© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Interaction of angiotensin II with the angiotensin type 2 receptor inhibits the cardiac transient outward potassium current
Department of Pharmacology, School of Medicine, Universidad Complutense, 28040, Madrid, Spain
* Corresponding author. Tel.: +34-91-394-1474; fax: +34-91-3941470. Email address: rcaballero{at}ift.csic.es
Received 3 October 2003; revised 9 December 2003; accepted 29 December 2003
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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Objective: The Ca2+-independent transient outward K+ current (Ito) plays a crucial role in shaping the cardiac action potential. In the present study, we examined whether angiotensin II (AngII) regulated the Ito as well as the putative intracellular cascade responsible for the effects. Methods: Ito was recorded in rat ventricular myocytes using the nystatin-perforated patch-clamp configuration. Results: AngII (0.1 µM) inhibited Ito (21.9±4.8% at +40 mV), but not the IK1, in a voltage- and time-independent manner. The inhibition decreased at concentrations higher than 1 µM resulting in a bell-shaped dose–response curve (IC50=3.1±1.5 µM). The blocking effects were abolished in the presence of the type 2 AngII receptor (AT2R) antagonist PD123319, but not in the presence of the selective type 1 AngII receptor (AT1R) antagonist candesartan. Moreover, the selective AT2R agonist CGP42112A completely reproduced the effects of AngII (20.5±2.4% of block at +40 mV), indicating that AngII-induced Ito block was mediated via stimulation of AT2R. Furthermore, selective stimulation of AT2R by CGP42112A significantly prolonged the rat atrial action potentials recorded using conventional microelectrode techniques. The AngII-induced inhibition of Ito was not modified by either N
-nitro-L-arginine-methyl ester (L-NAME) or eicosatetrayonic acid (ETYA), indicating that neither the nitric oxide (NO)–guanosine 3',5'-cyclic monophosphate (cGMP) system nor the arachidonic acid cascade was implicated in the effects of AngII on Ito. However, the AngII-induced Ito inhibition was completely abolished by the serine/threonine phosphatase type 2A (PP2A) inhibitors, okadaic acid and cantharidin, but not by the inactive analog of okadaic acid, 1-norokadaone. Intracellular application of PP2A decreased Kv4.2 currents recorded in transiently transfected Chinese hamster ovary cells (CHO). Conclusion: These results indicate that AngII activates PP2A through the stimulation of the AT2R, resulting in a decrease of the Ito amplitude.
KEYWORDS Angiotensin; K-channels; Protein phosphatases; Signal transduction
This article is referred to in the Editorial by E. Cerbai and A. Mugelli (pages 7–8) in this issue.
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Angiotensin II (AngII) regulates blood pressure, water-electrolyte balance, hormone secretion, cell growth and neuronal activity by interacting with two major specific receptors (AT1R and AT2R) on target organs [1]. Both receptors are usually present in adult human cardiac myocytes [2].
Several outward K+ currents determine the repolarization and refractoriness of the cardiac tissue. The Ca2+-independent, 4-aminopyridine-sensitive component of the transient outward current (Ito) is a rapidly activating and inactivating K+ current, which is present in several species including rat, dog and human [3]. Ito underlies the early phase of cardiac action potential repolarization in both human atria and ventricle, and influences the balance of inward and outward ionic currents during the plateau phase, modulating the voltage and time dependence of the action potential [3,4]. The density of Ito varies among species and, within particular species, it varies between different regions of the heart [3]. In humans, Ito is more abundantly expressed in the atrial tissue, Purkinje fibers and epicardium than in endocardium [4]. Moreover, in the ventricle, Ito recovers from inactivation faster in epicardial than in endocardial cells [4]. The effects of AngII on the neuronal transient type-A K+ current (IA) have already been described [5]. In neurons cultured from rat hypothalamus and brain stem, AngII produces an AT2R-mediated increase in the IA, an effect attributed empirically to the activation of phospholipase A2 (PLA2) and generation of arachidonic acid as well as to the activation of serine/threonine phosphatase type 2A (PP2A) [5]. However, AngII causes an AT1R-dependent inhibition of IA in the same cells, an effect involving the activation of protein kinase C [5]. Recently, it has been reported that the properties of Ito in canine endocardium and epicardium are under the tonic regulation of the renin–angiotensin system [6]. Incubation of epicardial myocytes with AngII for periods of 2–52 h inhibited the Ito, slowed the recovery from inactivation and shifted the voltage-dependence of inactivation to more positive potentials, so the resulting Ito resembled the Ito recorded in endocardial myocytes. Furthermore, AngII downregulates Kv4.3 mRNA and protein in neonatal rat myocytes [7]. However, the acute effects of AngII on cardiac Ito and the intracellular signaling pathways that couple cardiac AT1R and/or AT2R to changes in Ito density are currently unknown.
