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1 Department of Life Science, Pohang University of Science and Technology, Pohang, Republic of Korea; and 2 University Laboratory of Physiology, Oxford University, Oxford OX1 3PT, United Kingdom
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ABSTRACT |
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Effects of isoprenaline, carbachol, and Cs+ on intracellular Na+ activity (aiNa) and spontaneous action potentials were studied in multicellular and single cell preparations isolated from rabbit sinoatrial (SA) nodes. aiNa was measured with double-barreled Na+-selective microelectrodes and the fluorescent Na+-indicator sodium-binding benzofuran isophthalate (SBFI). In spontaneously beating cells, aiNa measured with Na+-selective microelectrodes and SBFI were 4.5 ± 1.2 mM (means ± SD, n = 21) in multicellular preparations and 4.0 ± 1.1 mM (n = 16) in single cells, respectively. Measurements of aiNa with microelectrodes showed that isoprenaline increased aiNa from 4.7 ± 1.2 to 5.5 ± 1.6 mM (n = 16, P < 0.01) and shortened the action potential cycle length (ACL) from 338 ± 46 to 269 ± 35 ms (n = 16, P < 0.01). However, increasing the action potential rate by pacing produced a much smaller increase in aiNa. Changes in aiNa and ACL produced by isoprenaline were blocked by Cs+. The selective hyperpolarization-activated inward current (If) blocker ZD-7288 decreased aiNa from 5.2 ± 1.0 to 4.6 ± 1.3 mM (n = 4, P < 0.01) and prolonged ACL from 394 ± 20 to 553 ± 68 ms (n = 4, P < 0.01). The If blocker substantially inhibited the increase in aiNa produced by isoprenaline. Carbachol and Cs+ decreased aiNa from 4.6 ± 1.4 to 3.9 ± 1.2 mM (n = 15, P < 0.01) and from 4.9 ± 1.0 to 3.9 ± 1.3 mM (n = 18, P < 0.01), respectively. In addition, carbachol and Cs+ prolonged ACL from 345 ± 44 to 587 ± 100 ms (n = 15, P < 0.01) and from 353 ± 30 to 464 ± 87 ms (n = 18, P < 0.01), respectively. However, carbachol and Cs+ almost did not change aiNa when SA node cells became quiescent in a 25.4 mM extracellular K+ concentration. The results suggest that isoprenaline, ZD-7288, carbachol, or Cs+ might have changed aiNa and action potential rate by possibly stimulating or inhibiting If carried by Na+. Measurements of aiNa with SBFI showed that isoprenaline, carbachol, and Cs+ produced aiNa changes that were similar to those measured with the microelectrodes.
rabbit heart; intracellular sodium ion activity; spontaneous depolarization; hyperpolarization-activated current; sinoatrial
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INTRODUCTION |
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SINOATRIAL NODE CELLS of the heart show a remarkable electrophysiological function characterized by slow and spontaneous diastolic depolarization. The diastolic depolarization is the basis of pacemaking activity of the sinoatrial node cells. Although recent studies have provided much information, the complicated ionic mechanisms underlying the pacemaker depolarization still remain unclear and controversial (23). A number of different ionic currents are known to contribute to generation of the pacemaker depolarization (23, 34). Those ionic currents include delayed rectifier K+ current (24), hyperpolarization-activated inward current (If), and Ca2+ current (17, 23). In addition, inward background current, Na+-K+-pump current, and Na+/Ca2+ exchanger current may also play a role in the pacemaker depolarization (23).
Sodium ions are known to be responsible for carrying If (3, 10, 22), inward background current (19), Na+-K+-pump current, and Na+/Ca2+ exchanger current. This suggests that during the normal activities of sinoatrial node cells, transmembrane Na+ movements may be substantial and play important roles in changing the cell membrane potentials, including the pacemaker depolarization. It has been suggested that the If does play an important role in the pacemaker depolarization (7, 10, 13, 37). However, If may be small under normal pacemaker activity of sinoatrial node cells (23).
Considering the various ionic currents carried by
Na+, it is possible to assume that
changes in the ionic currents and membrane potentials may alter
intracellular Na+ activity
(aiNa) of the sinoatrial node.
