Simultaneous measurements of intracellular cAMP and L-type Ca2+ current in single frog ventricular myocytes (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.)
Pflugers Archiv-european Journal of Physiology, 1997
It is widely believed that Ca release from the sarcoplasmic reticulum (SR) in heart muscle is due to ”Ca-induced Ca-release” (CICR), triggered by transmembrane Ca entry. However, in intact guinea-pig cells or cells dialysed with cAMP there may be an additional mechanism – SR release may be activated directly by membrane depolarisation without Ca entry. The first objective of the present study was to investigate whether this ”voltage-activated Ca release” (VACR) mechanism is present across species such as rabbit, rat and guinea-pig. The second objective was to characterise the dependence of a VACR mechanism on internal [cAMP]. Membrane current was measured with the whole-cell patch-clamp technique, intracellular [Ca] was monitored with Fura-2 (or a combination of Fluo-3/SNARF-1). Rapid changes of superfusate (within 100 ms) were made using a system which maintained cell temperature at 37°C. We used a train of conditioning pulses to ensure a standard SR load before each test pulse. In rabbit myocytes dialysed with 100 μM cAMP, 89.6 ± 7.0% of the control intracellular Ca (Cai) transient was still elicited by depolarisation during a switch to 5 mM Ni, which blocked pathways for Ca entry. This suggested that rabbit myocytes possess a VACR mechanism. The percentage of control Cai transient elicited by depolarisation in the presence of 5 mM Ni (i.e. magnitude of VACR) increased in a graded fashion with the pipette [cAMP] between zero and 100 μM. In rat myocytes dialysed with 50 μM cAMP, 64.4 ± 6.2% of SR release was activated by depolarisation in the presence of 5 mM Ni, suggesting the presence of a VACR mechanism. The extent to which VACR triggered SR release increased with the pipette [cAMP] between zero and 50 μM. In guinea-pig myocytes dialysed with 100 μM cAMP, 74.6 ± 3.6% of the control Cai transient was elicited by depolarisation in the presence of 5 mM Ni. The degree to which VACR triggered SR release was also graded with the pipette [cAMP] between zero and 100 μM. It therefore appears that each of the three species might possess a VACR mechanism which can be modulated by the internal [cAMP]. This may reflect an effect of cAMP to phosphorylate key proteins involved in excitation–contraction coupling. Under normal physiological conditions with a basal [cAMP] between 2 and 20 μM, VACR may play a role in triggering SR release. The role of VACR may increase under conditions which increase internal [cAMP].
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).
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.
Canadian Journal of Physiology and Pharmacology, 1984
The effects of procedures which diminish Ca2+ influx into myocardial cells on responses of isolated cardiac preparations to cAMP-independent histamine H1 receptor stimulation and cAMP-generating β-receptor stimulation were measured. The histamine response of guinea pig left atria, which appears to be primarily mediated by H1 receptors, was depressed to a greater extent than was the response of this preparation to isoproterenol by decreasing the extracellular Ca2+ concentration, and by the Ca2+ influx blocker D-600. Similarly, while the H1 agonist 2-pyridylethylamine dihydrochloride (PEA) produced increases in tension of a similar magnitude as the partial β-agonist salbutamol in both left atria and in papillary muscles, responses of both preparations to PEA were depressed to a significantly greater extent by decreasing the extracellular Ca2+ concentration than were responses to salbutamol. Overall, both the basal developed force of papillary muscles and the responses of these prepara...
