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Regulation by endothelin-1 of Na+-Ca2+ exchange current (INaCa) from guinea-pig isolated ventricular myocytes
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Research
doi: 10.1054/ceca.2001.0244, available online at http://www.idealibrary.com on
Regulation by endothelin-1 of Na;-Ca2; exchange current (INaCa) from guinea-pig isolated ventricular myocytes Y. H. Zhang, A. F. James, J. C. Hancox Department of Physiology and Cardiovascular Research Laboratories, School of Medical Sciences, Bristol, UK
Summary The cardiac Na;-Ca2; exchanger participates in Ca homeostasis, and Na;-Ca2; exchanger-mediated ionic current (INaCa) also contributes to the regulation of cardiac action potential duration. Moreover, INaCa can contribute to arrhythmogenesis under conditions of cellular Ca overload. Although it has been shown that the peptide hormone endothelin-1 (ET-1) can phosphorylate the cardiac Na;-Ca2; exchanger via protein kinase C (PKC), little is known about the effect of ET-1 on INaCa. In order to examine the effects of ET-1 on INaCa, whole-cell patch clamp measurements were made at 37⬚C from guinea-pig isolated ventricular myocytes. With major interfering currents inhibited, INaCa was measured as the current sensitive to nickel (Ni; 10 mM) during a descending voltage ramp. ET-1 (10 nM) significantly increased INaCa (~2-fold at 9100 mV). Application of a PKC activator (PMA; 1 M: phorbol 12-myristate 13-acetate), mimicked the effect of ET-1. In contrast, the PKC inhibitor chelerythrine (CLT, 1 M) abolished the stimulatory effect of ET-1. An inactive phorbol ester, 4-alpha-phorbol-12,13-didecanoate (4␣-PDD, 1 M) had no effect on INaCa. Collectively, these data indicate that ET-1 activated INaCa through a PKC-dependent pathway. In additional experiments, isoprenaline (ISO; which has also been reported to activate INaCa) was applied. The increase in INaCa density with ISO (1 M) was similar to that induced by ET-1 (10 nM). When INaCa was pre-stimulated by ET-1, application of ISO elicited no further increase in current and vice versa. ISO also had no additional effect on INaCa when the cells were pretreated with PMA. Application of CLT did not alter the response of INaCa to ISO. We conclude that ET-1 stimulated ventricular INaCa via a PKC-dependent mechanism under our recording conditions. Concentrations of ET-1 and ISO that stimulated INaCa to similar extents when applied separately were not additive when co-applied. The lack of synergy between the stimulatory effects of ET-1 and ISO may be important in protecting the heart from the potentially deleterious consequences of excessive stimulation of INaCa. © 2001 Harcourt Publishers Ltd.
INTRODUCTION Endothelin-1 (ET-1) is a peptide hormone produced within the heart by endothelial vascular and endocardial cells, and possibly also by cardiac myocytes [1]. ET-1 is thought to exert its action close to its site of production in a paracrine or autocrine manner [2]. ET-1 has a potent Received 12 June 2001 Revised 15 July 2001 Accepted 16 July 2001 Correspondence to: J. C. Hancox, Department of Physiology and Cardiovascular Research Laboratories, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK. Tel.: ;44 0117 928 9028; fax: ;44 0117 929 3194; e-mail: [email protected]
positive inotropic effect and it has been proposed that constitutive production of the hormone within the heart regulates myocardial contractility in a number of species, including human [3–6]. It has recently been suggested that ET-1 may have ventricular arrhythmogenic effects independent of coronary vasoconstriction in both normal and ischaemic diseased heart [7]. In addition, ET-1 can increase contractility in hypertrophied or failing heart preparations [8]. The stimulatory effects of ET-1 on the heart are generally considered to be mediated by activation of Gq-coupled receptors, which ultimately activate protein kinase C (PKC). Activation of Gq stimulates phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2), generating inositol 1,4,5-trisphosphate and 351
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diacylglycerol (DAG); in turn DAG subsequently mobilizes protein kinase C (PKC) [1]. Several mechanisms may contribute to the positive inotropic and arrhythmogenic effects of ET-1. These include: stimulation of Ca release from internal Ca stores [6], enhanced L-type Ca2; current through the activation of PKC [5,9], activation of the Na;-H; exchange, enhanced myofilament sensitivity to Ca2; [10], and also inhibition of the delayed rectifier K current [7,11]. Experiments with an inhibitor of the sarcolemmal Na2;-Ca2; exchanger, KB-R7943, have also raised the possibility that activation by ET-1 of sarcolemmal Na;Ca2; exchange contributes to positive inotropism with the agent [6]. The Na;-Ca2; exchanger (which is found in the cell membrane of almost all mammalian tissues) plays fundamental roles in the heart, including cellular Ca homeostasis and modulation of excitation-contraction (E-C) coupling [12–14]. With a stoichiometry widely held to be 3Na;:1Ca2;, the Na;-Ca2; exchanger is also electrogenic, and therefore generates ionic current (INaCa). INaCa contributes to shaping cardiac action potentials [13,14] and in pathological conditions such as Ca overload, Na;Ca2; exchanger-mediated currents are likely to underlie some arrhythmias involving afterdepolarizations [15–17]. Endogenous substances that up-regulate the activity of the exchanger may therefore have multiple effects on the heart, and the possible regulation of exchanger function by ET-1 is consequently of some potential importance. Iwamoto et al. [18,19] have provided evidence that the cloned cardiac isoform of the Na;-Ca2; exchanger can be phosphorylated by ET-1 via the stimulation of protein kinase C (PKC). In their studies, the extent of exchanger phosphorylation correlated well with 45Ca2; uptake, suggestive of enhanced exchanger activity [18,19]. Consistent with this, in Na-loaded sarcolemmal vesicles from rat heart, ET-1 significantly stimulated 45Ca2; uptake [20]. To-date there has been no study of the actions of ET-1 on INaCa recorded from cardiac myocytes. Direct measurement of INaCa has proved useful in the investigation of exchanger modulation by phenylephrine (PE), which up-regulates INaCa from rat ventricular myocytes through the activation of PKC [21]. Stimulation of mammalian INaCa by protein kinase A (PKA) has also been reported both for INaCa from cardiomyocytes and current carried by cloned exchanger (NCX1) expressed in Xenopus oocytes [22,23]. The objective of the present study was to study the effects of ET-1 on INaCa recorded from guineapig ventricular myocytes at 37⬚C. We report a stimulatory effect of ET-1 on INaCa, an action sensitive to inhibition of PKC. The interaction between ET-1 and isoprenaline (ISO), which activates the exchanger via PKA, was also investigated. Cell Calcium (2001) 30(5), 351–360
METHODS Cell isolation Ventricular myocytes were isolated from the hearts of male guinea-pigs (400–600 g) using enzymatic and mechanical dispersion as described previously [24]. Briefly, guinea-pigs were killed by cervical dislocation (a Home Office approved ‘Schedule 1’ method). The heart was then removed immediately and was cannulated and perfused (Langendorff perfusion system) with low Ca (150 M) Tyrode solution containing collagenase (Worthington, 0.6 mg ml91), protease (Sigma, 0.06 mg ml91) and bovine serum albumin (BSA, 0.2%), after a period of perfusion with a Ca-free solution. Cells were then released from both ventricles by mechanical dispersion. Following cell isolation, ventricular myocytes were kept at 4⬚C in high-K;, low Cl9 storage medium (Kraft-Brühe, KB medium) containing (in mM): 100 L-glutamate, 30 KCl, 5 Na-pyruvate, 20 taurine, 5 creatine, 5 succinic acid, 2 Na2ATP, 5 -OH butyrate, 20 glucose, 5 MgCl2, 1 EGTA, 10 HEPES ( pH adjusted to 7.2 with KOH). For experimental recording, an aliquot of cell suspension was placed on the glass bottom of a Perspex chamber on the stage of an inverted microscope (Nikon Diaphot) and left to settle for several minutes, before being exposed to Ca-containing external solution. Only cells that exhibited a clear rod-shaped and striated appearance were chosen for recording. Solutions Standard external Tyrode’s solution contained (in mM): 140 NaCl, 5 HEPES, 10 glucose, 4 KCl, 2.5 CaCl2, 1 MgCl2 (pH adjusted to 7.45 with NaOH). The extracellular solution for recording INaCa was K;-free Tyrode’s solution containing 10 M strophanthidin, 10 M nitrendipine and 1 mM BaCl2 to eliminate K, Na;-K; pump, Ca and background currents, respectively. External solutions were applied using a temperature-controlled rapid solution application device. The pipette solution contained (in mM): 70 Cs-aspartate, 40 CsCl, 20 NaCl, 4 Mg-ATP, 2.5 KH2PO4, 10 HEPES, 5 glucose, 10 BAPTA, 20 tetraethylammonium (TEA), 1 CaCl2, pH 7.2 (titrated with CsOH). The combination of 10 BAPTA and 1 CaCl2 gave a free pipette Ca concentration of ~20 nM (calculated with the Maxchelator program), although the [Ca2;] in the subsarcolemmal space is likely to have deviated significantly from this low value [25]. The compositions of these solutions are similar to those used in previous studies of INaCa [22,24]. INaCa was measured as the current sensitive to external Ni2; (10 mM). Control experiments indicated that the agents we have used in the present study (ET-1, ISO, PMA, CLT) had no effect on Ni2;-insensitive residual currents. Therefore, we used one barrel of the application © 2001 Harcourt Publishers Ltd