The
-adrenergic and cholinergic agonists (norepinephrine and
carbachol) are reported to regulate pacemaker activities of sinoatrial
node cells through modulation of
If (12, 14).
Cesium ions are known to inhibit pacemaker activities of sinoatrial
node cells by blocking If (6, 37). Can
changes of If
bring about a change in aiNa? Other Na+ currents such as inward
background Na+ currents and
Na+-K+-pump
currents may be assumed to play a role in the pacemaker activities.
Sinoatrial node cells may also have fast inward
Na+ currents (6). The magnitude of
these Na+ currents may differ from
region to region in the sinoatrial node (33). Therefore, changes in
these Na+ currents may also
produce changes in aiNa. Direct and simultaneous measurements of
aiNa and spontaneous action
potentials may provide information about changes in
aiNa when pacemaker activities are changed by
-adrenergic, cholinergic agonists or
Cs+.
The present study had two main aims. One was to measure aiNa in sinoatrial node cells of rabbit hearts using two different techniques: double-barreled Na+-selective microelectrodes and the fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI). aiNa was not measured in sinoatrial node cells, although it was measured in other cardiac tissues (15, 27-29). The other purpose was to test whether isoprenaline, carbachol, and Cs+ caused changes in aiNa in the sinoatrial node cells. This test might lead to some insights into the roles of Na+ influx in membrane potential changes, particularly in the pacemaker depolarization. The present study showed that isoprenaline, carbachol, and Cs+ changed both aiNa and pacemaker depolarization.
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METHODS |
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Multicellular preparation of sinoatrial node. Rabbits (1.0-1.5 kg) of either sex were killed by cervical dislocation. The hearts were excised rapidly and immersed in a dish containing oxygenated Tyrode solution. The sinoatrial node was dissected from each heart in oxygenated normal Tyrode solution at 36°C. The sinoatrial node with the crista terminalis was transferred into a perfusing chamber and was fixed on the bottom of the chamber with fine pins (27). The preparation was superfused with oxygenated Tyrode solution at 36 ± 0.5°C. A two-polar stimulating probe connected to a stimulator was placed on the crista terminalis to drive the preparation when needed. The Tyrode solution contained (in mM) 127 NaCl, 5.4 KCl, 1.0 MgCl2, 0.45 NaH2PO4, 21 NaHCO3, 1.8 CaCl2, and 5 glucose. The solution was gassed with 95% O2-5% CO2 and had a pH of 7.3-7.4.
Isolation of single sinoatrial node cells.
Single sinoatrial node cells were isolated from each rabbit heart using
a combination of enzymatic and mechanical dispersion (7). Briefly, an
albino rabbit that weighed <1.5 kg was killed by cervical
dislocation, and the heart with a long length of aorta was removed
rapidly. The heart was perfused at 37°C with a 1 mM Ca2+ solution for 4 min, a
Ca2+-free (with 200 µM EGTA)
solution for 6 min, and then an enzyme solution [50 U/ml elastase
(Sigma), 80 U/ml collagenase (Worthington)] for 20 min. The
sinoatrial node was then dissected, cut into two pieces, and incubated
in a new enzyme solution (50 U/ml elastase, 320 U/ml collagenase) for
10-20 min. Enzymes were then washed out by rinsing with
high-K+,
low-Cl
solution. Single
sinoatrial node cells were dispersed by smoothly agitating the tissue
with a glass pipette.
