The Selective Phosphodiesterase 4 Inhibitor Roflumilast and Phosphodiesterase 3/4 Inhibitor Pumafentrine Reduce Clinical Score and TNF Expression in Experimental Colitis in Mice
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Objective The specific inhibition of phosphodiesterase (PDE)4 and dual inhibition of PDE3 and PDE4 has been shown to decrease inflammation by suppression of pro-inflammatory cytokine synthesis. We examined the effect of roflumilast, a selective PDE4 inhibitor marketed for severe COPD, and the investigational compound pumafentrine, a dual PDE3/PDE4 inhibitor, in the preventive dextran sodium sulfate (DSS)-induced colitis model. Methods The clinical score, colon length, histologic score and colon cytokine production from mice with DSS-induced colitis (3.5% DSS in drinking water for 11 days) receiving either roflumilast (1 or 5 mg/kg body weight/d p.o.) or pumafentrine (1.5 or 5 mg/kg/d p.o.) were determined and compared to vehicle treated control mice. In the pumafentrine-treated animals, splenocytes were analyzed for interferon-γ (IFNγ) production and CD69 expression. Results Roflumilast treatment resulted in dose-dependent improvements of clinical score (weight loss, stool consistency and bleeding), colon length, and local tumor necrosis factor-α (TNFα) production in the colonic tissue. These findings, however, were not associated with an improvement of the histologic score. Administration of pumafentrine at 5 mg/kg/d alleviated the clinical score, the colon length shortening, and local TNFα production. In vitro stimulated splenocytes after in vivo treatment with pumafentrine showed a significantly lower state of activation and production of IFNγ compared to no treatment in vivo. Conclusions These series of experiments document the ameliorating effect of roflumilast and pumafentrine on the clinical score and TNF expression of experimental colitis in mice.Keywords:
Roflumilast
Phosphodiesterase 3
Phosphodiesterase inhibitor
Ex vivo
Phosphodiesterase 3
Contractility
PDE10A
Second messenger system
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Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes whose physiological role is the attenuation of the signaling mediated by the ubiquitous second messengers cAMP and cGMP. Given the myriad of physiological processes regulated by cAMP and cGMP, PDEs have long been studied as potential therapeutic targets. Although phosphodiesterase 3 (PDE3) activity is abundant in human cardiovascular tissues, and acute PDE3 inhibition, with agents such as milrinone, was beneficial in heart failure patients, prolonged treatments were associated with time-dependent reductions in hemodynamic effects and increased mortality. The molecular basis of this time-dependent reduction in efficacy has not been elucidated. In this context, we used a combination of approaches to determine PDE3 expression in human cardiovascular tissues and to elucidate the effects of prolonged elevations of cellular cAMP, as would occur with PDE3 inhibition, on this activity. Although our data confirms the expression of PDE3A in human blood vessel smooth muscle cells (HASMCs), we identify a previously unrecognized role for PDE3B in cAMP hydrolysis in human cardiovascular tissues. Specifically, although both PDE3A and PDE3B were expressed in HASMCs, their subcellular expression pattern and regulated expression by cAMP were distinct, with only expression of PDE3B being subject to cAMP-regulated expression. Thus, a paradigm emerges that allows for dual expression, with distinctive regulation, of both PDE3A and PDE3B proteins in cardiovascular tissues that may have profound significance for the rational design of molecules regulating this PDE activity.