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device for 10 mM Ni2; containing solution and one for the standard external solution for INaCa; the remaining three barrels were used for test agents required for particular experiments. Chemicals Chemicals used for cell isolation were from British Drug House (BDH Aristar-grade) or Sigma. ET-1 was dissolved in 1% acetic acid to make a stock solution of 0.1 mM. Phorbol 12-myristate 13-acetate (PMA, Sigma), chelerythrine (CLT, Sigma), 4-alpha-phorbol-12,13-didecanoate (4␣-PDD, Sigma) were dissolved in DMSO to make stock solutions of 1 mM. H-89 (Calbiochem) was dissolved in 50% ethanol to make a 100 mM stock solution. ISO (Sigma) was dissolved in distilled water to make appropriate stock solutions (1 mM). Stock solutions were stored at 920⬚C until use. These agents were then diluted to the final concentrations used in the experiments, immediately before use, by direct addition to bath solution. The final concentrations of the agents used in the present study were chosen with reference to previous studies [22,24] and [9,26]. We used maximally-effective concentrations to minimise the likelihood of failing to observe the involvement of particular pathways in the observed actions of ET-1 and ISO. Under our conditions, maximal responses of cells to the agents used occurred within 1–2 min following rapid application. Electrophysiological recording and data acquisition Recordings were made using the whole-cell mode of the patch clamp technique. Experiments were performed using an Axopatch 200A amplifier (Axon instruments, USA). Patch-pipettes (Corning 7052 glass, AM Systems) were pulled using a Brown Flaming P81 puller and firepolished to a final resistance of 1–2 M⍀ (Narishige MF 83 microforge, Japan). Protocols were generated and data recorded on-line with P-Clamp 6.0 software via an analogue-to-digital converter (Digidata 1200B, Axon Instruments, USA). Series resistance and membrane capacitance were compensated before commencement of recordings. Membrane capacitance was read from the dial of the recording amplifier to allow INaCa to be normalized to capacitance and expressed as current density during offline analysis. Capacitance values measured in this way have been found previously to correlate well with values obtained using a capacitative ‘surge’ measurement [27]. Data were expressed as mean
RESULTS ET-1 increases INaCa through a pathway involving PKC Similar to previous experiments from this laboratory [24], the present experiments were performed using a descending ramp protocol from ;80 mV to 9120 mV (dVdt91 : 0.4 V s91), applied from a holding potential of 980 mV (Fig. 1A, upper panel). Prior to the descending phase of the voltage ramp, the membrane potential was held at ;80 mV for 30 ms in order to avoid contamination of current during the ramp with any residual uncompensated capacitative current that might occur on depolarisation. Consecutive pulses were separated by a 14 second interpulse interval. The lower panel of Figure 1A shows representative current traces, illustrating the effect of ET-1 on INaCa. ET-1 (10 nM) increased both inward and outward current components following application. Figure 1B shows the mean current–voltage (I–V) relations for steady-state INaCa in control solution and in the presence of ET-1 (n:14; data expressed as current density). ET-1 (10 nM) produced a significant increase in Ni2;-sensitive INaCa across the range of membrane potentials tested and the reversal potential (Erev) for the current became more positive. With ET-1, INaCa at9100 mV was 2.50<0.2 fold that in control solution. However, the increase of INaCa at ;60 mV relative to control was less marked (1.35<0.08 fold that in control; P:0.01 between ;60 and 9100 mV; Fig. 1C). Thus, the action of ET-1 was most pronounced for inward INaCa. Voltage-dependent modulation of the cardiac Na;-Ca2; exchanger is also evident in published data regarding the -adrenergic signalling pathway [22–24,28]. ET-1 did not alter significantly the voltage-dependence or magnitude of Ni2;-insensitive current (Fig. 1D). Inward INaCa reflects the Naentry/Caexit (‘forward’ mode of the exchange); relevant to both Ca extrusion and generation of ionic current, such as the exchanger mediated transient inward current (ITI)[15,29]. Therefore, in line with other recent work from our laboratory [24], data obtained with subsequent experimental interventions were quantified and compared at a negative membrane potential (9100 mV), at which INaCa would be inwardly directed. In previous experiments on a cell line expressing NCX1, ET-1 increased 45Ca2; uptake through activation of PKC [18]. In addition, with experiments using a sarcolemmal vesicle preparation from rat heart, Ballard and Schaffer suggested that Gq protein agonists stimulated the Na;Ca2; exchanger through the activation of PKC [20]. However, they did not demonstrate blockade of the ET-1 effect by PKC inhibition in their study. To investigate whether or not the stimulatory effect on INaCa by ET-1 under our conditions was attributable to the activation of PKC, we used a phorbol ester, PMA and a specific inhibitor of PKC, chelerythrine (CLT). In order to ensure that any observed inhibitory action of CLT would be Cell Calcium (2001) 30(5), 351–360
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Fig. 1 ET-1 increases INaCa from guinea-pig ventricular myocytes. (A) Upper panel: ramp-pulse protocol used to elicit INaCa. Lower panel: representative current traces showing the effect of ET-1 (10 nM). (B) Plot of mean data showing the current–voltage (I–V) relation for 10 mM Ni2;-sensitive current (INaCa) from 14 cells. Asterisks denote potentials at which control and ET-1 plots were significantly different (P:0.01). (C) Histogram comparing the ratio between control INaCa and that in the presence of ET-1 at potentials of ;60 mV and 9100 mV (P:0.01). (D) I–V relation for the effect of ET-1 (10 nM) on Ni2;-insensitive current from 7 cells.
attributable to its known effects on PKC, rather than possible variation between preparations of cells in their response to ET-1, CLT was tested on batches of cells after confirming that these exhibited a robust response to ET-1. Without changing the amplitude of unstimulated INaCa, 1 M CLT almost completely abolished the stimulatory effect of 10 nM ET-1. Figure 2A shows experimental traces with control and CLT in the presence of ET-1. Figure 2B shows mean I–V plots for data from 7 cells. When treated with CLT together with ET-1, the mean INaCa was 1.20< 0.13 fold that in control at 9100 mV (P90.1, n:7). There was no significant difference between control and ET-1;CLT at any test potential (P90.1). In a similar fashion to ET-1, application of PMA (1 M) resulted in a significant increase in both inward and outward INaCa. Figure 3A shows the experimental traces (upper) and the normalized I–V relation (lower) for the effect of PMA (1 M) on INaCa. The mean data from 15 cells with PMA showed a 2.22<0.39 fold INaCa at 9100 mV (P:0.05) compared to control. These data are consistent with the stimulatory effect of ET-1 on INaCa involving the activation of PKC. However, a recent study reported that, Cell Calcium (2001) 30(5), 351–360
in a similar manner to the active form of the phorbol ester, the extracellular application of inactive phorbol ester, 4␣-phorbol-12,13-didecanoate (4␣-PDD) was able to elicit an increase in ICa,L recorded with perforated patch clamp in rat ventricular myocytes, suggesting that the effect of phorbol ester was independent of mobilization of PKC [9]. Therefore, in order to test further the role of PKC activation in mediating the ET-1 response, we investigated the effect of 4␣-PDD on INaCa amplitude. The upper panel of Figure 3B show representative current traces, whilst the lower panel shows I–V relations for control INaCa and current in the presence of 4␣-PDD (1 M). 4␣-PDD did not affect INaCa in our recording conditions. The mean value relative to control at 9100 mV was 1.41< 0.22 fold (P90.1, n:9). Collectively, the data in Figures 2 and 3 suggest that ET-1 stimulated INaCa in guinea-pig myocytes through the activation of PKC. Effects of ET-1 and ISO on INaCa when co-applied Recent studies provide evidence that the -adrenergic agonist ISO can increase the activity of the cardiac © 2001 Harcourt Publishers Ltd
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Fig. 2 A specific PKC inhibitor, chelerythrine (CLT) inhibits the action of ET-1 induced INaCa. (A) Raw current records, showing that in the presence of CLT (1 M) ET-1 could no longer increased INaCa. (B) Plots of mean I–V relations for INaCa from 7 cells show that CLT blocked the stimulatory effect of ET-1 on INaCa. There were no significant differences at any potential between control INaCa and that in the presence of ET-1;CLT (P:0.1).