Construction of double-barreled Na+-selective microelectrodes. Double-barreled microelectrodes can be made from two different types of glass tubings (26). Glass capillaries with outer and inner diameters of 1.5 and 1.15 mm, respectively, were cut for the ion-selective barrel in lengths of 8 cm. Another type of glass capillary with outer and inner diameters of 1.0 and 0.75 mm, respectively, for the conventional barrel was also cut in lengths of 8 cm. These two capillaries were then placed parallel to each other and glued with epoxy. The glass capillaries for the conventional barrel had an internal glass filament. The double-barreled capillaries were twisted and pulled using a horizontal puller (Narishige PD-5). The capillary for the conventional barrel was then heated at the center and bent at an angle of ~45°. The opening of the conventional barrel was tightly covered with heat-resistant tape, and the stem of the ion-selective barrel was put into a hole in the cover of a jar made of stainless steel (26). The jar with the double-barreled microelectrodes was placed in an oven and heated to a temperature of ~200°C. A small amount of silanization agent (tri-N-butylchlorosilane) was then added to the glass container in the jar and heated for 30 min. With this treatment, the ion-selective barrel became hydrophobic. The conventional barrel remained hydrophilic and was back-filled with 3 M KCl. The ion-selective barrel was filled with 100 mM NaCl, and the tip of the double-barreled microelectrode was carefully beveled (26). Finally, a column (50-200 µm) of the Na+ sensor (Fluka Chemical) was drawn up into the tip of the ion-selective barrel by applying negative pressure. In each experiment, the microelectrodes were calibrated as described previously (26).
The aiNa and membrane potential of the same sinoatrial node cell were measured using a double-barreled Na+-selective microelectrode. Measurement of aiNa and membrane potential with a Na+-selective microelectrode has been described in detail (26, 27). aiNa was displayed on a chart recorder at a slow speed. The action potential recorded by the conventional barrel was displayed on a oscilloscope and a chart recorder (model 2400; Gould, Cleveland, OH). The preparation was paced at a certain rate by the stimulating electrodes when a change in the rate of action potential was required.Measurement of intracellular Na+ with SBFI. Measurement of intracellular Na+ concentration with the fluorescent Na+ indicator SBFI has been described by Levi et al. (30). In short, freshly isolated sinoatrial node cells were incubated for 1.5 h at room temperature to be loaded with 15 µM SBFI-acetoxymethyl ester and 1 µM of 25% pluronic acid (both from Molecular Probes). Myocytes loaded with the indicator were moved to the experimental chamber and illuminated with ultraviolet light applied via the epifluorescence microscope. The 340- and 380-nm (Cairn Spectrophotometer System) excitation lights were transmitted to cells through the optic tube and a 400-nm dichroic mirror. Emitted light was collected by the objective and passed to a photomultiplier tube (PMT). The signal from the PMT was processed with input amplifiers and a ratio amplifier (30). The ratio of the light emitted with 340-nm excitation to that emitted with 380-nm excitation (340/380 ratio) represented the level of intracellular Na+ (31). Spindle-shaped cells showing spontaneous beating were used to measure the intracellular Na+ concentration. In most experiments with single cells, spontaneous action potentials were not measured because impalements of single cells with microelectrodes might have deteriorated their condition and Na+ could have leaked into the cells.
Data are expressed as means ± SD. Statistical significance was tested with paired Student's t-test, and P < 0.05 was taken to be significant unless otherwise indicated. The traces shown in this study are typical of at least three recordings unless otherwise indicated.| |
RESULTS |
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Intracellular
Na+ activities
of multicellular preparations and single cells.
Figure 1 shows the measurement of
aiNa and action potentials of
a sinoatrial node cell from a rabbit heart using a double-barreled
Na+-selective microelectrode. The
potentials of the Na+-selective
microelectrode and the conventional microelectrode in Tyrode solution
were set arbitrarily as zero in the recording. Figure 1,
A and
B, show that the impalement of a cell
with a double-barreled Na+-selective microelectrode
resulted in a large change (
132 mV) of the
Na+-selective microelectrode
potential (ENa)
and changes of transmembrane potential
(Vm) of the
conventional microelectrode. Figure 1C
shows the recording of aiNa
level that is the difference between
ENa and
Vm. In 21 measurements, the average level of
aiNa was 4.5 ± 1.2 mM, and
the average action potential cycle length (ACL) was 334 ± 40 ms. Our results showed that the tip of the double-barreled
Na+-selective microelectrode was
small enough to measure aiNa and action potential in multicellular preparations of sinoatrial nodes
of the rabbit hearts over a long period of time. In spontaneously beating cells, the microelectrodes could be maintained in one cell for
several hours. The stable and long impalements of the microelectrodes
allowed us to test effects of isoprenaline, carbachol, and
Cs+ on
aiNa and membrane potential of
the same cell in the sinoatrial node.
