A Specific Pattern of Phosphodiesterases Controls the cAMP Signals Generated by Different Gs-Coupled Receptors in Adult Rat Ventri
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Francesca Rochais, Aniella Abi-Gerges, K
参见附件。
INSERM U769 (F.R., A.A.-G., F.L., R.F., G.V.), Chatenay-Malabry, France
Univ-Paris-Sud (F.R., A.A.-G., F.L., R.F., G.V.), U769 Faculté de Pharmacie, Chatenay-Malabry, France
Division of Reproductive Biology (K.H., M.C.), Department of Gynecology and Obstetrics, Stanford University, California
Department of Pharmacology (D.M.F.C.), University of Cambridge, United Kingdom.
Abstract
Compartmentation of cAMP is thought to generate the specificity of Gs-coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca2+ current (ICa,L) on stimulation with 1-adrenergic receptors (1-ARs), 2-ARs, glucagon receptors (Glu-Rs) and prostaglandin E1 receptors (PGE1-Rs). All receptors but PGE1-R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide–gated (CNG) channels, stimulation of 1-AR and Glu-R increased cAMP, whereas 2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated 1-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE1-R and 2-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R, the measurements of ICa,L closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled 1-AR and 2-AR regulation of ICa,L, whereas only PDE4 controlled Glu-R regulation of ICa,L thus demonstrating that receptor–PDE coupling has functional implications downstream of cAMP. PGE1 had no effect on ICa,L even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to L-type Ca2+ channels. These results identify specific functional coupling of individual PDE families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.
Key Words: cAMP heart G-protein–coupled receptor phosphodiesterase
Introduction
Cardiac myocytes express a number of Gs-coupled receptors (GsPCRs) that raise intracellular cAMP levels and activate cAMP-dependent protein kinase (PKA) but exert different downstream effects. For instance, 1-adrenergic receptor (1-AR) stimulation produces a major and sustained increase in force of contraction, accelerates relaxation, and stimulates glycogen phosphorylase.1 2-AR stimulation also increases contractile force but does not activate glycogen phosphorylase2 and does not accelerate relaxation1,3 (see also ref. 2); glucagon receptor (Glu-R) stimulation activates phosphorylase and exerts positive inotropic and lusitropic effects, but the contractile effects fade with time.4 Finally, prostaglandin E1 (PGE1) has no effect on contractile activity or glycogen metabolism.5,6
Such observations led to the proposal that activation of different GsPCRs results in the accumulation of cAMP and phosphorylation of hormone target proteins in distinct compartments.7 The discovery of A-kinase anchoring proteins, responsible for the subcellular distribution of particulate PKA,8 and the development of new methodologies allowing to track local cAMP in living cells provided additional support for this concept.9–15
Because cAMP is a small and readily diffusible molecule, cAMP compartments can only exist under a restrictive set of conditions for the localization and activity of the different components of the cAMP signaling cascade, namely GsPCRs, adenylyl cyclases, PKA, and the cAMP phosphodiesterases (PDEs). Cardiac PDEs fall into four families: PDE1, which is activated by Ca2+/calmodulin; PDE2, which is stimulated by cGMP; PDE3, which is inhibited by cGMP; and PDE4. Whereas PDE1 and PDE2 can hydrolyze both cAMP and cGMP, PDE3 preferentially hydrolyzes cAMP, and PDE4 is specific for cAMP. Until now, only a limited number of studies have addressed directly the participation of PDEs to cAMP compartmentation and hormonal specificity in cardiac cells.9,10,12,15,16 In a recent study, we used recombinant cyclic nucleotide–gated (CNG) channels as cAMP biosensors in adult rat ventricular myocytes (ARVMs) to show that -adrenergic cAMP signals are localized by PKA-mediated activation of PDE3 or PDE4.13 However, PDE regulation of other hormonal cAMP signals is presently unknown. In the following, we address this point by comparing the cAMP signals generated by 1-AR, 2-AR, Glu-R, and PGE1-R, their regulation by individual PDE families, and the functional consequences on L-type Ca2+ channels.
Materials and Methods
Detailed methods are included in the online-only data supplement to this article, available at http://circres.ahajournals.org.
Total cAMP Determination
cAMP was measured by radioimmunossay after acetylation of the samples.20,21
PDE Assay
PDE activity was measured according to a modification of the two-step assay procedure method described by Thompson and Appleman17 with 1 μmol/L cAMP and 105 cpm [3H]-cAMP, as detailed previously.18
Electrophysiological Experiments
The whole-cell configuration of the patch-clamp technique was used to record the L-type calcium current (ICa,L) and the CNG current (ICNG). The extracellular Cs+-Ringer solution contained 1.8 mmol/L [Ca2+] and 1.8 mmol/L [Mg2+] for ICa,L recordings and nominal [Ca2+] and [Mg2+] plus 1 μmol/L nifedipine for ICNG recordings. Patch pipettes were filled with a Cs+ solution.
Data Analysis
ICa,L amplitude was measured as the difference between the peak inward current and the current at the end of the 400-ms duration pulse.19 ICNG amplitude was measured at the end of the 200-ms pulse. Capacitance and leak currents were not compensated. In 141 ARVMs, mean capacitance was 160.5±3.9 pF. ICNG density was calculated for each experiment as the ratio of current amplitude to cell capacitance. All the data are expressed as mean±SEM. When appropriate, the Student t test was used for statistical evaluation.
Results
PDE Activities in ARVMs
Figure 1 summarizes experiments in which the relative activity of PDE2, PDE3, and PDE4 in quiescent ARVMs was determined. Total cAMP hydrolytic activity was on average 109.8±16.6 pmol/min per mg protein (n=4) and was mainly attributable to PDE3 and PDE4, which represented 31% and 38% of the total, respectively. Under these assay conditions, PDE2 activity was minimal (Figure 1). Immunoprecipitation experiments showed that PDE4 activity is contributed by PDE4A, PDE4B, and PDE4D variants, whereas the PDE3A form is the predominant PDE3 expressed (data not shown). The relative activity of individual PDE families did not vary significantly after 24 and 48 hours of culture (data not shown). Moreover, the ratio of PDE3/PDE4 was similar to that measured in extracts of adult rat ventricle (data not shown). Thus, cultured ARVMs closely resemble the in vivo pattern of PDE expression.
