Spatiotemporal Dynamics of -Adrenergic cAMP Signals and L-Type Ca2+ Channel Regulation in Adult Rat Ventricular Myocytes: Role of Phosphodiesterases (original) (raw)
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Journal of General Physiology, 2001
Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotidegated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.. To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by Ca 2 ϩ -calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced Ca 2 ϩ influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the K I of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high K m for cAMP, whereas PDE type I has a low K m for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability.
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).
Journal of Molecular and Cellular Cardiology, 2019
Cyclic AMP phosphodiesterases (PDEs) are important modulators of the cardiac response to βadrenergic receptor (β-AR) stimulation. PDE3 is classically considered as the major cardiac PDE in large mammals and human, while PDE4 is preponderant in rodents. However, it remains unclear whether PDE4 also plays a functional role in large mammals. Our purpose was to understand the role of PDE4 in cAMP hydrolysis and excitation-contraction coupling (ECC) in the pig heart, a relevant pre-clinical model. Methods and Results Real-time cAMP variations were measured in isolated adult pig right ventricular myocytes (APVMs) using a Förster resonance energy transfer (FRET) biosensor. ECC was investigated in APVMs loaded with Fura-2 and paced at 1 Hz allowing simultaneous measurement of intracellular Ca 2+ and sarcomere shortening. The expression of the different PDE4 isoforms was assessed by Western blot in pig right ventricle and APVMs. Similarly to PDE3 inhibition with cilostamide (Cil), PDE4 inhibition with Ro 20-1724 (Ro) increased cAMP levels and inotropy under basal conditions. PDE4 inhibition enhanced the effects of the non-selective β-AR agonist isoprenaline (Iso) and the effects of Cil, and increased spontaneous diastolic Ca 2+ waves (SCWs) in these conditions. PDE3A, PDE4A, PDE4B and PDE4D isoforms are expressed in pig ventricle.. In APVMs isolated from a porcine model of repaired tetralogy of Fallot which leads to right ventricular failure, PDE4 inhibition also exerts inotropic and pro-arrhythmic effects. Conclusions Our results show that PDE4 controls ECC in APVMs and suggest that PDE4 inhibitors exert inotropic and pro-arrhythmic effects upon PDE3 inhibition or β-AR stimulation in our pre-clinical model. Thus, PDE4 inhibitors should be used with caution in clinics as they may lead to arrhythmogenic events upon stress.
T-type channels are expressed weakly or not at all in adult rat chromaffin cells (RCCs) and there is contrasting evidence as to whether they play a functional role in catecholamine secretion. Here we show that 3-5 days after application of pCPT-cAMP, most RCCs grown in serum-free medium expressed a high density of low-voltage-activated T-type channels without altering the expression and characteristics of high-voltage-activated channels. The density of cAMP-recruited T-type channels increased with time and displayed the typical biophysical and pharmacological properties of low-voltage-activated Ca 2+ channels: (1) steep voltage-dependent activation from −50 mV in 10 mM Ca 2+ , (2) slow deactivation but fast and complete inactivation, (3) full inactivation following short conditioning prepulses to −30 mV, (4) effective block of Ca 2+ influx with 50 µM Ni 2+ , (5) comparable permeability to Ca 2+ and Ba 2+ , and (6) insensitivity to common Ca 2+ channel antagonists. and the competitive antagonist of cAMP binding to PKA, Rp-cAMPS, had weak or no effect on the action of pCPT-cAMP. In line with this, the selective Epac agonist 8CPT-2Me-cAMP nicely mimicked the action of pCPT-cAMP and isoprenaline, suggesting the existence of a dominant Epac-dependent recruitment of T-type channels in RCCs that may originate from the activation of β-adrenoceptors. Stimulation of β-adrenoceptors occurs autocrinally in RCCs and thus, the neosynthesis of low-voltageactivated channels may represent a new form of 'chromaffin cell plasticity', which contributes, by lowering the threshold of action potential firing, to increasing cell excitability and secretory activity during sustained sympathetic stimulation and/or increased catecholamine circulation.
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).
