Cyclic Nucleotide Phosphodiesterase PDE1C1 in Human Cardiac Myocytes (original) (raw)
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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.
Molecular cloning and expression of human myocardial cGMP-inhibited cAMP phosphodiesterase
Proceedings of the National Academy of Sciences, 1992
We have cloned a cDNA for a myocardial cGMP-inhibited cAMP phosphodiesterase (cGI PDE) from a human heart cDNA library in A Zap II. The open reading frame [3.5 kilobases (kb)] of cDNA clone n.13.2 (7.7 kb) Abbreviations: cGI PDE, cGMP-inhibited cAMP phosphodiesterase; GST, glutathione S-transferase; IPTG, isopropyl f3-Dthiogalactopyranoside; SR, sarcoplasmic reticulum.
Journal of Clinical Investigation, 1997
known inhibitor of adenosine deaminase. Recently, EHNA was shown to block the activity of purified soluble cGMPstimulated phosphodiesterase (PDE2) from frog, human, and porcine heart with an apparent K i value of ف 1 M and with negligible effects on Ca 2 ϩ /calmodulin PDE (PDE1), cGMP-inhibited PDE (PDE3), and low K m cAMP-specific PDE (PDE4) (Méry, P.F., C. Pavoine, F. Pecker, and R. Fischmeister. 1995. Mol. Pharmacol. 48:121-130; Podzuweit, T., P. Nennstiel, and A. Muller. 1995. Cell. Signalling. 7:733-738). To investigate the role of PDE2 in the regulation of cardiac L-type Ca 2 ϩ current (I Ca ), we have examined the effect of EHNA on I Ca in freshly isolated human atrial myocytes. Extracellular application of 0.1-10 M EHNA induced an increase in the amplitude of basal I Ca ( ف 80% at 1 M) without modification of the current-voltage or inactivation curves. The maximal stimulatory effect of EHNA on I Ca was comparable in amplitude with the maximal effect of isoprenaline (1 M), and the two effects were not additive. The effect of EHNA was not a result of adenosine deaminase inhibition, since 2 Ј -deoxycoformycin ( , another adenosine deaminase inhibitor with no effect on PDE2, or adenosine (1-10 M) did not increase I Ca . In the absence of intracellular GTP, the substrate of guanylyl cyclase, EHNA did not increase I Ca . However, under similar conditions, intracellular perfusion with 0.5 M cGMP produced an 80% increase in I Ca . As opposed to human cardiomyocytes, EHNA (1-10 M) did not modify I Ca in isolated rat ventricular and atrial myocytes. We conclude that basal I Ca is controlled by PDE2 activity in human atrial myocytes. Both PDE2 and PDE3 may contribute to keep the cyclic nu-cleotides concentrations at minimum in the absence of adenylyl and/or guanylyl cyclase stimulation. ( J. Clin. Invest.
Biochemical and Biophysical Research Communications, 1996
The expression and activity of low Km, cGMP-inhibited cAMP phosphodiesterase (PDE3)4 were examined in rabbit and canine cardiac and skeletal muscle. In cardiac muscle, a cDNA probe whose sequence encompasses the catalytic domain of human myocardial PDE3 (PDE3A) hybridized predominantly with a 7.2-7.4 kb mRNA. No hybridization was observed in preparations from slow or fast twitch skeletal muscle. Likewise, PDE3 activity was present in cytosolic and microsomal fractions of cardiac muscle but was absent from cytosolic and microsomal fractions of slow twitch and fast twitch skeletal muscle. These results, which demonstrate the absence of PDE3 from slow and fast twitch mammalian skeletal muscle, further delineate the differences in beta-adrenergic receptor-mediated signal transduction pathways in cardiac and skeletal muscle.
2016
Rationale: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs). Objective: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments. Methods and Results: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation...