Fig. 3 Effect of phorbol esters on INaCa. (A) Application of PMA (1 M) stimulated INaCa. Upper panel shows representative current traces in control and in the presence of PMA. Lower panel: mean I–V relations for control and PMA from 15 cells. Asterisks denote potentials at which there was a significant difference (P90.05) between control and PMA. (B) Effects of an inactive form of phorbol ester, 4␣-PDD. Upper panel shows representative current traces with control and 4␣-PDD. 4␣-PDD (1M) did not increase the amplitude of INaCa. Lower panel: mean I–V plots from 9 cells. There was no significant difference between control and 4␣-PDD (P90.1 at all potentials).
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Fig. 4 Effect on INaCa of co-application of ET-1 and ISO. (A) Histogram presenting the current densities at ;60 mV (shaded) and at 9100 mV (blank) when ISO (1 M, left) and ET-1 (10 nM, right) were applied separately. Similar extents of stimulation were induced by these two agents. Significance level at ;60 mV was 0.059P90.02 and at 9100 mV was P 90.1. (B) Upper panel: representative current traces showing currents in control, following ET-1 and after subsequent exposure to ET-1;ISO. Current traces in the presence of ET-1 and in the presence of ET-1; ISO were closely superimposed. Lower panel: mean I–V plots from 7 cells. Both interventions (ET-1 alone and ET-1;ISO) were significantly different from control (* P:0.01; # P:0.01), whereas there were no significant differences between ET-1 and ET-1;ISO (P 90.1).
Na;-Ca2; exchanger isoform through the activation of a pathway involving PKA [22–24]. We were therefore interested to determine whether or not the stimulatory effects of ET-1 and ISO might be additive. Figure 4A compares the effects of ISO (1 M) and ET-1 (10 nM) on INaCa at two test potentials (;60 mV and 9100 mV). As in previous reports [22,24], ISO increased both inward and outward INaCa (Fig. 4A). There was no significant difference between the effects of 1 M ISO and 10 nM ET-1 at 9100 mV (P 90.1), and only a small difference in the effects of the two agents at ;60 mV (0.02:P:0.05). These data suggest that 1 M ISO and 10 nM ET-1 stimulate the exchanger to similar extents, when applied separately. Figure 4B shows the results of experiments in which ISO was applied to cells that had been first exposed to ET-1. The upper panel of Figure 4B shows representative current traces showing the action of ET-1 (10 nM) and the effect of subsequent ISO addition. The traces for ET-1 and ET-1 together with ISO are largely superimposed. The lower panel of Figure 4B shows the normalized I–V relations for ET-1 and ET-1 together with ISO (n:9). There were no significant differences between these two plots (P90.1). These data indicate that when cells were Cell Calcium (2001) 30(5), 351–360
first treated with ET-1, the subsequent application of ISO did not induce a further increase in INaCa. Similar results could also be obtained when the cells were first treated with ISO and then exposed to ISO together with ET-1 (data not shown). These results indicate that concentrations of ET-1 and ISO that produced similar increases of INaCa when applied separately, did not produce an additive response when the agents were co-applied. The relation between modulation by ISO and ET-1 of the Na;-Ca2; exchanger was further tested using the PKC activator PMA. Specifically, we investigated whether or not ISO might further increase INaCa following pretreatment with PMA. As shown in Figure 5A, application of PMA (1 M) significantly increased INaCa. However, when PMA (1 M) was applied together with ISO (1 M), ISO did not induce a further increase in INaCa. The mean data at 9100 mV for PMA only and ISO in the presence of PMA were: 2.22<0.39 (n:15) fold control INaCa and 2.15<0.06 (n:10) fold control INaCa, respectively (P 90.1 between these two interventions). Figure 5B shows the normalized I–V data for these experiments. The plots of PMA alone and PMA with ISO show no statistically significant difference from one another (P9 0.1). These results © 2001 Harcourt Publishers Ltd
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Fig. 5 Effect on INaCa of application of PMA followed by PMA;ISO. (A) Representative current traces showing the application of PMA (1 M) and PMA;ISO (1 M) on INaCa. (B) Mean I–V data plots from 15 cells showing that after the increase of INaCa with PMA, both interventions (PMA alone and PMA;ISO) were significantly different from control (*P:0.01; # P:0.01); however addition of ISO did not further increase INaCa beyond the level in the presence of PMA (P90.1 between PMA and PMA;ISO).
Fig. 6 Effect of a specific PKC inhibitor, chelerythrine (CLT) on ISO stimulated INaCa. (A) Current traces showing INaCa in control, ISO (1 M) and ISO; CLT (1 M). (B) Mean I–V data plots from 7 cells showing that in the presence of CLT, ISO still induced a stimulatory effect on INaCa. (*) P:0.01 between ISO and control; (#) P:0.01 between ISO;CLT and control. No significant differences between ISO and ISO;CLT (P90.1).
suggest that ISO was unable to increase INaCa further, when this had been previously stimulated by a PKC activator and are consistent with the observed interaction between ET-1 and ISO. We did not investigate effects of PMA on ISO-pretreated cells in this study; however, recently published data from our laboratory suggest that PMA does not alter INaCa in ISO pretreated cells [24]. Although modulation of INaCa by ISO is likely attributable to a PKA-dependent pathway [22,23], ISO can activate © 2001 Harcourt Publishers Ltd
PKC-dependent responses indirectly, at least under some conditions [30]. In order to be able to preclude involvement of such an effect in the lack of synergy between ISO and ET-1 or PMA, the response of INaCa to the application of ISO in the presence of the PKC inhibitor CLT was tested. The results from these experiments are shown in Figure 6. Figure 6A shows representative experimental traces. INaCa from cells incubated in CLT (1 M) could be stimulated by ISO (1 M). Mean I–V relations for INaCa Cell Calcium (2001) 30(5), 351–360
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with ISO and ISO in the presence of CLT were also largely superimposed (Figure 6B, n:7); there was no statistically significant difference between these two plots (P90.1). In contrast, when the cells were incubated with the PKAinhibitor, H-89, they no longer responded to ISO (1 M; n:9, data not shown; see also [22]). These data indicate clearly that ET-1 and ISO acted by different pathways (mediated by PKC and PKA respectively) to stimulate INaCa.
