Bimodal regulation of Na⁺–Ca²⁺ exchanger by β-adrenergic signaling pathway in shark ventricular myocytes

Results

Characterization of Na⁺–Ca²⁺ Exchange Current in Shark Ventricular Myocytes.

Fig. 1 A–C illustrates the procedure used to isolate the inward and outward components of current carried by the Na⁺–Ca²⁺ exchanger in a single voltage-clamped shark ventricular myocyte in which K⁺ and Ca²⁺ current were blocked (see legend). As the duration of clamp pulse was prolonged, the outward current activated at +60 mV slowly decayed, and the tail currents accompanying the repolarization of membrane to −80 mV became enhanced (Fig. 1 A). Exposure of myocytes to 5 mM Ni²⁺ blocked both the slowly decaying outward current and the accompanying inward tail current (Fig. 1 B). Subtraction of current envelopes obtained in the presence and absence of Ni²⁺ revealed a slowly decaying outward current on depolarization and expanding inward tail current on repolarization (Fig. 1 C), consistent with previous measurements of I _Na-Ca (13, 16, 17). The pulse protocol of Fig. 1 D employs a procedure that activates I _Na-Ca at +60 mV, then measures its voltage dependence with a repolarizing ramp pulse (17). The application of 5 mM Ni²⁺ blocked the outward current induced by depolarization to +60 mV by 56.7% ± 2.9% (n = 12; Table 1). The density of the outward exchanger current at 50 ms into the depolarizing pulse to +60 mV was 5.63 ± 0.8 pA/pF (n = 14), after subtraction of the Ni²⁺-resistant current. The voltage dependence of I _Na-Ca was measured by subtraction of currents induced by the ramp protocol in the presence and absence of Ni²⁺. It should be noted that, because the kinetics of the exchanger current may not be at their steady-state values during the ramp pulse, an accurate estimation of the voltage dependence of I _Na-Ca may be compromised. In addition, because the inward exchanger currents during the envelope pulse protocol decay by about 60% over 30 ms, it is likely that the 100-ms ramp protocol used might somewhat underestimate the inward exchanger current. Despite these constraints, the reversal potential could be reliably measured with this procedure. The Ni²⁺-blockable ramp current showed a reversal potential (E _rev) of −29.1 ± 5.7 mV (n = 9), providing for a calculated Ca²⁺ equilibrium potential (E _Ca) at +60 mV, using the equation where E _Na is 38.6 mV (extracellular Na⁺ concentration, [Na⁺]_o, = 270 mM and [Na⁺]_i = 60 mM). The calculated E _Ca for E _rev of −29.1 mV was about 72.5 mV. Under these conditions the effective [Ca²⁺]_i calculated from the Nernst equation was 7.1 μM at 2 mM [Ca²⁺]_o at the end of a 50 ms depolarizing pulse. Significant myocyte shortening was noted at these [Ca²⁺]_i values. There was considerable variability (−20 to −50 mV) in the reversal potential of I _Na-Ca after a 50-ms depolarizing pulse to +60 mV, resulting most likely from the variability in free cytosolic Ca²⁺ concentrations in different cells.

Modulation of Exchanger Activity by β-Adrenergic Signaling Pathway.

Application of 5 μM isoproterenol suppressed the magnitude of Ni²⁺-sensitive outward I _Na-Ca) induced by a 50-ms depolarizing pulse from −80 to +60 mV by 24.0% ± 7.2% (n = 3) (Fig. 2 A and B; see Table 1 for details). In the absence or presence of isoproterenol, 5 mM Ni²⁺ rapidly and reversibly suppressed the outward current. The slow kinetics of the isoproterenol-induced effect on I _Na-Ca is consistent with the activation of a second messenger-signaling pathway. To examine whether this effect was mediated by the binding of the hormone to the β-adrenoreceptor and the activation of the adenylate-cyclase dependent signaling pathway, adenylate cyclase was directly activated by forskolin. Forskolin mimicked the isoproterenol-suppressive effect on I _Na-Ca (Fig. 2 C and D), such that the outward I _Na-Ca generated at +60 mV was suppressed by 44.4% ± 0.95% (n = 2) (see Table 1 for details). Although the absolute outward current after Ni²⁺ exposure was smaller in the presence of isoproterenol or forskolin than in control myocytes (Fig. 2 A–D), the fraction of current inhibited by Ni²⁺ in control or isoproterenol-treated myocytes was similar (≈55%, Table 1). Thus, the Ni²⁺ effect does not appear to be modified by the phosphorylation state of the exchanger protein.

