
Na+-Ca2+ exchange. To evaluate the efficiency
of Ca2+ entry both through the exchanger and
ICaL as a trigger for Ca2+ release,
we have measured in this same group of cells the maximal rate of rise
as an indicator of the Ca2+ release flux and related this
to the Ca2+ entry (with potassium aspartatebased pipette
solution). These data are shown in Fig 11
. Solid symbols indicate data
obtained in control conditions; open symbols, after block of
ICaL. The top left panel shows the rate of
Ca2+ entry (in µmol · s-1
· F-1), obtained by dividing current densities by
valence and Faraday's constant. At +70 mV, Ca2+ entry in
the cell is almost exclusively through the exchanger and nearly as
large as Ca2+ entry through the Ca2+ channel at
0 mV, whereas at 0 mV, Ca2+ entry through the exchanger is
small. If we then look at the Ca2+ release flux at +70 mV
after block of ICaL (solid symbols in top middle
panel), it is also nearly as large as at 0 mV with
ICaL (open symbols). However, a major difference
is the time to this release, which is 53±8 ms with the exchanger at
+70 mV but only 11±3 ms with ICaL at 0 mV (top
right panel). These data imply that for the exchanger a larger amount
of Ca2+ entry is required before Ca2+ release
is triggered. We have further quantified trigger efficiency as the
relation between the peak rate of Ca2+ release and the
preceding Ca2+ influx, obtained by integrating
Ca2+ entry through ICaL or through
the exchanger, up to the time of peak dF/dt. This is shown in the
bottom panel of Fig 11
; solid symbols indicate
ICaL (measured at -10, 0, and +10 mV), and open
symbols indicate the Na+-Ca2+ exchange current
(measured at +50, +60, and +70 mV after block of
ICaL). Because the data are pooled for different
voltages, we have not curve-fitted the data, but it can be seen that
the relation is much steeper for ICaL than for
Na+-Ca2+ exchange. For five cells, the ratio
between peak dF/dt and the preceding Ca2+ entry was 54±5
(arbitrary units) for ICaL at 0 mV versus 15±4
for Na+-Ca2+ exchange at +70 mV. These data
indicate that the trigger efficiency of Ca2+ entry through
the exchanger is lower than of Ca2+ entry through the
L-type Ca2+ channel.

View larger version (21K):
[in this window]
[in a new window]
|
Figure 11. Relation between Ca2+ entry and
Ca2+ release in control conditions ( ) and after block of
the L-type Ca2+ channel ( ). Values are mean±SEM (pooled
data from five cells). Top left, Ca2+ entry was calculated
by dividing the Na+-Ca2+ exchange and
ICaL densities by the valence and Faraday's
constant; data are the same as in Fig 10A . Top middle, Ca2+
release was measured as the maximal value of the first derivative of
the fluo 3 signal; for each cell these values were normalized to the
maximal value of that cell. Top right, The delay to Ca2+
release was quantified as the time to the maximal value of the first
derivative. Bottom, Relation between Ca2+ release, peak
dF/dt, and preceding Ca2+ entry, measured as the integrated
Ca2+ entry up to the time of peak dF/dt. indicates
ICaL, measured at -10, 0, and +10 mV in six
cells; , reverse-mode Na+-Ca2+ exchange,
measured at +50, +60, and +70 mV after block of
ICaL; and a.u., arbitrary units. Experiments
were performed in potassium aspartatebased pipette solution;
[Na+]pip was 20 mmol/L.
|
|
Contribution of Reverse-Mode Na+-Ca2+
Exchange as Trigger for Ca2+ Release in the Presence of
ICaL
Even if the efficiency of Ca2+ entry through the
exchanger as trigger for release is low, it could still act as an
additional trigger to ICaL in normal conditions.
We have examined this issue by comparing Ca2+ release with
20 mmol/L [Na+]pip and with
0 mmol/L [Na+]pip in the presence
of ICaL. If reverse-mode
Na+-Ca2+ exchange contributes to the trigger,
then release should not decline, or at least decline less, at positive
voltages with 20 mmol/L [Na+]pip.
