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1 Department of Physiology, The role of
L-type calcium current
(ICa,L) in
impulse generation was studied in single sinoatrial nodal myocytes of
the rabbit, with the use of the amphotericin-perforated patch-clamp
technique. Nifedipine, at a concentration of 5 µM, was
used to block
ICa,L. At this
concentration, nifedipine selectively blocked
ICa,L for 81%
without affecting the T-type calcium current
(ICa,T), the fast sodium current, the delayed rectifier current
(IK), and the hyperpolarization-activated inward current. Furthermore, we did not
observe the sustained inward current. The selective action of
nifedipine on
ICa,L enabled us
to determine the activation threshold of
ICa,L, which was
around
nifedipine; delayed rectifier current; hyperpolarization-activated
current; T-type calcium current; fast sodium current; sustained inward
current
DIASTOLIC DEPOLARIZATION underlies automaticity of the
sinoatrial (SA) node (3) and starts when net ionic membrane current changes from outward to inward. Membrane current is composed of an
outward current, mainly carried by the delayed rectifier current (IK), and
several inward currents: the hyperpolarization-activated current
(If); two
calcium currents, the T-type calcium current (ICa,T) and the
L-type calcium current
(ICa,L); and a
background current
(Ib), which is
inward during diastole (18). Recently, a sustained inward sodium
current (Ist)
has been described, which could provide an inward current during early
diastolic depolarization (14). The relative contribution of these
currents to diastolic depolarization is still a matter of debate. At
present, there are three views on the ionic mechanism of pacemaking. In
the first view, the time course of decay of
IK together with
an inward Ib are
the dominant factors for the rate of diastolic depolarization (4,
34). In the second view,
If, which is
activated at a relatively negative membrane potential, plays a dominant
role (8, 11). In the third view, it is proposed that
ICa,T is active
during early diastolic depolarization (12, 15) and that the latter part
of the diastole is governed by
ICa,L, initiating the next action potential (12, 15, 26, 38).
The contribution of
ICa,L to
diastolic depolarization is generally considered to be minor, because
the "activation threshold" of
ICa,L is thought
to be around In this study we investigated the contribution of
ICa,L to the
impulse generation in single pacemaker cells isolated from the SA node
of the rabbit using the amphotericin-perforated patch-clamp technique.
Therefore, we used the dihydropyridine nifedipine, which is known to
block ICa,L (15,
21, 28) without affecting ICa,T (15). To
validate its selectivity as an
ICa,L blocker, we
first studied the effects of nifedipine on
ICa,L,
ICa,T, fast sodium current
(INa),
IK, and
If. We also
investigated the presence of
Ist. Selective
ICa,L blockade by
nifedipine enabled us to determine the activation threshold of
ICa,L more
precisely. Effects of selective ICa,L blockade on
pacemaker activity were studied by a combined voltage- and
current-clamp protocol. We demonstrate that
ICa,L contributes
already to the early phase of diastolic depolarization.
Cell Isolation
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
60 mV. As nifedipine (5 µM) abolished spontaneous
activity, we used a combined voltage- and current-clamp protocol to
study the effects of
ICa,L blockade on
repolarization and diastolic depolarization. This protocol mimics the
action potential such that the repolarization and subsequent diastolic
depolarization are studied in current-clamp conditions. Nifedipine
significantly decreased action potential duration at 50%
repolarization and reduced diastolic depolarization rate over the
entire diastole. Evidence was found that recovery from inactivation of
ICa,L occurs
during repolarization, which makes
ICa,L available already early in diastole. We conclude that
ICa,L contributes significantly to the net inward current during diastole and can modulate the entire diastolic depolarization.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
40 mV (18), whereas the pacemaker depolarization
occurs between 60 and 40 mV. However, the presumed small
contribution of
ICa,L to only the
last part of diastole is not in agreement with the marked bradycardic
effect of ICa,L blockers (13, 20, 25, 28), which on the other hand could be caused by a
specific action of the drug on
Ist (5, 14) or an
indirect effect on the
Na+/Ca2+
exchange current
(INaCa).
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-hydroxybutyric acid,
5.0 succinic acid, 2.0 Na2ATP, and
50 g/l polyvinylpyrrolidone (pH adjusted with KOH to 6.9) and gently
shaken. The KB solution was refreshed three times to remove the
dissociation solution. Thereafter, the strips were again triturated in
KB solution through a pipette (tip diameter of 0.8-1.2 mm) for
5-10 min. Samples of the resulting cell suspension (0.4 ml) were
placed in a recording chamber on the stage of an inverted microscope
(Nikon Diaphot) and superfused (0.6 ml/min) with normal Tyrode
solution. For our experiments, we selected spontaneously active spindle
and elongated spindlelike cells (7, 35). All experiments were performed at 35 ± 0.5°C. Temperature was maintained by a
translucent heating plate underneath the bottom of the recording
chamber (31) and continuously monitored. Animal care was in accordance
with institutional guidelines.