Thus, this study was undertaken to analyze the putative acute effects of AngII on the Ito recorded in isolated rat ventricular myocytes and to elucidate the intracellular signaling pathway involved in its effects. The results obtained demonstrate that AngII inhibits the Ito, an effect mediated by the stimulation of AT2R involving the activation of the PP2A signaling pathway.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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2.1. Transmembrane action potentials
Action potentials were recorded in left atria from male Wistar rats (
250 g) perfused with Tyrode solution (34 °C) and driven at 3 Hz using microelectrode techniques [8].
2.2. Myocyte isolation
Single ventricular myocytes were obtained by enzymatic dissociation using collagenase (type II, Worthington) as described elsewhere [9]. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (HIH Publication No. 85-23, Revised 1996).
The transient expression of Kv4.2 channels on CHO cells has been previously described [8]. Briefly, the cells were grown in Hams-F12 with 10% fetal bovine serum and transfected with the cDNA encoding the Kv4.2 (3 µg) channels together with the cDNA encoding the CD8 antigen (0.5 µg) by use of lipofectamine (Gibco). Before experimental use, cells were incubated with polystyrene microbeads precoated with anti-CD8 antibody (Dynabeads M450; Dynal, Norway).
2.3. Solutions and drugs
Tyrode's solution contained (mM): NaCl 137, KCl 5.4, MgCl2 1.0, CaCl2 1.8, NaH2PO4 0.42, NaHCO3 11 and dextrose 10. Myocytes were perfused with an external solution containing (mM): NaCl 90, TEA 50, KCl 4, HEPES 10, MgCl2 1, CoCl2 2, CaCl2 1 and dextrose 11 (pH 7.4 with NaOH). The CHO cells were perfused with an external solution containing (mM): NaCl 130, KCl 4, CaCl2 1, MgCl2 1, HEPES 10 and glucose 10 (pH=7.4 with NaOH). The internal solution contained (mM): K-aspartate 80, KCl 42, KH2PO4 10, MgATP 5, phosphocreatine 3, HEPES 5, EGTA 5 and nystatin 0.02 (pH 7.2 with KOH). Patch-clamp experiments were performed at room temperature (21–22 °C). AngII, PD123319, eicosatetrayonic acid (ETYA) (Sigma), candesartan (Astra-Zeneca), okadaic acid (OA) (Alomone Labs.), cantharidin and norokadaone (NOK) (Calbiochem) as powder were dissolved to yield 0.01 mM stock solutions and then diluted in the external solution. Serine/threonine phosphatase type 2A1 (Upstate) was dissolved in the internal solution to reach a final concentration of 10 U/ml.
2.4. Recording techniques
Ito was recorded using the perforated-nystatin patch-clamp configuration by using an Axopatch 200B and pCLAMP 9.0 software (Axon Instruments) [8]. Kv4.2 currents were recorded using the whole-cell configuration. The current recordings were sampled at 3–10 times the antialias filter setting. Micropipette resistance was kept below 3.5 M
when filled with internal solution and immersed in external solution. Capacitance and series resistance compensation were optimized and 80% compensation was usually obtained. Maximum Ito amplitude at +40 mV, cell capacitance and mean uncompensated access resistance averaged 1152±117 pA, 99.4±11.8 pF and 8.0±1.3 M, respectively (n=26).
2.5. Pulse protocols and analysis
The holding potential was maintained at –80 mV and the cycle time for any protocol was 30 s. To obtain current–voltage relationships and inactivation curves, 250-ms pulses, in 10 mV increments between –100 and +40 mV followed by a 250-ms test pulse to +50 mV, were applied. A 30-ms prepulse to –30 mV was always applied to inactivate INa. The Ito amplitude was measured as the difference between peak outward and end-pulse current, whereas the Kv4.2 current amplitude was measured at the peak. The inhibitory concentration at which 50% of block was achieved, IC50, and the Hill coefficient, nH, were obtained fitting the fractional block, f, at various AngII concentrations [D] to the Hill equation:
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The inactivation curves were fitted with a Boltzmann distribution:
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where A is the amplitude, Vh the midpoint of inactivation, Vm the test potential and k the slope factor.