The plot of peak dF/dt with 20 mmol/L
[Na+]pip and ICaL
present (solid symbols in Fig 6A
) already showed that the voltage
dependence of release follows the voltage dependence of
ICaL, except at +70 mV. Comparing
Ca2+ release observed with 20 mmol/L
[Na+]pip with Ca2+ release
observed with 0 mmol/L [Na+]pip,
panels B and D of Fig 3
showed that [Ca2+]i
at 20 ms, as a parameter for Ca2+ release in
this time period, declines with voltage whether or not reverse-mode
Na+-Ca2+ exchange is present. This is
further illustrated by the superimposed plots of
ICaL and [Ca2+]i for
these experiments, shown in Fig 12A
and 12B
. Although [Ca2+]i is higher at all
voltages with 20 mmol/L [Na+]pip,
compatible with a higher loading state of the SR in the presence of
increased [Na+]i,43 the voltage
dependence still follows the voltage dependence of
ICaL. Only at +70 mV is a deviation observed,
with release seen with 20 mmol/L
[Na+]pip but not with 0 mmol/L
[Na+]pip.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 12. Contribution of reverse-mode
Na+-Ca2+ exchange to triggering of
Ca2+ release in the presence of
ICaL. A, Superimposed plots of the increase
in [Ca2+]i at 20 ms after the depolarizing
step ( ) and of ICaL ( ;
ICaL has been scaled, with an inverse sign)
with 0 mmol/L [Na+]pip (pooled data from
six cells). The curves are superimposed throughout the full voltage
range. B, Same data for six cells studied with 20 mmol/L
[Na+]pip. indicates the increase in
[Ca2+]i at 20 ms after the depolarizing step;
, ICaL (ICaL
has been scaled, with an inverse sign). Only at +70 mV do the curves
have a different voltage dependence with more Ca2+ release.
C, Apparent efficiency of ICaL as trigger
for release calculated as the ratio between peak dF/dt as measure for
Ca2+ release and the preceding Ca2+ entry, ie,
the integrated nisoldipine-sensitive current up to the time of peak
dF/dt. Each data point is the mean of six cells. indicates 20
mmol/L [Na+]pip; , 0 mmol/L
[Na+]pip. Even with 20 mmol/L
[Na+]pip, the apparent trigger efficiency of
ICaL decreases with more positive
potentials, indicating that reverse mode does not contribute to the
trigger.
|
|
We also calculated the relation between peak dF/dt and the preceding
Ca2+ influx through ICaL as a
measure of the apparent efficiency of ICaL for 0
and 20 mmol/L [Na+]pip at -10
mV, +20 mV, and +50 mV. If reverse-mode
Na+-Ca2+ exchange adds substantially to the
trigger, the apparent efficiency of ICaL should
increase with voltage with 20 mmol/L
[Na+]pip, as the Ca2+ influx
through the exchanger increases >5-fold in this range of voltages (Fig 10
), or at least decrease less, compared with conditions with 0
mmol/L [Na+]pip. The results are shown
in Fig 12C
. Whether or not the pipette contains 20 mmol/L
Na+, the efficiency of ICaL
decreases with voltage in a similar way. This similar voltage
dependence of the apparent efficiency of ICaL in
the absence or presence of [Na+]pip indicates
that reverse-mode Na+-Ca2+ exchange does not
contribute substantially to the trigger for release in the presence of
ICaL for voltages below +60 mV.
 |
Discussion
|
|---|
The Ca
2+ release channel of the SR opens in response
to an increase
in Ca
2+ near the channel. Our data show that
important differences
exist between Ca
2+ entry through the
L-type Ca
2+ channel and
Ca
2+ entry through the
Na
+-Ca
2+ exchanger as a trigger for
Ca
2+ release. Reverse-mode Na
+-Ca
2+
exchange as a trigger is characterized
by (1) a low Ca
2+
entry rate except at very positive potentials
and (2) a low efficiency
with prolonged Ca
2+ entry before release
is triggered.
Furthermore, we have shown that the contribution
of reverse-mode
Na
+-Ca
2+ exchange to the triggering of
Ca
2+ release in the presence of
ICaL
can be detected only at potentials

+60 mV. These characteristics
predict that the role of reverse-mode
Na
+-Ca
2+
exchange as a trigger for Ca
2+ release during the action
potential
will be negligible.