Total cAMP Production in Response to Different GsPCRs in ARVMs
We next determined whether stimulation of different GsPCRs increased total cAMP in ARVMs (Figure 2). We used a combination of isoprenaline (ISO; 5 μmol/L) and the 2-AR antagonist ICI 118551 (ICI; 1 μmol/L) to stimulate 1-AR, a combination of ISO (5 μmol/L) in the presence of the 1-AR antagonist CGP 20712A (CGP; 1 μmol/L) to stimulate the 2-AR, glucagon (Glu; 1 μmol/L) to stimulate Glu-R, and PGE1 (1 μmol/L) to stimulate PGE1-R. Basal cAMP was more than doubled by ISO + ICI and Glu and increased by 70% by ISO + CGP. PGE1 had no effect on cAMP content under these experimental conditions. A 15-minute incubation with the broad-spectrum PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 100 μmol/L) increased the total cAMP content by 2-fold and dramatically potentiated the response to all agonists except PGE1. The effect of ISO + ICI and Glu were multiplied by 8 and 7, respectively, whereas that of ISO + CGP was increased 3-fold. cAMP content after PGE1 + IBMX was slightly increased when compared with IBMX alone, although it did not reach statistical significance.
Real-Time Monitoring of Subsarcolemmal cAMP Signals Elicited by Different GsPCRs in ARVMs
To further compare cAMP signals elicited by the different receptors, we used CNG channels that allow direct monitoring of cAMP variations beneath the plasma membrane in intact ARVMs.13 Figure 3 summarizes the results obtained with two CNGA2 mutants, which differ in their sensitivity to cAMP, E583M (EC50 for cAMP 10 μmol/L), and C460W/E583M (EC50 for cAMP 1 μmol/L).20 As depicted in Figure 3A, activation of 1-AR by a combination of ISO (5 μmol/L) and ICI (1 μmol/L) increased basal ICNG density 3-fold, whereas specific activation of 2-AR by ISO (5 μmol/L) plus CGP (1 μmol/L) had no effect. Similarly, activation of Glu-R by Glu (1 μmol/L) or PGE1-R by PGE1 (1 μmol/L) failed to activate ICNG (Figure 3A). These results indicate that cAMP concentration was below the activation threshold of E583M CNGA2 on stimulation of 2-AR, Glu-R, and PGE1-R. When using the C460W/E583M mutant (Figure 3B), activation of 1-AR increased basal ICNG density 6-fold. This effect was significantly higher (P<0.01) than that obtained in cells expressing E583M CNGA2, as expected from the relative affinities of the two channels toward cAMP. Interestingly, application of Glu now activated ICNG by 5-fold, but specific activation of 2-AR or PGE1-R still had no effect. Thus, the increased sensitivity of C460W/E583M CNGA2 allowed the detection of subsarcolemmal cAMP generated by activation of Glu-R but not by activation of 2-AR or PGE1-R.
Regulation of Subsarcolemmal cAMP Signals by PDEs
The above results suggest that cAMP signals elicited by the four different GsPCRs at the plasma membrane are not identical. We next investigated the contribution of PDEs to such specificity. Both CNGA2 mutants were used with essentially similar results, therefore the low-affinity E583M channel data are presented as supplemental Figures I and II. Preliminary experiments showed that IBMX (100 μmol/L) had no significant effect on ICNG from myocytes expressing E583M per C460W (n=6; data not shown). Figure 4A shows a typical experiment in which the amplitude of ICNG is depicted as a function of time, thus providing an instant readout of subsarcolemmal cAMP level in a single cardiac myocyte. 1-AR stimulation with ISO (5 μmol/L) + ICI (1 μmol/L) induced a substantial increase of ICNG, which was strongly potentiated by selective PDE3 inhibition with cilostamide (Cil; 1 μmol/L) or selective PDE4 inhibition with Ro 20-1724 (Ro; 10 μmol/L). Concomitant inhibition of all PDEs with IBMX (100 μmol/L) resulted in a similar potentiation of 1-AR stimulation to that obtained with selective PDE4 inhibition. At the end of each experiment, the cell was challenged with a saturating concentration (100 μmol/L) of the forskolin analog L-858051 to determine the maximal ICNG response. In Figure 4C and 4D, we investigated the role of PDEs in shaping the 2-AR cAMP response. As seen previously, application of ISO (5 μmol/L) + CGP (1 μmol/L) had no effect on ICNG. Selective inhibition of PDE3 during 2-AR stimulation with ISO + CGP induced a small and transient increase of ICNG. A somewhat stronger effect was observed on PDE4 inhibition, although it was also transient (Figure 4C). In contrast, concomitant inhibition of PDE3 and PDE4 unmasked a robust and sustained cAMP increase during 2-AR stimulation. These results indicate that PDE3 and PDE4 control cAMP elicited by 1-AR and 2-AR. Both PDEs are necessary to circumvent the cAMP rise elicited by 1-AR, whereas either PDE3 or PDE4 individually is sufficient to prevent cAMP to rise beneath membrane on 2-AR activation.
Figure 5A shows that inhibition of PDE3 had little effect on ICNG augmented by 1 μmol/L Glu, whereas a selective inhibition of PDE4 by Ro produced a strong stimulation of the current. Concomitant inhibition of both PDE3 and PDE4 by Cil + Ro, or of all PDEs by IBMX, resulted in a comparable effect as inhibition of PDE4 alone (Figure 5A and 5B). These results indicate that Glu-R cAMP signals at the membrane are controlled exclusively by PDE4. Although no increase in cAMP with PGE1 could be detected previously, we tested whether PDE inhibition could reveal a PGE1 effect on ICNG. As shown in Figure 5C, application of 1 μmol/L PGE1 did not affect ICNG. However, in the continuous presence of PGE1, simultaneous inhibition of PDE3 and PDE4 (by Cil + Ro) clearly activated the ICNG. IBMX was more efficient than Cil + Ro, indicating that other PDEs besides PDE3 and PDE4 might play a role under these conditions (Figure 5C). Thus, similar to 2-AR, cAMP mobilization by PGE1-R needs concomitant inhibition of PDE3 and PDE4 to be detected at the membrane. However, in contrast to 2-AR, other PDEs besides PDE3 and PDE4 regulate PGE1-R cAMP signals in cardiac cells.
