The effect of β-adrenergic stimulation on cardiac Na+/Ca2+ exchange has been controversial. To clarify the effect, we measured Na+/Ca2+ exchange current (INCX) in voltage-clamped guinea pig, mouse, and rat ventricular cells. When INCX was defined as a 5 mM Ni2+-sensitive current in guinea pig ventricular myocytes, 1 μM isoproterenol apparently augmented INCX by ∼32%. However, this increase was probably due to contamination of the cAMP-dependent Cl− current (CFTR-Cl− current, ICFTR-Cl), because Ni2+ inhibited the activation of ICFTR-Cl by 1 μM isoproterenol with a half-maximum concentration of 0.5 mM under conditions where INCX was suppressed. Five or ten millimolar Ni2+ did not inhibit ICFTR-Cl activated by 10 μM forskolin, an activator of adenylate cyclase, suggesting that Ni2+ acted upstream of adenylate cyclase in the β-adrenergic signaling pathway. Furthermore, in a low-extracellular Cl− bath solution, 1 μM isoproterenol did not significantly alter the amplitude of Ni2+-sensitive INCX at +50 mV, which is close to the reversal potential of ICFTR-Cl. No change in INCX amplitude was induced by 10 μM forskolin. When INCX was activated by extracellular Ca2+, it was not significantly affected by 1 μM isoproterenol in guinea pig, mouse, or rat ventricular cells. We concluded that β-adrenergic stimulation does not have significant effects on INCX in guinea pig, mouse, or rat ventricular myocytes.
- cystic fibrosis transmembrane conductance regulator
- nickel ion
the β-adrenergic receptor signaling cascade is a pivotal mechanism regulating cardiac contractility, and β-adrenergic receptor agonists such as isoproterenol produce positive inotropy, together with increasing cytosolic Ca2+ transient amplitude and rates of relaxation and intracellular Ca2+ decline. Major mechanisms underlying this positive inotropic action are enhanced activities of the sarcolemmal L-type Ca2+ channel, the sarcoplasmic reticulum Ca2+-ATPase, and the sarcoplasmic reticulum ryanodine receptor and a decreased myofilament Ca2+ sensitivity (3). However, the question of whether β-adrenergic receptor stimulation upregulates sarcolemmal Na+/Ca2+ exchange, which is the main Ca2+ extrusion system of the cardiac myocyte, has proved controversial.
Several groups have reported that the cardiac Na+/Ca2+ exchange protein is phosphorylated by PKA (16, 26, 27, 30). The augmentation of exchange activity through the β-adrenergic signaling pathway has also been reported. Linck et al. (19) found that Na+-dependent Ca2+ uptake of baby hamster kidney (BHK) cells expressing canine cardiac Na+/Ca2+ exchanger 1 (NCX1) was enhanced by 100 μM forskolin, an activator of adenylate cyclase, by ∼20%. In whole cell voltage-clamped guinea pig ventricular myocytes with other major currents inhibited, Perchenet et al. (25) and Zhang et al. (33) demonstrated that β-adrenergic stimulation enhanced Na+/Ca2+ exchange current (INCX) by ∼25–100%. Ruknudin et al. (26) found that PKA-activating reagents phosphorylated a cardiac isoform of the NCX expressed in Xenopus oocytes and increased both 45Ca2+ uptake (by ∼40%) and outward INCX (by >100%). They also observed PKA-dependent enhancement of Na+-dependent 45Ca2+ uptake by ∼40% in adult rat ventricular cardiomyocytes. Wei at al. (30) also reported that PKA phosphorylated the Na+/Ca2+ exchange protein and increased INCX by ∼500% in control and by ∼100% in failing ventricular cells from pig heart, suggesting that cardiac Na+/Ca2+ exchange is hyperphosphorylated in heart failure. To the contrary, no stimulatory effect of β-adrenergic receptor stimulation or PKA on exchange activity was detected in giant membrane patches excised from blebs of guinea pig ventricular cells (5), vesicle preparations of rat hearts (2), guinea pig ventricular cells (20), or BHK cells expressing the dog cardiac NCX1 (13).
The aim of this study was to clarify the effect of β-adrenergic stimulation on the native mammalian NCX. We measured INCX in voltage-clamped ventricular myocytes isolated from guinea pig, mouse, and rat hearts and studied the effect of β-adrenergic stimulation, with particular attention to the contribution of the PKA-activated Cl− current (CFTR-Cl− current, ICFTR-Cl) to the recorded membrane current. We concluded that β-adrenergic stimulation does not significantly affect INCX in the ventricular cells. When INCX was recorded using a protocol similar to the previous study using Ni2+ (25, 30, 33), a nonselective blocker of the exchanger, the amplitude of Ni2+-sensitive membrane current apparently increased after the application of isoproterenol, suggesting an increase in INCX. However, this potentiation was in fact attributable to an increase in ICFTR-Cl contaminating the Ni2+-sensitive current.
