Modified cAMP Derivatives: Powerful Tools in Heart Research (original) (raw)
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Circulation Research, 2008
Steady-state activation of cardiac -adrenergic receptors leads to an intracellular compartmentation of cAMP resulting from localized cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the time course of the cAMP changes in the different compartments, brief (15 seconds) pulses of isoprenaline (100 nmol/L) were applied to adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes beneath the membrane using engineered cyclic nucleotide-gated channels and within the cytosol with the fluorescence resonance energy transfer-based sensor, Epac2-camps. cAMP kinetics in the two compartments were compared to the time course of the L-type Ca 2ϩ channel current (I Ca,L ) amplitude. The onset and recovery of cAMP transients were, respectively, 30% and 50% faster at the plasma membrane than in the cytosol, in agreement with a rapid production and degradation of the second messenger at the plasma membrane and a restricted diffusion of cAMP to the cytosol. I Ca,L amplitude increased twice slower than cAMP at the membrane, and the current remained elevated for Ϸ5 minutes after cAMP had already returned to basal level, indicating that cAMP changes are not rate-limiting in channel phosphorylation/dephosphorylation. Inhibition of PDE4 (with 10 mol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 mol/L cilostamide) had a minor effect only on subsarcolemmal cAMP. However, when both PDE3 and PDE4 were inhibited, or when all PDEs were blocked using 3-isobutyl-l-methylxanthine (300 mol/L), cAMP signals and I Ca,L declined with a time constant Ͼ10 minutes. cAMP-dependent protein kinase inhibition with protein kinase inhibitor produced a similar effect as a partial inhibition of PDE4 on the cytosolic cAMP transient. Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed to isoprenaline showed a pronounced (up to Ϸ50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct -adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4. (Circ Res. 2008;102:1091-1100.)
Journal of Biological Chemistry, 2014
Background: Transient  1 AR activation remains at odds with long lasting cellular and physiological responses. Results: The agonist-occupied  1 AR continuously signals to adenylyl cyclase (AC) to produce cAMP in both cardiac myocytes and neurons for more than 8 h, which is masked by receptor-associated PDE4D8. Conclusion: Stimulation of  1 AR induces long-lasting cAMP production in the heart for ligand-induced physiological responses. Significance: We show a novel mechanism to understand persistent  1 AR signaling in the heart. Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRETbased biosensor and myocyte contraction assay, we show that the catecholamine-activated  1 adrenergic receptor ( 1 AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t1 ⁄ 2 of 3.9 h) in cardiac myocytes. However, the  1 AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation.  1 AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the  1 AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.
The Journal of Physiology, 2001
The beat rate and force of contraction of the heart are under the dual control of the sympathetic and parasympathetic systems . Both systems control reciprocally the synthesis of cAMP and, hence, the activity of cAMP-dependent protein kinase (PKA) and the phosphorylation of a multitude of regulatory proteins, including the L-type Ca 2+ channel for review see Hartzell, 1988;, phospholamban , the sarcoplasmic reticulum Ca 2+ release channel and contractile proteins . At the level of a single cardiac myocyte, a variation of intracellular cAMP level ([cAMP] i ) can be assessed indirectly by measuring the changes in the activity of a PKA substrate protein. Measurement of L-type Ca 2+ channel current (I Ca ) variations in response to cAMPelevating agents is a prototypical example of such an approach ; for review see Hartzell, 1. The cAMP fluorescent probe FlCRhR was used to monitor changes in intracellular cAMP concentration ([cAMP] i ) in isolated frog ventricular myocytes. The probe was introduced into the cell through a patch pipette which allowed simultaneous recording of the whole-cell L-type Ca 2+ current (I Ca ). Ratiometric imaging was used to monitor [cAMP] i changes in response to the b-adrenergic agonist isoprenaline (ISO) or to the direct adenylyl cyclase activator forskolin (FSK).