Phosphodiesterase 3
Milrinone
Cyclic nucleotide phosphodiesterase
PDE10A
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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 μm cAMP in the absence and presence of Ca2+/calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar Km and kcat 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 μm cAMP, in the absence of Ca2+/calmodulin. At 1.0 μm cAMP, in the presence of Ca2+/calmodulin, activation of Ca2+/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 [Ca2+] 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 PDE3-independent mechanisms. 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 μm cAMP in the absence and presence of Ca2+/calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar Km and kcat 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 μm cAMP, in the absence of Ca2+/calmodulin. At 1.0 μm cAMP, in the presence of Ca2+/calmodulin, activation of Ca2+/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 [Ca2+] 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 PDE3-independent mechanisms. PDE3 cyclic nucleotide phosphodiesterases are important in the regulation of intracellular cAMP content in cardiac myocytes (1Shakur Y. Holst L.S. Landstrom T.R. Movsesian M. Degerman E. Manganiello V. Prog. Nucleic Acids Res. Mol. Biol. 2001; 66: 241-277Crossref PubMed Google Scholar). Three isoforms of PDE3 have been identified in human myocardium (2Choi Y.H. Ekholm D. Krall J. Ahmad F. Degerman E. Manganiello V.C. Movsesian M.A. Biochem. J. 2001; 353: 41-50Crossref PubMed Scopus (0) Google Scholar, 3Wechsler J. Choi Y.H. Krall J. Ahmad F. Manganiello V.C. Movsesian M.A. J. Biol. Chem. 2002; 277: 38072-38078Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). They appear to be generated from the PDE3A gene by a combination of alternative transcriptional and post-transcriptional processing, and their amino acid sequences are identical except for the presence of different lengths of N-terminal sequences containing membrane association domains and sites for activation by phosphorylation (Fig. 1). PDE3A-136, present exclusively in microsomal fractions of human myocardium, contains two membrane association domains, "NHR1" and "NHR2," and three sites for phosphorylation and activation by cAMP-dependent protein kinase and protein kinase B. PDE3A-118, present in microsomal and cytosolic fractions of human myocardium, lacks NHR1 and the most upstream phosphorylation site, whereas PDE3A-94, also present in microsomal and cytosolic fractions of human myocardium, lacks NHR1, NHR2, and all three phosphorylation sites. The existence of these isoforms is likely to be important with respect to the intracellular compartmentation of cAMP metabolism. In cardiac myocytes, cAMP content is regulated differentially in intracellular compartments represented in microsomal and cytosolic fractions. Changes in cAMP content in these compartments correlate with changes in the phosphorylation of different substrates of cAMP-dependent protein kinase and with different physiologic responses (4Hayes J.S. 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In cardiac myocytes from rat hearts, different cyclic nucleotide phosphodiesterases have been shown to regulate cAMP-mediated signaling in spatially and functionally distinct intracellular compartments (11Zaccolo M. Pozzan T. Science. 2002; 295: 1711-1715Crossref PubMed Scopus (701) Google Scholar, 12Mongillo M. McSorley T. Evellin S. Sood A. Lissandron V. Terrin A. Huston E. Hannawacker A. Lohse M.J. Pozzan T. Houslay M.D. Zaccolo M. Circ. Res. 2004; 95: 67-75Crossref PubMed Scopus (311) Google Scholar). The fact that there are differences among PDE3 isoforms in human myocardium with respect to intracellular localization domains suggests that these isoforms may be involved in this compartmentation in this tissue. Furthermore, the N-terminal sequence differences among these isoforms might be expected to affect their catalytic activity and inhibitor sensitivity, as occurs with isoforms in the PDE4 family (13Bolger G.B. Erdogan S. Jones R.E. Loughney K. Scotland G. Hoffmann