DISCUSSION Modulation by ET-1 of INaCa Although there have been reports suggestive of phosphorylation and functional modulation of Na;-Ca2; exchanger protein by ET-1 in sarcolemmal vesicle preparations and CCL39 cells expressing NCX1 [18,20], until now there has been no study of the response to ET-1 of INaCa. Our data indicate that ET-1 can stimulate guinea-pig ventricular INaCa under whole-cell patch clamp conditions under which residual (Ni2;-insensitive) current was not altered by ET-1. The fact that the effect of ET-1 was mimicked by an active form of phorbol ester (PMA, which can directly activate PKC) and not by the inactive form, 4␣-PDD, suggests that under our experimental conditions, ET-1 exerted its modulatory effect on INaCa by PKC-dependent mechanism. In further support of this conclusion, the specific PKC inhibitor, chelerythrine abolished the effect of ET-1. These results are consistent with the generally accepted second messenger cascades in cardiac myocytes involving Gq-linked ET-1 receptors, stimulation of the hydrolysis of PLC and subsequent production of DAG which mobilizes PKC [1]. Our recording conditions were chosen to minimize contamination of INaCa by other currents: other Ca2; entry pathways were inhibited, and bulk (although not subsarcolemmal) [Ca2;] is expected to have been well-controlled [25]. The observed shift in Erev for Nisensitive INaCa with ET-1 and PKC activation is likely to have resulted from effects on the exchanger itself, since the magnitude of outward INaCa (and hence Ca2;-entry into the subsarcolemmal space) early during a descending voltage ramp can influence the recorded Erev of the current [25]. Taken collectively, and in the context of the existing literature, the most straightforward interpretation of our data is that the increased INaCa that we observed was directly linked to PKC mobilization following ET-1 application. Other neurotransmitters/hormones have been reported to modulate the Na;-Ca2; exchanger by binding to their receptors and activating a pathway involving PKC. For example, angiotensin-II stimulates the Na;-Ca2; exchanger by a PKC-dependent pathway [20]. The ␣1adrenergic agonist phenylephrine (PE) can also increase Na;-Ca2; exchanger activity in both sarcolemmal vesicles of rat preparations and intact cardiac myocytes studied Cell Calcium (2001) 30(5), 351–360
under whole-cell patch clamp mode [20,21]. Both these investigations of PE concluded that PKC mediates the transmitter’s effect, because selective PKC blockers abolished the response. However, in contrast with the present study, PMA failed to stimulate rat ventricular INaCa in the experiments of Stengl et al. [21]. The reasons for the apparent discrepancy in response to PMA and PKC-inhibition in that study are not entirely clear. It should be noted, however, that both recording conditions and animal species differ between the report by Stengl et al. [21] and our study, in which PMA activated INaCa. Given the facts (a) that Gq-protein-coupled agonists are important in producing a hypertrophic response and (b) that both the expression and activity of the Na;-Ca2; exchanger are significantly increased in hypertrophy [31,14], it is possible that agonism by ET-1 of Na;-Ca2; exchanger activity is relevant to the understanding of the mechanisms of hypertrophy and heart failure. Maximal responses of INaCa were obtained with 10 nM ET-1. This concentration is comparable to those used in other studies of ET-1 [11,32] and is relevant to the levels of ET-1 thought to occur within the myocardium (~40–65 pg/100 mg wet weight tissue [33]). It is therefore possible that modulation of the Na;-Ca2; exchanger as demonstrated in this study may contribute to the physiological effects of the peptide hormone. ET-1 exerts a positive inotropic effect in both normal and hypertrophied or failing heart preparations [1,6,8]. ET-1-induced increases in Ca transients and cell shortening in rabbit ventricular myocytes are sensitive to the Na;-Ca2; exchanger blocker, KB-R7943 [6]. Very recent data also link ET-1 and Ca2; entry via the reverse mode of the Na;-Ca2; exchange to the mechanism underlying the slow force response to stretch of papillary muscle [34]. Our data show activation by ET-1 of outward INaCa (i.e. reverse mode Na;-Ca2; exchange, in which Ca influx is induced), which is consistent with the observations in these published studies. The overall contribution of enhanced INaCa to the inotropic state of the heart may be complex, however, because ET-1 increases both modes of exchanger activity and will likely therefore influence both Ca entry and Ca efflux. The substantial stimulatory effect of ET-1 on inward INaCa that we observed may be of some functional significance. Under pathological conditions, inward INaCa contributes significantly to the arrhythmogenic transient inward current, ITI [15,29] and may therefore underlie some arrhythmias involving afterdepolarizations [15–17]. Some studies have shown beneficial effects on arrhythmias of ET-1 inhibition [35,36]. ET-1 blockers may therefore have potential as antiarrhythmic drugs [7]. One factor that may contribute to this is that, by inhibiting the stimulation of INaCa by ET-1, such agents would be expected to decrease the generation of ITI and associated pro-arrhythmia. © 2001 Harcourt Publishers Ltd
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Interaction between ET-1 and ISO In the present study, the stimulatory effects of ET-1 and ISO on INaCa were not additive. Several mechanisms could account for this observation. It is known that ET-1 can exert inhibitory as well as stimulatory effects; inhibitory effects appear to involve activation of the inhibitory G protein (Gi) [e.g. 26, 32, 37]. One feasible explanation for the lack of synergism between ET-1 and ISO might therefore be ET-1 receptor-linked activation of Gi, resulting in subsequent inhibition of adenylate cyclase. In this study, we did not investigate the involvement of Gi in the interaction between ET-1 and ISO (for example by incubating myocytes in pertussis toxin; PTX). However, under our conditions, application of PMA mimicked the action of ET-1 and PMA also showed a lack of synergism with ISO. Moreover, neither PMA nor ET-1 reduced the response of INaCa to ISO. The lack of synergism cannot be attributed to a Gi-mediated inhibitory effect, as PMA does not require Gi activation in order to exert its effect. Our experiments with CLT also indicate that the response of INaCa to ISO is not sensitive to the inhibition of PKC, and our results on the combined application of ISO and ET-1 therefore likely reflect the possibility that both inward and outward INaCa can only be augmented to a limited maximal extent. This may be likely, given that the concentrations of both ET-1 and ISO used in this study were chosen to be maximally effective, although the interaction between combinations of lower concentrations of ET-1 and ISO was not tested in the present study. Nevertheless, it is feasible that the lack of summation of ISO and ET-1 responses could result if pre-treatment with either ET-1 or ISO inhibits the response to subsequent application of the other agent via a mechanism that does not rely on Gi activation. A plausible explanation for the lack of summative response might be that PKA and PKC phosphorylate the same or closely juxtaposed sites. On the basis of immunoprecipitation data, Iwamoto et al. [19] identified three phosphorylation sites for PKC: Ser-249, Ser-250 and Ser-357 on the cardiac NCX1 protein. Although PKAdependent phosphorylation of NCX expressed in BHK cells and Xenopus oocytes has subsequently been observed [23,38], it is not yet known whether or not the phosphorylation site(s) is (are) the same as those acted on by PKC. Discriminating between the effects of PKC and PKA at the molecular level may be complex however, since mutations of all three serines to alanines (S249A, S250A, S357A) has been reported not to abolish the stim; ulatory effect of PMA on Na i -dependent 45Ca2; uptake [19]. One possibility is that, rather than directly phosphorylating the exchanger, protein kinases may activate a cytoplasmic regulatory protein, which in turn acts to stimulate exchanger activity [19,39]. It is not known whether or not such a regulatory protein is present in © 2001 Harcourt Publishers Ltd
cardiac myocytes, although a 13kDa regulatory protein has been reported for squid nerve fibres [39]. It has been suggested previously that the inhibition of the effects of ISO on ICa,L by ET-1 may be possible through the activation of Gq, rather than Gi since, in myocytes overexpressing the active subunit of Gq protein (G␣q) the response to ISO of ICa,L was significantly smaller than control and PTX failed to reverse the response of these myocytes to ISO [40], although the inhibitory response to ET-1 was sensitive to pertussis toxin in untransfected myocytes [26]. Thus Gq activation itself might impair the effects of activation of the -adrenoceptor-PKA cascade. If this were to explain our observations, the data with PMA together with ISO suggests that stages of the PKC cascade prior to PKC activation are not obligatory for such an action. On the basis of our data, it is not possible to further discriminate between, or speculate upon, the possible mechanisms underlying the lack of synergy between ET-1 and ISO. It remains important to establish the precise means by which activation of PKC and PKA upregulate exchanger activity – whether this be a direct result of exchanger phosphorylation or whether an accessory cytoplasmic protein is involved. The importance of the Na;-Ca2; exchanger to both calcium homeostasis and ionic current generation in the heart would seem to warrant further investigation of the actions of ET-1. Furthermore, detailed exploration of the mechanisms of interaction between PKC and PKA in modulating the cardiac Na;-Ca2; exchanger might provide new insights in understanding its regulation at the molecular level.
ACKNOWLEDGEMENTS This work was supported by British Heart Foundation project grant PG/98097, and by a Wellcome Trust fellowship to JCH. AFJ also acknowledges support from the British Heart Foundation (PG/98091). The authors thank Lesley Arberry for valuable assistance in ventricular myocyte isolation.