Electrophysiological Recording
Membrane potentials and membrane currents, except INa, were recorded using the amphotericin-perforated patch technique (17, 33) to prevent rundown of membrane currents by dilution of intracellular components. To minimize series resistance, INa was recorded in the whole cell mode with low-resistance electrodes of 1-2 M
. We only used cells that did not display rundown of membrane currents studied during the first 5 min. Electrodes were pulled from
borosilicate glass (1-mm outer diameter, with a glass fiber inside the
lumen) using a vertical one-stage patch-electrode puller and were heat polished. When using the amphotericin-perforated patch technique, we
dissolved 6 mg of amphotericin B (Sigma Chemical, St. Louis, MO) in 100 µl of dimethyl sulfoxide shortly before the experiment of which 10 µl of the solution were added to 3 ml of the electrode solution.
Measurements of action potentials and
ICa,L,
IK, and If were performed
in normal Tyrode solution and an electrode solution containing (in mM)
120 potassium gluconate, 20 KCl, 5 HEPES, 5 MgCl2, 0.6 CaCl2, 5 Na2ATP, 0.1 cAMP, and 5 EGTA (pH
adjusted with KOH to 7.2). For the measurement of
ICa,T and the
measurements of
ICa,L threshold,
the pipette solution contained (in mM) 140 CsCl, 5 EGTA, 1 MgCl2, 4 MgATP, and 5 HEPES (pH
adjusted with CsOH to 7.2). The external solution was
Na+ deficient in that the NaCl was
replaced with equimolar Tris · HCl. For
the measurement of
INa and
Ist, the pipette
solution contained (in mM) 110 CsOH, 20 CsCl, 5 EGTA, 1 MgCl2, 4 MgATP, and 5 HEPES (pH
adjusted with aspartic acid to 7.2). In these experiments, 5 mM CsCl
was added to the normal Tyrode solution in which the amount of
CaCl2 was lowered to 0.1 mM.
For the amphotericin-perforated patch technique, electrode tips were
immersed in normal electrode solution for 1 s and backfilled with the
electrode solution to which amphotericin was added. Electrode resistance ranged between 3 and 5 M. After being sealed to the membrane, series resistance dropped quickly to 8-12 M and
became stable within 10 min for a period of at least 1 h. Series
resistance was compensated for ~75%, resulting in a residual series
resistance of ~2-3 M. For the
INa measurements,
series resistance after compensation was ~0.5-2 M. Membrane
potential and membrane current were recorded with a custom-built
voltage-clamp amplifier. Command potentials for voltage-clamp and
stimulus pulses were obtained from a custom programmable pulse
generator. All potentials were corrected by subtracting the
pipette-to-bath liquid junction potential as calculated using the
JPCalc software package (1). Table 1
summarizes the ionic composition of pipette and bath solutions and the
subsequent liquid junction potential, which amounted to 14 mV for the
perforated patch recording of action potentials, ICa,L,
If, and
IK; 10 mV for the
perforated patch recording of ICa,T; and 15 mV
for the whole cell recording of
INa and
Ist. Data were
stored on videotape (Sony Betamax) using a pulse code modulation system
(Sony PCM501) modified to enable DC recordings. For off-line processing
on a Macintosh Quadra 650 personal computer (Apple Computer, Cupertino,
CA), a custom-written data acquisition and analysis program was used.
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Test Protocols and Data Analysis
Depolarizing and hyperpolarizing voltage-clamp recordings were digitized with a sample frequency of 2 and 500 Hz, respectively. When recording the INa, a sample frequency of 5 kHz was used. The instantaneous voltage-clamp recordings (Fig. 6) were digitized with a sample frequency of 2 kHz. To discriminate between drug effects and possible rundown, alternating depolarizing or hyperpolarizing voltage-clamp pulses were applied every 15 s to the cell both 2 min before the application of the drug and during drug administration. Only cells without detectable rundown of membrane currents were used. The protocol was as follows: after a conditioning prepulse of 0.5 s to40 mV, a test pulse to 0 mV of 0.5 s was applied, after which the voltage was clamped back to 40 mV for
0.5 s. Then the current-clamp mode was switched on again. After 15 s, a
similar pulse protocol was used with a hyperpolarizing test pulse of 1 s to 90 mV instead of the depolarizing pulse. To study drug effects
in more detail, another protocol in which depolarizing and
hyperpolarizing voltage-clamp steps from a holding potential of
40 mV to various potentials were applied intermitted this protocol. Steady-state currents and inward peak currents during the
test pulse as well as tail currents after the test pulse were recorded
and examined off-line. Currents are expressed as absolute values,
unless stated otherwise. Voltage-clamp protocols are described in more
detail in RESULTS.
Membrane capacitance was determined from the initial slope of the transmembrane voltage in response to the hyperpolarizing current pulses of 40 pA. Membrane capacitance was 53 ± 6 pF (mean ± SE, n = 23). For normalization, currents were expressed relative to membrane capacitance (pA/pF).
Because spontaneous activity was arrested by nifedipine even at a low concentration, a combined voltage- and current-clamp protocol was necessary to study drug effects on diastolic depolarization (see Figs. 9 and 10). Under control conditions, a depolarizing voltage-clamp step of ~50-ms duration to +10 mV mimicked the action potential, after which the amplifier was switched back to the current-clamp mode, leaving the repolarization and diastolic depolarization to occur naturally. Voltage-clamp pulses were applied at fixed intervals of 200 or 250 ms. The interval of pacing was chosen such that it was as close as possible and therefore only slightly shorter than the intrinsic interval.