Part of this study was presented at the 48th annual meeting of the Biophysical Society (31).
Ventricular myocytes were dissociated from guinea pig, mouse, and rat hearts. All procedures were approved by the Animal Research Committee of the Graduate School of Medicine, Kyoto University. The animals were deeply anesthetized by intraperitoneal injection of pentobarbital sodium (>0.1 mg/g body wt). For guinea pig hearts, the thorax was opened under artificial respiration and the aorta was cannulated to start retrograde perfusion of the heart. The heart was quickly excised and mounted on a Langendorff-type perfusion apparatus. The heart was first perfused with a control Tyrode solution and then with a nominally Ca2+-free control Tyrode solution until the heartbeat stopped, followed by Ca2+-free Tyrode solution containing 0.4 mg/ml collagenase (type I; Worthington Biochemical) for ∼15 min. Finally, the perfusate was switched to a high-K+, low-Cl− solution. The left ventricle and the septum were diced into ∼10 pieces and shaken gently in the high-K+, low-Cl− solution for ∼5 min. After filtration of the dispersed tissues, the resulting cell suspension was centrifuged (300 rpm, 5 min) and suspended in a modified DMEM solution.
Mouse and rat hearts were perfused with a cell isolation solution containing collagenase (type II, 1 mg/ml; Worthington Biochemical), trypsin (type I, 0.05 mg/ml; Sigma), protease (type XIV, 0.05 mg/ml; Sigma), and 0.2 mM CaCl2 for 10 min. The left ventricle and the septum were then diced and shaken gently for ∼15 min in the cell isolation solution noted above with BSA (fraction V, 1 mg/ml; Sigma). After filtration, the cell suspension was centrifuged and resuspended in the modified DMEM solution. The myocytes were used for experiments within 8 h.
The myocytes were voltage clamped using the whole cell voltage-clamp method with an Axopatch 200B amplifier (Axon Instruments). Current-voltage (I-V) relationships were measured by applying ramp pulses (change in voltage with time = 680 mV/s) as described in our previous study (10). Membrane current was filtered at 1 kHz and digitized at 2 kHz with a 12-bit analog-to-digital converter (ADM 670PCI; Micro Science, Tokyo, Japan), which was controlled using our original software. The ramp voltage pulse was applied every 2 or 6 s as indicated. The holding potential was −40 mV, and the experimental temperature was 36–37°C.
The control Tyrode solution contained (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.3 NaH2PO4, 5.5 glucose, and 5.0 HEPES (pH 7.4 with NaOH). The high-K+, low-Cl− solution contained (in mM) 25 KCl, 70 K+-glutamate, 10 KH2PO4, 10 taurine, 0.5 EGTA, 11 glucose, and 10 HEPES (pH 7.3 with KOH). The modified DMEM solution was prepared by adding 20 mM NaCl and 25 mM HEPES to DMEM (without NaHCO3; ICN Biomedicals), and pH was adjusted to 7.4 with NaOH. The cell isolation solution for mouse and rat myocytes contained (in mM) 130 NaCl, 5.4 KCl, 0.5 MgCl2, 0.33 NaH2PO4, 25 HEPES, 22 glucose, 1 l-glutamine, and 0.1 EGTA with 0.01 U/ml insulin (pH 7.4 with NaOH).
To record INCX or ICFTR-Cl, K+ channels, Ca2+ channels and Na+-K+ pump were inhibited using tetraethylammonium, Cs+, Ba2+, nicardipine, and ouabain in a manner similar to methods described in previous studies (10, 22). The compositions of bath and pipette solutions are listed in Table 1. The β-adrenergic receptor signaling cascade was activated by adding 1 μM isoproterenol or 10 μM forskolin to the bath.