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Effects of isoprenaline on
aiNa
and action potential.
Figure 3 shows the effects of 3 × 10
8 M isoprenaline on
aiNa and spontaneous action
potential of sinoatrial node cells. Figure 3,
A-C, represents typical
recordings of Vm (amplitude of spontaneous action potentials),
aiNa, and spontaneous action
potentials measured with a double-barreled Na+-selective microelectrode in a
multicellular preparation. Figure 3A
shows that isoprenaline at the concentration of 3 × 10
8 M produced little
change in action potential amplitude. Isoprenaline increased
aiNa from 5.0 to 5.7 mM (Fig.
3B). After isoprenaline was washed
out, aiNa recovered to the
initial level. In 16 tests, isoprenaline increased
aiNa from 4.7 ± 1.2 to 5.5 ± 1.6 mM. Figure
3C shows the spontaneous action
potentials before and during exposure to isoprenaline. Isoprenaline
shortened ACL from 338 ± 46 to 269 ± 35 ms
(n = 16) and increased the slope of
the spontaneous depolarization. In the spontaneous action potentials,
prominent pacemaker depolarization fused smoothly into the upstroke
(see also Figs. 4 and 7). This suggests that the cell used in the
experiment might locate at the central area of the sinoatrial node
(16). Figure 3D shows the effect of isoprenaline
(10
6 M) on
aiNa measured with SBFI in a
single sinoatrial node cell. Isoprenaline increased
aiNa from 4.1 to 5.2 mM. In
seven tests, isoprenaline
(10
7-10
6
M) increased aiNa from 3.8 ± 1.0 to 7.4 ± 1.6 mM. The effect of isoprenaline on
aiNa in single cells was
similar to that in multicellular preparations.
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Effects of isoprenaline and stimulation rate on aiNa and action potential. As shown in Fig. 3, isoprenaline increased the rate of spontaneous action potential and aiNa. An important question arises as to whether the increase in aiNa (Fig. 3) was produced by an increase in the rate of action potential by isoprenaline. This question was tested in the experiments shown in Fig. 4.
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7 M) caused an increase
in aiNa from 5.4 to 6.5 mM,
and the increase of stimulation rate from 3 Hz to 4 Hz by pacing
produced a small change in
aiNa. In three tests,
isoprenaline increased aiNa from 4.9 ± 0.4 to 5.7 ± 0.6 mM
(P < 0.01), and the increase of stimulation rate by pacing increased
aiNa from 4.9 ± 0.4 to 5.2 ± 0.5 mM (P < 0.01).
The aiNa increase produced by
stimulation rate was much smaller than that produced by isoprenaline.
Figure 4C represents
the action potentials recorded at points
a-c as indicated in Fig.
4B. Isoprenaline shortened ACL from
335 ms (Fig. 4C,
trace a) to 250 ms (Fig.
4C, trace
b) so that the rate of the spontaneous action
potential could be increased. The isoprenaline increased the rate of
spontaneous action potentials from 3 Hz (Fig.
4C, trace
a) to 4 Hz (Fig. 4C, trace b). As shown in Fig.
4C, trace
b, isoprenaline markedly increased the slope of the
pacemaker depolarization. This produced no clear fusion of the
pacemaker depolarization with the upstroke. As a result, the membrane
potential smoothly depolarized from maximum diastolic potential to
action potential peak.
Figure 4C, trace
c shows the action potentials recorded at
point c in Fig.
4B during stimulation of the
sinoatrial node at 4 Hz by external electrodes. This action potential
rate of 4 Hz that was increased from the control rate of 3 Hz by pacing
was identical to the action potential rate increased by
10
7 M isoprenaline (Fig.
4C, trace
b). As the action potentials produced by isoprenaline
(Fig. 4C, trace
b) were compared with those produced by pacing (Fig.
4C, trace
c), we found interesting differences. First, the
slope of pacemaker depolarization produced by isoprenaline was much
steeper than that produced by pacing. Second, there was no clear fusion
between the pacemaker depolarization and the upstroke. Third, upstroke
of the action potential produced by isoprenaline seemed much slower
than that produced by pacing, although the rates of action potentials
were identical.
