INTRODUCTION

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INTRACELLULAR SODIUM ION CONCENTRATIONS ([Na⁺]_i) are much lower than extracellular concentrations. This lower [Na⁺]_i is maintained by a Na⁺-K⁺ pump in the cell membrane. The large Na⁺ gradient across the cell membrane is an important driving force for several membrane transporters, such as the Na⁺/Ca²⁺ exchanger, the Na⁺/H⁺ exchanger, the Na⁺-HCO₃ cotransporter, the Na⁺-glucose carrier, and the inward Na⁺ currents during action potentials (15). Changes in the transmembrane Na⁺ gradient could affect the action potential, intracellular pH, Ca²⁺ sensitivity of myofilaments, intracellular Ca²⁺ concentration ([Ca²⁺]_i), electrical conduction, and the contractile force of heart cells. Intracellular Na⁺ overload could cause cardiac arrhythmia (19). Therefore, it is of fundamental importance to understand the regulation of the [Na⁺]_i when assessing physiological and pathophysiological processes in the heart.

The stimulation of ₁-adrenergic receptors sets in motion various steps of the cardiac excitation-contraction coupling cascade, which regulates the cardiac rhythm, conduction, and force of contraction (28). It has been reported that the stimulation of ₁-adrenergic receptors by phenylephrine decreased intracellular Na⁺ activity (a_Naⁱ) in multicellular preparations of cardiac tissues (9, 37). In Purkinje fibers (26, 37) and papillary muscle (6), ₁-adrenergic receptor agonists stimulated the Na⁺-K⁺ pump, which would decrease a_Naⁱ. In ventricular myocytes perfused with a high-Na⁺ solution, ₁-adrenergic receptor agonists and protein kinase C (PKC) stimulated the Na⁺-K⁺ pump current (8, 33). The increase in the Na⁺-K⁺ pump current was mediated by the _1b-subtype of the receptor (35). However, the effect of the stimulation of ₁-adrenergic receptors on a_Naⁱ has not yet been examined in single cardiac myocytes. In addition, the signaling pathways involved are also uncertain.

₁-Adrenergic receptors in mammalian hearts are linked to two signal transducing pathways, i.e., the inositol trisphosphate (IP₃) and the diacylglycerol pathways (1, 2). The first pathway mobilizes Ca²⁺ from intracellular nonmitochondrial stores, whereas the latter activates diacylglycerol-dependent PKC (1). It has been reported that this PKC regulates positive inotropy (25), negative inotropy (13), the Na⁺-H⁺ exchanger (7, 32), and the Na⁺-K⁺ pump (33) on stimulation of ₁-adrenergic receptors. However, it is not clear that activation of PKC is involved in the a_Naⁱ decrease by ₁-adrenoceptor in cardiac cells.

The aim of our study was to elucidate the mechanism by which ₁-adrenergic receptor stimulation changes a_Naⁱ in single ventricular myocytes. We looked for connections between changes in a_Naⁱ and intracellular signaling pathways. In particular, our study was concerned with the role of PKC in the change of a_Naⁱ in ventricular myocytes in which an intact cytosol was maintained.

	METHODS

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Cell isolation. Single ventricular myocytes were isolated from each guinea pig heart with the use of a method described previously (10). Briefly, guinea pigs were killed by cervical dislocation, and their hearts were removed rapidly. The heart was retrogradely perfused at 37°C with a 750 µM Ca²⁺ solution and a Ca²⁺-free solution followed by an enzyme solution. The enzyme solution contained 150 µM Ca²⁺, collagenase type I, and protease type XIV. The heart was then flushed with a 150 µM Ca²⁺ solution. The ventricles were removed and chopped into small pieces, which were then shaken in a flask containing a 150 µM Ca²⁺ solution. The cell suspension was then left to sediment. The supernatant was replaced with a 500 µM Ca²⁺ solution. The cells were kept at room temperature.

Experimental solutions, perfusions, and reagents. Myocytes in the experimental chamber were continuously superfused at room temperature (24-26^°C) with Tyrode solution containing 10 mM glucose, 5 mM HEPES, 140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂, titrated to pH 7.4 with 4 M NaOH. The experimental chamber had a volume of 150 µl, and the flow rate of the Tyrode solution was 2 ml/min. Miniature solenoid valves (LFAA 1201618H; Lee Products, Bucks, UK) selected the solution entering the chamber, and the superfusate within the chamber could be changed within 5 s. The solution level in the chamber was controlled with a suction system. The chamber and solenoid valves were mounted on the sliding stage of a Nikon Diaphot microscope that sat on an antivibration table (Newport). Atenolol (5 µM; a -adrenergic receptor antagonist) was always included in the 50 µM phenylephrine solution to prevent possible stimulation of -adrenergic receptors by phenylephrine. Atenolol alone at a concentration of 5 µM did not affect [Na⁺]_i.