R. Wilkinson I. Farrell C. Houslay M.D. Biochem. J. 1997; 328: 539-548Crossref PubMed Scopus (173) Google Scholar, 14Huston E. Lumb S. Russell A. Catterall C. Ross A.H. Steele M.R. Bolger G.B. Perry M.J. Owens R.J. Houslay M.D. Biochem. J. 1997; 328: 549-558Crossref PubMed Scopus (90) Google Scholar). Any such differences are likely to be pertinent to the inotropic actions of PDE3 inhibitors, because these drugs affect cAMP hydrolysis in microsomal and cytosolic fractions of cardiac myocytes with different potencies (15Shakur Y. Fong M. Hensley J. Cone J. Movsesian M.A. Kambayashi J. Yoshitake M. Liu Y. Cardiovasc. Drugs Ther. 2002; 16: 417-427Crossref PubMed Scopus (62) Google Scholar, 16Movsesian M.A. J. Card Fail. 2003; 9: 475-480Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). These considerations may also have some influence on cGMP-mediated signaling in cardiac muscle. The cAMP hydrolytic activity of PDE3 is inhibited competitively by cGMP, and inhibition of cAMP hydrolysis by cGMP has been shown to contribute to several actions of cGMP, including the stimulation of renin secretion, the potentiation of vasodilatory responses to adrenomedullin, the inhibition of tumor necrosis factor-α-induced NF-κB-dependent inflammatory responses in vascular smooth muscle cells, and the potentiation of delayed rectifier K+ currents in sino-atrial cells (17Kurtz A. Gotz K.H. Hamann M. Wagner C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4743-4747Crossref PubMed Scopus (86) Google Scholar, 18Aizawa T. Wei H. Miano J.M. Abe J. Berk B.C. Yan C. Circ. Res. 2003; 93: 406-413Crossref PubMed Scopus (113) Google Scholar, 19Fung E. Fiscus R.R. J. Cardiovasc. Pharmacol. 2003; 41: 849-855Crossref PubMed Scopus (10) Google Scholar, 20Shimizu K. Shintani Y. Ding W.G. Matsuura H. Bamba T. Br. J. Pharmacol. 2002; 137: 127-137Crossref PubMed Scopus (31) Google Scholar). cGMP can raise intracellular cAMP content in cardiac myocytes by inhibiting PDE3 activity (21Patel K.N. Yan L. Gandhi A. Scholz P.M. Weiss H.R. Basic Res. Cardiol. 2001; 96: 34-41Crossref PubMed Scopus (6) Google Scholar), and this may contribute to the potentiation of L-type Ca2+ currents in cardiac myocytes by cGMP-raising agents (22Lohmann S.M. Fischmeister R. Walter U. Basic Res. Cardiol. 1991; 86: 503-514Crossref PubMed Scopus (168) Google Scholar, 23Mery P.F. Lohmann S.M. Walter U. Fischmeister R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1197-1201Crossref PubMed Scopus (412) Google Scholar, 24Mery P.F. Pavoine C. Belhassen L. Pecker F. Fischmeister R. J. Biol. Chem. 1993; 268: 26286-26295Abstract Full Text PDF PubMed Google Scholar, 25Vandecasteele G. Verde I. Rucker-Martin C. Donzeau-Gouge P. Fischmeister R. J. Physiol. (Lond.). 2001; 533: 329-340Crossref Scopus (130) Google Scholar). Differences in the sensitivities of PDE3 isoforms in different intracellular compartments to inhibition by cGMP might therefore be important in cGMP-mediated signaling in cardiac myocytes. Our goal was to gain insight into the possible role of PDE3 isoforms in the compartmental regulation of cAMP metabolism in human myocardium. We characterized the catalytic activity and inhibitor sensitivity of these isoforms, and we examined their contribution to total and to cGMP-regulated cAMP hydrolytic activity in cytosolic and microsomal fractions of human myocardium. Expression of Recombinant PDE3 Isoforms—Baculovirus-containing constructs of PDE3A-136, PDE3A-118, and PDE3A-94 with the FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) inserted immediately upstream of the stop codon were prepared as described previously (3Wechsler J. Choi Y.H. Krall J. Ahmad F. Manganiello V.C. Movsesian M.A. J. Biol. Chem. 2002; 277: 38072-38078Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 26Shakur Y. Takeda K. Kenan Y. Yu Z.X. Rena G. Brandt D. Houslay M.D. Degerman E. Ferrans V.J. Manganiello V.C. J. Biol. Chem. 2000; 275: 38749-38761Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Virus pools were prepared by infecting 4.5 × 108 Sf9 cells suspended in 450 ml of Sf-900 II SFM medium (Invitrogen). Unless otherwise stated, all steps were carried out at 27 °C. When cell death reached 50%, cell suspensions were sedimented at 3,000 rpm (JA-20; Beckman Instruments, Fullerton, CA), and the supernatant was collected. 