To characterize action potentials, several action potential parameters were determined: action potential amplitude, action potential duration between 50% depolarization and 50% repolarization (APD50), action potential duration between 50% depolarization and 100% repolarization (APD100), diastolic depolarization rate (DDR), maximum upstroke velocity (dV/dtmax), and maximum diastolic potential (MDP). Diastolic depolarization rate was measured by fitting a straight line over the 50- or 75-ms time interval starting at the MDP + 1 mV (DDR50 and DDR75, respectively). MDP + 1 mV was used rather than MDP because the time at which the MDP + 1 mV was reached could more reliably be determined than the time at which the MDP was reached.
Statistics
For statistical analysis we used the mean values of the action potential parameters of 10 subsequent action potentials. All results are presented as means ± SE. Statistical significance was determined by a Student's t-test for paired or unpaired observations, where appropriate. A probability of P < 0.05 was considered significant.Drugs
Nifedipine (Sigma Chemical) was freshly dissolved in 97% ethanol, after which the solution was 1,000 times diluted in normal Tyrode solution. Because of the photosensitivity of the drug, the room was darkened during the experiment. E-4031 {1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonyl aminobenzoyl)piperidine} was a kind gift from Eisai. The agent was dissolved in distilled water at 1,000 times the concentration used. Batches of both stock solutions were stored at20°C until use.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Specificity of Nifedipine as ICa,L Blocker
We first performed a series of voltage-clamp experiments to study the effects of nifedipine on six membrane ionic currents possibly involved in pacemaking: ICa,L, ICa,T, INa, Ist, IK, and If.Effect of nifedipine on
ICa,L.
The effect of nifedipine on
ICa,L was studied
using depolarizing voltage-clamp pulses
(P1) of 500-ms duration that
were applied from a holding potential of 40 mV (Fig.
1A,
top). On depolarization, ICa,L activates
rapidly (15), which results in a net inward current (Fig.
1A, bottom
left). Inactivation of
ICa,L coincides with the activation of
IK resulting in a
less negative net membrane current, eventually becoming outward.
Nifedipine (5 µM) drastically reduces the inward current (Fig.
1A, bottom
right) and unmasks an instantaneous current step
together with a slowly activating outward current.
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32 ± 5.4 mV
(n = 8). This
Ib most likely
consists of various time-independent currents, e.g.,
Na+-K+
pump (27),
Na+/Ca2+
exchanger, background Cl
current, background Na+ current,
and a small leakage current flowing through the seal resistance
[see Verheijck et al. (34)]. For each individual experiment, the slope conductance and reversal potential of
Ib were
determined. For each experiment new
I-V relations were made by subtracting
the calculated Ib
from the peak current. Figure 1C shows
the average "corrected" I-V
relations in the absence and presence of nifedipine
(n = 8). From these corrected
I-V relations it is apparent that they
have the same voltage dependence. The similar voltage dependence can be
appreciated even more when the corrected
I-V relation in the presence of
nifedipine is scaled up by a constant factor of 5.3 (Fig.
1D). From these experiments it can
be concluded that 5 µM nifedipine blocks 81% of
ICa,L.
Effect of nifedipine on
ICa,T.
To explore the effect of nifedipine on
ICa,T, we first
had to isolate the current from other membrane currents. Activation of
INa was prevented
by replacing the extracellular NaCl with an equimolar concentration of
Tris · HCl. Furthermore,
IK was blocked by
replacing potassium with cesium in the intracellular solution. The
removal of extracellular Na+ and
intracellular K+ also strongly
reduces If. Under
these conditions, we employed the method of Bean (2) to dissect the two
types of Ca2+ currents, i.e., to
use two different holding potentials. We used holding potentials of
90 and 50 mV similar to those used by Hagiwara et al.
(15). After appropriate correction for liquid junction potential, their
holding potentials amount to 93 and 53 mV, respectively.
In the experiment shown in Fig.
2A, a
depolarizing voltage-clamp pulse
(P1) of 150-ms duration was
applied from alternate holding potentials of 90 and 50 mV
to minimize the effect of possible "rundown" during the time
course of the experiment. During steps from 50 mV to more
positive test potentials,
ICa,L activates rapidly. Also,
ICa,T may
activate to a small extent. When stepping from the more negative
holding potential of 90 mV, both
ICa,L and
ICa,T activate
(Fig. 2A,
left). The difference in the current traces between the two different holding potentials then consists of
only ICa,T
(bottom current trace of Fig. 2A)
(15). The I-V relation of the thus
obtained ICa,T is
illustrated in Fig. 2B (open circles).
In four experiments, the amplitude of
ICa,T measured at
the peak I-V value at 20 mV was
1.9 ± 0.4 pA/pF, which compares well with the maximum current
density of 2.14 pA/pF reported by Hagiwara et al. (15). Nifedipine (5 µM) blocked the inward current when stepping from 50 to
20 mV (Fig. 2A,
right). From the more negative
holding potential, the test pulse still elicited an inward current. The
difference in currents generated at the two holding potentials
represents ICa,T
and appears to be insensitive to nifedipine (Fig.