Abstract RATIONALE: β-Adrenoceptor activation contributes to sudden death risk in heart failure. Chronic β-adrenergic stimulation, as occurs in patients with heart failure, causes potentially arrhythmogenic reductions in slow delayed-rectifier K(+) current (IKs). OBJECTIVE: To assess the molecular mechanisms of IKs downregulation caused by chronic β-adrenergic activation, particularly the role of exchange protein directly activated by cAMP (Epac). METHODS AND RESULTS: Isolated guinea pig left ventricular cardiomyocytes were incubated in primary culture and exposed to isoproterenol (1 μmol/L) or vehicle for 30 hours. Sustained isoproterenol exposure decreased IKs density (whole cell patch clamp) by 58% (P<0.0001), with corresponding decreases in potassium voltage-gated channel subfamily E member 1 (KCNE1) mRNA and membrane protein expression (by 45% and 51%, respectively). Potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) mRNA expression was unchanged. The β1-adrenoceptor antagonist 1-[2-((3-Carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol dihydrochloride (CGP-20712A) prevented isoproterenol-induced IKs downregulation, whereas the β2-antagonist ICI-118551 had no effect. The selective Epac activator 8-pCPT-2'-O-Me-cAMP decreased IKs density to an extent similar to isoproterenol exposure, and adenoviral-mediated knockdown of Epac1 prevented isoproterenol-induced IKs/KCNE1 downregulation. In contrast, protein kinase A inhibition with a cell-permeable highly selective peptide blocker did not affect IKs downregulation. 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate-AM acetoxymethyl ester (BAPTA-AM), cyclosporine, and inhibitor of nuclear factor of activated T cell (NFAT)-calcineurin association-6 (INCA6) prevented IKs reduction by isoproterenol and INCA6 suppressed isoproterenol-induced KCNE1 downregulation, consistent with signal-transduction via the Ca(2+)/calcineurin/NFAT pathway. Isoproterenol induced nuclear NFATc3/c4 translocation (immunofluorescence), which was suppressed by Epac1 knockdown. Chronic in vivo administration of isoproterenol to guinea pigs reduced IKs density and KCNE1 mRNA and protein expression while inducing cardiac dysfunction and action potential prolongation. Selective in vivo activation of Epac via sp-8-pCPT-2'-O-Me-cAMP infusion decreased IKs density and KCNE1 mRNA/protein expression. CONCLUSIONS: Prolonged β1-adrenoceptor stimulation suppresses IKs by downregulating KCNE1 mRNA and protein via Epac-mediated Ca(2+)/calcineurin/NFAT signaling. These results provide new insights into the molecular basis of K(+) channel remodeling under sustained adrenergic stimulation.
Pfl�gers Archiv European Journal of Physiology, 1998
The β-agonist isoproterenol (ISO) reduces the Na/K pump current (I p ) via β-adrenergic receptors when the intracellular calcium concentration ([Ca 2+ ] i ) is below 150 nM [8]. In the present study, the intracellular signaling pathway was investigated with whole-cell patch-clamp of isolated guinea pig ventricular myocytes. The inhibitory effect of ISO could be mimicked by external application of the membrane-permeant cAMP analog chlorophenylthio-cAMP (0.5 mM), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX, 100 µM), or the adenylyl cyclase activator forskolin (50 µM). Intracellular application of the synthetic peptide inhibitor of protein kinase A (PKA), PKI (5 µM), prevented the effect of ISO. These results suggest that the inhibitory effect of ISO on I p is mediated via a phosphorylation step induced by a cAMP-dependent PKA pathway. Neither the non-specific protein kinase inhibitor H 7 (100 µM) nor the protein phosphatase inhibitor calyculin A (0.5 µM) had any effect on I p in the absence of ISO. However, H 7 could increase I p and calyculin A could reduce it in the presence of ISO (1 µM and 12 nM respectively). These results indicate that there is a low basal level of phosphorylation which makes the effects of H 7 and calyculin A difficult to detect in the absence of an ISO-induced increase in phosphorylation level.