Regulation of the L-Type Ca2+ Current by GsPCRs and PDEs
Collectively, the above results show that different PDEs are involved in the regulation of cAMP generated by distinct GsPCRs in cardiac myocytes. To evaluate the functional implication of these findings, we examined the regulation of a major sarcolemmal cAMP target, the L-type Ca2+ current (ICa,L), by GsPCRs and PDEs. The experiments were performed at 24 hours, as with CNG channel experiments. Selective inhibition of PDE3 by Cil (1 μmol/L) or PDE4 by Ro (10 μmol/L) had no effect on basal ICa,L (data not shown). Because the concentration of ISO (5 μmol/L) used in the 1-AR stimulation of ICNG produced a maximal stimulation of ICa,L (121.3±22.2% over control; n=9; data not shown), a lower dose of ISO (1 nmol/L, with 1 μmol/L ICI) was used in subsequent experiments to assess the contribution of PDE isoforms to the 1-AR regulation of ICa,L. In these conditions, selective inhibition of PDE3 by Cil or PDE4 by Ro resulted in a marked potentiation of the prestimulated ICa,L (Figure 6A and 6B). As shown in Figure 6C and 6D, 2-AR stimulation with ISO (5 μmol/L) and CGP (1 μmol/L) produced a small but significant stimulation of ICa,L. This effect could be further potentiated by PDE3 inhibition with Cil and more markedly by PDE4 blockade with Ro. These results indicate that PDE3, and more prominently PDE4, are integral components of ICa,L regulation by 1-AR and 2-AR. In another series of experiments, the regulation of ICa,L by Glu was investigated. At high (1 μmol/L) concentration, Glu maximally stimulated ICa,L, and addition of PDE inhibitors had no effect (data not shown). At the concentration of 10 nmol/L, Glu slightly decreased ICa,L, and PDE3 inhibition by Cil had no effect. However, application of Ro to block PDE4 clearly increased ICa,L (Figure 7A and 7B). Finally, as shown in Figure 7C and 7D, PGE1 had no effect on ICa,L, even when either PDE3 or PDE4 was blocked. However, concomitant inhibition of PDE3 and PDE4 increased ICa,L in the presence of PGE1, but this effect was not different from the effect of the inhibitors on the basal ICa,L (Figure 7D). These results confirm the specific and functional coupling of Glu-R to PDE4 and show that the cAMP compartment generated by activation of PGE1-R is not in contact with L-type Ca2+ channels.
Discussion
Although a number of recent studies have underscored the role of PDEs in tailoring hormonal cAMP signals,9–13,21–23 none have addressed directly whether distinct hormonal cAMP signals are controlled by different PDEs. To gain some insight into this question, we used complementary biochemical and electrophysiological techniques to resolve cAMP at the cellular, subsarcolemmal, and local levels. We show that four canonical GsPCRs expressed in ARVMs generate different cAMP signals because of a specific contribution of individual PDE families. For all receptors but PGE1-R, the nature of this functional coupling determines the regulation of a major downstream cAMP target, the L-type Ca2+ channels.
In agreement with previous studies, we found that PDE3 and PDE4 represent the major cAMP hydrolytic activities in rodent cardiomyocytes.12,13,24 Although there is a general agreement that 1-AR increases cAMP in adult rat cardiac preparations,3,25–27 the ability of 2-AR to do so is a matter of debate.3,25,26 Although Glu increases cAMP in various cardiac preparations,4 recent studies failed to detect it.27,28 Thus, we checked whether stimulation of the different receptors led to detectable increases in total cAMP in ARVMs. We found that stimulation of 1-AR, Glu-R, and to a lesser extent 2-AR, increased total cAMP content, and that IBMX greatly accentuated these effects. In contrast, PGE1 alone did not increase cAMP, although a small effect could be observed in the presence of IBMX. This result is at variance with those from Buxton and Brunton29 in rabbit cardiomyocytes, in which ISO and PGE1 similarly increased cAMP. However, in addition to species differences, the experimental conditions used here are different because PGE1 was used at a 10-fold lower concentration (1 versus 10 μmol/L) and application lasted 5-fold less time (3 versus 15 minutes).
We next used CNG channels to monitor cAMP triggered by distinct routes near the plasma membrane.11,13 We found that activation of 1-AR and Glu-R elicit readily detectable cAMP at the membrane, whereas 2-AR and PGE1-R do not. In heterologous expression systems and in rat olfactory epithelium, CNG channels associate preferentially with noncaveolar lipid rafts.30 If this holds true in ARVMs, failure to detect cAMP elicited by 2-AR and PGE1-R could be attributable to localization of these receptors in caveolae.31–34 However, this would not compromise the role of PDEs in preventing cAMP diffusion out of such membrane compartments. Indeed, we found that IBMX, at a 100 μmol/L concentration, increased the response of ICNG not only to 1-AR and Glu-R stimulation, but also revealed cAMP generated by 2-AR and PGE1-R stimulation. Given the Ki of IBMX for the different PDEs,35 at 100 μmol/L, this inhibitor should block PDE1, PDE2, PDE3, and PDE4 activities by, respectively, 98%, 93%, 97%, and 87%. To delineate the specific contribution of PDE4, we used Ro, which, at 10 μmol/L, should inhibit 71% of its activity without affecting the other PDEs.35 We found that PDE4 hydrolyzes cAMP produced by all the receptors tested, thus limiting PKA activation and Ca2+ channel phosphorylation on 1-AR, 2-AR, and Glu-R activation. These results are consistent with previous studies showing that PDE4 is critically involved in the regulation of ICa,L and contractility by catecholamines and Glu.21,28,36 PDE4 isoforms are likely to play this ubiquitous role because of their molecular diversity and specific subcellular localization.37–39 However, whether specific PDE4 isoforms are associated with different receptors remains unclear. Recent data in neonatal cardiomyocytes reported an important role for PDE4B2 in controlling norepinephrine-induced cAMP increase.12 However, in the same model, other studies suggest that PDE4D isoforms are preferentially associated with the 2-AR. For instance, PDE4D5 and, to a lesser extent, PDE4D3 are recruited to the 2-AR by -arrestin on ISO challenge.22,40 Moreover, Xiang et al16 showed that PDE4D selectively affects 2-AR versus 1-AR positive chronotropism. Whether these findings apply to ARVMs remains to be established.