Effects of isoprenaline on
aiNa
and action potential in presence of
Cs+ and ZD-7288.
If the increase in aiNa by
isoprenaline was due to an increase of
If, the increase
in aiNa should be
substantially blocked by an inhibitor of the inward If,
Cs+ or ZD-7288. Figure
5 shows the effects of isoprenaline on
aiNa and spontaneous action
potentials in the absence and presence of
Cs+ or the
If blocker
ZD-7288. Figure 5, A and
B, represent action potential
amplitude and aiNa,
respectively, in a multicellular preparation. The sinoatrial node cells
were first exposed to 6 mM Cs+,
which decreased aiNa by ~1.5
mM. This result is consistent with the view that
Cs+ inhibited
If carried by
Na+. When the decreased
aiNa was stabilized, 10
6 M isoprenaline was
added to cells still exposed to
Cs+. The addition of isoprenaline
did not change aiNa, indicating that the inward
If was blocked by
Cs+. After washout of
Cs+,
aiNa returned to the control
level. Under the control condition, isoprenaline
(10
6 M) increased
aiNa. Five experiments showed results that were similar to those shown in Fig. 5,
A and
B.
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Effects of carbachol on
aiNa
and action potential.
Figure 6,
A-C, shows the effects of a
muscarinic agonist, carbachol, on
aiNa and spontaneous action
potential in a multicellular preparation of sinoatrial node. Figure 6,
A and
B, represents action potential
amplitude and aiNa, respectively. Carbachol
(10
6 M) decreased
aiNa by ~1.2 mM, which
returned to the control level after washout of carbachol (Fig.
6B). Figure 6C, traces
a and b, represent the
spontaneous action potentials recorded at the points indicated in Fig.
6B. The recordings of spontaneous
action potentials indicate that carbachol decreased the slope of the
pacemaker depolarization and, thereby, the rate of spontaneous action
potentials. In 15 tests, carbachol
(10
6-5 × 10
6 M) decreased
aiNa from 4.6 ± 1.4 to 3.9 ± 1.2 mM (P < 0.01). Carbachol
decreased the slope of spontaneous depolarization and prolonged action
potential cycle length from 345 ± 44 to 587 ± 100 ms
(P < 0.01).
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6 M) on
aiNa in a single cell measured
with the fluorescent Na+ indicator
SBFI. Carbachol substantially decreased
aiNa, which recovered to the
control level after washout of carbachol. This result agrees with that
obtained using Na+-selective
microelectrodes. In six tests with single cells, carbachol (10
6 M) decreased
aiNa from 3.6 ± 1.7 to 2.8 ± 1.4 mM (P < 0.01).
Effects of Cs+ on aiNa and action potential. Figure 7 shows the effects of Cs+ (2 and 6 mM) on aiNa and spontaneous action potentials of sinoatrial node cells. Figure 7, A and B, shows action potential amplitude and aiNa, respectively, in a multicellular preparation. Cs+ (6 mM) depolarized maximum diastolic membrane potential and decreased aiNa. Figure 7C, traces a and b, represents spontaneous action potentials recorded at points a and b as indicated in Fig. 7B. Cs+ decreased the slope of pacemaker depolarization and the rate of the action potentials. The recordings of action potentials (Fig. 7C, traces a and b) show that fusions of the pacemaker depolarization with the upstroke were smooth. This suggests that the cell used in this experiment might locate at the central area of the sinoatrial node. In 18 tests with 6 mM Cs+, Cs+ decreased the aiNa from 4.9 ± 1.0 to 3.9 ± 1.3 mM (P < 0.01) and prolonged ACL from 353 ± 30 to 464 ± 87 ms (P < 0.01). In the experiments with single cells, Cs+ (6 mM) also decreased aiNa, as shown in Fig. 7D. In six tests with single cells, Cs+ (6 mM) decreased aiNa from 3.8 ± 1.8 to 2.0 ± 0.4 mM (P < 0.01). Although most experiments were done with 6 mM Cs+, some experiments were carried out with 2 mM Cs+. Cs+ decreased aiNa and depolarized maximum diastolic potentials in a dose-dependent manner, as shown in Fig. 7E. Figure 7E shows an example in which 2 and 6 mM Cs+ decreased aiNa by 0.8 and 1.2 mM, respectively, and depolarized diastolic membrane potentials in the same preparation. Thus the effects of Cs+ were dose dependent in both changes of aiNa and maximum diastolic potential.
