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 (I_f), 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 I_f (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 I_f does play an important role in the pacemaker depolarization (7, 10, 13, 37). However, I_f 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 (aⁱ_Na) 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 I_f (12, 14). Cesium ions are known to inhibit pacemaker activities of sinoatrial node cells by blocking I_f (6, 37). Can changes of I_f bring about a change in aⁱ_Na? 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 aⁱ_Na. Direct and simultaneous measurements of aⁱ_Na and spontaneous action potentials may provide information about changes in aⁱ_Na when pacemaker activities are changed by -adrenergic, cholinergic agonists or Cs⁺.

The present study had two main aims. One was to measure aⁱ_Na 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). aⁱ_Na 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 aⁱ_Na 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 aⁱ_Na and pacemaker depolarization.

	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).

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.

	RESULTS

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Intracellular Na⁺ activities of multicellular preparations and single cells. Figure 1 shows the measurement of aⁱ_Na 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 (E_Na) and changes of transmembrane potential (V_m) of the conventional microelectrode. Figure 1C shows the recording of aⁱ_Na level that is the difference between E_Na and V_m. In 21 measurements, the average level of aⁱ_Na 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 aⁱ_Na 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 aⁱ_Na and membrane potential of the same cell in the sinoatrial node.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Simultaneous and continuous measurement of intracellular Na+ activity (aiNa) and spontaneous action potential (Vm) with a double-barreled Na+-selective microelectrode. A: Na+-selective microelectrode potential (ENa). B: amplitude of Vm. C: intracellular Na+ activity (aiNa; ENa  Vm). Voltages (ENa and ENa  Vm) of A and B were filtered by using 2 identical low-pass filters (27). In B, an increase in speed of chart recorder shows Vm. Experiment was done with a multicellular preparation of sinoatrial node. A double-barreled Na+-selective microelectrode should be used because sinoatrial node cells are electrically heterogeneous (25).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Calibration of fluorescent sodium-binding benzofuran isophthalate (SBFI) for intracellular Na+ concentration ([Na+]i). A: a single cell was calibrated for [Na+]i using an in situ calibration method in which intracellular Na+ was equilibrated with extracellular Na+ using a calibration solution. Calibration solution contained 2 µm gramicidin (G), 40 µm monensin (M), and 100 µm strophanthidin (S), as indicated below trace in A. Before calibration, strophanthidin (106 M) was applied to see whether cell properly responded to inhibition of Na+-K+ pump. After Na+ concentration was changed to 2, 5, or 20 mM, time to reach a stable level was varied. At 20 mM extracellular Na+ concentration, time was somewhat slow and the stable level was used to construct a calibration curve. B: typical calibration curve of SBFI for Na+; relationship between stable levels of ratio signals and Na+ concentrations.

Effects of isoprenaline on aⁱ_Na and action potential. Figure 3 shows the effects of 3 × 10⁸ M isoprenaline on aⁱ_Na and spontaneous action potential of sinoatrial node cells. Figure 3, A-C, represents typical recordings of V_m (amplitude of spontaneous action potentials), aⁱ_Na, 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 × 10⁸ M produced little change in action potential amplitude. Isoprenaline increased aⁱ_Na from 5.0 to 5.7 mM (Fig. 3B). After isoprenaline was washed out, aⁱ_Na recovered to the initial level. In 16 tests, isoprenaline increased aⁱ_Na 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 (10⁶ M) on aⁱ_Na measured with SBFI in a single sinoatrial node cell. Isoprenaline increased aⁱ_Na from 4.1 to 5.2 mM. In seven tests, isoprenaline (10⁷-10⁶ M) increased aⁱ_Na from 3.8 ± 1.0 to 7.4 ± 1.6 mM. The effect of isoprenaline on aⁱ_Na in single cells was similar to that in multicellular preparations.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of isoprenaline (Iso) on aiNa and Vm. A: continuous recording of Vm. B: effect of Iso on aiNa. C: Vm recorded during control condition (a) and Iso exposure (b). D: recording of aiNa measured with fluorescent Na+ indicator SBFI in a single cell showing an increase of aiNa produced by Iso.

Effects of isoprenaline and stimulation rate on aⁱ_Na and action potential. As shown in Fig. 3, isoprenaline increased the rate of spontaneous action potential and aⁱ_Na. An important question arises as to whether the increase in aⁱ_Na (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.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effects of Iso and 4-Hz stimulation on aiNa and Vm. A: continuous recording of Vm. B: effects of 107 M Iso and 4-Hz stimulation on aiNa. C: Vm recorded during control condition (a), Iso exposure (b), and 4-Hz stimulation (c). Experiment was done with a multicellular preparation.