Comparison With Previous Studies
Several early studies (reviewed in Reference 1515 ) had indicated
that mechanical activity, ie, force development or shortening, at
positive potentials could be related to
Na+-Ca2+ exchange. However, in a series of
studies on single cells, no Ca2+ release was observed at
these positive potentials.48,22 One possible explanation
was a temperature effect, since most of these studies were done at room
temperature. A first study to document Ca2+ release
triggered by Ca2+ entry through the exchanger was done at
35°C16; in a comparative study, Vornanen et
al17 demonstrated the importance of working at a
physiological temperature. In the study of Vornanen
et al and in the study of Wasserstrom and Vites,18 the
voltage dependence of peak shortening was sigmoidal. Contractions at
positive potentials were linked to Ca2+ release triggered
by Na+-Ca2+ exchange; they were observed only
at 35°C to 37°C.17,18 In the study of Vornanen et al,
it was noted that the time to peak shortening at very positive
potentials was larger than that at +10 mV and that application of
Ca2+ channel block increased the time to peak
shortening.17 A delay for Na+-Ca2+
exchangetriggered release after Ca2+ channel block was
also observed by Nuss and Houser,16 and it was suggested
that reverse-mode Na+-Ca2+ exchange was less
effective as a trigger than was
ICaL.16,17,21 Our data confirm that
Ca2+ entry through Na+-Ca2+
exchange can trigger release, whereas our analysis of the
kinetics of the [Ca2+]i transients and of the
relation between Ca2+ release and Ca2+ entry
establishes the low intrinsic efficiency of this mechanism.
Our experimental findings differ substantially from the results
published by Bridge, Levi, and colleagues,2528 who used a
different approach to study the contribution of Ca2+ entry
via the exchanger. These investigators used a rapid switch device to
block ICaL just before the action potential or
depolarizing test pulse to minimize changes in loading of the SR. With
this approach, they found that the amplitude of cell shortening and of
the [Ca2+]i transient during a test pulse to
+10 mV or during the action potential was decreased, but only to a
moderate extent and without the slowing in the rate of upstroke that we
have shown in the present study. The major difference between their
approach and our approach is that we used a steady-state block of
ICaL. The advantage of our approach is that one
can obtain a more complete block of ICaL, which
can otherwise pose problems because of the voltage dependence of block.
A possible drawback is that loading of the SR might have been
significantly decreased and that this decrease in the SR content would
be responsible for the observed changes in the
[Ca2+]i transient. In our conditions,
Ca2+ loading was not severely reduced and cannot account
for our findings. With 20 mmol/L Na+ in the
pipette, significant loading of the SR occurs via the
Na+-Ca2+ exchanger during the conditioning
prepulses, and this was not affected by ICaL
block, as demonstrated during caffeine-induced Ca2+ release
(Fig 1
). In addition, if the Ca2+ content had been reduced,
[Ca2+]i transients at all potentials would
have been reduced, but in contrast, [Ca2+]i
transients at very positive potentials were only marginally affected
(see Fig 4
). Last, we and others have studied
[Ca2+]i transients for different degrees of
Ca2+ loading of the SR with ICaL as
the trigger for release.33,37,39,44 With decreased
Ca2+ loading, the amplitude and rate of rise of the
[Ca2+]i transient decreased, but a delay as
seen in the present study was not observed.
It has also been reported previously that intracellular Cs+
would reduce Ca2+ release from the SR.18,39,40
Such a difference must be quantitative, since many investigators have
observed Ca2+ release from the SR with
Cs+-containing pipette solution, and it has been proposed
that especially Na+-Ca2+ exchangedependent
release would be sensitive to inhibition by Cs+. Our data
seem to support this view, although at present it is unclear what
causes this difference. In species such as the rat, with large
transient outward K+ currents, the use of Cs+
would improve voltage control and possibly reduce spurious activation
of Ca2+ channels during large depolarization, thereby
reducing Ca2+ release. However, this does not apply to the
guinea pig myocytes. Ni2+-sensitive currents with either
K+- or Cs+-based pipette solutions were
comparable, which would seem to exclude the possibility that reduced
Ca2+ release may be related to an inhibition of
Na+-Ca2+ exchange itself. A remaining
possibility is that intracellular Cs+ reduces
Ca2+ release itself; block of K+ channels in