2.6. Statistical methods
Results are expressed as mean±S.E.M. Data were compared using ANOVA followed by Newman–Keuls test. P<0.05 was considered statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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3.1 Effects of angiotensin II on Ito currents
Fig. 1A shows the effects of 0.1 µM AngII on the Ito recorded using the whole-cell configuration of the patch-clamp technique. Under these conditions, the effects of AngII were inconsistent, varying between different cells. In the experiment shown, AngII did not modify the current elicited by a 250-ms pulse from –80 to +40 mV. Fig. 1B shows the effects of AngII on the Ito recorded using the nystatin-perforated patch-configuration. Under these conditions, AngII consistently inhibited the current amplitude in six cells by 21.9±4.8% and, thus, the nystatin-perforated patch configuration was used to perform all the experiments shown in this paper. Control Ito traces were fitted to a bi-exponential function yielding two time constants of inactivation and AngII did not modify the inactivation kinetics (P>0.05) (Table 1). The inhibition reached steady state after 6–7 min and was slowly reversible upon washout with AngII-free solution (Fig. 1C). The blocking effects of AngII were concentration-dependent between 0.001 and 1 µM (Fig. 1D). Fitting the data at this range of concentrations to a Hill equation (nH=1) the IC50 and the maximum blockade (Bmax) obtained were 24.1±3.4 nM and 30.1±3.9%, respectively (dashed line). Fitting the data to the Hill equation, and assuming that Bmax will reach 100% an IC50 value of 3.1±1.5 µM was obtained (continuous line). However, when the AngII concentration was increased to 5 and 10 µM, the blockade significantly decreased, resulting in a bell-shaped dose response curve (P<0.05).
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Fig. 2A shows Ito and inward rectifier current (IK1) traces elicited using the protocol shown at the top. Ito current–voltage relationships in the absence and presence of 0.1 µM AngII are shown in Fig. 2B together with the fractional block plotted as a function of the membrane potential. AngII-induced block appeared at 0 mV coinciding with the activation of the current and resulted to be voltage-independent (14.4±9.3% at 0 mV, n=6, P>0.05 vs. blockade at +40 mV). We also analyzed the effects of AngII on the steady-state inactivation of the current. The inactivation curves were constructed by plotting the peak current amplitude obtained with the test pulse to +50 mV as a function of the potential of the preceding pulse (Fig. 2C) resulting in a Vh of –35.5±5.3 mV (k=3.5±0.5 mV, n=6). AngII did not modify the voltage-dependence of current inactivation (Table 1). Furthermore, when the fraction of block was plotted as a function of the voltage of the preceding pulse, it was not different at voltages between –100 and –50 mV (18.7±7.9% and 18.9±6.5%, respectively; P>0.05). Fig. 2D shows that AngII slightly, but non-significantly, increased the IK1 (10.8±6.2% at –120 mV) and, thus, this effect was not studied further.
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3.2. Effects of AngII were mediated by AT2R but not AT1R
Fig. 3A shows Ito traces elicited by applying pulses from –80 to +40 mV in the absence and the presence of 0.01 µM candesartan, a selective AT1R antagonist. In five cells, candesartan did not modify the current amplitude, the kinetics or the voltage-dependence of Ito inactivation. In the presence of candesartan, 1 µM AngII inhibited Ito at +40 mV by 26.6±6.5% (n=5) without modifying the time course of inactivation (Fig. 3B). This effect was not statistically different from that obtained in the absence of candesartan (28.4±6.5% at +40 mV, P>0.05). Fig. 3C and D shows that candesartan did not modify any of the effects produced by 1 µM AngII alone. The blockade appeared at 0 mV coinciding with the activation of the current, and was voltage-independent between 0 and +40 mV. AngII, both in the absence and the presence of candesartan, inhibited Ito current at +50 mV when prepulses between –100 and –40 mV were applied, without modifying the voltage-dependence of Ito inactivation (Vh=–32.1±1.0 vs. –33.1±0.6 mV, n=5). All these results indicated that the inhibition of the Ito produced by AngII was not mediated via AT1R stimulation.