Fluorescence measurements and in vivo calibration. Freshly isolated ventricular myocytes were incubated for 1.5 h at room temperature with 15 µM SBFI-AM and 1 µM pluronic acid. Myocytes loaded with the indicator were then moved to the experimental chamber and illuminated with ultraviolet light applied via an epifluorescence microscope. Excitation was at 340 and 380 nm (Cairn Spectrophotometer System) via 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 then processed by a ratio amplifier (20). The ratio of the light emitted with the 340-nm excitation to that emitted with the 380-nm excitation (340/380 ratio) represents the level of intracellular Na⁺ (21).

Electrophysiological measurements. Membrane potential was measured with conventional microelectrodes pulled from filamented thin-wall glass tubing of 1.5-mm outer diameter and 1.2-mm inner diameter (World Precision Instruments). They were filled with filtered 300 mM KCl and had a resistance of between 25 and 40 M. Membrane potential was measured with an Axoclamp 2A amplifier (Axon Instruments). Action potentials were elicited at 0.25-0.3 Hz by 2-ms depolarizing current pulses passed through the microelectrode. To hold the membrane potential at either 40 or 85 mV constantly, the cells were voltage-clamped using the switch-clamp mode of the Axoclamp 2A amplifier. [Na⁺]_i and membrane potential were simultaneously recorded on a chart recorder (Gould 3400 series).

Statistics. All quantitative data are expressed as means ± SE. The results were analyzed for differences using ANOVA. We calculated EC₅₀ with the Microcal Origin for Windows software program. Differences were considered significant when P values were <0.05.

	RESULTS

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Effect of PMA on a_Naⁱ. Figure 1 shows the effects of the PKC activator PMA on a_Naⁱ and membrane potential in a single guinea pig ventricular myocyte driven at 0.25-0.3 Hz. PMA is generally used to selectively activate the PKC and has been known to activate the enzyme maximally at ~100 nM, so we used this concentration in the present study. The top and bottom traces in Fig. 1A represent a_Naⁱ and membrane potential, respectively. Application of 100 nM PMA for 5 min decreased a_Naⁱ from 4.3 to 3.1 mM and slightly hyperpolarized the diastolic membrane potential. The changes in a_Naⁱ and membrane potential induced by the drug usually stabilized within 5 min, so we applied the drug for 5 min. In 12 myocytes tested, 100 nM PMA decreased the a_Naⁱ from 6.5 ± 0.4 to 5.0 ± 0.4 mM (P < 0.01). In 9 of 12 myocytes tested, 100 nM PMA slightly hyperpolarized the diastolic membrane potential. Two myocytes showed no change, and the remaining myocyte showed slight depolarization. Figure 1, B and C, shows the concentration dependency of the PMA effect on a_Naⁱ. Figure 1B shows superimposed recordings of a_Naⁱ changes produced by different concentrations of PMA. PMA decreased a_Naⁱ at concentrations >0.01 nM. Figure 1C shows a concentration-response curve for the decrease in a_Naⁱ caused by PMA. A maximal a_Naⁱ decrease of 1.5 ± 0.2 mM (n = 12) was observed at a PMA concentration of 100 nM; the PMA concentration required for a half-maximal response was 0.46 ± 0.13 nM (n = 3-5 for each concentration of PMA).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of 4-phorbol 12-myristate 13-acetate (PMA) on intracellular Na+ activity (aNai) and membrane potential (Vm) in guinea pig ventricular myocytes stimulated at 0.25-0.3 Hz. A: aNai (top) and membrane potential (bottom) traces for a myocyte stimulated at 0.3 Hz was treated with 100 nM PMA for 5 min. B: concentration-dependent effect of PMA on the decrease in aNai. Superimposed traces show changes in aNai when sodium-binding benzofuran isophthalate (SBFI)-loaded myocytes stimulated at 0.3 Hz were treated with PMA at the concentrations of 0.01, 0.1, 10, and 1,000 nM for 5 min. C: concentration-response curve for the decreases in aNai caused by PMA. Results are expressed as mean (±SE) decreases in aNai (mM). Each data point represents 3-5 cells. The time periods of exposure to PMA in A and B are indicated by the lines below the top traces. Data shown in A are representative of 12 separate experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effects of 4-phorbol 12-myristate 13-acetate (4-PMA), staurosporine (STAU), bisindolylmaleimide I (GF-109203X), and PMA on aNai and membrane potential. A: a myocyte stimulated at 0.25 Hz was treated with 100 nM 4-PMA. B: a myocyte stimulated at 0.3 Hz was first exposed to 100 nM PMA and then treated with 10 nM staurosporine. C: a myocyte stimulated at 0.3 Hz was first exposed to 5 µM GF-109203X and then treated with 100 nM PMA. The periods of exposure to 4-PMA, staurosporine, GF-109203X, and PMA in A, B, and C are indicated by the lines below the top traces. Data shown in A, B, and C are representative of 6, 3, and 5 separate experiments, respectively.