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7

Effects of isoprenaline on aⁱ_Na and action potential in presence of Cs⁺ and ZD-7288. If the increase in aⁱ_Na by isoprenaline was due to an increase of I_f, the increase in aⁱ_Na should be substantially blocked by an inhibitor of the inward I_f, Cs⁺ or ZD-7288. Figure 5 shows the effects of isoprenaline on aⁱ_Na and spontaneous action potentials in the absence and presence of Cs⁺ or the I_f blocker ZD-7288. Figure 5, A and B, represent action potential amplitude and aⁱ_Na, respectively, in a multicellular preparation. The sinoatrial node cells were first exposed to 6 mM Cs⁺, which decreased aⁱ_Na by ~1.5 mM. This result is consistent with the view that Cs⁺ inhibited I_f carried by Na⁺. When the decreased aⁱ_Na was stabilized, 10⁶ M isoprenaline was added to cells still exposed to Cs⁺. The addition of isoprenaline did not change aⁱ_Na, indicating that the inward I_f was blocked by Cs⁺. After washout of Cs⁺, aⁱ_Na returned to the control level. Under the control condition, isoprenaline (10⁶ M) increased aⁱ_Na. Five experiments showed results that were similar to those shown in Fig. 5, A and B.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effects of Iso on aiNa and Vm in absence and presence of Cs+ and ZD-7288. A: continuous recording of Vm. B: effect of 106 M Iso on aiNa in presence and absence of Cs+. C: Vm recorded during control condition (a), Cs+ exposure (b), Cs+ + Iso exposure (c), and Iso exposure (d). Experiment (A-C) was done with a multicellular preparation. D: continuous recording of Vm in a multicellular preparation. Note 1-h break in recording. E: effect of 2 × 108 M Iso on aiNa in absence and presence of ZD-7288. Note that aiNa decreased during 1-h break period (onset of ZD-7288 effect). F: aiNa measured with fluorescent SBFI in a single cell; effect of Iso on aiNa in absence and presence of Cs+.

Effects of carbachol on aⁱ_Na and action potential. Figure 6, A-C, shows the effects of a muscarinic agonist, carbachol, on aⁱ_Na and spontaneous action potential in a multicellular preparation of sinoatrial node. Figure 6, A and B, represents action potential amplitude and aⁱ_Na, respectively. Carbachol (10⁶ M) decreased aⁱ_Na 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 (10⁶-5 × 10⁶ M) decreased aⁱ_Na 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).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effects of carbachol (Car) on aiNa and Vm. A: continuous recording of Vm. B: effect of Car on aiNa. C: Vm recorded during control condition (a) and Car exposure (b). Experiment (A-C) was done with a multicellular preparation. D: aiNa measured with fluorescent SBFI in a single cell; effect of Car on aiNa.

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Effects of Cs⁺ on aⁱ_Na and action potential. Figure 7 shows the effects of Cs⁺ (2 and 6 mM) on aⁱ_Na and spontaneous action potentials of sinoatrial node cells. Figure 7, A and B, shows action potential amplitude and aⁱ_Na, respectively, in a multicellular preparation. Cs⁺ (6 mM) depolarized maximum diastolic membrane potential and decreased aⁱ_Na. 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 aⁱ_Na 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 aⁱ_Na, as shown in Fig. 7D. In six tests with single cells, Cs⁺ (6 mM) decreased aⁱ_Na 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 aⁱ_Na 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 aⁱ_Na 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 aⁱ_Na and maximum diastolic potential.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effects of Cs+ on aiNa and Vm. A: continuous recording of Vm. B: effect of Cs+ on aiNa. C: Vm recorded during control condition (a) and Cs+ exposure (b). D: aiNa measured with fluorescent SBFI in a single cell; effect of Cs+ on aiNa. E: effects of 2 and 6 mM Cs+ on maximum diastolic potential (MDP) and aiNa. Experiments (A-C, E) were done with multicellular preparations.

Effects of carbachol or Cs⁺ on aⁱ_Na in quiescent sinoatrial node cells. The results in Figs. 6 and 7 suggest that carbachol and Cs⁺ inhibited inward Na⁺ movement, possibly hyperpolarization-activated I_f during the pacemaker depolarization of sinoatrial node cells. If the cells become depolarized and quiescent, the hyperpolarization-activated I_f is expected to be absent. Under this condition, carbachol and Cs⁺ should not decrease aⁱ_Na. 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 aⁱ_Na 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 (V_m) to 34 mV and decreased aⁱ_Na. When aⁱ_Na and V_m stabilized, the addition of carbachol or Cs⁺ produced little or no change in aⁱ_Na. Four experiments showed similar results.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effects of carbachol (Car) or Cs+ on aiNa and Vm under control condition and 25.4 mM extracellular K+ concentration ([K+]o). A: continuous recording of Vm. B: effect of Car on aiNa. A' and B' were continuous recordings of traces in A and B, respectively. A': 25.4 mM [K+]o ceased Vm. B': 25.4 mM [K+]o decreased aiNa and prevented a decrease of aiNa caused by Car or Cs+ under control conditions. Experiment was done with a multicellular preparation.