Nearly complete (95%) and selective PDE3 inhibition should be achieved by 1 μmol/L Cil.35 Using this inhibitor, we found that PDE3 controls the cAMP signals generated by 1-AR, 2-AR, and PGE1-R but not Glu-R. However, on ICa,L response, PDE3 controlled the effects of 1-AR and 2-AR but had no impact on Glu-R or PGE1-R stimulation. We showed previously that PDE3 controlled cAMP diffusion and consequently ICa,L activation in cardiac myocytes subjected to -adrenergic stimulation.13,21,36,41,42 Our findings contrast with results obtained in neonatal rat ventricular myocytes in which PDE3 was found to have a moderate effect on 1-AR (with norepinephrine)–induced cAMP.12 Moreover, in right ventricular strips from adult rat, PDE3 inhibition had no effect on the contractile response to the 1-AR agonist dobutamine but potentiated that of Glu.27 In parallel cAMP assays, these authors found that PDE3 inhibition also had no effect on the response to dobutamine but revealed a substantial cAMP increase caused by Glu. Although obvious differences in developmental stage, cardiac territory, or rat strains might account for some of the discrepancies, one should emphasize that CNG channels provide a readout of subsarcolemmal cAMP, whereas other methods measure cytosolic or total cAMP. Thus, one could speculate that on 1-AR stimulation, PDE3 controls the membrane cAMP pool but not a cytosolic one possibly involved in contractility (sarcoplasmic reticulum-associated PDE3 for instance43). In the case of Glu-R, the opposite would be true: PDE3 would not control subsarcolemmal cAMP generated by Glu-R but would prevent the access of the second messenger to other, nonsarcolemmal inotropic targets. For such a hypothesis to be valid, one must further speculate either that totally segregated cAMP pathways are triggered by the different receptors (including PKA targets), or that differential regulation of PDE3 is triggered by receptor activation. The latter mechanism was found in frog heart but not in rat.44
Although inhibition of either PDE3 or PDE4 potentiated the 1-AR response, and selective inhibition of PDE4 only potentiated the response to Glu, concomitant inhibition of both low Km cAMP PDEs was necessary to observe a substantial cAMP accumulation on 2-AR and PGE1-R stimulations. This indicates that both PDE3 and PDE4 are functionally associated with 2-AR and PGE1-R, but that one is sufficient to lower cAMP below the detection threshold of CNG channels. When examining the regulation of ICa,L by 2-AR, single inhibition of PDE3 or PDE4 potentiated the effect of ISO+CGP. This may reflect the 3- to 10-fold higher sensitivity of PKA versus CNG channels to cAMP. In contrast, single inhibition of PDE3 or PDE4 failed to reveal any effect of PGE1 on ICa,L. Although concomitant inhibition of both PDEs readily stimulate ICa,L in these cells and may mask a PGE1 effect (Figure 7D), the data suggest that PGE1-R lie in a distinct compartment from L-type Ca2+ channels.
On stimulation of 1-AR, 2-AR, and Glu-R, inhibition of PDE1–4 by IBMX had a similar effect on ICNG, as did concomitant PDE3 and PDE4 inhibition. This suggests that no other PDE, besides PDE3 and PDE4, played a significant role under our experimental conditions. However, a very different situation occurred with PGE1. In this case, inhibition of PDE1 through PDE4 by IBMX had a greater effect than concomitant inhibition of PDE3 and PDE4. Thus, other PDEs, most likely PDE1 or PDE2, may also regulate PGE1-R–mediated intracellular cAMP signals.
Although this study was performed on healthy cardiac myocytes, it is tempting to speculate that changes in the organization of the cAMP compartments might take place in pathological conditions, such as heart failure. Cardiac hypertrophy and failure are associated with a profound remodeling of the cAMP signaling pathway in cardiomyocytes.45 As for changes in PDE expression or activity, the results published so far are limited and contradictory. In rapid pacing-induced heart failure in dog, PDE3 mRNA and activity were decreased, whereas PDE4D was unchanged.46 In the same model, a modest reduction in total PDE activity was found in the left ventricular subendocardium but not in the epicardium, suggesting regional differences.47 In human, sarcoplasmic reticulum–associated PDE3 activity was shown to be unchanged in heart failure,43 although total PDE3 activity appears reduced and PDE3A protein expression downregulated in end-stage failing human heart.48 In rat, the expression and activity of PDE3 and PDE4 are strongly augmented in hypertension-induced hypertrophy.49 Finally, a recent study shows that PDE4D deficiency favors ryanodine receptor aberrant phosphorylation and promotes heart failure in mice.50 Although more work is needed to obtain a clear picture of the PDE rearrangement occurring in hypertrophy and heart failure, this study suggests that PDE alteration may affect cAMP compartmentation, leading to untargeted hormonal cAMP signals, aberrant phosphorylation of targets proteins and, in fine, contribute to cardiac dysfunction.
Acknowledgments
This work was supported by an INSERM fellowship (M.C.), Fondation de France (G.V.), French Ministry of Education and Research (F.R. and A.A.-G.), Association Franaise contre les Myopathies (F.R.), National Institutes of Health (RO1 HD 20788 to M.C. and K.H.), and by European Union Contract n°LSHM-CT-2005-018833/EUGeneHeart (R.F.). We thank Patrick Lechêne (INSERM U769) for skilful technical assistance and Dr Frank Lezoualc’h for critical reading of this manuscript.