Effect of phenylephrine on a_Naⁱ in the absence and presence of staurosporine, GF-109203X, or PMA. We next examined the effects of the ₁-adrenergic receptor agonist phenylephrine on a_Naⁱ and membrane potential in single cardiac cells as shown in Fig. 3. Phenylephrine is generally used to selectively activate the ₁-adrenoceptor and has been known to activate the receptor maximally at about 50 µM, so we used this concentration in the present study. As shown in Fig. 3A, exposure of the myocyte to 50 µM phenylephrine decreased a_Naⁱ from 6.0 to 4.5 mM and slightly hyperpolarized the diastolic membrane potential. Because the changes in a_Naⁱ and membrane potential induced by the drug usually stabilized in 5 min, we applied the drug for 5 min. After washout of the phenylephrine, the decreased a_Naⁱ slowly returned to its initial value. In 11 myocytes tested, 50 µM phenylephrine decreased a_Naⁱ from 6.1 ± 0.3 to 4.6 ± 0.3 mM (P < 0.01), which is similar to the decrease in a_Naⁱ produced by 100 nM PMA; 8 myocytes showed a slight hyperpolarization in diastolic membrane potential, 2 myocytes showed no change, and the other myocyte showed slight depolarization. It has been known that stimulation of the ₁-adrenergic receptor results in stimulation of the Na⁺/H⁺ exchanger (7, 32), which might increase a_Naⁱ. Therefore, we tested whether stimulation of the Na⁺/H⁺ exchanger would in part offset the decrease in a_Naⁱ induced by phenylephrine. In five myocytes pretreated with 10 µM MIA, a specific inhibitor of the Na⁺/H⁺ exchanger, 50 µM phenylephrine decreased a_Naⁱ by 1.6 ± 0.2 mM (data not shown). This value was not significantly different from the a_Naⁱ decrease caused by 50 µM phenylephrine in myocytes that had not been pretreated with MIA (1.5 ± 0.2 mM, n = 11, P = 0.61). The results suggest that the ₁-adrenergic receptor stimulation of the Na⁺-H⁺ exchanger did not cause a significant increase in a_Naⁱ under our conditions.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effects of phenylephrine (PE) on aNai and membrane potential in the absence and presence of staurosporine or GF-109203X. A: a myocyte stimulated at 0.25 Hz was treated with 50 µM phenylephrine in the presence of 5 µM atenolol (ATE) for 5 min. B: a myocyte stimulated at 0.3 Hz was treated with 50 µM phenylephrine in the presence of 25 nM staurosporine. C: a myocyte stimulated at 0.3 Hz was treated with 50 µM phenylephrine in the presence of 5 µM GF-109203X. The periods of exposure to phenylephrine, atenolol, staurosporine, and GF-109203X in A, B, and C are indicated by the lines below the top traces. Data shown in A, B, and C are representative of 11, 5, and 5 separate experiments, respectively.

Effect of PMA on a_Naⁱ in the presence of TTX and vice versa. In cardiac muscle cells, intracellular Na⁺ is regulated by Na⁺ influx through Na⁺ channels, the Na⁺/H⁺ exchanger, the Na⁺/Ca²⁺ exchanger, Na⁺-HCO₃ cotransport, and Na⁺ efflux through the Na⁺-K⁺ pump. Thus the change in a_Naⁱ induced by phenylephrine and PMA might be due to alterations in Na⁺ influx or the Na⁺-K⁺ pump. To determine whether the decrease in the a_Naⁱ caused by PMA was due to a change in Na⁺ influx through Na⁺ channels, we tested the effect of TTX on a_Naⁱ in the presence of PMA. As shown in Fig. 4A, application of 100 nM PMA decreased a_Naⁱ from 8.4 to 5.6 mM. When a_Naⁱ stabilized at the lower level and 5 µM TTX was added, the a_Naⁱ was further decreased significantly by 1.0 ± 0.1 mM (n = 6, P < 0.01). Also, pretreatment with 5 µM TTX did not prevent the decrease in a_Naⁱ caused by 100 nM PMA; addition of 100 nM PMA significantly decreased a_Naⁱ by 1.5 ± 0.3 mM (Fig. 4B, n = 9, P < 0.01) in myocytes pretreated with 5 µM TTX. This value is similar to the decrease in a_Naⁱ induced by PMA in the absence of TTX (Figs. 1A and 4A). The results indicate that the decrease in a_Naⁱ mediated by PKC is not due to a change in Na⁺ movement through Na⁺ channels.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of PMA and tetrodotoxin (TTX) on aNai and membrane potential in guinea pig ventricular myocytes. A: a myocyte stimulated at 0.25 Hz was treated with 5 µM TTX in the presence of 100 nM PMA. B: a myocyte stimulated at 0.25 Hz was treated with 100 nM PMA in the presence of 5 µM TTX. The periods of exposure to TTX and PMA in A and B are indicated by the lines below the top traces. Data shown in A and B are representative of 6 and 9 separate experiments, respectively.