2A, bottom
traces, and Fig.
2B). Notice that the different current traces under the normal and nifedipine conditions follow a
similar time course. The absence of an effect of
nifedipine on
ICa,T was
confirmed in the three other cells.
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Effect of nifedipine on
INa.
We then questioned whether the nodal cells contained
INa and if so,
whether INa was
sensitive to nifedipine in these cells. Therefore,
INa was isolated
from other transmembrane currents by
1) reducing
ICa,L by lowering
the extracellular Ca2+
concentration to 0.1 mM (14), 2)
blockade of If by
adding 5 mM CsCl to the external solution, and
3) blockade of
IK by replacing potassium with cesium in the intracellular solution (cf. Table 1). Only
two of eight cells tested showed
INa. Directly
after seal breakthrough, we were able to record action potentials for 1 min after which spontaneous electrical activity ceased. For these two
cells, the first recorded action potentials showed a high maximum rate
of rise (16 and 19 V/s). These two cells were used to study the effect
of nifedipine on
INa. Figure
3 shows the results of an experiment in
which a depolarizing voltage-clamp pulse of 50 mV
(P1) of 100-ms duration was
applied from a holding potential of 95 mV. The bottom trace of
Fig. 3A shows the current recording in
response to the test pulse. Activation of
INa produces a
large inward current that inactivates rapidly and is followed by a
steady-state current during the rest of the test pulse. Nifedipine neither affected the inward current nor the steady-state current during
P1 (Fig.
3A, bottom
right). Figure 3B
shows the absence of an effect of nifedipine on
INa to different
test potentials. The absence of an effect of nifedipine on
INa was confirmed
in the other cell that showed
INa.
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Effect of nifedipine on
Ist.
The experimental protocol used to study
INa could also be
used to investigate whether the nodal cells contained
Ist and if so,
whether Ist was
sensitive to nifedipine in these cells. According to Guo et al. (14),
Ist could only be
separated from
ICa,L by reducing
the extracellular Ca2+
concentration, as we did, thereby substantially decreasing the amplitude of
ICa,L but
enhancing Ist.
The much slower activation and inactivation of
Ist compared with
INa enables
discrimination of
Ist from
INa. In the
experiment of Fig. 3 we applied a voltage-clamp step from a holding
potential of 95 to 50 mV, a voltage at which Ist should be
clearly visible as a slowly inactivating current (14). However,
directly after the inactivation of
INa, a
steady-state current was reached (Fig.
3A, bottom left
trace), which was not altered by the administration
of 5 µM nifedipine (Fig. 3A,
bottom right trace), demonstrating
the absence of
Ist in our
preparation. Similar observations were made at the other potentials
tested as illustrated in Fig. 3C,
which shows the steady-state current measured after 15 ms
(Iss,15, cf. Fig.
3A), in both the presence and
absence of nifedipine. Similar results were obtained for the other cell
that showed INa.
95 mV.
In response to each depolarizing voltage-clamp pulse, the membrane
current reached a steady-state level immediately after the capacitative
current transient. Thus this experiment clearly demonstrates the
absence of both
INa and Ist. Similar
results were obtained in the five other cells. Figure 4B illustrates the average
I-V relation of the
Iss,15 (cf. Fig. 4A) and clearly shows a linear
I-V relation in the voltage range of
90 to 0 mV. Such a linear relationship would not be expected if
Ist were present
(14).
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Effect of nifedipine on
IK.
The effect of nifedipine on
IK was
investigated using depolarizing voltage-clamp pulses
(P1) of 500-ms duration that
were applied from a holding potential of 40 mV (Fig.
5A,
top trace). The bottom traces of
Fig. 5A show current recordings in
response to these depolarizing test pulses
(P1). Activation of
IK produces, in
conjunction with an outward
Ib (34), an
outwardly directed current. We previously demonstrated that in the
preparation we used,
IK only consists
of the rapid component of IK
(IK,r)
(34). Tail currents following depolarizing pulses
(Itail) are
predominantly due to deactivation of
IK (10, 32, 34).
In this experiment, 5 µM nifedipine blocked the inward transient
current completely and produced an outward shift of the holding current
at 40 mV of 12 pA. It did not affect the currents at the end of
the depolarizing voltage-clamp step or their time course.
Itail were
reduced slightly without a change in their time course.
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40 and 10 mV, but this shift was not significant at any
of the potentials tested.
Figure 5C summarizes the effect of
nifedipine (5 µM) on
Itail to
different test potentials (n = 11) and
shows the absence of an effect of nifedipine on
Itail at any
potential measured.
From the data presented in Fig. 5 it cannot be excluded that nifedipine
affects IK at
voltages at which diastolic depolarization occurs (from about 60
to 40 mV). Therefore, the effect of nifedipine on
IK was studied in
more detail in an additional series of experiments. In those
experiments we used a protocol in which
IK was fully activated, whereas other voltage-dependent currents were absent because
they were either fully inactivated
(ICa,T and
ICa,L) or not
activated (If).