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Fig. 4A shows Ito traces recorded in control conditions, in the presence of 1 µM PD123319, a selective AT2R antagonist, and in the presence of 0.1 µM AngII plus PD123319. PD123319 significantly increased the current (21.1±11.3 % at +40 mV, n=4, P<0.05), without modifying the inactivation time course of Ito. Interestingly, in the presence of PD123319, AngII failed to inhibit the Ito (1.6±0.03% of block at +40 mV, n=4, P>0.05). PD123319 increased the current between +10 and +40 mV, and addition of AngII did not induce further modifications at this range of potentials (Fig. 4B). Furthermore, neither PD123319 alone nor AngII plus PD123319 modified the voltage-dependence of inactivation (Fig. 4C). These results indicated that the effects of AngII on Ito were prevented by PD123319, suggesting that they were mediated via the AT2R. To confirm this observation, we studied the effects of CGP42112A, a selective AT2R agonist, on Ito. CGP42112A (1 µM) inhibited the Ito at +40 mV by 20.5±2.4% (n=4, P>0.05 vs. AngII block) without modifying the time course of inactivation (Fig. 4D, Table 1) and this effect resulted to be voltage-independent (Fig. 4E). Moreover, CGP42112A did not modify the voltage-dependence of inactivation (Fig. 4F, Table 1) and the blockade did not change at potentials between –100 mV (12.4±4.7%) and –50 mV (13.7±5.0%, P>0.05). Fig. 1E shows the concentration-dependent effects of CGP42112A. Inhibitory effects reached a maximum at 1 µM, whereas when the CGP42112A concentration was increased to 5 and 10 µM the blockade significantly decreased, resulting in a bell-shaped dose response curve (P<0.05). All these results clearly demonstrated that the selective activation of AT2R completely reproduced the effects of AngII.
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3.3. Effects on transmembrane action potentials
The effects of 1 µM CGP42112A on the action potentials recorded in isolated rat atria were also examined. Fig. 5A shows action potentials recorded in the absence and the presence of CGP42112A. It can be appreciated that the direct stimulation of the AT2R did not modify the resting membrane potential (–82.3±0.9 mV) or the action potential amplitude (111.9±1.6 mV, n=6). In contrast, CGP42112A slightly, but significantly, lengthened the action potential duration measured at 20%, 50% and 90% of repolarization (Fig. 5B).
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3.4 Signaling pathway involved in the effects of AngII on Ito
Stimulation of the AT2R activates three major cascades of intracellular events: (1) regulation of the nitric oxide (NO)–guanosine 3',5'-cyclic monophosphate (cGMP) system, (2) stimulation of PLA2 and release of arachidonic acid, and (3) activation of protein phosphatases and protein dephosphorylation [10]. Thus, in the first group of experiments, the inhibitory effect of AngII on Ito was examined in the presence of the NO-synthase inhibitor L-NAME (100 µM) (Fig. 6A). Superfusion of L-NAME alone did not modify the current amplitude (not shown). Moreover, there were no significant differences in the Ito inhibition produced by AngII and AngII plus L-NAME (21.9±4.8% vs. 17.8±3.5% at +40 mV, n=4, P>0.05). We next analyzed the effects of AngII in the presence of ETYA, a general inhibitor of arachidonic acid metabolism [11]. Superfusion of ETYA (10 µM) inhibited the Ito by 21.6±5.8% at +40 mV (n=4) (Fig. 6B). When AngII (0.1 µM) was superfused in the presence of ETYA (Fig. 6C), the blockade induced (24.3±3.8% at +40 mV, n=5) was not different from that produced by AngII (P>0.05). These data strongly indicated that neither the NO–cGMP system nor arachidonic acid metabolism was involved in the effects of AngII on Ito. Thus, we investigated the possible involvement of protein phosphatases in the effects of AngII on Ito.