Footnotes
Original received April 5, 2005; resubmission received January 12, 2006; revised resubmission received March 2, 2006; accepted March 9, 2006.
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INSERM U769 (F.R., A.A.-G., F.L., R.F., G.V.), Chatenay-Malabry, France
Univ-Paris-Sud (F.R., A.A.-G., F.L., R.F., G.V.), U769 Faculté de Pharmacie, Chatenay-Malabry, France
Division of Reproductive Biology (K.H., M.C.), Department of Gynecology and Obstetrics, Stanford University, California
Department of Pharmacology (D.M.F.C.), University of Cambridge, United Kingdom.
Abstract
Compartmentation of cAMP is thought to generate the specificity of Gs-coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca2+ current (ICa,L) on stimulation with 1-adrenergic receptors (1-ARs), 2-ARs, glucagon receptors (Glu-Rs) and prostaglandin E1 receptors (PGE1-Rs). All receptors but PGE1-R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide–gated (CNG) channels, stimulation of 1-AR and Glu-R increased cAMP, whereas 2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated 1-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE1-R and 2-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R, the measurements of ICa,L closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled 1-AR and 2-AR regulation of ICa,L, whereas only PDE4 controlled Glu-R regulation of ICa,L thus demonstrating that receptor–PDE coupling has functional implications downstream of cAMP. PGE1 had no effect on ICa,L even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to L-type Ca2+ channels. These results identify specific functional coupling of individual PDE families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.
Key Words: cAMP heart G-protein–coupled receptor phosphodiesterase
Introduction
Cardiac myocytes express a number of Gs-coupled receptors (GsPCRs) that raise intracellular cAMP levels and activate cAMP-dependent protein kinase (PKA) but exert different downstream effects. For instance, 1-adrenergic receptor (1-AR) stimulation produces a major and sustained increase in force of contraction, accelerates relaxation, and stimulates glycogen phosphorylase.1 2-AR stimulation also increases contractile force but does not activate glycogen phosphorylase2 and does not accelerate relaxation1,3 (see also ref. 2); glucagon receptor (Glu-R) stimulation activates phosphorylase and exerts positive inotropic and lusitropic effects, but the contractile effects fade with time.4 Finally, prostaglandin E1 (PGE1) has no effect on contractile activity or glycogen metabolism.5,6
Such observations led to the proposal that activation of different GsPCRs results in the accumulation of cAMP and phosphorylation of hormone target proteins in distinct compartments.7 The discovery of A-kinase anchoring proteins, responsible for the subcellular distribution of particulate PKA,8 and the development of new methodologies allowing to track local cAMP in living cells provided additional support for this concept.9–15
Because cAMP is a small and readily diffusible molecule, cAMP compartments can only exist under a restrictive set of conditions for the localization and activity of the different components of the cAMP signaling cascade, namely GsPCRs, adenylyl cyclases, PKA, and the cAMP phosphodiesterases (PDEs). Cardiac PDEs fall into four families: PDE1, which is activated by Ca2+/calmodulin; PDE2, which is stimulated by cGMP; PDE3, which is inhibited by cGMP; and PDE4. Whereas PDE1 and PDE2 can hydrolyze both cAMP and cGMP, PDE3 preferentially hydrolyzes cAMP, and PDE4 is specific for cAMP. Until now, only a limited number of studies have addressed directly the participation of PDEs to cAMP compartmentation and hormonal specificity in cardiac cells.9,10,12,15,16 In a recent study, we used recombinant cyclic nucleotide–gated (CNG) channels as cAMP biosensors in adult rat ventricular myocytes (ARVMs) to show that -adrenergic cAMP signals are localized by PKA-mediated activation of PDE3 or PDE4.13 However, PDE regulation of other hormonal cAMP signals is presently unknown. In the following, we address this point by comparing the cAMP signals generated by 1-AR, 2-AR, Glu-R, and PGE1-R, their regulation by individual PDE families, and the functional consequences on L-type Ca2+ channels.
Materials and Methods
Detailed methods are included in the online-only data supplement to this article, available at http://circres.ahajournals.org.
Total cAMP Determination
cAMP was measured by radioimmunossay after acetylation of the samples.20,21
PDE Assay
PDE activity was measured according to a modification of the two-step assay procedure method described by Thompson and Appleman17 with 1 μmol/L cAMP and 105 cpm [3H]-cAMP, as detailed previously.18
Electrophysiological Experiments
The whole-cell configuration of the patch-clamp technique was used to record the L-type calcium current (ICa,L) and the CNG current (ICNG). The extracellular Cs+-Ringer solution contained 1.8 mmol/L [Ca2+] and 1.8 mmol/L [Mg2+] for ICa,L recordings and nominal [Ca2+] and [Mg2+] plus 1 μmol/L nifedipine for ICNG recordings. Patch pipettes were filled with a Cs+ solution.
Data Analysis
ICa,L amplitude was measured as the difference between the peak inward current and the current at the end of the 400-ms duration pulse.19 ICNG amplitude was measured at the end of the 200-ms pulse. Capacitance and leak currents were not compensated. In 141 ARVMs, mean capacitance was 160.5±3.9 pF. ICNG density was calculated for each experiment as the ratio of current amplitude to cell capacitance. All the data are expressed as mean±SEM. When appropriate, the Student t test was used for statistical evaluation.
Results
PDE Activities in ARVMs
Figure 1 summarizes experiments in which the relative activity of PDE2, PDE3, and PDE4 in quiescent ARVMs was determined. Total cAMP hydrolytic activity was on average 109.8±16.6 pmol/min per mg protein (n=4) and was mainly attributable to PDE3 and PDE4, which represented 31% and 38% of the total, respectively. Under these assay conditions, PDE2 activity was minimal (Figure 1). Immunoprecipitation experiments showed that PDE4 activity is contributed by PDE4A, PDE4B, and PDE4D variants, whereas the PDE3A form is the predominant PDE3 expressed (data not shown). The relative activity of individual PDE families did not vary significantly after 24 and 48 hours of culture (data not shown). Moreover, the ratio of PDE3/PDE4 was similar to that measured in extracts of adult rat ventricle (data not shown). Thus, cultured ARVMs closely resemble the in vivo pattern of PDE expression.