From a holding potential of 40 mV, a conditioning voltage-clamp
step to +20 mV was used to fully activate
IK (Fig. 6A,
top trace). Thereafter, repolarizing
steps to various test potentials
(P1) were made. Currents were
measured directly after the surge of the capacitative transient on the
test potential. In this way, an instantaneous
I-V relation was obtained in which IK was fully
present. Figure 6A
(bottom trace) shows two
representative current traces recorded under normal conditions and
after the administration of 5 µM nifedipine. Nifedipine reduces the
peak inward current during the conditioning voltage-clamp step to +20 mV but does not alter the steady-state level at the end of the pulse.
During P1 the control and
nifedipine current traces completely overlap and have a similar time
course. Figure 6B shows the lack of
effect of 5 µM nifedipine on the instantaneous
I-V relationship (n = 8). Because no effect
of nifedipine was observed on the instantaneous I-V relationship of
IK (Fig.
6B), we conclude that nifedipine does not affect
IK.
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Effect of nifedipine on
If.
Next, the effect of nifedipine on the
If was
investigated. Figure
7A
(bottom trace) shows representative
current recordings in response to 2-s hyperpolarizing voltage-clamp
steps (P1) from a holding
potential of 40 mV to 50 and 70 mV (Fig.
7A, top trace). On hyperpolarization an inward current is
activated that predominantly consists of
If (11) and
deactivates after return to the holding potential
(If tail). In
this experiment and eight others, 5 µM nifedipine did not alter the
quasi-steady-state current (If steady state)
or If tail (Fig.
7B). Therefore, we conclude that
nifedipine does not affect
If.
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Activation Threshold of ICa,L
From the above experiments, we conclude that 5 µM nifedipine blocks ICa,L without affecting ICa,T, INa, IK, and If. Furthermore, we demonstrate the absence of Ist in our cells. The highly selective action of nifedipine on ICa,L enabled us to determine the activation threshold for ICa,L from the data obtained in the experiments in which the effect of nifedipine on ICa,T was evaluated (Fig. 2). In these experiments, given the selective blockade of ICa,L by nifedipine, the time-dependent difference current (control nifedipine) in response to voltage-clamp steps from a holding potential
of 90 mV should be
ICa,L. Possible
errors caused by a change in passive membrane properties between the
control and nifedipine recordings were avoided by capacitive current
subtraction performed on individual membrane current recordings. The
capacitive transient elicited on stepping back from the test potentials
to the holding potential was added to the capacitive transient during the test pulse. This resulted in a complete removal of the capacitive transient during the test step and thus in an uncontaminated recording of the activation of
ICa,L (Fig.
8A). This procedure is allowed, because stepping back from the test potential to the holding potential does not activate time-dependent currents under our experimental conditions: 1) activation of
INa is prevented
by replacement of the extracellular NaCl with an equimolar
concentration of Tris · HCl;
2)
IK is blocked by
replacement of potassium with cesium in the intracellular solution, and
3)
If is blocked by
the removal of extracellular Na+
and intracellular K+.
Figure 8A
shows current recordings in response to a series of depolarizing test
pulses (P1) from a holding
potential of 90 mV, under control conditions (Fig.
8A, middle left), and in
the presence of 5 µM nifedipine (Fig. 8A, middle
right). The bottom panel of Fig.
8A shows the nifedipine-sensitive
difference current (control nifedipine) reflecting
ICa,L. From this
figure it becomes clear that a depolarizing voltage-clamp step to
60 mV can already activate some
ICa,L. Figure
8B shows the average
I-V relation of the difference current
reflecting ICa,L
(n = 4). The
I-V relation reaches its most negative
value of 12.2 ± 0.8 pA/pF at 10 mV.
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To our knowledge this is the first time that direct evidence is
presented that
ICa,L can be
activated at potentials as negative as 60 mV and thus may be
able to serve as an inward current during the entire diastolic depolarization.
Effect of ICa,L Blockade on Electrical Activity of SA Nodal Myocytes
From the results presented above, we conclude that Ist is absent in our preparation and that 5 µM nifedipine blocks ICa,L in nodal myocytes for 81%, without affecting ICa,T, IK, and If as well as INa, when present. In higher concentrations, nifedipine slightly blocked If (data not shown). Therefore, it was justified to use 5 µM nifedipine as a tool in studying the role of ICa,L in diastolic depolarization. To study the effect of ICa,L blockade on electrical activity, we had to use a combined voltage- and current-clamp protocol, because spontaneous electrical activity was arrested at 5 µM nifedipine. A voltage-clamp pulse (P1, Fig. 9A) was applied to depolarize the cell to such an extent that after the release of the voltage clamp, repolarization and the subsequent diastolic depolarization followed a time course as under free running conditions (see also Table 2). It should be noted that the cycle length of the spontaneously beating cell is always somewhat (on the average 17 ms, Table 2) longer than the cycle of the paced cell. This is due to the protocol being used: when a SA node cell is paced, one is obliged to use a slightly shorter pacing interval than the intrinsic interval. This explains why the upstroke of the second paced action potential starts earlier than the upstroke of the second spontaneous action potential (Fig. 9A). Careful examination of Fig. 9A further shows that the repolarization phases of the spontaneously initiated second action potential and the driven second action potential are also very similar. Table 2 shows that, under control conditions, the combined voltage- and current-clamp protocol does not alter any of the action potential parameters compared with spontaneous electrical activity (n = 11).