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In the mammalian heart, more than 90% of the protein phosphatase activity consists of serine/threonine phosphatases PP1 and PP2A [12]. Thus, we analyzed the effects of AngII in the absence and the presence of 10 nM OA, which at this concentration acts as a selective PP2A inhibitor [12]. Superfusion of OA did not modify the Ito (Fig. 7A), but completely reversed the inhibition produced by AngII (20.5±3.2% at +40 mV, n=5) (Fig. 7B). AngII significantly inhibited the current between +10 and +40 mV in a voltage-independent manner and OA reversed the effects produced by AngII (Fig. 7C). Neither AngII alone nor AngII plus OA modified the voltage-dependence of Ito-inactivation (Fig. 7D, Table 1). These results suggested that the AT2R-mediated inhibition of AngII on Ito involved the activation of PP2A. We next studied the effects of AngII in the presence of the inactive OA analogue, 1-norokadaone (NOK, 10 nM) and cantharidin (1 µM), which inhibits PP1 and PP2A [12]. NOK did not modify the current amplitude (2.4±1.3%, n=3) (Fig. 8A) and the AngII-induced inhibition of Ito was similar in the absence and in the presence of NOK (18.2±2.2%, n=3, P>0.05). Cantharidin did not modify the Ito and AngII failed to inhibit the current in the presence of this inhibitor (Fig. 8B, n=4). These results confirmed that the AT2R-mediated inhibition of Ito induced by AngII involves the activation of PP2A.
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In some cell types, serine/threonine phosphatases counter-regulate the effects mediated by the various protein kinases in response to AT1R activation [1]. To analyze whether the activation of PP2A was preceded by the activation of AT1R-protein kinase C (PKC) signaling pathway, experiments were performed in the presence of staurosporine, a PKC inhibitor. Fig. 8C demonstrated that staurosporine did not modify the Ito and that AngII decreased the current in a similar way in the presence (23.1±0.03%, n=4) and in the absence of staurosporine (22.7±1.2%, P>0.05).
3.5. Effects on Kv4.2 channels
The effects of OA (10 nM) were analyzed on Kv4.2 channels transiently transfected on CHO cells. OA slightly decreased the current amplitude (15.9±5.8% at +40 mV, n=4) without modifying the fast and slow time constant of inactivation (6.4±0.8 and 37.0±3.2 ms, respectively) (Fig. 8D). Finally, we studied the effects of the intracellular application of PP2A. In these experiments, the tip of the pipette was filled with PP2A-free internal solution, in order to obtain "control" current records. Fig. 8E shows current traces obtained in the same cell just after seal breaking and when steady-state effects of 10 U/ml PP2A were achieved. PP2A reduced Kv4.2 currents elicited by 250-ms pulses to +40 mV by 32.9±11.7% (n=4) without any time-dependent effect, i.e., it simply scaled down the current.
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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The main findings of the present study are as follows: (1) AngII inhibited the Ito exhibiting a bell-shaped dose–response relationship but did not modify the IK1; (2) the AngII-induced inhibition was mediated by the selective activation of the AT2R and was associated with the activation of PP2A; and (3) the selective activation of AT2R produced a lengthening of the rat atrial action potential duration.
To our knowledge, this is the first detailed study of the acute effects of AngII on the cardiac Ito. Previously, Yu et al. [6] found that superfusion of AngII did not modify the Ito recorded in canine ventricular myocytes using the whole-cell patch-clamp. The exact reason for this discrepancy is unclear, but it could be attributable to the patch-clamp configuration used. When the Ito was recorded using the whole-cell patch-clamp, the effects of AngII were non-repetitive and inconsistent. However, using the nystatin-perforated patch configuration a consistent inhibition of the Ito was observed, suggesting that, to achieve the AngII effects, it is necessary to preserve the intracellular milieu. Similarly, the AngII-induced increase in L-type Ca2+ current was also observed only when the perforated patch configuration was used [13].
The effect of AngII was voltage-independent and concentration-dependent between 0.001 and 1 µM. At higher concentrations, the blocking effect decreased resulting in a bell-shaped dose–response curve. This behavior has already been described with AngII in other preparations and was attributed to the desensitization of the AT1R [14]. Although desensitization and internalization of AT2R was not observed in embryonic mouse fibroblasts [15], our results would suggest that AT2R, as with other G protein-coupled receptors, displays desensitization in adult rat cardiac myocytes. Another possible explanation was that, at high concentrations, activation of AT1R contributed to the reduction of block. However, a bell-shaped dose response curve was also observed when the selective AT2R agonist was tested, a result that reinforced the hypothesis that AngII-induced Ito inhibition was due to the selective AT2R activation at all the concentrations tested.