British Journal of Pharmacology, 1999
The eects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I Ca) and intracellular cyclic AMP concentration ([cAMP] i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the dierent cardiac PDE subtypes was con®rmed by RT ± PCR. 2 IBMX (100 mM), a broad-spectrum PDE inhibitor, increased basal I Ca by 120% and [cAMP] i by 70%, similarly to a saturating concentration of the b-adrenoceptor agonist isoprenaline (1 mM). However, MIMX (1 mM), a PDE1 inhibitor, EHNA (10 mM), a PDE2 inhibitor, cilostamide (0.1 mM), a PDE3 inhibitor, or Ro 20-1724 (0.1 mM), a PDE4 inhibitor, had no eect on basal I Ca and little stimulatory eects on [cAMP] i (20 ± 30%). 3 Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any eect on I Ca or [cAMP] i. While all combinations tested signi®cantly increased [cAMP] i (40 ± 50%), only cilostamide (0.1 mM)+Ro20-1724 (0.1 mM) produced a signi®cant stimulation of I Ca (50%). Addition of EHNA (10 mM) to this mix increased I Ca to 110% and [cAMP] i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 mM) or isoprenaline (1 mM). 4 When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I Ca by (&40% and had negligible eect on [cAMP] i , each selective PDE inhibitor induced a clear stimulation of [cAMP] i and an additional increase in I Ca. Maximal eects on I Ca were &8% for MIMX (3 mM), &20% for EHNA (1 ± 3 mM), &30% for cilostamide (0.3 ± 1 mM) and &50% for Ro20-1724 (0.1 mM). 5 Our results demonstrate that PDE1-4 subtypes regulate I Ca in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal I Ca , all four PDE subtypes determine the response of I Ca to a stimulus activating cyclic AMP production, with the rank order of potency PDE44PDE34PDE24PDE1.
Journal of Biological Chemistry, 2005
Three isoforms of PDE3 (cGMP-inhibited) cyclic nucleotide phosphodiesterase regulate cAMP content in different intracellular compartments of cardiac myocytes in response to different signals. We characterized the catalytic activity and inhibitor sensitivity of these isoforms by using recombinant proteins. We determined their contribution to cAMP hydrolysis in cytosolic and microsomal fractions of human myocardium at 0.1 and 1.0 M cAMP in the absence and presence of Ca 2؉ /calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar K m and k cat values for cAMP and are equal in their sensitivities to inhibition by cGMP and cilostazol. In microsomes, PDE3A-136, PDE3A-118, and PDE3A-94 comprise the majority of cAMP hydrolytic activity under all conditions. In cytosolic fractions, PDE3A-118 and PDE3A-94 comprise >50% of the cAMP hydrolytic activity at 0.1 M cAMP, in the absence of Ca 2؉ /calmodulin. At 1.0 M cAMP, in the presence of Ca 2؉ /calmodulin, activation of Ca 2؉ /calmodulin-activated (PDE1) and other non-PDE3 phosphodiesterases reduces their contribution to <20% of cAMP hydrolytic activity. cGMP inhibits cAMP hydrolysis in microsomal fractions by inhibiting PDE3 and in cytosolic fractions by inhibiting both PDE3 and PDE1. These findings indicate that the contribution of PDE3 isoforms to the regulation of cAMP hydrolysis in intracellular compartments of human myocardium and the effects of PDE3 inhibition on cAMP hydrolysis in these compartments are highly dependent on intracellular [Ca 2؉ ] and [cAMP], which are lower in failing hearts than in normal hearts. cGMP may amplify cAMP-mediated signaling in intracellular compartments of human myocardium by PDE3-dependent and PDE3independent mechanisms.
Modified cAMP Derivatives: Powerful Tools in Heart Research
Current Medicinal Chemistry, 2011
Receptor-mediated changes in intracellular cyclic AMP concentration play critical role in the autonomic control of the heart, including regulation of a variety of ion channels via mechanisms involving protein kinase A, EPAC, or direct actions on cyclic nucleotide gated ion channels. In case of any ion channel, the actual signal transduction cascade can be identified by using properly modified cAMP derivatives with altered binding and activating properties. In this study we focus to structural modifications of cAMP resulting in specific activator and blocking effects on PKA or EPAC. Involvement of the cAMP-dependent signal transduction pathway in controlling rapid delayed rectifier K + current was studied in canine ventricular myocytes using these specific cAMP analogues. Adrenergic stimulation increased the density of IKr in canine ventricular cells, which effect was mediated by a PKA-dependent but EPAC-independent pathway. It was also shown that intracellular application of large concentrations of cAMP failed to fully activate PKA comparing to the effect of isoproterenol, forskolin, or PDE-resistant cAMP derivatives. This difference was fully abolished following inhibition of phosphodiesterase by IBMX. These results are in line with the concept of compartmentalized release, action, and degradation of cAMP within signalosomes.