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Figure 9B shows that nifedipine (5 µM) predominantly reduces the early phase of repolarization and therefore reduces APD50. The drug also slows diastolic depolarization from the start of diastole, while APD100 and MDP remain unaltered. Table 2 summarizes the effect of 5 µM nifedipine on action potential parameters studied with the combined voltage- and current-clamp protocol. Data are obtained by averaging (for each individual experiment) 10 subsequent action potentials under control conditions and during the administration of nifedipine. Nifedipine significantly shortened APD50 by 17% and significantly reduced DDR50 and DDR75 by 29% and 26%, respectively.
Availability of Inward Current During Diastole
In the previous section it was demonstrated that selective but partial blockade of ICa,L reduced diastolic depolarization rate during the entire diastole (Fig. 9B). To study the "dynamic" availability of the inward current during repolarization and diastolic depolarization, we performed a combined current- and voltage-clamp protocol comparable to that described in the previous section. During repolarization and diastole, extra voltage-clamp steps to 0 mV of 10-ms duration (P2) were applied to fully activate ICa,L at different times after the onset of P1 (Fig. 10A). The bottom panel shows current traces in response to the combined voltage- and current-clamp protocol as described in the top panel of Fig. 10A. Under control conditions, an inwardly directed peak current (superimposed on a larger outward current) can already be elicited after ~40 ms, which becomes net inward ~10 ms before the MDP is reached. After the administration of 5 µM nifedipine, which only affects ICa,L, the amount of inward current is drastically reduced and no net inward current could be elicited during either repolarization or diastolic depolarization. Figure 10B summarizes the effect of nifedipine on the inward peak current at different times after the onset of P1 (n = 5). Under control conditions, inwardly directed peak currents can be elicited after 40 ms, which is considerably earlier than the time at which the MDP is reached (67 ± 4 ms). Nifedipine (5 µM) blocked most of the inwardly directed current throughout repolarization and diastolic depolarization. These experiments demonstrate that ICa,L is available to serve as an inward current during the entire diastolic depolarization.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Selective Effect of Nifedipine on ICa,L
In this study we investigated the role of ICa,L by blockade with nifedipine in the generation of electrical activity in rabbit SA nodal myocytes. Binding studies revealed that among the available calcium channel blockers, dihydropyridines like nifedipine have the highest affinity for calcium channels (23, 24, 29), suggestive for a high selectivity. Although the potency of nifedipine as a ICa,L blocker is widely accepted, much less is known about its selectivity. Concentrations of 2-10 µM are commonly used to block ICa,L completely (12, 15, 16). Hagiwara et al. (15) demonstrated a complete blockade of ICa,L by 2 µM nifedipine in rabbit SA nodal myocytes. They also showed that concentrations as high as 2 µM did not affect ICa,T. We studied the effects of 5 µM nifedipine on ICa,L, ICa,T, INa, IK, and If and demonstrated that 5 µM nifedipine reduces ICa,L by 81% without affecting ICa,T, INa, IK, and If. The lower blocking effect of nifedipine on ICa,L in our study compared with Hagiwara et al. (15) can at least in part be explained by the correction we made for the presence of a time-independent Ib, which results in a more reliable estimate of the blocking effect of nifedipine (Fig. 1, C and D). Our results further show that nifedipine blocked ICa,L at all test potentials to an equal extent.Contribution of ICa,L to Impulse Generation
Effect of nifedipine on diastolic depolarization. Doerr et al. (12) used an "action potential clamp" method to measure the contribution of ICa,L to the action potential in rabbit SA nodal myocytes. This technique can be more easily used in cells with low membrane resistance like ventricular and atrial cells. In SA node cells, membrane resistance is high. Thus small offsets in command voltage may introduce considerable errors in measurements of small currents during diastole and make this method not reliable in measuring the contribution of ICa,L to diastolic depolarization. This problem is avoided in the method we used in this study. With the combined voltage- and current-clamp method, the cell is only depolarized with a voltage-clamp pulse and the subsequent repolarization and diastolic depolarization are in the current-clamp mode. It can be argued that the depolarizing voltage-clamp step will slightly alter the activation process of ICa,L. However, the duration of the voltage-clamp step used is such that ICa,L will reach a level at the end of the step similar to that reached during the spontaneously pacing action potential. This is likely to occur because the repolarization after the voltage-clamp step is similar to the repolarization during spontaneous activity (Fig. 10A).Another argument against our method might be that alterations in the repolarization process induced by nifedipine also affect the time course of activation of other currents, which could influence the rate of diastolic depolarization. From Fig. 9B, it is clear that nifedipine predominantly affects the initial repolarization rate and hardly affects the remaining repolarization rate. It is therefore not likely that the deactivation process of IK, the dominant current during repolarization, has been altered. Because the last phase of repolarization is not affected, activation of If, which occurs at negative potentials, is also not likely to be altered. Therefore, the method used seems to be justified and seems to be the best way to directly measure the contribution of ICa,L to diastolic depolarization.