The blocking effects of AngII were abolished by PD123319, but not by candesartan, indicating that they were mediated via the stimulation of AT2R. These results were confirmed using the selective AT2R agonist CGP42112A that reproduced the effects of AngII. In human hearts, AT2R expression may be equal to or even exceed that of the AT1R. Furthermore, AT2R expression increases in pathological conditions, such as cardiac hypertrophy, myocardial infarction, chronic atrial fibrillation (CAF) and heart failure (HF) [1,10,16]. The signaling mechanisms of AT2R involve the regulation of the NO–cGMP system, the activation of the PLA2 and release of arachidonic acid and the activation of protein phosphatases [10]. In our experiments, the AngII-inhibition of Ito was not modified by L-NAME or ETYA, indicating that neither the activation of the NO–cGMP system nor the stimulation of the arachidonic acid cascade is involved in the effects of AngII on Ito. In contrast, at concentrations (10 nM) at which it selectively inhibits PP2A [12], OA completely reversed the effects of AngII, which suggested that this effect was mediated by the stimulation of serine/threonine phosphatases. Experiments performed with cantharidin and NOK, as positive and negative controls, respectively, further confirmed the involvement of the PP2A in the AT2R-mediated inhibition of the Ito by AngII. In some cell types, tyrosine and/or serine/threonine phosphatases reverse, or at least counter-regulate, the effects mediated by the various protein kinases in response to AT1R activation [1]. Even when our results demonstrated that the effects of AngII are due to the selective activation of AT2R, which do not activate PKC [1,5], we studied the effects of AngII in the presence of staurosporine, an inhibitor of all PKC isoforms (and other intracellular kinases). The results confirmed that AT1R-PKC phosphorylation did not precede the inhibitory effects of AT2R-PP2A. A previous report demonstrated that 2,3-butanedione monoxime, that activates PP1 and PP2A, inhibits Ito in rat ventricular myocytes [17]. This effect was reversed by cAMP-dependent stimulation, supporting the hypothesis that the inhibition of Ito was due to dephosphorylation of the channel. Indeed, both rKv4.2 and hKv4.3 genes, which encode for the 
-subunit of the channels that underlie the Ito in rat and human myocardium, respectively, display consensus sites for protein kinases, including PKC, PKA and MAPK [18–20]. Furthermore, the present results demonstrated that Kv4.2 currents decreased when PP2A was intracellularly applied reproducing the effects observed on Ito after AT2R stimulation. Since it has been currently assumed that CHO cells did not constitutively express KChIP2 proteins [21], our results could be interpreted considering that PP2A dephosphorylates the protein kinase site of the Kv4.2 -subunits to decrease the Ito. However, very recently, it has been reported that CHO cells expressed a potential endogenous KChIP-like protein [22]; thus, it is also possible that effects of AngII are the consequence of the dephosphorylation of the KChIP2 β-subunit instead the Kv4.2 pore forming subunit of the channel. Furthermore, we cannot rule out the possibility that PP2A did not dephosphorylate the channel - and/or β-subunits directly but rather another protein that in turn modulates the channel.
In neurons cultured from newborn rat hypothalamus and brain stem, AngII increased the IA through stimulation of AT2R that activated PLA2 and PP2A pathways [5,23]. It is important to note the opposing actions of AngII on the transient outward current described in cardiac myocytes and neurons (IA), even when the signaling pathway could be similar. It is known that IA and Ito are primarily encoded by Kv4 -subunits that immunoprecipitates with KChiP2 subunits. Given this, it would seem unlikely that direct desphosphorylation of the Kv4 -subunit and/or KChIP2 accessory subunits underlies the AngII-mediated augmentation of IA and the attenuation of Ito. However, it should be stressed that multiples modes of modulation of ion channels by a single enzyme has been described [24], an effect that might explain different effects of PP2A on IA and Ito. An alternative hypothesis might be that PP2A dephosphorylated a modulatory protein, perhaps distinct in neurons versus heart cells. Recently, it has been identified a novel protein, the CD26-related protein DPPX, that is an important component of the channels mediating the IA in many neurons and that is a key regulator of the processing, membrane expression and function of native IA [25]. However, at present time, it remains to be elucidated whether this subunit may also contribute to the Ito channels in the heart.