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Effects of carbachol or
Cs+ on
aiNa in
quiescent sinoatrial node cells.
The results in Figs. 6 and 7 suggest that carbachol and
Cs+ inhibited inward
Na+ movement, possibly
hyperpolarization-activated If
during the pacemaker depolarization of sinoatrial node cells. If the
cells become depolarized and quiescent, the hyperpolarization-activated If is expected to
be absent. Under this condition, carbachol and Cs+ should not decrease
aiNa. Figure
8 shows the effects of carbachol or
Cs+ in spontaneously beating and
quiescent sinoatrial node cells (multicellular preparation). Figure
8B shows that carbachol substantially decreased aiNa in
spontaneously beating cells. The cells were then exposed to 25.4 mM
K+ and became quiescent (Fig. 8,
A' and
B'). The high extracellular K+ concentration depolarized the
membrane potential
(Vm) to
34 mV and decreased
aiNa. When
aiNa and
Vm stabilized,
the addition of carbachol or Cs+
produced little or no change in
aiNa. Four experiments showed
similar results.
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DISCUSSION |
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Intracellular Na+ activity of sinoatrial node cells. In the present study, intracellular Na+ activity (aiNa) of multicellular preparations and single cells of rabbit sinoatrial node was first measured with double-barreled Na+-selective microelectrodes and the fluorescent Na+ indicator SBFI. The aiNa values measured with the two different methods were similar. The levels of aiNa might have been dependent on the region in the sinoatrial node and the rate of spontaneous beating of the node cells. The peripheral cells of sinoatrial node had TTX-sensitive Na+ currents, and the central cells had little TTX sensitivity (33). This suggests that the Na+ influx in central cells was less than that in the peripheral cells, producing different aiNa levels in the two regions. Our study did not suggest any differences between aiNa of central and peripheral cells of the sinoatrial node. However, our current techniques might not be able to detect these differences if they exist. This problem remains to be studied.
The two methods were suitable for measuring aiNa of cells in rabbit sinoatrial node. The levels of aiNa in the rabbit sinoatrial node were broadly in agreement with the aiNa values reported for ventricular cells of rabbit hearts. Na+-selective microelectrodes were used to measure aiNa in rabbit ventricular cells, and aiNa values reported were 5.8 mM (28) and 8.4 mM (8). These values were slightly higher than the value (4.5 mM) obtained from rabbit sinoatrial node cells. With the use of the fluorescent Na+ indicator SBFI, an aiNa value of 2.9 mM was reported in rabbit ventricular myocytes (29). This aiNa level was similar to the average aiNa value (4.0 mM) obtained from spontaneously beating cells of sinoatrial node in the present study. It is necessary to be cautious when comparing aiNa levels measured with different methods and under different experimental conditions. In the present study, aiNa was measured in sinoatrial node cells that were spontaneously beating. The rates of spontaneous beating were variable among sinoatrial nodes and among experimental conditions, such as multicellular preparation and single cells isolated from the sinoatrial node. It is not clear whether the sinoatrial node cells used in our study were in the center, the periphery, or the junctional area between the two regions of the sinoatrial node. The different origin of the cells used might contribute to variability of aiNa and their changes as produced by isoprenaline, carbachol, or Cs+. Judging from the action potential shapes, cells used might be identified for their location: central or peripheral region.
-Adrenergic receptor, intracellular
Na+ activity,
and pacemaker potential.