Measurement of cAMP in the cardiac conduction system of rats
Journal of Histochemistry & Cytochemistry, 1995
To characterize differences in regional cAMP production in the cardiac conduction system, 18 rats were anesthetized with pentobarbital (65 mg/kg IP) and randomized into a control (n = 9) and a stimulated group (n = 9). The stimulated group received aminophylline (20 mg/kg SC) and isoproterenol (16 micrograms/kg SC). The concentration of cAMP in freeze-dried, micro dissected pieces (1-3 micrograms) of cardiac tissue was measured using a new microanalytical method. The cAMP contents in right atrium, atrioventricular node, His bundle, and left ventricle (fmol/microgram dry weight, mean +/- SE) were 38.9 +/- 2.5, 39.0 +/- 4.3, 46.4 +/- 6.1, and 41.4 +/- 3.3 in controls and 72.9 +/- 6.7, 86.1 +/- 2.9, 115.0 +/- 11.5, and 79.5 +/- 7.3 in the stimulated group, respectively. Basal cAMP levels were similar throughout the heart, whereas isoproterenol increased cAMP levels in all regions (p < 0.01). Furthermore, cAMP levels in His bundle, after isoproterenol, were higher than in any other r...
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.
The Journal of Physiology, 1994
1. An increase in the rate of stimulation induces an augmentation of L-type Ca2+ currents (Ica) and concomitant slowing of current decay in rat ventricular cells. This facilitation is quasi immediate (1-3 s), graded with the rate of stimulation, and occurs only from negative holding potentials. We investigated this effect using trains of stimulation at 1 Hz and the whole-cell patch-clamp technique (18-22°C). 2. The decay of Ica is normally bi-exponential and comprises fast and slow current components (Ica fe and Ica,sc respectively). Facilitation of Ica was observed only when ica,fc was predominant. 3. Facilitation developed during the run-up of 'Ca with the interconversion of Ica,sc into ica,fC and vanished during the run-down of 'Ca with the loss of 'ca,fc. Ni2" (300 /AM) and nifedipine (1 M) suppressed facilitation owing to the preferential inhibition of Ica,fc-4. Facilitation of Ica was not altered (when present) or favoured (when absent) by the cAMP-dependent phosphorylation of Ca2" channels promoted by isoprenaline or by intracellular application of cAMP or of the catalytic subunit of protein kinase A (C-sub). A similar effect was observed when the dihydropyridine agonist Bay K 8644 was applied. In both cases, facilitation was linked to a preferential increase of 'ca,fe 5. Following intracellular application of inhibitors of protein kinase A in combination with a non-hydrolysable ATP analogue, Ica consisted predominantly of Ica,sc and no facilitation was observed. The calmodulin antagonist naphthalenesulphonamide had no effect on facilitation. 6. When Bay K 8644 was applied in combination with isoprenaline, cAMP or C-sub, the decay of Ica was slowed with the predominant development of Ica,s'c and facilitation of Ica was nearly abolished. Facilitation also depended on extracellular Ca2+, and was suppressed when Ba2" replaced Ca2+ as the permeating ion. 7. When no EGTA was included in the patch pipette, facilitation was not further enhanced but a use-dependent decrease of Ica frequently occurred. When BAPTA was used in place of EGTA, the rate of inactivation of Ica was reduced and facilitation was abolished. 8. In conclusion, the facilitation of Ica that reflects a voltage-driven interconversion of Ica,fe into Icasc is also regulated by Ca2' and by cAMP-dependent phosphorylation. The presence of the gating pattern typified by 'Ca fe is required. Ca21 may exert its effect near the inner pore of the Ca21 channel protein and control the distribution between the closed states of the two gating pathways. Voltage-gated dihydropyridine-sensitive (L-type) Ca21 messengers is fundamental in cellular physiology (Trautwein channels are multimeric transmembrane proteins crucially & Kameyama, 1986; Tsien, Bean, Hess, Lansman, Nilius & involved in the development and control of heart Nowycky, 1986; Hartzell, 1988; Pelzer et al. 1990). L-type contractility (for review see Hartzell, 1988; Pelzer, Pelzer & Ca21 channels are also subject to modulation by a variety McDonald, 1990). Their opening is promoted primarily by of exogenous synthetic ligands, termed Ca2+channel membrane depolarization but their regulation by neuroantagonists/agonists, such as dihydropyridines (Tsien et al. transmitters, hormones, drugs and intracellular second 1986; Pelzer et al. 1990).
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.