It should also be stressed that the selective AT2R antagonist PD123319 increased the Ito, an effect that could be attributed primarily to a direct interaction of the drug with the channel protein. However, PD123319 did not increase the current generated by Kv4.2 channels expressed transiently in CHO cells (not shown). Since it has not been previously described that PD123319 acted in any tissue as an "inverse agonist" of AT2R, a second possibility to explain the results could be that the drug antagonized an agonist-independent or constitutive activity of the AT2R [1]. This basal activity has already been described for other G protein-coupled receptors, such as thyroid-stimulating hormone and muscarinic receptors [1]. Following this hypothesis, it is expected that the OA inhibition of a constitutive PP2A activity would also increase the Ito. Our results demonstrated that OA did not modify the Ito, whereas slightly inhibited the Kv4.2 currents. Thus, it is possible that the decrease of the current amplitude produced by OA by a direct interaction with the channel neutralize the increase of the Ito that would produce the inhibition on the basal activity of the AT2R-PP2A.
Our results demonstrated that selective stimulation of AT2R produced an inhibition of Ito and a prolongation of the atrial action potential duration in rats. It would be of interest to analyze whether selective AT2R activation also produces these effects in human cardiomyocytes. Some cardiac diseases, like cardiac hypertrophy, HF and CAF, are accompanied by an increase in the cardiac and systemic levels of AngII, which leads to a protective upregulation of AT2R and a downregulation of the AT1R [10,16]. Some of these patients are in treatment with selective AT1R antagonists, which allow to the selective stimulation of AT2R by AngII. However, Ito is downregulated in CAF, HF and cardiac hypertrophy [26,27]. Thus, more studies are needed to elucidate the possible proarrhythmic and/or antiarrhythmic effect of the inhibition produced by AngII on Ito via AT2R here demonstrated.
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Acknowledgements |
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This work was supported by Comisión Interministerial de Ciencia y Tecnología (SAF99-0069/2002-02304), Comunidad Autónoma de Madrid (08.4/0038.1/2001), Fondo de Investigaciones Sanitarias (01/1130) and Pfizer Foundation Grants. We thank Guadalupe Pablo for her excellent technical support.
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Notes |
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Time for primary 14 days
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References |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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- De Gasparo M., Catt K.J., Inagami T., Wright J.W., Unger T.H. International union of pharmacology: XXIII. The angiotensin II receptors. Pharmacol. Rev. (2000) 52:415–472.
[Abstract/Free Full Text] - Regitz-Zagrosek V., Friedel N., Heymann A., et al. Regulation, chamber localization, and subtype distribution of angiotensin II receptors in human hearts. Circulation (1995) 91:1461–1471.
[Abstract/Free Full Text] - Nerbonne J.M. Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J. Physiol. (2000) 525:285–298.
[Abstract/Free Full Text] - Oudit G.Y., Kassiri Z., Sah R., Ramirez R.J., Zobel C., Backx P.H. The molecular physiology of the cardiac transient outward potassium current (Ito) in normal and diseased myocardium. J. Mol. Cell Cardiol. (2001) 33:851–872.[CrossRef][Web of Science][Medline]
- Sumners C., Gelband C.H. Neuronal ion channel signalling pathways: modulation by angiotensin II. Cell. Signal. (1998) 10:303–311.[CrossRef][Web of Science][Medline]
- Yu H., Gao J., Wang H., et al. Effects of the renin–angiotensin system on the current Ito in epicardial and endocardial ventricular myocytes from the canine heart. Circ. Res. (2000) 86:1062–1068.
[Abstract/Free Full Text] - Zhang T.-T., Takimoto K., Stewart A., Zhu C., Levitan E. Independent regulation of cardiac Kv4.3 potassium channel expression by angiotensin II and phenylephrine. Circ. Res. (2001) 88:476–482.
[Abstract/Free Full Text] - Caballero R., Delpón E., Valenzuela C., Longobardo M., González T., Tamargo J. Direct effects of candesartan and eprosartan on human cloned potassium channels involved in cardiac repolarization. Mol. Pharmacol. (2001) 59:825–836.