In particular, when recording ICFTR-Cl (see Fig. 1), bath solution 1 and pipette solution 1 were used. INCX was inhibited by removing Na+ and Ca2+ from the pipette solution (addition of 10 mM EGTA) and Ca2+ from the bath solution. To record Ni2+-sensitive INCX (cf. Figs. 2, 4, and 5), bath solution 2 or 3 and pipette solution 2 were used. Free Ca2+ concentration (0.8 μM) was calculated with WinMAXC (Ref. 4; http://www.stanford.edu/∼cpatton/maxc.html). INCX was defined as the 5 mM Ni2+-sensitive current recorded under these experimental conditions. For recording extracellular Ca2+-induced INCX, bath solution 4 and pipette solution 3 were used (see Figs. 6 and 7). Free Ca2+ concentration was 0.8 μM. The bath solution was changed from one containing 0.2 mM EGTA and no CaCl2 to one containing no EGTA and 0.5 (guinea pig and mouse INCX) or 2 (rat INCX) mM Ca2+ with a rapid solution switcher (8, 9). Extracellular Ca2+ was applied for a short period (5 s) every 30 s to avoid intracellular accumulation of Ca2+ and depletion of Na+ via Na+/Ca2+ exchange.
Membrane current was normalized by cell capacitance (pA/pF) and presented as mean (SD). Statistical analysis was performed using one-way repeated-measures ANOVA with the Student-Newman-Keuls test (StatView, SAS Institute). P < 0.05 was considered significant.
Inhibition of isoproterenol-activated ICFTR-Cl by Ni2+.
In guinea pig ventricular myocytes, β-adrenergic stimulation activates ICFTR-Cl, whose I-V relationship is similar to that for INCX (1, 12, 21). However, in the previous reports studying the effects of β-adrenergic stimulation on INCX in intact myocytes, activation of ICFTR-Cl was not systematically investigated. In this study, we first tested whether Ni2+, which has often been used for isolating INCX, affects ICFTR-Cl. In Fig. 1A, ICFTR-Cl was activated by applying 1 μM isoproterenol, and membrane currents at +50 and −100 mV were plotted against time. The I-V relationship of the isoproterenol-activated current (the difference between b and a in Fig. 1B) showed characteristics of ICFTR-Cl, and the reversal potential [−34 mV (SD 6) (n = 7)] was close to the calculated equilibrium potential of Cl− (−33 mV). Under these experimental conditions, INCX was effectively inhibited by removing Na+ and Ca2+ from the pipette solution (addition of 10 mM EGTA) and by omitting Ca2+ from the extracellular solution. Inhibition of INCX was confirmed by the finding that 5 mM Ni2+ did not attenuate membrane current in the absence of isoproterenol (n = 3, data not shown). In contrast, superfusion of 5 mM Ni2+ almost completely and reversibly inhibited ICFTR-Cl induced by isoproterenol. The half-maximal concentration of Ni2+ required to inhibit ICFTR-Cl was 0.5 mM (Fig. 1C).
It is notable that Ni2+ exerted an inhibitory action after a time lag (∼30 s), whereas no delay was observed upon removing Ni2+ (see also Fig. 3A). This fact suggests that the Ni2+ action was not due to the competition with Cl− at the channel pore. Indeed, 5 mM (as shown in Fig. 1D; n = 4) or 10 mM Ni2+ (n = 4) did not attenuate ICFTR-Cl, which is induced by forskolin, an activator of adenylate cyclase. Therefore, we concluded that the site of Ni2+ action is upstream of adenylate cyclase in the β-adrenergic signaling cascade, perhaps at the β-adrenergic receptor or related G protein (Gs). The above experimental findings suggest that if INCX is isolated as the Ni2+-sensitive current, then INCX is overestimated during β-adrenergic stimulation because of contamination of ICFTR-Cl.
Potentiation of Ni2+-sensitive current by isoproterenol.
The effect of β-adrenergic stimulation on INCX was studied with a protocol similar to previous studies by applying 5 mM Ni2+ (25, 30, 33) as shown in Fig. 2. Application of 5 mM Ni2+ rapidly and reversibly inhibited INCX under control conditions. One micromolar isoproterenol increased the membrane conductance such that the amplitude of five millimolar Ni2+-sensitive current was larger by 77% in the presence of isoproterenol (d − e in Fig. 2B) than in control (a − b in Fig. 2B). No significant difference was observed between the reversal potential of the Ni2+-sensitive current before and during isoproterenol application. In 29 cells, the degree of isoproterenol activation of the Ni2+-sensitive current ranged from −5% to 135% [32% (SD 35)], consistent with previous reports (25, 33). However, it should be noted that Ni2+ inhibition in the presence of isoproterenol has both rapid and delayed components in the majority of cells, whereas only the rapid effect is apparent in the control (Fig. 2A).