	DISCUSSION

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Intracellular Na⁺ activity of sinoatrial node cells. In the present study, intracellular Na⁺ activity (aⁱ_Na) 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 aⁱ_Na values measured with the two different methods were similar. The levels of aⁱ_Na 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 aⁱ_Na levels in the two regions. Our study did not suggest any differences between aⁱ_Na 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.

-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, I_f (11, 20). If the I_f 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 aⁱ_Na and the rate of spontaneous action potentials. However, it is possible that the increase in aⁱ_Na 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 aⁱ_Na (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 aⁱ_Na. This possibility was tested by comparison of the aⁱ_Na increase produced by isoprenaline with the aⁱ_Na 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 aⁱ_Na (Fig. 4, trace B). In other words, the increase in aⁱ_Na produced by isoprenaline was much greater than the increase in aⁱ_Na produced by pacing. Therefore, our results suggest that the major increase in aⁱ_Na produced by isoprenaline was due to an increase of inward Na⁺ movement, possibly I_f, during pacemaker depolarization but not simply by an increase in the rate of action potentials. However, the magnitude of increase in aⁱ_Na produced by isoprenaline or pacing might depend on the origin of the cells. In the cells from the periphery, aⁱ_Na 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, I_f, 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 I_f carried by Na⁺ is inhibited by stimulation of muscarinic receptor, carbachol is expected to decrease aⁱ_Na. Our study showed that the muscarinic agonist carbachol decreased aⁱ_Na and the slope of the pacemaker depolarization.

4

Cs⁺, ZD-7288, I_f current, intracellular Na⁺ activity, and pacemaker potential. Cs⁺ is known to block I_f in sinoatrial node cells and Purkinje fibers of mammalian hearts (4, 6). ZD-7288 was reported to selectively block I_f in sinoatrial cells (1, 32). The reports are supported by our results because Cs⁺ or ZD-7288 decreased aⁱ_Na and the slope of the pacemaker depolarization. In the present study, Cs⁺ (2 and 6 mM) decreased aⁱ_Na and depolarized maximum diastolic potential in a concentration-dependent manner. The effects of 2 mM Cs⁺ on aⁱ_Na 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 I_f and 6 mM Cs⁺ affected some other ionic currents, the aⁱ_Na decrease by 6 mM Cs⁺ might be caused by changes in I_f as well as other ionic currents. Our study showed that both 2 and 6 mM Cs⁺ blocked the increase in aⁱ_Na produced by isoprenaline.

Change in aⁱ_Na by I_f: Calculation by OXSOFT heart model. An important question to be resolved is whether changes in I_f 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, g_b,Na to give an intracellular Na⁺ concentration during steady rhythmic activity similar to that recorded experimentally. We found that with g_b,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 aⁱ_Na) was produced by the model. Figure 9 shows, first, the result of increasing the values of the Na⁺ (g_f,Na) and K⁺ (g_f,K) components of I_f conductance by 50%. This produced an increase in intracellular Na⁺ similar to that recorded experimentally with isoprenaline. The increase in aⁱ_Na was ~0.8 mM and required ~1.5 min to approach a new steady state. By contrast, halving the conductance of the I_f channels reduced aⁱ_Na by ~0.8 mM, which was similar to that recorded experimentally with Cs⁺ block. These results show that changes in I_f of the magnitude known to be produced by isoprenaline and Cs⁺ would be sufficient to generate the changes in intracellular Na⁺ recorded experimentally.

View larger version (10K): [in this window] [in a new window]   Fig. 9.   Computed changes in aiNa using single sinus node cell model in the OXSOFT heart program, version 4.8. A: voltage changes during spontaneous activity in model. B: changes in aiNa. C: hyperpolarization-activated inward current (If). Conductance (gf) was first increased, producing an increase in aiNa similar to that produced experimentally by Iso. When gf was reduced, aiNa decreased in a way similar to that produced by Cs+ block of If. Changes in spontaneous rate in model were similar to those produced experimentally by isoprenaline and Cs+.