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5. Woo SH, Lee CO. Effects of endothelin-1 on Ca2; signaling in guinea-pig ventricular myocytes: role of protein kinase C. J Mol Cell Cardiol 1999; 31: 631–643. 6. Yang HT, Sakurai K, Sugawara H, Watanabe T, Norota I, Endoh M. Role of Na;/Ca2; exchange in endothelin-1-induced increases in Ca2; transient and contractility in rabbit ventricular myocytes: pharmacological analysis with KB-R7943. Br J Pharmacol 1999; 126: 1785–1795. 7. Duru F, Barton M, Luscher TF, Candinas R. Endothelin and cardiac arrhythmias: do endothelin antagonists have a therapeutic potential as antiarrhythmic drugs? Cardiovasc Res 2001; 49: 272–280. 8. Pieske B, Beyermann B, Breu V, et al. Functional effects of endothelin and regulation of endothelin receptors in isolated human nonfailing and failing myocardium. Circulation 1999; 99: 1802–1809. 9. He JQ , Pi Y, Walker JW, Kamp TJ. Endothelin-1 and photoreleased diacylglycerol increase L-type Ca2; current by activation of protein kinase C in rat ventricular myocytes. J Physiol 2000; 524: 807–820. 10. Kramer BK, Smith TW, Kelly RA. Endothelin and increased contractility in adult rat ventricular myocytes. Role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na;-H; exchanger. Circ Res 1991; 68: 269–279. 11. James AF, Ramsey JE, Reynolds AM, Hendry BM, Shattock MJ. Effects of ET-1 on K; currents from rat ventricular myocytes. Biochem Biophys Res Comm 2001; 284: 1048–1055. 12. Eisner DA, Lederer WJ. Na-Ca exchange: stoichiometry and electrogenicity. Am J Physiol 1985; 248: C189–C202. 13. Janvier NC, Boyett MR. The role of Na-Ca exchange current in the cardiac action potential. Cardiovasc Res 1996; 32: 69–84. 14. Blaustein MP, Lederer WJ. Sodium/calcium exchange: its physiological implications. Physiol Rev 1999; 79: 763–854. 15. Fozzard HA. Afterdepolarizations and triggered activity. Basic Res Cardiol 1992; 87 (Suppl 2): 105–113. 16. Luo CH, Rudy Y. A dynamic model of the cardiac ventricular action potential. II. Afterdepolarizations, triggered activity, and potentiation. Circ Res 1994; 74: 1097–1113. 17. Ming Z, Aronson R, Nordin C. Mechanism of current-induced early afterdepolarizations in guinea pig ventricular myocytes. Am J Physiol 1994; 267: H1419–H1428. 18. Iwamoto T, Wakabayashi S, Shigekawa M. Growth factorinduced phosphorylation and activation of aortic smooth muscle Na;/Ca2; exchanger. J Biol Chem 1995; 270: 8996–9001. 19. Iwamoto T, Pan Y, Nakamura TY, Wakabayashi S, Shigekawa M. Protein kinase C-dependent regulation of Na;/Ca2; exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation. Biochemistry 1998; 37: 17230–17238. 20. Ballard C, Schaffer S. Stimulation of the Na;-Ca2; exchanger by Phenylephrine, Angiotensin II and Endothelin 1. J Mol Cell Cardiol 1996; 28: 11–17. 21. Stengl M, Mubagwa K, Carmeliet E, Flameng W. Phenylephrineinduced stimulation of Na;/Ca2; exchange in rat ventricular myocytes. Cardiovasc Res 1998; 38: 703–710. 22. Perchenet L, Hinde AK, Patel KC, Hancox JC, Levi AJ. Stimulation of Na/Ca exchange by the beta-adrenergic/protein kinase A pathway in guinea-pig ventricular myocytes at 37 degrees C. Pf lugers Arch 2000; 439: 822–828. 23. Ruknudin A, He S, Lederer WJ, Schulze DH. Functional differences between cardiac and renal isoforms of the rat
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Research
doi: 10.1054/ceca.2001.0244, available online at http://www.idealibrary.com on
Regulation by endothelin-1 of Na;-Ca2; exchange current (INaCa) from guinea-pig isolated ventricular myocytes Y. H. Zhang, A. F. James, J. C. Hancox Department of Physiology and Cardiovascular Research Laboratories, School of Medical Sciences, Bristol, UK
Summary The cardiac Na;-Ca2; exchanger participates in Ca homeostasis, and Na;-Ca2; exchanger-mediated ionic current (INaCa) also contributes to the regulation of cardiac action potential duration. Moreover, INaCa can contribute to arrhythmogenesis under conditions of cellular Ca overload. Although it has been shown that the peptide hormone endothelin-1 (ET-1) can phosphorylate the cardiac Na;-Ca2; exchanger via protein kinase C (PKC), little is known about the effect of ET-1 on INaCa. In order to examine the effects of ET-1 on INaCa, whole-cell patch clamp measurements were made at 37⬚C from guinea-pig isolated ventricular myocytes. With major interfering currents inhibited, INaCa was measured as the current sensitive to nickel (Ni; 10 mM) during a descending voltage ramp. ET-1 (10 nM) significantly increased INaCa (~2-fold at 9100 mV). Application of a PKC activator (PMA; 1 M: phorbol 12-myristate 13-acetate), mimicked the effect of ET-1. In contrast, the PKC inhibitor chelerythrine (CLT, 1 M) abolished the stimulatory effect of ET-1. An inactive phorbol ester, 4-alpha-phorbol-12,13-didecanoate (4␣-PDD, 1 M) had no effect on INaCa. Collectively, these data indicate that ET-1 activated INaCa through a PKC-dependent pathway. In additional experiments, isoprenaline (ISO; which has also been reported to activate INaCa) was applied. The increase in INaCa density with ISO (1 M) was similar to that induced by ET-1 (10 nM). When INaCa was pre-stimulated by ET-1, application of ISO elicited no further increase in current and vice versa. ISO also had no additional effect on INaCa when the cells were pretreated with PMA. Application of CLT did not alter the response of INaCa to ISO. We conclude that ET-1 stimulated ventricular INaCa via a PKC-dependent mechanism under our recording conditions. Concentrations of ET-1 and ISO that stimulated INaCa to similar extents when applied separately were not additive when co-applied. The lack of synergy between the stimulatory effects of ET-1 and ISO may be important in protecting the heart from the potentially deleterious consequences of excessive stimulation of INaCa. © 2001 Harcourt Publishers Ltd.
INTRODUCTION Endothelin-1 (ET-1) is a peptide hormone produced within the heart by endothelial vascular and endocardial cells, and possibly also by cardiac myocytes [1]. ET-1 is thought to exert its action close to its site of production in a paracrine or autocrine manner [2]. ET-1 has a potent Received 12 June 2001 Revised 15 July 2001 Accepted 16 July 2001 Correspondence to: J. C. Hancox, Department of Physiology and Cardiovascular Research Laboratories, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK. Tel.: ;44 0117 928 9028; fax: ;44 0117 929 3194; e-mail: [email protected]
positive inotropic effect and it has been proposed that constitutive production of the hormone within the heart regulates myocardial contractility in a number of species, including human [3–6]. It has recently been suggested that ET-1 may have ventricular arrhythmogenic effects independent of coronary vasoconstriction in both normal and ischaemic diseased heart [7]. In addition, ET-1 can increase contractility in hypertrophied or failing heart preparations [8]. The stimulatory effects of ET-1 on the heart are generally considered to be mediated by activation of Gq-coupled receptors, which ultimately activate protein kinase C (PKC). Activation of Gq stimulates phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2), generating inositol 1,4,5-trisphosphate and 351
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diacylglycerol (DAG); in turn DAG subsequently mobilizes protein kinase C (PKC) [1]. Several mechanisms may contribute to the positive inotropic and arrhythmogenic effects of ET-1. These include: stimulation of Ca release from internal Ca stores [6], enhanced L-type Ca2; current through the activation of PKC [5,9], activation of the Na;-H; exchange, enhanced myofilament sensitivity to Ca2; [10], and also inhibition of the delayed rectifier K current [7,11]. Experiments with an inhibitor of the sarcolemmal Na2;-Ca2; exchanger, KB-R7943, have also raised the possibility that activation by ET-1 of sarcolemmal Na;Ca2; exchange contributes to positive inotropism with the agent [6]. The Na;-Ca2; exchanger (which is found in the cell membrane of almost all mammalian tissues) plays fundamental roles in the heart, including cellular Ca homeostasis and modulation of excitation-contraction (E-C) coupling [12–14]. With a stoichiometry widely held to be 3Na;:1Ca2;, the Na;-Ca2; exchanger is also electrogenic, and therefore generates ionic current (INaCa). INaCa contributes to shaping cardiac action potentials [13,14] and in pathological conditions such as Ca overload, Na;Ca2; exchanger-mediated currents are likely to underlie some arrhythmias involving afterdepolarizations [15–17]. Endogenous substances that up-regulate the activity of the exchanger may therefore have multiple effects on the heart, and the possible regulation of exchanger function by ET-1 is consequently of some potential importance. Iwamoto et al. [18,19] have provided evidence that the cloned cardiac isoform of the Na;-Ca2; exchanger can be phosphorylated by ET-1 via the stimulation of protein kinase C (PKC). In their studies, the extent of exchanger phosphorylation correlated well with 45Ca2; uptake, suggestive of enhanced exchanger activity [18,19]. Consistent with this, in Na-loaded sarcolemmal vesicles from rat heart, ET-1 significantly stimulated 45Ca2; uptake [20]. To-date there has been no study of the actions of ET-1 on INaCa recorded from cardiac myocytes. Direct measurement of INaCa has proved useful in the investigation of exchanger modulation by phenylephrine (PE), which up-regulates INaCa from rat ventricular myocytes through the activation of PKC [21]. Stimulation of mammalian INaCa by protein kinase A (PKA) has also been reported both for INaCa from cardiomyocytes and current carried by cloned exchanger (NCX1) expressed in Xenopus oocytes [22,23]. The objective of the present study was to study the effects of ET-1 on INaCa recorded from guineapig ventricular myocytes at 37⬚C. We report a stimulatory effect of ET-1 on INaCa, an action sensitive to inhibition of PKC. The interaction between ET-1 and isoprenaline (ISO), which activates the exchanger via PKA, was also investigated. Cell Calcium (2001) 30(5), 351–360