20 ml of this supernatant were added to 2.0 × 108 Sf9 cells suspended in 200 ml of SFM medium. When cell death in this population reached 20%, cell suspensions were sedimented at 1,200 rpm (JA-20; Beckman Instruments) for 10 min at 4 °C. Pelleted cells were washed twice with ice-cold phosphate-buffered saline, resuspended, and incubated for 15 min in ice-cold buffer containing 50 mm TES, 3The abbreviations used are:TESN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acidrtrecombinant 0.5 m NaCl, 0.1 mm EDTA (pH 7.4), 1% Nonidet P-40, 5% glycerol, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin. Lysates were prepared by hand homogenization (glass-Teflon, 10-20 strokes). After an additional 15 min on ice, suspensions were sedimented at 14,000 rpm (JA-20; Beckman Instruments) for 15 min at 4 °C. The supernatant fractions were used for the experiments described below. N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid recombinant Preparation of Subcellular Fractions of Human Myocardium—Human left ventricular myocardium was obtained from the explanted hearts of patients with dilated cardiomyopathy undergoing cardiac transplant. Cytosolic and KCl-washed microsomal fractions were prepared by homogenization and differential sedimentation as described previously (3Wechsler J. Choi Y.H. Krall J. Ahmad F. Manganiello V.C. Movsesian M.A. J. Biol. Chem. 2002; 277: 38072-38078Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Each preparation was made from tissue from at least three different hearts. Immunoprecipitation of PDE3 from Subcellular Fractions—Undiluted rabbit polyclonal antibody (3 μl) raised against the C terminus of human PDE3A (2Choi Y.H. Ekholm D. Krall J. Ahmad F. Degerman E. Manganiello V.C. Movsesian M.A. Biochem. J. 2001; 353: 41-50Crossref PubMed Scopus (0) Google Scholar) was incubated with 30 μl of packed G-Sepharose beads (Amersham Biosciences) for 3 h at 4°C. Beads with bound antibody were sedimented at 4,000 rpm for 3 min in a Hermle Z180m centrifuge (Labnet, Edison, NJ) and washed with ice-cold buffer containing 20 mm Tris, 150 mm NaCl, and 0.1% Nonidet P-40 (pH 7.4, 4 °C). This process was repeated three times. Cytosolic fractions containing 50 pmol/min of cAMP hydrolytic activity (measured at 0.1 μm cAMP as described below) were added to the antibody/G-Sepharose beads and co-incubated overnight at 4 °C. Beads were removed by sedimentation at 14,000 rpm for 4 min at 4 °C. Western Blotting—Lysates of Sf9 cells were dissolved in SDS buffer, subjected to SDS-PAGE (10% acrylamide), and transferred electrophoretically to polyvinylidine fluoride membranes (2Choi Y.H. Ekholm D. Krall J. Ahmad F. Degerman E. Manganiello V.C. Movsesian M.A. Biochem. J. 2001; 353: 41-50Crossref PubMed Scopus (0) Google Scholar). PDE3 was visualized with horseradish peroxidase-conjugated anti-FLAG monoclonal antibody M2 (Sigma) and ECL reagent (Amersham Biosciences). Quantitation of rtPDE3 Isoforms—C-terminal FLAG-tagged rtPDE3 protein was quantified by enzyme-linked immunosorbent assay. Varying amounts of Sf9 cell lysates were incubated overnight in wells of Nunc Immuno-polystyrene plates with Maxisorb surfaces (VWR Scientific, Denver, CO) at 4 °C. After blocking with 50 mg/ml bovine serum albumin in Tris-buffered saline, wells were incubated with horseradish peroxidase-conjugated anti-FLAG monoclonal antibody M2. ABTS (2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) diammonium salt) reagent (Sigma) was added at 0.22 mg/ml, and color development (millioptical density/min) was monitored over a 20-min time course. C-terminal FLAG-tagged bacterial alkaline phosphatase (Sigma) was used as a standard. For all determinations, calculations of the amount of FLAG-tagged protein were based on readings in the range through which the rate of color development varied linearly with the amount of protein per well. Quantitation of Cyclic Nucleotide Phosphodiesterase Activity and Its