Total cAMP Production in Response to Different GsPCRs in ARVMs
We next determined whether stimulation of different GsPCRs increased total cAMP in ARVMs (Figure 2). We used a combination of isoprenaline (ISO; 5 μmol/L) and the 2-AR antagonist ICI 118551 (ICI; 1 μmol/L) to stimulate 1-AR, a combination of ISO (5 μmol/L) in the presence of the 1-AR antagonist CGP 20712A (CGP; 1 μmol/L) to stimulate the 2-AR, glucagon (Glu; 1 μmol/L) to stimulate Glu-R, and PGE1 (1 μmol/L) to stimulate PGE1-R. Basal cAMP was more than doubled by ISO + ICI and Glu and increased by 70% by ISO + CGP. PGE1 had no effect on cAMP content under these experimental conditions. A 15-minute incubation with the broad-spectrum PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 100 μmol/L) increased the total cAMP content by 2-fold and dramatically potentiated the response to all agonists except PGE1. The effect of ISO + ICI and Glu were multiplied by 8 and 7, respectively, whereas that of ISO + CGP was increased 3-fold. cAMP content after PGE1 + IBMX was slightly increased when compared with IBMX alone, although it did not reach statistical significance.
Real-Time Monitoring of Subsarcolemmal cAMP Signals Elicited by Different GsPCRs in ARVMs
To further compare cAMP signals elicited by the different receptors, we used CNG channels that allow direct monitoring of cAMP variations beneath the plasma membrane in intact ARVMs.13 Figure 3 summarizes the results obtained with two CNGA2 mutants, which differ in their sensitivity to cAMP, E583M (EC50 for cAMP 10 μmol/L), and C460W/E583M (EC50 for cAMP 1 μmol/L).20 As depicted in Figure 3A, activation of 1-AR by a combination of ISO (5 μmol/L) and ICI (1 μmol/L) increased basal ICNG density 3-fold, whereas specific activation of 2-AR by ISO (5 μmol/L) plus CGP (1 μmol/L) had no effect. Similarly, activation of Glu-R by Glu (1 μmol/L) or PGE1-R by PGE1 (1 μmol/L) failed to activate ICNG (Figure 3A). These results indicate that cAMP concentration was below the activation threshold of E583M CNGA2 on stimulation of 2-AR, Glu-R, and PGE1-R. When using the C460W/E583M mutant (Figure 3B), activation of 1-AR increased basal ICNG density 6-fold. This effect was significantly higher (P<0.01) than that obtained in cells expressing E583M CNGA2, as expected from the relative affinities of the two channels toward cAMP. Interestingly, application of Glu now activated ICNG by 5-fold, but specific activation of 2-AR or PGE1-R still had no effect. Thus, the increased sensitivity of C460W/E583M CNGA2 allowed the detection of subsarcolemmal cAMP generated by activation of Glu-R but not by activation of 2-AR or PGE1-R.
Regulation of Subsarcolemmal cAMP Signals by PDEs
The above results suggest that cAMP signals elicited by the four different GsPCRs at the plasma membrane are not identical. We next investigated the contribution of PDEs to such specificity. Both CNGA2 mutants were used with essentially similar results, therefore the low-affinity E583M channel data are presented as supplemental Figures I and II. Preliminary experiments showed that IBMX (100 μmol/L) had no significant effect on ICNG from myocytes expressing E583M per C460W (n=6; data not shown). Figure 4A shows a typical experiment in which the amplitude of ICNG is depicted as a function of time, thus providing an instant readout of subsarcolemmal cAMP level in a single cardiac myocyte. 1-AR stimulation with ISO (5 μmol/L) + ICI (1 μmol/L) induced a substantial increase of ICNG, which was strongly potentiated by selective PDE3 inhibition with cilostamide (Cil; 1 μmol/L) or selective PDE4 inhibition with Ro 20-1724 (Ro; 10 μmol/L). Concomitant inhibition of all PDEs with IBMX (100 μmol/L) resulted in a similar potentiation of 1-AR stimulation to that obtained with selective PDE4 inhibition. At the end of each experiment, the cell was challenged with a saturating concentration (100 μmol/L) of the forskolin analog L-858051 to determine the maximal ICNG response. In Figure 4C and 4D, we investigated the role of PDEs in shaping the 2-AR cAMP response. As seen previously, application of ISO (5 μmol/L) + CGP (1 μmol/L) had no effect on ICNG. Selective inhibition of PDE3 during 2-AR stimulation with ISO + CGP induced a small and transient increase of ICNG. A somewhat stronger effect was observed on PDE4 inhibition, although it was also transient (Figure 4C). In contrast, concomitant inhibition of PDE3 and PDE4 unmasked a robust and sustained cAMP increase during 2-AR stimulation. These results indicate that PDE3 and PDE4 control cAMP elicited by 1-AR and 2-AR. Both PDEs are necessary to circumvent the cAMP rise elicited by 1-AR, whereas either PDE3 or PDE4 individually is sufficient to prevent cAMP to rise beneath membrane on 2-AR activation.
Figure 5A shows that inhibition of PDE3 had little effect on ICNG augmented by 1 μmol/L Glu, whereas a selective inhibition of PDE4 by Ro produced a strong stimulation of the current. Concomitant inhibition of both PDE3 and PDE4 by Cil + Ro, or of all PDEs by IBMX, resulted in a comparable effect as inhibition of PDE4 alone (Figure 5A and 5B). These results indicate that Glu-R cAMP signals at the membrane are controlled exclusively by PDE4. Although no increase in cAMP with PGE1 could be detected previously, we tested whether PDE inhibition could reveal a PGE1 effect on ICNG. As shown in Figure 5C, application of 1 μmol/L PGE1 did not affect ICNG. However, in the continuous presence of PGE1, simultaneous inhibition of PDE3 and PDE4 (by Cil + Ro) clearly activated the ICNG. IBMX was more efficient than Cil + Ro, indicating that other PDEs besides PDE3 and PDE4 might play a role under these conditions (Figure 5C). Thus, similar to 2-AR, cAMP mobilization by PGE1-R needs concomitant inhibition of PDE3 and PDE4 to be detected at the membrane. However, in contrast to 2-AR, other PDEs besides PDE3 and PDE4 regulate PGE1-R cAMP signals in cardiac cells.