METHODS Cell isolation Ventricular myocytes were isolated from the hearts of male guinea-pigs (400–600 g) using enzymatic and mechanical dispersion as described previously [24]. Briefly, guinea-pigs were killed by cervical dislocation (a Home Office approved ‘Schedule 1’ method). The heart was then removed immediately and was cannulated and perfused (Langendorff perfusion system) with low Ca (150 M) Tyrode solution containing collagenase (Worthington, 0.6 mg ml91), protease (Sigma, 0.06 mg ml91) and bovine serum albumin (BSA, 0.2%), after a period of perfusion with a Ca-free solution. Cells were then released from both ventricles by mechanical dispersion. Following cell isolation, ventricular myocytes were kept at 4⬚C in high-K;, low Cl9 storage medium (Kraft-Brühe, KB medium) containing (in mM): 100 L-glutamate, 30 KCl, 5 Na-pyruvate, 20 taurine, 5 creatine, 5 succinic acid, 2 Na2ATP, 5 -OH butyrate, 20 glucose, 5 MgCl2, 1 EGTA, 10 HEPES ( pH adjusted to 7.2 with KOH). For experimental recording, an aliquot of cell suspension was placed on the glass bottom of a Perspex chamber on the stage of an inverted microscope (Nikon Diaphot) and left to settle for several minutes, before being exposed to Ca-containing external solution. Only cells that exhibited a clear rod-shaped and striated appearance were chosen for recording. Solutions Standard external Tyrode’s solution contained (in mM): 140 NaCl, 5 HEPES, 10 glucose, 4 KCl, 2.5 CaCl2, 1 MgCl2 (pH adjusted to 7.45 with NaOH). The extracellular solution for recording INaCa was K;-free Tyrode’s solution containing 10 M strophanthidin, 10 M nitrendipine and 1 mM BaCl2 to eliminate K, Na;-K; pump, Ca and background currents, respectively. External solutions were applied using a temperature-controlled rapid solution application device. The pipette solution contained (in mM): 70 Cs-aspartate, 40 CsCl, 20 NaCl, 4 Mg-ATP, 2.5 KH2PO4, 10 HEPES, 5 glucose, 10 BAPTA, 20 tetraethylammonium (TEA), 1 CaCl2, pH 7.2 (titrated with CsOH). The combination of 10 BAPTA and 1 CaCl2 gave a free pipette Ca concentration of ~20 nM (calculated with the Maxchelator program), although the [Ca2;] in the subsarcolemmal space is likely to have deviated significantly from this low value [25]. The compositions of these solutions are similar to those used in previous studies of INaCa [22,24]. INaCa was measured as the current sensitive to external Ni2; (10 mM). Control experiments indicated that the agents we have used in the present study (ET-1, ISO, PMA, CLT) had no effect on Ni2;-insensitive residual currents. Therefore, we used one barrel of the application © 2001 Harcourt Publishers Ltd
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device for 10 mM Ni2; containing solution and one for the standard external solution for INaCa; the remaining three barrels were used for test agents required for particular experiments. Chemicals Chemicals used for cell isolation were from British Drug House (BDH Aristar-grade) or Sigma. ET-1 was dissolved in 1% acetic acid to make a stock solution of 0.1 mM. Phorbol 12-myristate 13-acetate (PMA, Sigma), chelerythrine (CLT, Sigma), 4-alpha-phorbol-12,13-didecanoate (4␣-PDD, Sigma) were dissolved in DMSO to make stock solutions of 1 mM. H-89 (Calbiochem) was dissolved in 50% ethanol to make a 100 mM stock solution. ISO (Sigma) was dissolved in distilled water to make appropriate stock solutions (1 mM). Stock solutions were stored at 920⬚C until use. These agents were then diluted to the final concentrations used in the experiments, immediately before use, by direct addition to bath solution. The final concentrations of the agents used in the present study were chosen with reference to previous studies [22,24] and [9,26]. We used maximally-effective concentrations to minimise the likelihood of failing to observe the involvement of particular pathways in the observed actions of ET-1 and ISO. Under our conditions, maximal responses of cells to the agents used occurred within 1–2 min following rapid application. Electrophysiological recording and data acquisition Recordings were made using the whole-cell mode of the patch clamp technique. Experiments were performed using an Axopatch 200A amplifier (Axon instruments, USA). Patch-pipettes (Corning 7052 glass, AM Systems) were pulled using a Brown Flaming P81 puller and firepolished to a final resistance of 1–2 M⍀ (Narishige MF 83 microforge, Japan). Protocols were generated and data recorded on-line with P-Clamp 6.0 software via an analogue-to-digital converter (Digidata 1200B, Axon Instruments, USA). Series resistance and membrane capacitance were compensated before commencement of recordings. Membrane capacitance was read from the dial of the recording amplifier to allow INaCa to be normalized to capacitance and expressed as current density during offline analysis. Capacitance values measured in this way have been found previously to correlate well with values obtained using a capacitative ‘surge’ measurement [27]. Data were expressed as mean
RESULTS ET-1 increases INaCa through a pathway involving PKC Similar to previous experiments from this laboratory [24], the present experiments were performed using a descending ramp protocol from ;80 mV to 9120 mV (dVdt91 : 0.4 V s91), applied from a holding potential of 980 mV (Fig. 1A, upper panel). Prior to the descending phase of the voltage ramp, the membrane potential was held at ;80 mV for 30 ms in order to avoid contamination of current during the ramp with any residual uncompensated capacitative current that might occur on depolarisation. Consecutive pulses were separated by a 14 second interpulse interval. The lower panel of Figure 1A shows representative current traces, illustrating the effect of ET-1 on INaCa. ET-1 (10 nM) increased both inward and outward current components following application. Figure 1B shows the mean current–voltage (I–V) relations for steady-state INaCa in control solution and in the presence of ET-1 (n:14; data expressed as current density). ET-1 (10 nM) produced a significant increase in Ni2;-sensitive INaCa across the range of membrane potentials tested and the reversal potential (Erev) for the current became more positive. With ET-1, INaCa at9100 mV was 2.50<0.2 fold that in control solution. However, the increase of INaCa at ;60 mV relative to control was less marked (1.35<0.08 fold that in control; P:0.01 between ;60 and 9100 mV; Fig. 1C). Thus, the action of ET-1 was most pronounced for inward INaCa. Voltage-dependent modulation of the cardiac Na;-Ca2; exchanger is also evident in published data regarding the -adrenergic signalling pathway [22–24,28]. ET-1 did not alter significantly the voltage-dependence or magnitude of Ni2;-insensitive current (Fig. 1D). Inward INaCa reflects the Naentry/Caexit (‘forward’ mode of the exchange); relevant to both Ca extrusion and generation of ionic current, such as the exchanger mediated transient inward current (ITI)[15,29]. Therefore, in line with other recent work from our laboratory [24], data obtained with subsequent experimental interventions were quantified and compared at a negative membrane potential (9100 mV), at which INaCa would be inwardly directed. In previous experiments on a cell line expressing NCX1, ET-1 increased 45Ca2; uptake through activation of PKC [18]. In addition, with experiments using a sarcolemmal vesicle preparation from rat heart, Ballard and Schaffer suggested that Gq protein agonists stimulated the Na;Ca2; exchanger through the activation of PKC [20]. However, they did not demonstrate blockade of the ET-1 effect by PKC inhibition in their study. To investigate whether or not the stimulatory effect on INaCa by ET-1 under our conditions was attributable to the activation of PKC, we used a phorbol ester, PMA and a specific inhibitor of PKC, chelerythrine (CLT). In order to ensure that any observed inhibitory action of CLT would be Cell Calcium (2001) 30(5), 351–360
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Fig. 1 ET-1 increases INaCa from guinea-pig ventricular myocytes. (A) Upper panel: ramp-pulse protocol used to elicit INaCa. Lower panel: representative current traces showing the effect of ET-1 (10 nM). (B) Plot of mean data showing the current–voltage (I–V) relation for 10 mM Ni2;-sensitive current (INaCa) from 14 cells. Asterisks denote potentials at which control and ET-1 plots were significantly different (P:0.01). (C) Histogram comparing the ratio between control INaCa and that in the presence of ET-1 at potentials of ;60 mV and 9100 mV (P:0.01). (D) I–V relation for the effect of ET-1 (10 nM) on Ni2;-insensitive current from 7 cells.