The time course of the Ni2+ action is summarized in Fig. 3. The inhibition of ICFTR-Cl by Ni2+ (Fig. 3A) was characterized by a time lag of ∼30 s on average, and the inhibition time course was variable among cells. No time lag was apparent in the inhibition of INCX by Ni2+ in the absence of isoproterenol (Fig. 3B). During β-adrenergic stimulation induced by 1 μM isoproterenol, Ni2+ rapidly attenuated the membrane current in a manner similar to that of control, followed by a plateau and further inhibition in 19 of 29 cells. The duration of the plateau was close to the observed time lag of Ni2+ inhibition of ICFTR-Cl. The delayed component was not clear in the other 10 cells. The amplitude of Ni2+-sensitive current during isoproterenol application was significantly larger than that of the control in the group with the delayed component [by 47.4% (SD 35.3); n = 19] but not in the group without the delayed component (Fig. 3D). In the latter case, longer application might be needed to reach a full Ni2+ inhibition. The delayed component of Ni2+ action suggested that Ni2+ also attenuated ICFTR-Cl under this experimental condition.
To decrease the possible contamination of INCX by ICFTR-Cl, we carried out the same protocol using a lower-Cl− bath solution (bath solution 3) and measured the amplitude of INCX at a membrane potential near the reversal potential for ICFTR-Cl (Fig. 4). One micromolar isoproterenol did not affect membrane current at +50 mV but increased it at −100 mV (Fig. 4A). Lowering extracellular Cl− concentration shifted the reversal potential of the isoproterenol-induced current to positive potentials (c − b in Fig. 4B) [45 mV (SD 5.5); n = 14], which was clearly different from that of the Ni2+-sensitive current in control (b − a in Fig. 4B) [−45 mV (SD 3.9); n = 14]. The amplitude of the Ni2+-sensitive current during isoproterenol application was not significantly different from that in control, when the amplitude was measured at +50 mV (Fig. 4C). The amplitude of the Ni2+-sensitive current measured at −100 mV tended to be larger in the presence of isoproterenol than that in the control. However, the difference was not statistically significant (data not shown), although isoproterenol significantly increased the membrane current amplitude at −100 mV by 90% (SD 60) (n = 14). The Ni2+ inhibition of isoproterenol action might be weaker under the experimental condition of low extracellular Cl−.
The effect of β-adrenergic stimulation on INCX was studied further with forskolin (Fig. 5). Because forskolin-activated ICFTR-Cl was not attenuated by Ni2+ (Fig. 1D), the contamination of ICFTR-Cl in the recorded Ni2+-sensitive INCX should be negligible when adenylate cyclase is activated by forskolin. Ten micromolar forskolin increased membrane conductance, probably because of activation of ICFTR-Cl (Fig. 5A). However, the I-V relationship of Ni2+-sensitive INCX during forskolin application was almost identical to control (Fig. 5B), and no statistical significance was found between the amplitude of the Ni2+-sensitive current before and during forskolin application (Fig. 5C).
No potentiation of INCX by isoproterenol.
The above findings indicated that β-adrenergic receptor stimulation does not increase INCX. However, Ni2+ has multiple nonspecific effects and could conceivably interfere with the detection of an underlying β-adrenergic activation of INCX. To rule out this possibility, INCX was isolated as an extracellular Ca2+-activated current as previously described (6, 22) instead of using Ni2+ (Fig. 6). Figure 6A shows 0.5 mM Ca2+-induced outward INCX at a holding potential of −40 mV. One micromolar isoproterenol induced an inward shift of the holding current and increased membrane conductance due to activation of ICFTR-Cl. It should be noted that INCX (b − a, Fig. 6A) was outward at the membrane potentials examined because of a higher Na+ concentration in the pipette solution and was clearly different from ICFTR-Cl (c − a, Fig. 6A). Isoproterenol did not change INCX (d − c, Fig. 6A). Figure 6B shows the time courses of Ca2+-induced INCX and membrane current in the absence of extracellular Ca2+, which reflects ICFTR-Cl. INCX at the end of isoproterenol application tended to be smaller than the control [95.2% (SD 13.7) of control, n = 10], but the difference was not statistically significant (Fig. 6C). We conclude from this and the above-described experiments that β-adrenergic stimulation does not significantly affect INCX in guinea pig ventricular myocytes.
The effect of β-adrenergic stimulation on INCX was studied further in murine and rat ventricular myocytes using the same protocol as described in Fig. 6 and as shown in Fig. 7. In these ventricular cells, 1 μM isoproterenol did not activate ICFTR-Cl, consistent with the results in previous studies (18, 24). No significant difference was found between the amplitude of INCX before and during isoproterenol application in either murine or rat myocytes. We concluded that β-adrenergic stimulation has no significant effect on INCX in guinea pig, mouse, or rat ventricular cells.