Fig. 3 shows that both isoproterenol and forskolin significantly suppressed the inward and outward I _Na-Ca at most potentials tested, and shifted its reversal potential toward more negative potentials (Fig. 3 A and B). The changes in E _rev (ΔE _rev, Table 1) in the presence of isoproterenol or forskolin were −22.9 ± 1.8 mV (n = 3) and −29.6 ± 3.7 (n = 2), respectively. These findings suggest that stimulation of the β-adrenergic/adenylate cyclase pathway results in substantial reduction (≈2.5-fold with isoproterenol and ≈3.1-fold with forskolin) in [Ca²⁺]_i in shark ventricular myocytes. The apparent enhancement of I _Na-Ca in the small range of potentials spanning its reversal potential (Fig. 3 A and B) occurs, in part, from the reduction of [Ca²⁺]_i, as the Ca²⁺ influx on the exchanger is suppressed at more positive potentials. Consistent with this idea, in myocytes where [Ca²⁺]_i was buffered no apparent enhancement of I _Na-Ca was observed (17).

Bimodal Regulation of Na⁺–Ca²⁺ Exchanger by Isoproterenol.

To quantitate the efficacy of the β-adrenergic regulatory effect on the Ca²⁺ influx or efflux mode of the exchanger, we used the “envelope” pulse protocol, by which both the inward and outward component of Ca²⁺ transport on the exchanger could be independently assessed. Fig. 4 illustrates the effect of isoproterenol on the Ni²⁺-sensitive envelope of the exchanger-activated currents. In the presence of isoproterenol both the inward and outward current components were reduced (Fig. 4 B). Clearly, the decreased Ca²⁺ influx via the Na⁺–Ca²⁺ exchanger into the myocyte would by itself reduce the Ca²⁺ efflux on the exchanger during the repolarization period. Nevertheless, the efficacy of Ca²⁺ efflux on the exchanger in the presence of isoproterenol appeared to be enhanced when corrected for the reduced Ca²⁺ influx. This was reflected in the larger ratio of the inward to outward (I _inward/I _outward) I _Na-Ca in the isoproterenol-treated myocytes (Fig. 4 C). We also measured the magnitude of Ni²⁺-sensitive outward current of each episode at 25 ms after the onset of depolarization. The amplitude of the peak Ni²⁺-sensitive inward current was measured at 3 ms after the start of repolarization because the fast capacitive transients may contaminate the ionic current in the early part of the tail currents. In quantifying the Ca²⁺ influx and efflux modes of the exchanger, we found that as the duration of the depolarizing pulse increased gradually the ratio of I _inward/I _outward in control and isoproterenol-treated myocytes increased, albeit with different slopes (Fig. 4 C). It is likely that the higher [Ca²⁺]_i during the longer depolarizing pulses facilitates the Ca²⁺ efflux mode of the exchanger, through activation of its Ca²⁺ regulatory site (18, 19). In isoproterenol-treated myocytes, in fact, the slope [(I _inward/I _outward)/s] of the relation (reflecting the efficacy of Ca²⁺ efflux via the exchanger, Fig. 4 C) was larger (11.0 ± 1.2, n = 3) than that (7.22 ± 1.0, n = 7) found in control myocytes. Quantification of the total Ni²⁺-sensitive charge (Q) transported by the exchanger in the inward and outward direction similarly showed that isoproterenol increased the fraction of inward to outward charge (Q _inward/Q _outward) by ≈2-fold, Fig. 4 D. This finding therefore suggests more efficient Ca²⁺ extrusion by the exchanger in the presence of isoproterenol.

Fig. 5 illustrates the effect of isoproterenol on the time course of rise in [Ca²⁺]_i, constructed from digital integration of I _Na-Ca traces of Fig. 4 A and B, using a 3 Na⁺ to 1 Ca²⁺ exchanger stoichiometry. As the duration of depolarizing pulse became longer both the Ca²⁺ influx and efflux via the exchanger appeared to increase (Fig. 5 A). In the presence of isoproterenol the peak [Ca²⁺]_i was much smaller than that found in control solution (Fig. 5 A and B), consistent with suppression of Ca²⁺ influx via the exchanger. However, the decay of intracellular Ca²⁺ transients during repolarization appeared to be faster in isoproterenol-treated myocytes (see normalized traces, Fig. 5 C), consistent with idea that isoproterenol also enhances the efficacy of Ca²⁺ extrusion via the exchanger.