attributable to its known effects on PKC, rather than possible variation between preparations of cells in their response to ET-1, CLT was tested on batches of cells after confirming that these exhibited a robust response to ET-1. Without changing the amplitude of unstimulated INaCa, 1 M CLT almost completely abolished the stimulatory effect of 10 nM ET-1. Figure 2A shows experimental traces with control and CLT in the presence of ET-1. Figure 2B shows mean I–V plots for data from 7 cells. When treated with CLT together with ET-1, the mean INaCa was 1.20< 0.13 fold that in control at 9100 mV (P90.1, n:7). There was no significant difference between control and ET-1;CLT at any test potential (P90.1). In a similar fashion to ET-1, application of PMA (1 M) resulted in a significant increase in both inward and outward INaCa. Figure 3A shows the experimental traces (upper) and the normalized I–V relation (lower) for the effect of PMA (1 M) on INaCa. The mean data from 15 cells with PMA showed a 2.22<0.39 fold INaCa at 9100 mV (P:0.05) compared to control. These data are consistent with the stimulatory effect of ET-1 on INaCa involving the activation of PKC. However, a recent study reported that, Cell Calcium (2001) 30(5), 351–360
in a similar manner to the active form of the phorbol ester, the extracellular application of inactive phorbol ester, 4␣-phorbol-12,13-didecanoate (4␣-PDD) was able to elicit an increase in ICa,L recorded with perforated patch clamp in rat ventricular myocytes, suggesting that the effect of phorbol ester was independent of mobilization of PKC [9]. Therefore, in order to test further the role of PKC activation in mediating the ET-1 response, we investigated the effect of 4␣-PDD on INaCa amplitude. The upper panel of Figure 3B show representative current traces, whilst the lower panel shows I–V relations for control INaCa and current in the presence of 4␣-PDD (1 M). 4␣-PDD did not affect INaCa in our recording conditions. The mean value relative to control at 9100 mV was 1.41< 0.22 fold (P90.1, n:9). Collectively, the data in Figures 2 and 3 suggest that ET-1 stimulated INaCa in guinea-pig myocytes through the activation of PKC. Effects of ET-1 and ISO on INaCa when co-applied Recent studies provide evidence that the -adrenergic agonist ISO can increase the activity of the cardiac © 2001 Harcourt Publishers Ltd
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Fig. 2 A specific PKC inhibitor, chelerythrine (CLT) inhibits the action of ET-1 induced INaCa. (A) Raw current records, showing that in the presence of CLT (1 M) ET-1 could no longer increased INaCa. (B) Plots of mean I–V relations for INaCa from 7 cells show that CLT blocked the stimulatory effect of ET-1 on INaCa. There were no significant differences at any potential between control INaCa and that in the presence of ET-1;CLT (P:0.1).
Fig. 3 Effect of phorbol esters on INaCa. (A) Application of PMA (1 M) stimulated INaCa. Upper panel shows representative current traces in control and in the presence of PMA. Lower panel: mean I–V relations for control and PMA from 15 cells. Asterisks denote potentials at which there was a significant difference (P90.05) between control and PMA. (B) Effects of an inactive form of phorbol ester, 4␣-PDD. Upper panel shows representative current traces with control and 4␣-PDD. 4␣-PDD (1M) did not increase the amplitude of INaCa. Lower panel: mean I–V plots from 9 cells. There was no significant difference between control and 4␣-PDD (P90.1 at all potentials).
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Fig. 4 Effect on INaCa of co-application of ET-1 and ISO. (A) Histogram presenting the current densities at ;60 mV (shaded) and at 9100 mV (blank) when ISO (1 M, left) and ET-1 (10 nM, right) were applied separately. Similar extents of stimulation were induced by these two agents. Significance level at ;60 mV was 0.059P90.02 and at 9100 mV was P 90.1. (B) Upper panel: representative current traces showing currents in control, following ET-1 and after subsequent exposure to ET-1;ISO. Current traces in the presence of ET-1 and in the presence of ET-1; ISO were closely superimposed. Lower panel: mean I–V plots from 7 cells. Both interventions (ET-1 alone and ET-1;ISO) were significantly different from control (* P:0.01; # P:0.01), whereas there were no significant differences between ET-1 and ET-1;ISO (P 90.1).
Na;-Ca2; exchanger isoform through the activation of a pathway involving PKA [22–24]. We were therefore interested to determine whether or not the stimulatory effects of ET-1 and ISO might be additive. Figure 4A compares the effects of ISO (1 M) and ET-1 (10 nM) on INaCa at two test potentials (;60 mV and 9100 mV). As in previous reports [22,24], ISO increased both inward and outward INaCa (Fig. 4A). There was no significant difference between the effects of 1 M ISO and 10 nM ET-1 at 9100 mV (P 90.1), and only a small difference in the effects of the two agents at ;60 mV (0.02:P:0.05). These data suggest that 1 M ISO and 10 nM ET-1 stimulate the exchanger to similar extents, when applied separately. Figure 4B shows the results of experiments in which ISO was applied to cells that had been first exposed to ET-1. The upper panel of Figure 4B shows representative current traces showing the action of ET-1 (10 nM) and the effect of subsequent ISO addition. The traces for ET-1 and ET-1 together with ISO are largely superimposed. The lower panel of Figure 4B shows the normalized I–V relations for ET-1 and ET-1 together with ISO (n:9). There were no significant differences between these two plots (P90.1). These data indicate that when cells were Cell Calcium (2001) 30(5), 351–360
first treated with ET-1, the subsequent application of ISO did not induce a further increase in INaCa. Similar results could also be obtained when the cells were first treated with ISO and then exposed to ISO together with ET-1 (data not shown). These results indicate that concentrations of ET-1 and ISO that produced similar increases of INaCa when applied separately, did not produce an additive response when the agents were co-applied. The relation between modulation by ISO and ET-1 of the Na;-Ca2; exchanger was further tested using the PKC activator PMA. Specifically, we investigated whether or not ISO might further increase INaCa following pretreatment with PMA. As shown in Figure 5A, application of PMA (1 M) significantly increased INaCa. However, when PMA (1 M) was applied together with ISO (1 M), ISO did not induce a further increase in INaCa. The mean data at 9100 mV for PMA only and ISO in the presence of PMA were: 2.22<0.39 (n:15) fold control INaCa and 2.15<0.06 (n:10) fold control INaCa, respectively (P 90.1 between these two interventions). Figure 5B shows the normalized I–V data for these experiments. The plots of PMA alone and PMA with ISO show no statistically significant difference from one another (P9 0.1). These results © 2001 Harcourt Publishers Ltd
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Fig. 5 Effect on INaCa of application of PMA followed by PMA;ISO. (A) Representative current traces showing the application of PMA (1 M) and PMA;ISO (1 M) on INaCa. (B) Mean I–V data plots from 15 cells showing that after the increase of INaCa with PMA, both interventions (PMA alone and PMA;ISO) were significantly different from control (*P:0.01; # P:0.01); however addition of ISO did not further increase INaCa beyond the level in the presence of PMA (P90.1 between PMA and PMA;ISO).
Fig. 6 Effect of a specific PKC inhibitor, chelerythrine (CLT) on ISO stimulated INaCa. (A) Current traces showing INaCa in control, ISO (1 M) and ISO; CLT (1 M). (B) Mean I–V data plots from 7 cells showing that in the presence of CLT, ISO still induced a stimulatory effect on INaCa. (*) P:0.01 between ISO and control; (#) P:0.01 between ISO;CLT and control. No significant differences between ISO and ISO;CLT (P90.1).
suggest that ISO was unable to increase INaCa further, when this had been previously stimulated by a PKC activator and are consistent with the observed interaction between ET-1 and ISO. We did not investigate effects of PMA on ISO-pretreated cells in this study; however, recently published data from our laboratory suggest that PMA does not alter INaCa in ISO pretreated cells [24]. Although modulation of INaCa by ISO is likely attributable to a PKA-dependent pathway [22,23], ISO can activate © 2001 Harcourt Publishers Ltd
PKC-dependent responses indirectly, at least under some conditions [30]. In order to be able to preclude involvement of such an effect in the lack of synergy between ISO and ET-1 or PMA, the response of INaCa to the application of ISO in the presence of the PKC inhibitor CLT was tested. The results from these experiments are shown in Figure 6. Figure 6A shows representative experimental traces. INaCa from cells incubated in CLT (1 M) could be stimulated by ISO (1 M). Mean I–V relations for INaCa Cell Calcium (2001) 30(5), 351–360
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with ISO and ISO in the presence of CLT were also largely superimposed (Figure 6B, n:7); there was no statistically significant difference between these two plots (P90.1). In contrast, when the cells were incubated with the PKAinhibitor, H-89, they no longer responded to ISO (1 M; n:9, data not shown; see also [22]). These data indicate clearly that ET-1 and ISO acted by different pathways (mediated by PKC and PKA respectively) to stimulate INaCa.