We studied the effect of β-adrenergic stimulation on INCX in voltage-clamped guinea pig, mouse, and rat ventricular cells. No significant activation of INCX by β-adrenergic stimulation was detected. When INCX was defined as Ni2+-sensitive current in guinea pig ventricular myocytes, it was apparently increased by isoproterenol. However, this augmentation was most likely due to the activation of ICFTR-Cl, which is also sensitive to Ni2+. Our results support previous studies (2, 5, 13, 20) and a recent report of studies of rabbit ventricular cells by Ginsburg and Bers (11).
Using guinea pig ventricular cells, Perchenet et al. (25) and Zhang et al. (33) reported the enhancement of INCX by β-adrenergic stimulation. We were able to confirm their experimental results using a similar experimental protocol with Ni2+ (Fig. 2). However, the apparent enhancement of INCX was probably due to the activation of ICFTR-Cl in light of the following findings. 1) Ni2+ inhibited the activation of ICFTR-Cl by 1 μM isoproterenol, with a half-maximum concentration of 0.5 mM, but not activation by 10 μM forskolin. 2) Forskolin did not augment the amplitude of INCX. 3) The amplitude of INCX was not affected by isoproterenol when measured near the reversal potential of ICFTR-Cl. 4) Isoproterenol did not significantly affect the extracellular Ca2+-induced INCX. The fact that the I-V relationships in control and in the presence of both 1 μM isoproterenol and 10 mM Ni2+ were superimposable in the experiments reported by Perchenet et al. (Ref. 25, Fig. 1) suggests that ICFTR-Cl was suppressed by Ni2+ in their experiments. They ruled out the involvement of ICFTR-Cl in their study, because 9-anthracenecarboxylic acid and glibenclamide did not affect the increases in INCX (25). However, the dose of the blockers and the experimental protocols were not clearly stated in their study. We did not study these blockers in the present study, because Ni2+ clearly attenuated the activation of ICFTR-Cl by isoproterenol. One difference between our study and that of Perchenet et al. (25) is the effect of forskolin on INCX. Although they found an enhancement of Ni2+-sensitive INCX by forskolin, we failed to detect this. The reason for this discrepancy is not yet clear. However, our finding is consistent with the view that the apparent activation of INCX was due to the contamination of ICFTR-Cl, because ICFTR-Cl activated by forskolin was not attenuated by Ni2+ (Fig. 1D).
Wei et al. (30) reported that isoproterenol enhanced INCX (5 mM Ni2+-sensitive current) in pig heart by ∼500%. If ICFTR-Cl is also activated by β-adrenergic stimulation in pig ventricular cells, the increase in INCX is likely to be overestimated. In mouse and rat ventricular myocytes, we could not detect significant effects of isoproterenol on INCX and isoproterenol did not activate a membrane current similar to ICFTR-Cl. The lack of isoproterenol-activated Cl− current in mouse and rat ventricular cells is consistent with previous reports (18, 24). In these myocytes, agonists to purinergic receptors activate the Cl− current (17, 29, 32).
The cardiac Na+/Ca2+ exchange protein may indeed be phosphorylated by PKA (16, 26, 27), and its activity may be enhanced by PKA in heterogeneous expression systems (19, 26). However, the enhancement might be due to secondary modulation of Na+- or Ca2+-dependent regulation (14, 15) because intracellular Na+ and Ca2+ were not tightly controlled in these experiments. Further study is needed to clarify the differences between the results in heterogeneous expression systems and those in native cells. To the contrary, the current through the amphibian cardiac NCX, which has a potential nucleotide-binding site (a Walker A motif), seems to be attenuated by the activation of the β-adrenergic receptor (7, 13).
Ni2+ appears to act upstream of adenylate cyclase in the β-adrenergic signaling pathway. We (21, 23) and Tareen et al. (28) previously reported that extracellular Na+ is pivotal for the action of isoproterenol on both ICFTR-Cl and L-type Ca2+ current. Replacement of extracellular Na+ with other cations attenuated the stimulatory action of isoproterenol but did not affect ICFTR-Cl activated by forskolin or cAMP (28) as is the case with Ni2+. The underlying mechanisms of Ni2+ action and the removal of extracellular Na+ in β-adrenergic signaling may be similar, although the precise mechanism is not clear at present.
This study was supported by a Grant-in-Aid for Scientific Research and the Leading Project for Biosimulation from the Ministry of Education, Culture, Sports, Science and Technology (to S. Matsuoka).
We thank Drs. Donald W. Hilgemann and Akinori Noma for encouragement and advice, Dr. Takao Shioya for advice about the cell isolation method in mouse hearts, and Dr. Trevor Powell for careful reading of the manuscript and valuable comments.
↵* X. Lin and H. Jo contributed equally to this work.
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