The stimulation of the
-adrenergic receptor with its agonist,
isoprenaline, increased the action potential rate by enhancing the
hyperpolarization-activated inward current,
If (11, 20). If
the If is carried
by Na+, stimulation of the
-adrenergic receptor should increase intracellular Na+ activity. Indeed, our
experiments showed that stimulation of the receptor by isoprenaline
increased aiNa and the rate of
spontaneous action potentials. However, it is possible that the
increase in aiNa by
isoprenaline might be due to an increase in the rate of spontaneous
action potentials. In other cardiac tissues such as ventricular cells and Purkinje fibers, an increase in stimulation rate raised
aiNa (2, 5). In these tissues,
Na+ enters the cells mainly
through fast Na+ channels during
action potentials, and, thereby, an increase in the rate of action
potentials should increase the amount of Na+ influx per unit time. In
sinoatrial node cells, Na+ might
enter the cells during the upstroke of spontaneous action potentials,
and an increase in action potential rate would increase aiNa. This possibility was
tested by comparison of the
aiNa increase produced by
isoprenaline with the aiNa
increase produced by an increase of action potential rate (Fig. 4). The
increase in action potential rate produced by isoprenaline was
identical to the increase in action potential rate produced by pacing.
A simple increase in action potential rate by pacing produced a small
increase in aiNa (Fig. 4,
trace B). In other words, the
increase in aiNa produced by
isoprenaline was much greater than the increase in aiNa produced by pacing.
Therefore, our results suggest that the major increase in
aiNa produced by isoprenaline
was due to an increase of inward
Na+ movement, possibly
If, during
pacemaker depolarization but not simply by an increase in the rate of
action potentials. However, the magnitude of increase in
aiNa produced by isoprenaline
or pacing might depend on the origin of the cells. In the cells from
the periphery, aiNa increase might be also contributed by a rise in
Na+ influx through fast
(TTX-sensitive) Na+ channels.
Muscarinic receptor, intracellular
Na+ activity,
and pacemaker potential.
In contrast to
-adrenergic-receptor activation, muscarinic-receptor
activation was reported to inhibit the hyperpolarization-activated current, If,
resulting in a decrease of the slope of pacemaker depolarization (9,
11). Therefore, stimulation of muscarinic receptor by an agonist, such
as carbachol, slowed the heart rate. If
If carried by
Na+ is inhibited by stimulation of
muscarinic receptor, carbachol is expected to decrease
aiNa. Our study showed that
the muscarinic agonist carbachol decreased
aiNa and the slope of the
pacemaker depolarization.
4
M) did not prevent the aiNa
decrease by carbachol in sinoatrial node cells (data not shown).
Therefore, it is unlikely that the decrease in
aiNa by carbachol might be due
to a stimulation of the
Na+-K+
pump in sinoatrial node cells.
Cs+, ZD-7288, If current, intracellular Na+ activity, and pacemaker potential. Cs+ is known to block If in sinoatrial node cells and Purkinje fibers of mammalian hearts (4, 6). ZD-7288 was reported to selectively block If in sinoatrial cells (1, 32). The reports are supported by our results because Cs+ or ZD-7288 decreased aiNa and the slope of the pacemaker depolarization. In the present study, Cs+ (2 and 6 mM) decreased aiNa and depolarized maximum diastolic potential in a concentration-dependent manner. The effects of 2 mM Cs+ on aiNa and maximum diastolic potential were qualitatively similar to those of 6 mM Cs+ (Fig. 7); therefore, we used 6 mM Cs+ in most experiments. If 2 mM Cs+ was enough to block If and 6 mM Cs+ affected some other ionic currents, the aiNa decrease by 6 mM Cs+ might be caused by changes in If as well as other ionic currents. Our study showed that both 2 and 6 mM Cs+ blocked the increase in aiNa produced by isoprenaline.