The combined voltage- and current-clamp experiments show that 5 µM nifedipine reduced the rate of the diastolic depolarization during the entire diastole. This finding is in good agreement with the findings of Doerr et al. (12) who showed that the D-600-sensitive current is present during entire diastolic depolarization. Recently, however, Zaza et al. (39) also used the action potential clamp method and showed that the net effect of nifedipine is not a reduction in inward current but a reduction in outward current. They provide evidence that the reduction in outward current is indirectly caused by a reduction in the intracellular calcium level which in turn reduces a calcium-dependent potassium conductance. Their results thus suggest that nifedipine would not reduce but enhance diastolic depolarization rate. However, we directly show that nifedipine does reduce diastolic depolarization rate, which is to be expected from a blockade of an inward current. It should be noted that the experiments of Zaza et al. (39) do not exclude this action of ICa,L during diastolic depolarization because they show the net effect of a reduction in both inward and outward currents.
Activation threshold of
ICa,L.
The effect of nifedipine on diastolic depolarization can be explained
either by a nonselective blockade of the drug, an indirect effect on
INaCa caused by a
reduction in intracellular Ca2+
(30), or by activation of
ICa,L earlier in
diastole and at more negative potentials than generally is assumed. The
activation threshold of
ICa,L is thought
to be around 40 mV (18), although a more negative threshold of 50
mV has been reported as well (4, 22). However, in the latter
experiments (4), a contribution of
ICa,T could not
be excluded.
40 mV, If
will activate and on depolarization,
ICa,T will be
activated as well. In most studies, therefore, a holding potential of
approximately 40 mV is used, thus fixing the threshold for
activation to that potential. The selective action of nifedipine on
ICa,L enabled us
to use a more negative holding potential (90 mV) to evaluate the
threshold for activation of
ICa,L (Fig. 8).
We clearly show that
ICa,L can indeed
be activated at potentials between 60 and 40 mV, i.e.,
the voltage range in which diastolic depolarization takes place.
In the experiments in which we determined the threshold for activation
of ICa,L, a more
negative holding potential (90 mV, Fig. 8) was used in
comparison with the experiments in which we tested the effect of
nifedipine on
ICa,L (40
mV, Fig. 1). At the holding potential of 90 mV, the most
negative value of the normalized I-V
relation was 12.2 ± 0.8 pA/pF
(n = 4), which is slightly more
negative (not significant) than the value of 11.8 ± 1.8 pA/pF (n = 8) obtained at a holding
potential of 40 mV. This slightly more negative value is most
likely due to a larger number of available
ICa,L channels at
more negative holding potentials. This may also explain that a holding
potential of 90 mV induces a 10-mV voltage shift of the most
negative current value of the I-V
relationship. Direct evidence of the holding potential-dependent voltage shift was obtained in the same cells because two different holding potentials were used (90 and 50
mV). At a holding potential of 90 mV used to
determine the activation threshold of
ICa,L (Fig. 8),
the most negative current value of the
I-V relationship was observed at a
test potential of 10 mV. At a holding potential of 50 mV,
the most negative current value of the
I-V relationship was observed at a
test potential of 0 mV (data not shown). This demonstrates that the
voltage at which the most negative current value of the
ICa,L-V
relationship occurs depends on the holding potential used.
Contribution of ICa,L to diastolic depolarization. In the SA node model of Wilders et al. (37), in which the activation and inactivation curves of ICa,L reported by Hagiwara et al. (15) are used, ICa,L amounts to ~0.5 pA or 0.0147 pA/pF at the MDP level, i.e., one or a few ICa,L channels are in the conducting state at the MDP level (36). This indicates that also on the basis of previously measured properties of ICa,L, ICa,L contributes to the first part of diastolic depolarization.
The rapid availability of ICa,L during repolarization and diastole is demonstrated in Fig. 10. During P1 the inward peak current will mainly consist of ICa,L, because P1 starts at potentials between45 and 40 mV. Inward currents elicited by
P2 during the last phase of
repolarization and the onset of diastole could, due to the more
negative membrane potential, also consist of a small fraction of
ICa,T (15).
However, from the steady-state inactivation relations of
ICa,T and
ICa,L found by
Hagiwara et al. (15), it can be derived that even in steady state at
the average MDP level (59 mV, see Table 2), only 7% of
ICa,T is available as opposed to
ICa,L, which is
only modestly inactivated (1%) at this potential. This means that only
a very small part of inward-going peak currents elicited on
P2 can be attributed to activation
of ICa,T.