Regulation of the L-Type Ca2+ Current by GsPCRs and PDEs
Collectively, the above results show that different PDEs are involved in the regulation of cAMP generated by distinct GsPCRs in cardiac myocytes. To evaluate the functional implication of these findings, we examined the regulation of a major sarcolemmal cAMP target, the L-type Ca2+ current (ICa,L), by GsPCRs and PDEs. The experiments were performed at 24 hours, as with CNG channel experiments. Selective inhibition of PDE3 by Cil (1 μmol/L) or PDE4 by Ro (10 μmol/L) had no effect on basal ICa,L (data not shown). Because the concentration of ISO (5 μmol/L) used in the 1-AR stimulation of ICNG produced a maximal stimulation of ICa,L (121.3±22.2% over control; n=9; data not shown), a lower dose of ISO (1 nmol/L, with 1 μmol/L ICI) was used in subsequent experiments to assess the contribution of PDE isoforms to the 1-AR regulation of ICa,L. In these conditions, selective inhibition of PDE3 by Cil or PDE4 by Ro resulted in a marked potentiation of the prestimulated ICa,L (Figure 6A and 6B). As shown in Figure 6C and 6D, 2-AR stimulation with ISO (5 μmol/L) and CGP (1 μmol/L) produced a small but significant stimulation of ICa,L. This effect could be further potentiated by PDE3 inhibition with Cil and more markedly by PDE4 blockade with Ro. These results indicate that PDE3, and more prominently PDE4, are integral components of ICa,L regulation by 1-AR and 2-AR. In another series of experiments, the regulation of ICa,L by Glu was investigated. At high (1 μmol/L) concentration, Glu maximally stimulated ICa,L, and addition of PDE inhibitors had no effect (data not shown). At the concentration of 10 nmol/L, Glu slightly decreased ICa,L, and PDE3 inhibition by Cil had no effect. However, application of Ro to block PDE4 clearly increased ICa,L (Figure 7A and 7B). Finally, as shown in Figure 7C and 7D, PGE1 had no effect on ICa,L, even when either PDE3 or PDE4 was blocked. However, concomitant inhibition of PDE3 and PDE4 increased ICa,L in the presence of PGE1, but this effect was not different from the effect of the inhibitors on the basal ICa,L (Figure 7D). These results confirm the specific and functional coupling of Glu-R to PDE4 and show that the cAMP compartment generated by activation of PGE1-R is not in contact with L-type Ca2+ channels.
Discussion
Although a number of recent studies have underscored the role of PDEs in tailoring hormonal cAMP signals,9–13,21–23 none have addressed directly whether distinct hormonal cAMP signals are controlled by different PDEs. To gain some insight into this question, we used complementary biochemical and electrophysiological techniques to resolve cAMP at the cellular, subsarcolemmal, and local levels. We show that four canonical GsPCRs expressed in ARVMs generate different cAMP signals because of a specific contribution of individual PDE families. For all receptors but PGE1-R, the nature of this functional coupling determines the regulation of a major downstream cAMP target, the L-type Ca2+ channels.
In agreement with previous studies, we found that PDE3 and PDE4 represent the major cAMP hydrolytic activities in rodent cardiomyocytes.12,13,24 Although there is a general agreement that 1-AR increases cAMP in adult rat cardiac preparations,3,25–27 the ability of 2-AR to do so is a matter of debate.3,25,26 Although Glu increases cAMP in various cardiac preparations,4 recent studies failed to detect it.27,28 Thus, we checked whether stimulation of the different receptors led to detectable increases in total cAMP in ARVMs. We found that stimulation of 1-AR, Glu-R, and to a lesser extent 2-AR, increased total cAMP content, and that IBMX greatly accentuated these effects. In contrast, PGE1 alone did not increase cAMP, although a small effect could be observed in the presence of IBMX. This result is at variance with those from Buxton and Brunton29 in rabbit cardiomyocytes, in which ISO and PGE1 similarly increased cAMP. However, in addition to species differences, the experimental conditions used here are different because PGE1 was used at a 10-fold lower concentration (1 versus 10 μmol/L) and application lasted 5-fold less time (3 versus 15 minutes).
We next used CNG channels to monitor cAMP triggered by distinct routes near the plasma membrane.11,13 We found that activation of 1-AR and Glu-R elicit readily detectable cAMP at the membrane, whereas 2-AR and PGE1-R do not. In heterologous expression systems and in rat olfactory epithelium, CNG channels associate preferentially with noncaveolar lipid rafts.30 If this holds true in ARVMs, failure to detect cAMP elicited by 2-AR and PGE1-R could be attributable to localization of these receptors in caveolae.31–34 However, this would not compromise the role of PDEs in preventing cAMP diffusion out of such membrane compartments. Indeed, we found that IBMX, at a 100 μmol/L concentration, increased the response of ICNG not only to 1-AR and Glu-R stimulation, but also revealed cAMP generated by 2-AR and PGE1-R stimulation. Given the Ki of IBMX for the different PDEs,35 at 100 μmol/L, this inhibitor should block PDE1, PDE2, PDE3, and PDE4 activities by, respectively, 98%, 93%, 97%, and 87%. To delineate the specific contribution of PDE4, we used Ro, which, at 10 μmol/L, should inhibit 71% of its activity without affecting the other PDEs.35 We found that PDE4 hydrolyzes cAMP produced by all the receptors tested, thus limiting PKA activation and Ca2+ channel phosphorylation on 1-AR, 2-AR, and Glu-R activation. These results are consistent with previous studies showing that PDE4 is critically involved in the regulation of ICa,L and contractility by catecholamines and Glu.21,28,36 PDE4 isoforms are likely to play this ubiquitous role because of their molecular diversity and specific subcellular localization.37–39 However, whether specific PDE4 isoforms are associated with different receptors remains unclear. Recent data in neonatal cardiomyocytes reported an important role for PDE4B2 in controlling norepinephrine-induced cAMP increase.12 However, in the same model, other studies suggest that PDE4D isoforms are preferentially associated with the 2-AR. For instance, PDE4D5 and, to a lesser extent, PDE4D3 are recruited to the 2-AR by -arrestin on ISO challenge.22,40 Moreover, Xiang et al16 showed that PDE4D selectively affects 2-AR versus 1-AR positive chronotropism. Whether these findings apply to ARVMs remains to be established.