Cellular Signalling, 2008
Transcripts for the PDE4A10 cyclic AMP phosphodiesterase isoform are present in a wide variety of rat tissues including the heart. Sequence comparisons between the putative human and mouse promoters revealed a number of conserved regions including both an Sp1 and a CREB-binding site. The putative mouse PDE4A10 promoter was amplified from genomic DNA and sub-cloned into a luciferase reporter vector for investigation of activity in neonatal cardiac myocytes. Transfection with this construct identified a high level of luciferase expression in neonatal cardiac myocytes. Surprisingly, this activity was down-regulated by elevation of intracellular cAMP through a process involving PKA, but not EPAC, signalling. Such inhibition of the rodent PDE4A10 promoter activity in response to elevated cAMP levels is in contrast to the PDE4 promoters so far described. Site-directed mutagenesis revealed that the Sp1 binding site at promoter position −348 to −336 is responsible for the basal constitutive expression of murine PDE4A10. The conserved CREB-binding motif at position −370 to −363 also contributes to basal promoter activity but does not in itself confer cAMP inhibition upon the PDE4A10 promoter. EMSA analysis confirmed the authenticity of CREB and Sp1 binding sites. The transcriptional start site was identified to be an adenine residue at position −55 in the mouse PDE4A10 promoter. We present evidence that this novel down-regulation of PDE4A10 is mediated by the transcription factor ICER in a PKA dependent manner. The pool of cAMP in cardiac myocytes that down-regulates PDE4A10 is regulated by β-adrenoceptor coupled adenylyl cyclase activity and via hydrolysis determined predominantly by the action of PDE4 (cAMP phosphodiesterase-4) and not PDE3 (cAMP phosphodiesterase-3). We suggest that increased cAMP may remodel cAMP-mediated signalling events by not only increasing the expression of specific PDE4 cAMP phosphodiesterases but also by down-regulating specific isoforms, such as is shown here for PDE4A10 in cardiac myocytes.
Biochimica et Biophysica Acta (BBA) - General Subjects, 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.
PLoS ONE, 2010
Left ventricular hypertrophy leads to heart failure and represents a high risk leading to premature death. Cyclic nucleotides (cAMP and cGMP) play a major role in heart contractility and cyclic nucleotide phosphodiesterases (PDEs) are involved in different stages of advanced cardiac diseases. We have investigated their contributions in the very initial stages of left ventricular hypertrophy development. Wistar male rats were treated over two weeks by chronic infusion of angiotensin II using osmotic mini-pumps. Left cardiac ventricles were used as total homogenates for analysis. PDE1 to PDE5 specific activities and protein and mRNA expressions were explored. Rats developed arterial hypertension associated with a slight cardiac hypertrophy (+24%). cAMP-PDE4 activity was specifically increased while cGMP-PDE activities were broadly increased (+130% for PDE1; +76% for PDE2; +113% for PDE5) and associated with increased expressions for PDE1A, PDE1C and PDE5A. The cGMP-PDE1 activation by Ca 2+ /CaM was reduced. BNP expression was increased by 3.5-fold, while NOX2 expression was reduced by 66% and AMP kinase activation was increased by 64%. In early cardiac hypertrophy induced by angiotensin II, all specific PDE activities in left cardiac ventricles were increased, favoring an increase in cGMP hydrolysis by PDE1, PDE2 and PDE5. Increased cAMP hydrolysis was related to PDE4. We observed the establishment of two cardioprotective mechanisms and we suggest that these mechanisms could lead to increase intracellular cGMP: i) increased expression of BNP could increase ''particulate'' cGMP pool; ii) increased activation of AMPK, subsequent to increase in PDE4 activity and 59AMP generation, could elevate ''soluble'' cGMP pool by enhancing NO bioavailability through NOX2 downregulation. More studies are needed to support these assumptions. Nevertheless, our results suggest a potential link between PDE4 and AMPK/NOX2 and they point out that cGMP-PDEs, especially PDE1 and PDE2, may be interesting therapeutic targets in preventing cardiac hypertrophy.