The inward currents elicited by P2
during the last phase of repolarization and the onset of diastole may
also consist of a small fraction of
INa. We found
INa in only two
of eight cells that we used to study the effect of nifedipine on
INa. The two cells containing
INa showed a high
dV/dtmax
(16 and 19 V/s), which is much higher than the average
dV/dtmax
of 6.1 V/s observed in the cells in which the availability of
ICa,L was
studied. This indicates that the cells used to study the availability
of ICa,L presumably do not contain
INa. More
importantly, we demonstrated that nifedipine does not affect
INa or
ICa,T. Therefore,
the nifedipine-sensitive current in Fig. 10 can be fully attributed to
ICa,L. The
envelope of peak currents elicited on
P2 shows the dynamic recovery from inactivation of
ICa,L. Under
control conditions, inwardly directed peak current could be activated
40 ms after P1, which is
considerably earlier than the time at which the MDP is reached (67 ms
after P1). Experiments like the
one shown in Fig. 10 demonstrate that recovery from inactivation occurs
already early during diastole, which supports the idea that
ICa,L, if
activated, can contribute a significant inward current during the
entire diastolic depolarization.
Attributing the reduction in diastolic depolarization rate after the
administration of nifedipine to a reduction of
ICa,L, we now can
make a back-of-the-envelope estimate of its contribution to diastole.
Administration of 5 µM nifedipine decreased
DDR75 by 26%. At this
concentration
ICa,L is reduced
by 81%. Thus we may conclude that
ICa,L contributes
to 32% of the net inward current during diastole. In the
SA node model of Wilders et al. (37), the average contribution of
ICa,L to the net
membrane current calculated also over the first 75 ms of diastolic
depolarization was also 32%. A similar contribution of
ICa,L of ~30%
to this early phase of diastolic depolarization can be observed in the SA node model of Demir et al. (6).
It must be noted, however, that because of the slowing of the diastolic
depolarization rate when nifedipine is present, membrane potential will
remain more negative for a longer time. This will result in a smaller
IK because of a
decrease in driving force, a more pronounced activation of
If, and a larger
inward current due to
Ib. These effects
will increase net inward current during diastole, thereby counteracting
to some extent the effect of the reduction of
ICa,L. Therefore,
it is likely that the 32% contribution of
ICa,L to the net
inward current during diastole is an underestimation. On the other
hand, blockade of
ICa,L will
indirectly lower the Na+/Ca2+
exchange current
(INaCa). Thus
the reduction in inward current during diastole will in part also be
due to a reduction in
INaCa.
We now can also compare the contribution of several inward currents to
diastolic depolarization. Van Ginneken and Giles (31) calculated that
the amplitude of
If during
diastolic depolarization is similar to that of the net inward current.
For Ib we
recently measured a conductance of 39.5 pS/pF and a reversal potential of 32 mV (34). During diastolic depolarization from 60 to 40 mV, Ib
will thus generate an average inward current of 0.71 pA/pF, which is
about five times the net inward current during diastolic
depolarization. The summed inward currents
If and
Ib, together with
ICa,L and
ICa,T,
necessitate a large repolarizing outward current, most likely
IK,
counterbalancing all but a small portion of total inward current. It is
this resulting net inward current that underlies diastolic depolarization.
The above considerations demonstrate that it is not possible to dissect
the process of diastolic depolarization into fixed contributions of
several distinct ionic membrane currents. Rather, they demonstrate that
all currents are important. Inhibition or stimulation of a single
pacemaker current inevitably influences the other pacemaker currents.
| |
ACKNOWLEDGEMENTS |
|---|
The authors thank Dr. Tobias Op't Hof (Academic Medical Center, University of Amsterdam, Department of Experimental Cardiology, The Netherlands) for stimulating interest and helpful discussions and Jan Bourier for technical assistance.
| |
FOOTNOTES |
|---|
This work was supported by a combined Grant 900516093 from the Netherlands Organization for Scientific Research and the Netherlands Heart Foundation.
Address for reprint requests: E. E. Verheijck, Academic Medical Center, Univ. of Amsterdam, Dept. of Physiology, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands (E-mail: e.verheijck{at}amc.uva.nl).
Received 19 November 1996; accepted in final form 9 November 1998.
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Discussion
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L. Protas, D. DiFrancesco, and R. B. Robinson L-type but not T-type calcium current changes during postnatal development in rabbit sinoatrial node Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1252 - H1259. [Abstract] [Full Text] [PDF]
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 T. M. Vinogradova, Y.-Y. Zhou, K. Y. Bogdanov, D. Yang, M. Kuschel, H. Cheng, and R.-P. Xiao Sinoatrial Node Pacemaker Activity Requires Ca2+/Calmodulin-Dependent Protein Kinase II Activation Circ. Res., October 27, 2000; 87(9): 760 - 767. [Abstract] [Full Text] [PDF]
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T. Mitsuiye, Y. Shinagawa, and A. Noma Sustained Inward Current During Pacemaker Depolarization in Mammalian Sinoatrial Node Cells Circ. Res., July 21, 2000; 87(2): 88 - 91. [Abstract] [Full Text] [PDF]
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K. Y. Bogdanov, T. M. Vinogradova, and E. G. Lakatta Sinoatrial Nodal Cell Ryanodine Receptor and Na+-Ca2+ Exchanger : Molecular Partners in Pacemaker Regulation Circ. Res., June 22, 2001; 88(12): 1254 - 1258. [Abstract] [Full Text] [PDF]
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