the SR membrane45 may affect the rate of release or
decrease Ca2+ uptake and affect the Ca2+ load
of the SR. The higher Cl- concentration with CsCl-based
pipette solution could also affect Cl- transport across
the SR membrane.46
Ca2+ Entry Through the Na+-Ca2+
Exchanger as Trigger for Ca2+ Release
Ca2+ entry through the
Na+-Ca2+ exchanger is capable of triggering
Ca2+ release from the SR but differs substantially from the
Ca2+ current. A striking characteristic of Ca2+
release triggered by Na+-Ca2+ exchange is the
delay between the onset of depolarization and the actual release, a
delay that is inversely related to the rate of Ca2+ entry
through the exchanger. A similar delay is never observed with
Ca2+ release triggered by ICaL and
is most likely related to fundamental differences between
Ca2+ entry through both pathways. We have shown that with
20 mmol/L [Na+]pip and large
depolarizations, Ca2+ entry rate through the
Na+-Ca2+ exchanger on the whole-cell level can
reach values comparable to the rate of Ca2+ entry through
ICaL. However, Ca2+ entry through
the exchanger appears to be intrinsically less efficient, since a
larger amount of Ca2+ entry is required to obtain
comparable Ca2+ release. This lower efficiency is most
likely related to differences in the Ca2+ entry at the
molecular level. As shown experimentally, the Ca2+ flux
through a single L-type Ca2+ channel versus the rate of
Ca2+ entry through a single
Na+-Ca2+ exchange molecule is 2 to 3 orders of
magnitude larger, ie,
0.3 pA for the L-type Ca2+ channel
at 0 mV47 and 0.6 to 1.3 fA for the outward exchange
current (current evoked by switch from 0 to 40 mmol/L
[Na+] in the cytosolic solution in a giant excised patch
at 37°C48). This parameter may be critical to
obtain high [Ca2+] near the Ca2+ release
channel. In addition to the amplitude of the single-channel
Ca2+ influx, the localization in relation to the release
channel will determine the local increase in Ca2+ near the
SR. In studies with labeled antibodies, it was shown that
Na+-Ca2+ exchange proteins could be found over
the entire sarcolemma, although the density in T tubules seemed to be
higher than in the external sarcolemma.49,50 Even with
Na+-Ca2+ exchange proteins localized in the
vicinity of Ca2+ release channels, the lower molecular
Ca2+ entry rate may still be a limiting factor. In their
modeling of [Ca2+] in the junctional cleft between
sarcolemma and SR feet, Langer and Peskoff51 propose that
11 L-type Ca2+ channel and 100
Na+-Ca2+ exchange proteins have access to this
restricted space. With Ca2+ influx via the L-type
Ca2+ channel, [Ca2+] at the Ca2+
release channels in the cleft at 1 ms after the depolarizing step is 3
orders of magnitude higher than the concentration reached by
reverse-mode Na+-Ca2+ exchange after 10 ms
(assuming 16 mmol/L [Na+]i); this
model predicts that Ca2+ release triggered by
Na+-Ca2+ exchange would be delayed, as we have
observed. In the model, simultaneous influx through the
Ca2+ channel further reduces Ca2+ entry through
the exchanger. However, this may be less pronounced near
Ca2+ channels with a longer first latency.
Contribution of Reverse-Mode Na+-Ca2+
Exchange to Ca2+ Release From the SR
Whereas the data show that reverse-mode
Na+-Ca2+ exchange can trigger release in
specific experimental conditions, this trigger cannot simply be assumed
to be additive to ICaL in normal conditions. To
facilitate the study of Na+-Ca2+ exchange as a
trigger for release, we worked with high
[Na+]pip, block of
ICaL, and a wide range of voltages. These
experimental conditions will lead to an overestimation of the
contribution of the exchanger to triggered Ca2+ release
during the action potential. First, [Na+]i in
guinea pig ventricular myocytes in normal conditions is
<10 mmol/L.52 Second, large depolarizations,
as required for the exchanger to trigger Ca2+ release with
a delay of <50 ms, are reached only very briefly during the normal
action potential. Last, in the presence of ICaL,
early activation of Ca2+ release is likely to interrupt
Ca2+ entry through the Na+-Ca2+
exchanger very early. Even ICaL itself may
lessen the influx through the exchanger.51 Grantham and
Cannell29 have recorded significant outward
Na+-Ca2+ exchange currents during action
potential clamp recording after block of
ICaL. However, these authors have also pointed
out that in the presence of ICaL and
ICaL-triggered release, Ca2+ entry
through the exchanger will be reduced. Aside from these considerations,
direct evaluation of Ca2+ release triggered by
ICaL with 20 mmol/L versus 0
mmol/L [Na+]pip failed to reveal a
significant contribution of reverse-mode
Na+-Ca2+ exchange to the triggering of
Ca2+ release in the presence of ICaL
(Fig 12
).