[Abstract/Free Full Text] - Heredia M.P., Fernandez-Velasco M., Benito G., Delgado C. Neuropeptide Y increases 4-aminopyridine-sensitive transient outward potassium current in rat ventricular myocytes. Br. J. Pharmacol. (2002) 135:1701–1706.[CrossRef][Web of Science][Medline]
- Matsubara H. Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases. Circ. Res. (1998) 83:1182–1191.
[Abstract/Free Full Text] - Zhu M., Natarajan R., Nadler J.L., Moore J.M., Gelband C.H., Sumners C. Angiotensin II increases neuronal delayed rectifier K+ current: role of 12-lipoxygenase metabolites of arachidonic acid. J. Neurophysiol. (2000) 84:2494–2501.
[Abstract/Free Full Text] - Herzig S., Neumann J. Effects of serine/threonine protein phosphatases on ion channels in excitable membranes. Physiol. Rev. (2000) 80:173–210.
[Abstract/Free Full Text] - Aiello E.A., Cingolani H.E. Angiotensin II stimulates cardiac L-type Ca2+ current by a Ca2+- and protein kinase C-dependent mechanism. Am. J. Physiol. (2001) 280:H1528–H1536.[Web of Science]
- Widdop R.E., Matrougui K., Levy B.I., Henrion D. AT2 receptor-mediated relaxation is preserved after long-term AT1 receptor blockade. Hypertension (2002) 40:516–520.
[Abstract/Free Full Text] - Csikos T., Balmforth A.J., Grojec M., Gohlke P., Culman J., Unger T. Angiotensin AT2 receptor degradation is prevented by ligand occupation. Biochem. Biophys. Res. Commun. (1998) 243:142–147.[CrossRef][Web of Science][Medline]
- Goette A., Arndt M., Röcken C., et al. Regulation of angiotensin II receptor subtypes during atrial fibrillation in humans. Circulation (2000) 101:2678–2681.
[Abstract/Free Full Text] - Xiao Y.F., McArdle J.J. Activation of protein kinase A partially reverses the effects of 2,3 butanedione monoxime on the transient outward K+ current of rat ventricular myocytes. Life Sci. (1995) 57:335–343.[CrossRef][Web of Science][Medline]
- Dilks D., Ling H.P., Cockett M., Sokol P., Numann R. Cloning and expression of the human Kv4.3 potassium channel. J. Neurophysiol. (1999) 81:1974–1977.
[Abstract/Free Full Text] - Anderson A.E., Adams J.P., Qian Y., Cook R.G., Pfaffinger P.J., Sweatt D. Kv4.2 phosphorylation by cyclic AMP-dependent protein kinase. J. Biol. Chem. (2000) 275:5337–5346.
[Abstract/Free Full Text] - Adams J.P., Anderson A.E., Varga A.W., et al. The A-type potassium channel Kv4.2 is a substrate for the mitogen-activated protein kinase ERK. J. Neurochem. (2000) 75:2277–2287.[CrossRef][Web of Science][Medline]
- An W.F., Bowlby M.R., Betty M., et al. Modulation of A-type potassium channels by a family of calcium sensors. Nature (2000) 403:553–556.[CrossRef][Medline]
- Patel S.P., Campbell D.L., Morales M.J., Strauss H.C. Heterogeneous expression of KChIP2 isoforms in the ferret heart. J. Physiol. (2002) 539:649–656.
[Abstract/Free Full Text] - Kang J., Sumners C., Posner P. Angiotensin II type 2 receptor-modulated changes in potassium currents in cultured neurons. Am. J. Physiol. (1993) 265:C607–C616.[Web of Science][Medline]
- Nitabach M., Llamas A., Thomson I., Collins K., Holmes T. Phosphorylation-dependent and phosphorylation-independent modes of modulation of Shaker family voltage-gated potassium channels by Src family protein tyrosine kinases. J. Neurosci. (2002) 22:7913–7922.
[Abstract/Free Full Text] - Nadal M., Ozaita A., Amarillo Y., et al. CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K channels. Neuron (2003) 37:449–461.[CrossRef][Web of Science][Medline]
- Nattel S., Li D. Ionic remodeling in the heart. Pathophysiological significance and new therapeutic opportunities for atrial fibrillation. Circ. Res. (2000) 87:440–447.
[Abstract/Free Full Text] - Tomaselli G.F., Marbán E. Electrophysiological remodeling in hypertrophy and heart failure. Cardiovasc. Res. (1999) 42:270–283.
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