It was reported that Cs+ hyperpolarized maximum diastolic potential and thereby might stimulate Na+-K+ pump in sinoatrial node cells of guinea pigs (36). In our study, however, Cs+ did not hyperpolarize maximum diastolic potential but rather slightly depolarized this potential (Fig. 7). Furthermore, Cs+ decreased substantially the slope of the pacemaker potential and slowed down the rate of action potentials (Fig. 7). In the Purkinje fibers of canine hearts, Cs+ also depolarized maximum diastolic potential (4). If Cs+ stimulates the Na+-K+ pump, it should hyperpolarize the maximum diastolic potential. The different results might be due to the difference in animal species. It is possible that the effect of Cs+ on the central cells (dominant pacemaker cells) of sinoatrial nodes is different from those on the peripheral cells (subsidiary pacemaker cells). However, our study showed that Cs+ decreased aiNa and spontaneous action potentials in both central and peripheral cells (Figs. 5 and 7). The cells showing smooth fusion of the pacemaker depolarization with the upstroke (Figs. 3, 4, and 7) might be located in the central region of the sinoatrial node (16). On the other hand, the cells showing distinct fusion of the pacemaker depolarization with the upstroke might be peripheral cells of the sinoatrial node. It has been suggested that If does not play an important role in the pacemaker activity of the central cells of sinoatrial node (33). This implies that inward Na+ movement (presumably If) is not present during the pacemaker depolarization of central cells of the sinoatrial node. Our study showed that isoprenaline increased aiNa and that the slope of the pacemaker potential in the sinoatrial cells had smooth fusion of the pacemaker depolarization with the upstroke. Therefore, our results suggest that inward Na+ movement, possibly If, is present and plays a role in the pacemaker activity in the center area of the sinoatrial node. In summary, our results are consistent with the view that isoprenaline, ZD-7288, carbachol, and Cs+ might change aiNa and action potential rate by possibly stimulating or inhibiting the hyperpolarization-activated current, If. However, the changes in aiNa might be also contributed by other ionic currents such as outward K+ current (18), background Na+ current (19), and Na+/Ca2+ exchange current. Although Cs+ predominantly blocked If, it might not be a specific blocker of If (23). In addition, the fact that spontaneous firing still occurred in the presence of 2 or 6 mM Cs+ or ZD-7288 indicates that If cannot be the only current generating spontaneous depolarization. Although ZD-7288 did not completely block the increase of aiNa by isoprenaline, the selective If blocker inhibited the major part of the aiNa increase. Therefore, the change in If might make a significant contribution to the change in aiNa. It is important to make a distinction between demonstrating the If responsible for aiNa changes by Na+ entry and identifying the other ionic currents responsible for the pacemaker activity. Although several inward currents have been proposed to contribute to the pacemaker activity, there is no agreement on which of the currents makes the major contribution to the activity (23). Therefore, it may be difficult to identify the relative contribution of the inward currents in detail.Change in aiNa by If: Calculation by OXSOFT heart model. An important question to be resolved is whether changes in If would be expected to produce changes in intracellular Na+ of the magnitude recorded in our experiments. To answer this question, we therefore performed computations using the single rabbit sinus node cell model of Noble et al. (35) as implemented in the OXSOFT heart program, version 4.8. First, we adjusted the magnitude of the background Na+ conductance, gb,Na to give an intracellular Na+ concentration during steady rhythmic activity similar to that recorded experimentally. We found that with gb,Na set to 0.0001 nS (which is one-half the value used in the original model), a steady-state Na+ concentration of 4.65 mM (3.5 mM in aiNa) was produced by the model. Figure 9 shows, first, the result of increasing the values of the Na+ (gf,Na) and K+ (gf,K) components of If conductance by 50%. This produced an increase in intracellular Na+ similar to that recorded experimentally with isoprenaline. The increase in aiNa was ~0.8 mM and required ~1.5 min to approach a new steady state. By contrast, halving the conductance of the If channels reduced aiNa by ~0.8 mM, which was similar to that recorded experimentally with Cs+ block. These results show that changes in If of the magnitude known to be produced by isoprenaline and Cs+ would be sufficient to generate the changes in intracellular Na+ recorded experimentally.
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ACKNOWLEDGEMENTS |
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We thank Dr. Won-Kyung Ho for advice on preparation of single cells from rabbit sinoatrial node.
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FOOTNOTES |
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This work was supported by the Basic Science Research Institute Program (Project BSRI-97-4435), Biotech 2000 Program from the Ministry of Science and Technology, Korea, and Korea Science and Engineering Foundation (KOSEF 97-0401-02).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: C. O. Lee, Dept. of Life Science, Pohang Univ. of Science and Technology, Pohang, Republic of Korea.
Received 2 March 1998; accepted in final form 8 September 1998.
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