Comparing the effect of isoproterenol and KB-R7943 on the envelope of the exchanger currents and their ratios showed that 10 μM KB-R7943 was as effective as Ni²⁺ in suppressing both the inward and outward I _Na-Ca (Fig. 6), such that the ratio of I _inward to I _outward in KB-R7943-treated myocytes was the same as that of control cells (data not shown). These findings suggest that the larger ratio of inward to outward exchanger currents observed in isoproterenol-treated myocytes results from a unique regulatory mechanism of the Ca²⁺ efflux mode of the exchanger.

Discussion

Our results describe a mechanism by which β-adrenergic agonists enhance the efficacy of Ca²⁺ efflux even when Ca²⁺ influx through the Na⁺–Ca²⁺ exchanger is reduced. The bimodal regulation of the exchanger in shark ventricular myocytes provides a possible mechanism for the intriguing findings that in intact ventricular strips, β-adrenergic agonists, while potentiating the phasic component of contraction, suppress the maintained component of tension (11). It has been previously suggested (13, 17) that activation of Ca²⁺ channel produces the initial rapid influx of Ca²⁺, whereas the later phase of contraction is supported by the slower influx of Ca²⁺ via the Na⁺–Ca²⁺ exchanger. In this scheme the simultaneous adrenergic-induced enhancement of I _Ca and the suppression of Ca²⁺ influx on the exchanger may provide for the observed enhancement of the rate of rise of twitch early in the action potential (20) and for the dissociation of contraction from the duration of depolarization later on in the action potential (4, 11). In addition, the adrenergic enhancement of the efficacy of Ca²⁺ efflux via the Na⁺–Ca²⁺ exchanger, described here, may contribute significantly to the decay of intracellular Ca²⁺ and acceleration of relaxation. Such differential and temporal modulation of Ca²⁺ influx and efflux may have both developmental and evolutionary significance for hormonal regulation of cellular Ca²⁺ homeostasis. This mechanism may have particularly evolved in hearts, where sarcoplasmic reticulum (SR) is poorly developed and physiological Ca²⁺ release pools are absent (13). In this respect, in the amphibian and neonatal mammalian hearts in which SR is also poorly developed, the β-adrenergic agonists appeared to similarly enhance the twitch and suppress KCl-induced contractures (21, 22), consistent with the enhancement of I _Ca and suppression of Ca²⁺ influx on the exchanger (17, 23). In frog ventricular myocytes, the ratio of the inward to outward exchanger current did not increase, nor did the slope of the relationship between the ratio and the duration of the depolarization change (17). In guinea-pig ventricular myocytes incubated at 37°C, β-adrenergic agonists were shown to stimulate both the Ca²⁺ influx and efflux mode of the exchanger via the protein kinase A (PKA)-mediated signaling pathway (24). We have been unable to reproduce these data in rat myocytes at room temperature. Nevertheless, the enhancement of exchanger current in this report was not accompanied by a shift in E _rev, as Ca²⁺ was highly buffered by the dialyzing solution (24).

In the present result we show that β-adrenergic signaling pathway induces a shift of E _rev toward more negative potential (Fig. 2 A and B). We have proposed that isoproterenol- and forskolin-induced decrease in [Ca²⁺]_i may be responsible for the negative shift in the E _rev, because in highly Ca²⁺-buffered cells (9 mM [EGTA]_i and 6.17 mM CaCl₂; free [Ca²⁺]_i = 200 nM) the negative shift in E _rev was prevented (17), even though isoproterenol continued to suppress the outward Na⁺–Ca²⁺ exchanger current (17). Because E _rev may also change in response to the alteration of exchanger stoichiometry (25) we considered whether a change in stoichiometry could be responsible for such a shift. If the stoichiometry for Na⁺ to Ca²⁺ increases from 3:1 to 4:1 because of the reduction of [Ca²⁺]_i, the reversal potential would shift toward positive potentials, as predicted theoretically and reported experimentally (25). Therefore, the negative shift in the E _rev in the presence of isoproterenol is unlikely to be related to the change in the exchanger stoichiometry.