Nearly complete (95%) and selective PDE3 inhibition should be achieved by 1 μmol/L Cil.35 Using this inhibitor, we found that PDE3 controls the cAMP signals generated by 1-AR, 2-AR, and PGE1-R but not Glu-R. However, on ICa,L response, PDE3 controlled the effects of 1-AR and 2-AR but had no impact on Glu-R or PGE1-R stimulation. We showed previously that PDE3 controlled cAMP diffusion and consequently ICa,L activation in cardiac myocytes subjected to -adrenergic stimulation.13,21,36,41,42 Our findings contrast with results obtained in neonatal rat ventricular myocytes in which PDE3 was found to have a moderate effect on 1-AR (with norepinephrine)–induced cAMP.12 Moreover, in right ventricular strips from adult rat, PDE3 inhibition had no effect on the contractile response to the 1-AR agonist dobutamine but potentiated that of Glu.27 In parallel cAMP assays, these authors found that PDE3 inhibition also had no effect on the response to dobutamine but revealed a substantial cAMP increase caused by Glu. Although obvious differences in developmental stage, cardiac territory, or rat strains might account for some of the discrepancies, one should emphasize that CNG channels provide a readout of subsarcolemmal cAMP, whereas other methods measure cytosolic or total cAMP. Thus, one could speculate that on 1-AR stimulation, PDE3 controls the membrane cAMP pool but not a cytosolic one possibly involved in contractility (sarcoplasmic reticulum-associated PDE3 for instance43). In the case of Glu-R, the opposite would be true: PDE3 would not control subsarcolemmal cAMP generated by Glu-R but would prevent the access of the second messenger to other, nonsarcolemmal inotropic targets. For such a hypothesis to be valid, one must further speculate either that totally segregated cAMP pathways are triggered by the different receptors (including PKA targets), or that differential regulation of PDE3 is triggered by receptor activation. The latter mechanism was found in frog heart but not in rat.44
Although inhibition of either PDE3 or PDE4 potentiated the 1-AR response, and selective inhibition of PDE4 only potentiated the response to Glu, concomitant inhibition of both low Km cAMP PDEs was necessary to observe a substantial cAMP accumulation on 2-AR and PGE1-R stimulations. This indicates that both PDE3 and PDE4 are functionally associated with 2-AR and PGE1-R, but that one is sufficient to lower cAMP below the detection threshold of CNG channels. When examining the regulation of ICa,L by 2-AR, single inhibition of PDE3 or PDE4 potentiated the effect of ISO+CGP. This may reflect the 3- to 10-fold higher sensitivity of PKA versus CNG channels to cAMP. In contrast, single inhibition of PDE3 or PDE4 failed to reveal any effect of PGE1 on ICa,L. Although concomitant inhibition of both PDEs readily stimulate ICa,L in these cells and may mask a PGE1 effect (Figure 7D), the data suggest that PGE1-R lie in a distinct compartment from L-type Ca2+ channels.
On stimulation of 1-AR, 2-AR, and Glu-R, inhibition of PDE1–4 by IBMX had a similar effect on ICNG, as did concomitant PDE3 and PDE4 inhibition. This suggests that no other PDE, besides PDE3 and PDE4, played a significant role under our experimental conditions. However, a very different situation occurred with PGE1. In this case, inhibition of PDE1 through PDE4 by IBMX had a greater effect than concomitant inhibition of PDE3 and PDE4. Thus, other PDEs, most likely PDE1 or PDE2, may also regulate PGE1-R–mediated intracellular cAMP signals.
Although this study was performed on healthy cardiac myocytes, it is tempting to speculate that changes in the organization of the cAMP compartments might take place in pathological conditions, such as heart failure. Cardiac hypertrophy and failure are associated with a profound remodeling of the cAMP signaling pathway in cardiomyocytes.45 As for changes in PDE expression or activity, the results published so far are limited and contradictory. In rapid pacing-induced heart failure in dog, PDE3 mRNA and activity were decreased, whereas PDE4D was unchanged.46 In the same model, a modest reduction in total PDE activity was found in the left ventricular subendocardium but not in the epicardium, suggesting regional differences.47 In human, sarcoplasmic reticulum–associated PDE3 activity was shown to be unchanged in heart failure,43 although total PDE3 activity appears reduced and PDE3A protein expression downregulated in end-stage failing human heart.48 In rat, the expression and activity of PDE3 and PDE4 are strongly augmented in hypertension-induced hypertrophy.49 Finally, a recent study shows that PDE4D deficiency favors ryanodine receptor aberrant phosphorylation and promotes heart failure in mice.50 Although more work is needed to obtain a clear picture of the PDE rearrangement occurring in hypertrophy and heart failure, this study suggests that PDE alteration may affect cAMP compartmentation, leading to untargeted hormonal cAMP signals, aberrant phosphorylation of targets proteins and, in fine, contribute to cardiac dysfunction.
Acknowledgments
This work was supported by an INSERM fellowship (M.C.), Fondation de France (G.V.), French Ministry of Education and Research (F.R. and A.A.-G.), Association Franaise contre les Myopathies (F.R.), National Institutes of Health (RO1 HD 20788 to M.C. and K.H.), and by European Union Contract n°LSHM-CT-2005-018833/EUGeneHeart (R.F.). We thank Patrick Lechêne (INSERM U769) for skilful technical assistance and Dr Frank Lezoualc’h for critical reading of this manuscript.
Footnotes
Original received April 5, 2005; resubmission received January 12, 2006; revised resubmission received March 2, 2006; accepted March 9, 2006.
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