Although we have presented strong evidence that the
contribution of reverse-mode Na+-Ca2+ exchange
to the triggering of Ca2+ release is likely to be minor
during a normal action potential, it is clear that (reverse-mode)
Na+-Ca2+ exchange will significantly influence
excitation-contraction coupling. Earlier observations on
contractions16,17,40 and the data shown in Fig 3
demonstrate the important differences between contractions and
[Ca2+]i transients with low or high
[Na+]i. The plot of peak
[Ca2+]i shown in Fig 3B
corresponds to the
plots of peak shortening published previously. With high
[Na+]i, [Ca2+]i
transients are larger and will be prolonged during maintained
depolarizations. This can be related to a higher Ca2+
content of the SR,43 with a larger fractional
release,37,39,53 and to decreased Ca2+ efflux
or net Ca2+ influx during maintained depolarizations. For
very strong depolarizations at
+60 mV, as can only be obtained during
voltage clamp, reverse-mode Na+-Ca2+ exchange
will also trigger release. At these potentials, influx via the
exchanger may increase the Ca2+ load of the SR before
Ca2+ release is triggered and further enhance
Ca2+ release.54
In conclusion, our data support the view that in normal conditions,
ICaL will be the major trigger for
Ca2+ release from the SR. Na+-Ca2+
exchange will modulate Ca2+ release from the SR as both
reverse and forward mode contribute to the net Ca2+ flux in
and out of the cell and thereby determine the Ca2+ content
of the SR. Reverse-mode Na+-Ca2+ exchange may
possibly become more important as a trigger in certain conditions, eg,
when [Na+]i is increased (such as during
block of the Na+-K+ pump), in the presence of a
decreased Ca2+ current, or possibly with an increased
expression of the Na+-Ca2+ exchanger (such as
has been described during cardiac hypertrophy or
failure).55 Even in these conditions, it is expected to be
less efficient than ICaL, as was also recently
reported for transgenic mice overexpressing a mutant of the
Na+-Ca2+ exchanger with high
activity.56 Lower efficiency of reverse-mode
Na+-Ca2+ exchange as a trigger for
Ca2+ release is most likely related to lower molecular
Ca2+ entry rates and spatial organization.
 |
Selected Abbreviations and Acronyms
|
|---|
| cyt (as subscript) |
= |
cytosolic solution |
| ICaL |
= |
L-type Ca2+ current |
| pip (as subscript) |
= |
pipette solution |
| SR |
= |
sarcoplasmic reticulum |
|
 |
Acknowledgments
|
|---|
The study was supported by a grant from the Fund for Scientific
Research
(FWO) and the Bekales Foundation. Dr Sipido is a postdoctoral
researcher
of the FWO, Belgium. The stay of Ms Maes was supported by
the
Born Bunge Foundation. We thank Drs Carmeliet, Mubagwa, and
Callewaert
for critical comments on the manuscript and Dr Verdonck for
helpful
discussions on [Na
+]
i regulation and
measurements of Na
+-K
+ pump currents.
Received March 13, 1997;
accepted September 2, 1997.
 |
References
|
|---|
-
Anderson K, Lai FA, Liu QY, Rousseau E, Erickson
HP, Meissner G. Structural and functional characterization of the
purified cardiac ryanodine receptor-Ca2+ release channel
complex. J Biol Chem. 1989;264:13291335.[Abstract/Free Full Text]
-
Fabiato A. Time and calcium dependence of activation
and inactivation of calcium-induced release of calcium from the
sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell.
J Gen Physiol. 1985;85:247289.[Abstract/Free Full Text]
-
Fabiato A. Simulated calcium current can both cause
calcium loading in and trigger calcium release from the sarcoplasmic
reticulum of a skinned canine cardiac Purkinje cell. J Gen
Physiol. 1985;85:291320.[Abstract/Free Full Text]
-
London B, Krueger JW. Contraction in voltage-clamped,
internally perfused single heart cells. J Gen Physiol. 1986;88:475505.[Abstract/Free Full Text]
-
Cannell MB, Berlin JR, Lederer WJ. Effect of membrane
potential changes on the calcium transient of single rat cardiac muscle
cells. Science. 1987;238:14191423.[Abstract/Free Full Text]
-
Beuckelmann DJ, Wier WG. Mechanism of release of
calcium from sarcoplasmic reticulum of guinea-pig cardiac cells.
J Physiol (Lond). 1988;405:233255.[Abstract/Free Full Text]
-
Nabauer M, Callewaert G, Cleemann L, Morad M.