We also compared the regulation of exchanger by KB-R7943 with that of isoproterenol. KB-R7943 has been reported to block the outward Na⁺–Ca²⁺ exchanger current more potently than the inward current under unidirectional ionic conditions, possibly by binding to the Ca²⁺-binding conformation of the exchanger (26). Under the bidirectional ionic conditions, KB-R7943 has been reported to inhibit the inward and outward exchanger currents with equal potency (27). Fig. 6 shows that 10 μM KB-R7943 blocked both the inward and outward exchanger current as effectively as did Ni²⁺. However, when the suppressions of outward exchanger current by isoproterenol and KB-R7943 were equivalent, the shift in the E _rev was smaller with KB-R7943 compared with isoproterenol, suggesting less reduction in [Ca²⁺]_i with this drug. In addition, the ratio of the inward to outward exchanger current, and the slope of the relation (reflecting the efficacy of Ca²⁺ efflux via the exchanger, Fig. 4 C) was larger in isoproterenol than in control or KB-R7943-treated cells. The data support the idea that suppression of influx mode of the exchanger is not a prerequisite for the enhancement of Ca²⁺ efflux mode of the exchanger, suggesting that isoproterenol may independently enhance the efficacy of the Ca²⁺ efflux mode of the exchanger.

The mechanism for the enhancement of the efficacy of Ca²⁺ efflux by isoproterenol, even at lower [Ca²⁺]_i, is not as yet clear. One possibility to consider is that PKA-induced phosphorylation may increase the affinity of the exchanger for its ionic substrates. An ATP-dependent enhancement of Na⁺–Ca²⁺ exchanger by an increase in the affinity of the exchanger for its substrates and the regulatory site has been already reported in the squid giant axon and cardiac myocytes (28–30). The effect of ATP in the squid giant axon requires internal Ca²⁺, hydrolyzable ATP, and the phosphorylation process, suggesting involvement of a Ca²⁺-dependent protein kinase (31, 32). The outward exchanger current in CHO cells expressing Na⁺–Ca²⁺ exchanger was also stimulated by hyperphosphorylation process, but the mechanism does not appeared to be mediated by protein kinases (33). Although phosphorylation has been implicated previously in adult mammalian exchanger (34) and a possible PKA-dependent phosphorylation site has been identified on the cloned protein (35), experiments on giant excised patches from cardiac myocytes suggest that the ATP effect is mediated by phosphatidylinositol 4,5-bisphosphate (PIP₂) generated from phosphatidylinositol (PI) (36). In frog heart, the existence of a nine-amino acid exon, containing a putative nucleotide-binding domain (23) not present in the mammalian heart, appears to be responsible for the cAMP-dependent suppression of the exchanger (17, 23). Whether a similar structural component is present in the shark heart isoform of the exchanger remains to be determined. The functional data, presented here, suggest that the shark exchanger may also contain additional regulatory sites that serve as molecular determinants responsible for the unusual bimodal regulation of the exchanger.

We have also considered other possible mechanisms that, if activated by β-adrenergic pathway, could produce an apparent enhancement of Ca²⁺ efflux. In mammalian hearts, the PKA-dependent phosphorylation of phospholamban and the subsequent stimulation of the Ca²⁺ pump (5, 6), in addition to decreased myofilaments Ca²⁺ sensitivity (7–9), are thought to mediate the relaxant properties of β-adrenergic pathway. Supposing that the acceleration of Ca²⁺-ATPase in the sarcoplasmic reticulum or plasma membrane reduces [Ca²⁺]_i during relaxation, the level of Ca²⁺ efflux via the Na⁺–Ca²⁺ exchanger might be underestimated because of the decreased [Ca²⁺]_i by such mechanisms. On the other hand, a decrease in myofilament sensitivity to Ca²⁺ by the PKA pathway might increase [Ca²⁺]_i, thereby accelerating Ca²⁺ efflux via the exchanger.

The possible bimodal regulation of the exchanger by PKA-mediated phosphorylation in parallel with enhancement of Ca²⁺ current may allow for the temporal shift in delivery of contractile Ca²⁺ from a slow (exchanger-mediated) to a faster (Ca²⁺ channel-mediated) pathway. β-Adrenergic-induced suppression of the Ca²⁺ influx and enhancement of the extrusion mode of the exchanger, in shark heart lacking significant intracellular Ca²⁺ release pools, may have evolved to meet the evolutionary requirement for faster contraction and accelerated relaxation in animals subjected to fight, fright, and flight. Whether such a PKA-dependent-regulatory mechanism can be made to operate by genetic manipulation of the mammalian Na⁺–Ca²⁺ exchanger when the exchanger is overexpressed (37, 38) in heart failure remains to be tested.