Molecular cloning and expression of human myocardial cGMP-inhibited cAMP phosphodiesterase (original) (raw)
Related papers
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.
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.
Cyclic Nucleotide Phosphodiesterase PDE1C1 in Human Cardiac Myocytes
Journal of Biological Chemistry, 2007
Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K m values of ϳ1 M and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 M, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP-and Ca 2؉mediated signaling in these cells.
Toward the identification of the cardiac cGMP inhibited-phosphodiesterase catalytic site
Journal of computer-aided molecular design, 1998
Cyclic nucleotide phosphodiesteras (PDEs) comprise a complex group of enzymes; five major PDE families or classes with distinctive properties have been identified. Among these a great deal of interest has recently been focused on the so called cGMP-inhibited low K(m) cAMP phosphodiesterase (cGI PDE) or PDE III. A number of positive inotropic agents, including the well-known milrinone, display a specific inhibition of PDE III as primary mechanism of action. Recent studies have been carried out to develop a pharmacophore model of the PDE III active site. We therefore performed molecular modelling and 3D-SAR studies so as to better define structural requirements for potent and selective enzymatic inhibition. The DISCO (DIStance COmparison) strategy has been applied on a set of compounds taken from literature and a milrinone analogue previously synthesized by us, all of which are characterized by a marked inotropic effect but with varying degrees of enzyme selectivity. A common pharmaco...
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1994
The human platelet cilostamide- and cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) was rapidly purified approximately 19,000-fold to apparent homogeneity using single step affinity chromatography on the isothiocyanate derivative of cilostamide coupled to aminoethyl agarose. Within 24 h, 30 micrograms of enzyme protein was obtained from 20 ml of packed platelets. Vmax for cAMP and cGMP was 6.1 and 0.9 mumol/min per mg protein, respectively. Several polypeptides (110/105, 79, 62, 55/53 kDa) were identified after SDS-PAGE, all of which were immunologically related to cGI-PDE and represented approx. 5, 20, 50 and 20% of the total protein, respectively. Limited proteolysis of the cGI-PDE with chymotrypsin produced a major fragment of approximately 47 kDa (and at least two smaller peptides) with catalytic activity and sensitivity to cGMP and OPC 3911 similar to controls. Phosphorylation of the cGI-PDE by cAMP-dependent protein kinase (A-kinase) resulted in maximal incorporation of 0.6-1.8 mol of 32P/mol 110/105 and 79 kDa polypeptides; much lower and variable amounts of phosphate were incorporated into the 62 and 55/53 kDa polypeptides. After digestion of cGI-PDE with several proteinases a number of peptides were isolated and sequenced. Most of the peptide sequences obtained could be aligned within the carboxy terminal domain of the deduced sequence of the human cardiac cGI-PDE. These and other results suggest that the subunit size of the intact platelet cGI-PDE is 110 kDa and that proteolytic fragments of 79, 62 and 55/53 kDa are produced during purification. The smaller fragments (62 and 55/53 kDa) contain the catalytic domain; the larger fragments (110 and 79 kDa) also contain the regulatory domain with phosphorylation sites for A-kinase.
Genomics, 1996
Two distinct PDE3 [cyclic GMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE)] isoforms, cGIP1 and cGIP2, have been identified. Here we report cloning of the cDNA and gene encoding human (H)cGIP1 (classified as PDE3B). The cDNA encodes a protein of 1112 amino acids (approximately 123 kDa). Northern blots indicate that its mRNA is expressed in several adipose tissue depots. The human PDE3B gene is composed of 16 exons spanning more than 114 kb and was localized to chromosome 11p15 by in situ hybridization. Exon/intron boundaries were determined, and genetic polymorphism, confirmed by single-strand conformational polymorphism of DNA from 25 healthy subjects, was demonstrated in exon 4 at nucleotide 1389 (A/G). Two polymorphic dinucleotide repeat sequences were identified in introns 5 and 12.
Biochemistry, 1998
The mechanism of discrimination between cGMP and cAMP in the catalytic site of the cGMPbinding cGMP-specific phosphodiesterase (BTPDE5A1 or PDE5) has been investigated. A hydropathy analysis of the catalytic domains of different families of PDEs suggests that substrate selectivity of PDEs could result from the pattern of hydrophobic/hydrophilic residues in a short segment surrounding a conserved Glu that has been shown to be critical for cGMP binding in the catalytic domain of PDE5. This implies that the substrate selectivity of PDE5 could be altered by replacing the residues within this segment that are conserved in cGMP-specific PDEs with the conserved residues in the corresponding positions of cAMPspecific PDEs. The A769T/L771R, W762L/Q765Y, and W762L/Q765Y/A769T/L771R mutant PDE5s were expressed in High Five cells, and their substrate selectivities were compared with that of wild-type PDE5. The results indicate that the substrate-binding site of PDE5 contains positive elements for accommodating cGMP, as well as negative elements that discriminate against binding of cAMP, and that the cGMP/cAMP selectivity of PDE5 can be shifted 106-fold by substituting four residues of PDE5 with the residues in the corresponding positions of PDE4. H]cAMP were purchased from Amersham Corp. cGMP, cAMP, histone VIII-S, C. atrox snake venom, 3-isobutyl-1-methylxanthine (IBMX), and zaprinast were obtained from Sigma. Hydroxyapatite was from Bio-Rad.