Regulation of calcium release is gated by calcium current, not gating
charge, in cardiac myocytes. Science. 1989;244:800803.[Abstract/Free Full Text]
-
duBell WH, Houser SR. Voltage and beat dependence of
Ca2+ transient in feline ventricular myocytes.
Am J Physiol. 1989;257:H746H759.[Abstract/Free Full Text]
-
Wibo M, Bravo G, Godfraind T. Postnatal maturation of
excitationcontraction coupling in rat ventricle in relation to the
subcellular localization and surface density of
1,4-dihydropyridine and ryanodine receptors.
Circ Res. 1991;68:662673.[Abstract/Free Full Text]
-
Carl SL, Felix K, Caswell AH, Brandt NR, Ball WJJ,
Vaghy PL, Meissner G, Ferguson DG. Immunolocalization of sarcolemmal
dihydropyridine receptor and sarcoplasmic reticular
triadin and ryanodine receptor in rabbit ventricle and atrium. J
Cell Biol. 1995;129:672682.
-
Stern MD. Theory of excitation-contraction coupling in
cardiac muscle. Biophys J. 1992;63:497517.[Abstract/Free Full Text]
-
Wier WG, Egan TM, Lopez-Lopez JR, Balke CW. Local
control of excitation-contraction coupling in rat heart cells.
J Physiol (Lond). 1994;474:463471.[Abstract/Free Full Text]
-
Lopez-Lopez JR, Shacklock PS, Balke CW, Wier WG. Local
calcium transients triggered by single L-type calcium channel currents
in cardiac cells. Science. 1995;268:10421045.[Abstract/Free Full Text]
-
Cannell MB, Cheng H, Lederer WJ. The control of calcium
release in heart muscle. Science. 1995;268:10451049.[Abstract/Free Full Text]
-
Levi AJ, Brooksby P, Hancox JC. One hump or two?: the
triggering of calcium release from the sarcoplasmic reticulum and the
voltage dependence of contraction in mammalian cardiac muscle.
Cardiovasc Res. 1993;27:17431757.[Free Full Text]
-
Nuss HB, Houser SR. Sodium-calcium exchange-mediated
contractions in feline ventricular myocytes. Am
J Physiol. 1992;263:H1161H1169.[Abstract/Free Full Text]
-
Vornanen M, Shepherd N, Isenberg G. Tension-voltage
relations of single myocytes reflect Ca release triggered by Na/Ca
exchange at 35 degrees C but not 23 degrees C. Am J
Physiol. 1994;267:C623C632.[Abstract/Free Full Text]
-
Wasserstrom JA, Vites A-M. The role of
Na+-Ca2+ exchange in activation of
excitation-contraction coupling in rat ventricular
myocytes. J Physiol (Lond). 1997;493:529542.[Medline]
[Order article via Infotrieve]
-
Leblanc N, Hume JR. Sodium current-induced release of
calcium from cardiac sarcoplasmic reticulum. Science. 1990;248:372376.[Abstract/Free Full Text]
-
Lipp P, Niggli E. Sodium current-induced calcium
signals in isolated guinea-pig ventricular myocytes.
J Physiol (Lond). 1994;474:439446.[Abstract/Free Full Text]
-
Sham JSK, Cleemann L, Morad M. Gating of the cardiac
Ca2+ release channel: the role of Na+ current
and Na+-Ca2+ exchange. Science. 1992;255:850853.[Abstract/Free Full Text]
-
Bouchard RA, Clark RB, Giles WR. Role of sodium-calcium
exchange in activation of contraction in rat ventricle. J
Physiol (Lond). 1993;472:391413.[Abstract/Free Full Text]
-
Sipido KR, Carmeliet E, Pappano AJ. Na+
current and Ca2+ release from the sarcoplasmic reticulum
during action potentials in guinea-pig ventricular
myocytes. J Physiol (Lond). 1995;489:117.[Medline]
[Order article via Infotrieve]
-
Cannell MB, Grantham CJ, Main MJ, Evans AM. The roles
of the sodium and calcium current in triggering calcium release from
the sarcoplasmic reticulum. Ann N Y Acad Sci. 1996;779:443450.[Medline]
[Order article via Infotrieve]
-
Levi AJ, Spitzer KW, Kohmoto O, Bridge JHB.
Depolarization-induced Ca entry via Na-Ca exchange triggers SR release
in guinea pig cardiac myocytes. Am J Physiol. 1994;266:H1422H1433.