DISCUSSION Modulation by ET-1 of INaCa Although there have been reports suggestive of phosphorylation and functional modulation of Na;-Ca2; exchanger protein by ET-1 in sarcolemmal vesicle preparations and CCL39 cells expressing NCX1 [18,20], until now there has been no study of the response to ET-1 of INaCa. Our data indicate that ET-1 can stimulate guinea-pig ventricular INaCa under whole-cell patch clamp conditions under which residual (Ni2;-insensitive) current was not altered by ET-1. The fact that the effect of ET-1 was mimicked by an active form of phorbol ester (PMA, which can directly activate PKC) and not by the inactive form, 4␣-PDD, suggests that under our experimental conditions, ET-1 exerted its modulatory effect on INaCa by PKC-dependent mechanism. In further support of this conclusion, the specific PKC inhibitor, chelerythrine abolished the effect of ET-1. These results are consistent with the generally accepted second messenger cascades in cardiac myocytes involving Gq-linked ET-1 receptors, stimulation of the hydrolysis of PLC and subsequent production of DAG which mobilizes PKC [1]. Our recording conditions were chosen to minimize contamination of INaCa by other currents: other Ca2; entry pathways were inhibited, and bulk (although not subsarcolemmal) [Ca2;] is expected to have been well-controlled [25]. The observed shift in Erev for Nisensitive INaCa with ET-1 and PKC activation is likely to have resulted from effects on the exchanger itself, since the magnitude of outward INaCa (and hence Ca2;-entry into the subsarcolemmal space) early during a descending voltage ramp can influence the recorded Erev of the current [25]. Taken collectively, and in the context of the existing literature, the most straightforward interpretation of our data is that the increased INaCa that we observed was directly linked to PKC mobilization following ET-1 application. Other neurotransmitters/hormones have been reported to modulate the Na;-Ca2; exchanger by binding to their receptors and activating a pathway involving PKC. For example, angiotensin-II stimulates the Na;-Ca2; exchanger by a PKC-dependent pathway [20]. The ␣1adrenergic agonist phenylephrine (PE) can also increase Na;-Ca2; exchanger activity in both sarcolemmal vesicles of rat preparations and intact cardiac myocytes studied Cell Calcium (2001) 30(5), 351–360
under whole-cell patch clamp mode [20,21]. Both these investigations of PE concluded that PKC mediates the transmitter’s effect, because selective PKC blockers abolished the response. However, in contrast with the present study, PMA failed to stimulate rat ventricular INaCa in the experiments of Stengl et al. [21]. The reasons for the apparent discrepancy in response to PMA and PKC-inhibition in that study are not entirely clear. It should be noted, however, that both recording conditions and animal species differ between the report by Stengl et al. [21] and our study, in which PMA activated INaCa. Given the facts (a) that Gq-protein-coupled agonists are important in producing a hypertrophic response and (b) that both the expression and activity of the Na;-Ca2; exchanger are significantly increased in hypertrophy [31,14], it is possible that agonism by ET-1 of Na;-Ca2; exchanger activity is relevant to the understanding of the mechanisms of hypertrophy and heart failure. Maximal responses of INaCa were obtained with 10 nM ET-1. This concentration is comparable to those used in other studies of ET-1 [11,32] and is relevant to the levels of ET-1 thought to occur within the myocardium (~40–65 pg/100 mg wet weight tissue [33]). It is therefore possible that modulation of the Na;-Ca2; exchanger as demonstrated in this study may contribute to the physiological effects of the peptide hormone. ET-1 exerts a positive inotropic effect in both normal and hypertrophied or failing heart preparations [1,6,8]. ET-1-induced increases in Ca transients and cell shortening in rabbit ventricular myocytes are sensitive to the Na;-Ca2; exchanger blocker, KB-R7943 [6]. Very recent data also link ET-1 and Ca2; entry via the reverse mode of the Na;-Ca2; exchange to the mechanism underlying the slow force response to stretch of papillary muscle [34]. Our data show activation by ET-1 of outward INaCa (i.e. reverse mode Na;-Ca2; exchange, in which Ca influx is induced), which is consistent with the observations in these published studies. The overall contribution of enhanced INaCa to the inotropic state of the heart may be complex, however, because ET-1 increases both modes of exchanger activity and will likely therefore influence both Ca entry and Ca efflux. The substantial stimulatory effect of ET-1 on inward INaCa that we observed may be of some functional significance. Under pathological conditions, inward INaCa contributes significantly to the arrhythmogenic transient inward current, ITI [15,29] and may therefore underlie some arrhythmias involving afterdepolarizations [15–17]. Some studies have shown beneficial effects on arrhythmias of ET-1 inhibition [35,36]. ET-1 blockers may therefore have potential as antiarrhythmic drugs [7]. One factor that may contribute to this is that, by inhibiting the stimulation of INaCa by ET-1, such agents would be expected to decrease the generation of ITI and associated pro-arrhythmia. © 2001 Harcourt Publishers Ltd
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Interaction between ET-1 and ISO In the present study, the stimulatory effects of ET-1 and ISO on INaCa were not additive. Several mechanisms could account for this observation. It is known that ET-1 can exert inhibitory as well as stimulatory effects; inhibitory effects appear to involve activation of the inhibitory G protein (Gi) [e.g. 26, 32, 37]. One feasible explanation for the lack of synergism between ET-1 and ISO might therefore be ET-1 receptor-linked activation of Gi, resulting in subsequent inhibition of adenylate cyclase. In this study, we did not investigate the involvement of Gi in the interaction between ET-1 and ISO (for example by incubating myocytes in pertussis toxin; PTX). However, under our conditions, application of PMA mimicked the action of ET-1 and PMA also showed a lack of synergism with ISO. Moreover, neither PMA nor ET-1 reduced the response of INaCa to ISO. The lack of synergism cannot be attributed to a Gi-mediated inhibitory effect, as PMA does not require Gi activation in order to exert its effect. Our experiments with CLT also indicate that the response of INaCa to ISO is not sensitive to the inhibition of PKC, and our results on the combined application of ISO and ET-1 therefore likely reflect the possibility that both inward and outward INaCa can only be augmented to a limited maximal extent. This may be likely, given that the concentrations of both ET-1 and ISO used in this study were chosen to be maximally effective, although the interaction between combinations of lower concentrations of ET-1 and ISO was not tested in the present study. Nevertheless, it is feasible that the lack of summation of ISO and ET-1 responses could result if pre-treatment with either ET-1 or ISO inhibits the response to subsequent application of the other agent via a mechanism that does not rely on Gi activation. A plausible explanation for the lack of summative response might be that PKA and PKC phosphorylate the same or closely juxtaposed sites. On the basis of immunoprecipitation data, Iwamoto et al. [19] identified three phosphorylation sites for PKC: Ser-249, Ser-250 and Ser-357 on the cardiac NCX1 protein. Although PKAdependent phosphorylation of NCX expressed in BHK cells and Xenopus oocytes has subsequently been observed [23,38], it is not yet known whether or not the phosphorylation site(s) is (are) the same as those acted on by PKC. Discriminating between the effects of PKC and PKA at the molecular level may be complex however, since mutations of all three serines to alanines (S249A, S250A, S357A) has been reported not to abolish the stim; ulatory effect of PMA on Na i -dependent 45Ca2; uptake [19]. One possibility is that, rather than directly phosphorylating the exchanger, protein kinases may activate a cytoplasmic regulatory protein, which in turn acts to stimulate exchanger activity [19,39]. It is not known whether or not such a regulatory protein is present in © 2001 Harcourt Publishers Ltd
cardiac myocytes, although a 13kDa regulatory protein has been reported for squid nerve fibres [39]. It has been suggested previously that the inhibition of the effects of ISO on ICa,L by ET-1 may be possible through the activation of Gq, rather than Gi since, in myocytes overexpressing the active subunit of Gq protein (G␣q) the response to ISO of ICa,L was significantly smaller than control and PTX failed to reverse the response of these myocytes to ISO [40], although the inhibitory response to ET-1 was sensitive to pertussis toxin in untransfected myocytes [26]. Thus Gq activation itself might impair the effects of activation of the -adrenoceptor-PKA cascade. If this were to explain our observations, the data with PMA together with ISO suggests that stages of the PKC cascade prior to PKC activation are not obligatory for such an action. On the basis of our data, it is not possible to further discriminate between, or speculate upon, the possible mechanisms underlying the lack of synergy between ET-1 and ISO. It remains important to establish the precise means by which activation of PKC and PKA upregulate exchanger activity – whether this be a direct result of exchanger phosphorylation or whether an accessory cytoplasmic protein is involved. The importance of the Na;-Ca2; exchanger to both calcium homeostasis and ionic current generation in the heart would seem to warrant further investigation of the actions of ET-1. Furthermore, detailed exploration of the mechanisms of interaction between PKC and PKA in modulating the cardiac Na;-Ca2; exchanger might provide new insights in understanding its regulation at the molecular level.
ACKNOWLEDGEMENTS This work was supported by British Heart Foundation project grant PG/98097, and by a Wellcome Trust fellowship to JCH. AFJ also acknowledges support from the British Heart Foundation (PG/98091). The authors thank Lesley Arberry for valuable assistance in ventricular myocyte isolation.
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