Cardiac myocyte–secreted cAMP exerts paracrine action via adenosine receptor activation
Journal of Clinical Investigation, 2014
These data suggest that extracellular cAMP protects the heart from adrenergically induced hypertrophy and fibrosis and that this is mediated through its metabolite adenosine acting mainly on CM A 1 R and CF A 2 R. Exogenous cAMP confers diametral changes of intracellular cAMP in CMs and CFs. A quantitative PCR (qPCR) analysis of adenosine receptor expression in primary rat neonatal CMs or CFs (purity of isolates >90% each) revealed that the A 1 R subtype is virtually the exclusive adenosine receptor in CMs (Figure 2A), whereas A 2A R and A 2B R dominate in CFs (ref. 19 and Figure 2E). A 3 R expression was marginal in CMs and CFs (Figure 2, A and E). Similar results were obtained from adult mouse CMs (AMCMs) or adult mouse CFs (AMCFs) (Supplemental Figure 5, A and B). We then determined in what way distinct adenosine receptor profiles of cardiac cells affect their response to exogenously added cAMP. Since adenosine receptors couple to G s that regulate the activity of adenylyl cyclase, quantitation of intracellular cAMP was chosen to determine the effects extracellular cAMP exerts through adenosine. Isolated CMs or CFs were infected with an adenoviral vector for the expression of a fluorescence resonance energy transfer-based (FRET-based) cAMP sensor, and FRET was measured under various conditions in real time. Intriguingly, intracellular cAMP formation in CMs induced by β-adrenergic stimulation (Iso) was efficiently prevented by exogenous cAMP (Figure 2, B-D, and Supplemental Video 1). This effect was blocked by an A 1 R antagonist (DPCPX, 100 nM) and by the nonspecific adenosine receptor antagonist (DPSPX, 10 nM), but not by antagonists against A 2A R (SCH-442416, 100 nM), A 2B R (PSB-1115, 500 nM), or A 3 R (VUF 5574, 100 nM) (Figure 2, B-D). Next, this experimental setup was applied to CFs. As above, β-adrenergic stimulation by Iso increased intracellular cAMP, but, in contrast to CMs, exogenous cAMP enhanced formation of intracellular cAMP (Figure 2, F-H, Supplemental Video 2, and Supplemental Figure 6A for concentration response curves).
Pflugers Archiv-european Journal of Physiology, 1990
Voltage-gated Ca channels have been reported to be regulated by membrane potential, phosphorylation and binding of specific agonists or antagonists such as dihydropyridines. We report here evidence that cyclic AMP (cAMP) modulates the activation of Ca-channel current by the dihydropyridine agonist Bay K 8644. Bay K 8644 (racemate) alone induces a primary voltage-dependent, potentiating effect on peak current amplitude and accelerates the current decay. In contrast, in the presence of cAMP activators, we observed a striking slowing of the decay in addition to the increase in peak current. The agonist (−)-Bay K 8644, but not the antagonist (+)-Bay K 8644, when applied in combination with cAMP, forskolin or isoproterenol, mimics the effect of the racemate. We have interpreted the results presented here in respect of a cAMP-dependent modulation of Bay K 8644 effects on cardiac Ca-channel currents. It may open the new perspective that dephosphorylated and phosphorylated Ca channels have distinct pharmacology.
Biophysical Journal, 2006
The space between the t-tubule invagination and the sarcoplasmic reticulum (SR) membrane, the dyad, in ventricular myocytes has been predicted to experience very high [Ca2+] for short periods of time during a Ca2+ transient. The dyadic space accommodates many protein kinases responsible for the regulation of Ca2+ handling proteins of the cell. We show in vitro that cAMP-dependent protein kinase (PKA) is inhibited by high [Ca2+] through a shift in the ratio of CaATP/MgATP toward CaATP. We further generate a three-dimensional mathematical model of Ca2+ and ATP diffusion within dyad. We use this model to predict the extent to which PKA would be inhibited by an increased CaATP/MgATP ratio during a Ca2+ transient in the dyad in vivo. Our results suggest that under normal physiological conditions a myocyte paced at 1 Hz would experience up to 55% inhibition of PKA within the cardiac dyad, with inhibition averaging 5% throughout the transient, an effect which becomes more pronounced as the myocyte contractile frequency increases (at 7 Hz, PKA inhibition averages 28% across the dyad throughout the duration of a Ca2+ transient).