Effect of PMA on a_Naⁱ in the presence of strophanthidin or a high extracellular K⁺ concentration. In further experiments, we tested whether the decrease in a_Naⁱ caused by PMA was related to a change in Na⁺ movement through the Na⁺-K⁺ pump. Figure 5A shows the effect of PMA on a_Naⁱ in the presence of the Na⁺-K⁺ pump inhibitor strophanthidin. Application of 100 µM strophanthidin produced a substantial increase in a_Naⁱ that stabilized somewhat after 3 min. Adding PMA in the presence of strophanthidin did not significantly decrease a_Naⁱ (the a_Naⁱ decrease of 0.1 ± 0.1 mM, n = 7, P > 0.05). Note the decrease in a_Naⁱ caused by PMA under the control conditions (Figs. 1A and 4A). In other words, when the Na⁺-K⁺ pump was inhibited by strophanthidin, the decrease in a_Naⁱ caused by PMA was blocked.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of PMA on aNai and membrane potential in guinea pig ventricular myocytes pretreated with strophanthidin (STRO) and 24 mM extracellular K+ concentration (High [K+]o). A: a myocyte stimulated at 0.3 Hz was treated with 100 nM PMA in the presence of 100 µM strophanthidin. B: a myocyte stimulated at 0.3 Hz was treated with 100 nM PMA in the presence of 24 mM [K+]o. The periods of exposure to strophanthidin, PMA, and high [K+]o in A and B are indicated by the lines below the top traces. Data shown in A and B are representative of 7 and 3 separate experiments, respectively.

	DISCUSSION

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Maintenance of a low a_Naⁱ and a large transmembrane Na⁺ gradient is important for the electrophysiological functions of a variety of tissues including cardiac muscles. In cardiac muscles, the large transmembrane Na⁺ gradient provides the driving force for inward Na⁺ movement and for the transport of other ions and molecules such as Ca²⁺, H⁺, HCO₃, sugar, and amino acids. Therefore, a change in a_Naⁱ could bring about changes in the intracellular concentrations of these ions and chemicals. For example, a small increase in a_Naⁱ causes a concomitant rise in intracellular Ca²⁺ through the operation of the Na⁺/Ca²⁺ exchange process, which in turn increases the contractile force of cardiac muscle cells (16).

Regulation of a_Naⁱ by an intracellular signaling pathway. a_Naⁱ is maintained at a low level by the Na⁺-K⁺ pump and can be regulated by the activation of a receptor on the cell surface membrane. Activation of -adrenoceptors decreases a_Naⁱ in rabbit ventricular myocytes (5) and in cardiac Purkinje fibers (3, 18, 24, 34). Furthermore, it has been shown that cAMP also decreases a_Naⁱ in cardiac Purkinje fibers (24). Therefore, the decrease in a_Naⁱ may be mediated via the cAMP-dependent protein kinase A pathway. It has been suggested that phosphorylation of the Na⁺-K⁺-ATPase stimulates the Na⁺-K⁺ pump and thus produces a decrease in a_Naⁱ.

Mechanism of the a_Naⁱ decrease. In the diacylglycerol-dependent PKC signaling pathway, PKC phosphorylates a target protein that then induces physiological responses such as changes in a_Naⁱ, [Ca²⁺]_i, and contractile force (4, 11, 12, 22). The level of the a_Naⁱ is mainly dependent on Na⁺ influx through Na⁺ channels and Na⁺ efflux effected by the Na⁺-K⁺ pump. Therefore, it is conceivable that PKC might decrease a_Naⁱ by phosphorylation of membrane proteins such as Na⁺ channels and the Na⁺-K⁺ pump. We could conclude that the decrease in a_Naⁱ effected by PMA is not related to a change in Na⁺ influx through Na⁺ channels because the pretreatment with TTX did not prevent the decrease in a_Naⁱ caused by PMA.