Inhibition—Cyclic nucleotide hydrolytic activity in Sf9 cell lysates was quantified at 30 °C by using the two-step snake venom method with [3H]cAMP and [3H]cGMP as substrates (2Choi Y.H. Ekholm D. Krall J. Ahmad F. Degerman E. Manganiello V.C. Movsesian M.A. Biochem. J. 2001; 353: 41-50Crossref PubMed Scopus (0) Google Scholar). EGTA, CaCl2, and calmodulin (provided by Donald Blumenthal, University of Utah) were included as indicated in the text. Km values for cAMP and cGMP hydrolytic activity and Ki values for inhibition of cAMP hydrolytic activity by the competitive inhibitor cilostazol were calculated by nonlinear regression using the equation v/Vmax = [S]/(Km(1 + ([inhibitor]/Ki)) + [S]). Values for kcat were calculated using the equation kcat = Vmax[PDE3]. PDE3 activity in subcellular fractions of human myocardium was quantified by measuring cAMP hydrolysis as described above in the absence and presence of cilostazol. To minimize possible inhibition of other subfamilies of PDE3 at maximally inhibitory concentrations of cilostazol, these measurements were made at concentrations of cilostazol that inhibited cAMP hydrolytic activity by rtPDE3A submaximally; PDE3 activity was calculated by dividing the amount of activity inhibited by cilostazol in subcellular fractions by the fractional inhibition of rtPDE3 activity at the same cilostazol concentration. Catalytic Activity and Inhibitor Sensitivity of PDE3 Isoforms—Our first objective was to determine whether there were differences among the three PDE3A isoforms in human myocardium with respect to catalytic activity. To do this, we characterized the catalytic activity of FLAG-tagged recombinant forms of PDE3A-136, PDE3A-118, and PDE3A-94. The apparent molecular weights of the expressed proteins were consistent with published values (3Wechsler J. Choi Y.H. Krall J. Ahmad F. Manganiello V.C. Movsesian M.A. J. Biol. Chem. 2002; 277: 38072-38078Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and there was no evidence of proteolysis of any of the isoforms (Fig. 2). There were no significant differences among the three PDE3 isoforms with respect to Km for cAMP (TABLE ONE). Our quantitation of rtPDE3 isoforms by enzyme-linked immunosorbent assay allowed a comparison of kcat values for these different isoforms, and we found no significant differences among them in this regard. The variance among preparations with respect to values for kcat was large, however, and the possibility of small differences among the isoforms with respect to kcat cannot be excluded.TABLE ONECatalytic activity of rtPDE3 isoforms Each value represents the mean ± S.D. for three to five different preparations of FLAG-tagged rtPDE3A.IsoformKm, cAMPkcat, cAMPnmmin–1PDE3A-13688 ± 1129 ± 11PDE3A-11893 ± 1630 ± 18PDE3A-9479 ± 2131 ± 7 Open table in a new tab We proceeded to examine the sensitivities of the cAMP hydrolytic activity of these isoforms to the competitive inhibitors cGMP and cilostazol. Because cGMP and cAMP are competitive substrates for PDE3, the sensitivity of its cAMP hydrolytic activity to inhibition by cGMP is reflected in the Km value for cGMP. There were no significant differences among the three PDE3A isoforms in this regard (TABLE TWO). In the case of cilostazol, which is not a substrate, we measured the Ki values for the inhibition of cAMP hydrolysis. As with cGMP, all three PDE3A isoforms were comparable in their sensitivity to inhibition by cilostazol (TABLE TWO). Similar results were obtained for inhibition by the competitive inhibitor milrinone (data not shown).TABLE TWOSensitivities of rtPDE3 isoforms to inhibition by cGMP and cilostazolKm, cGMPKi, cilostazolnmnmrtPDE3A-13630 ± 550 ± 10rtPDE3A-11820 ± 239 ± 11rtPDE3A-9428 ± 356 ± 6 Open table in a new tab Contribution of PDE3 Isoforms to cAMP Hydrolytic Activity in Human Myocardium—Our second objective was to examine the contribution of PDE3 isoforms to cAMP hydrolytic activity in subcellular fractions of human myocardium. This was done under several conditions. First, we considered that changes in intracellular [Ca2+] in diastole and systole might affect the contribution of Ca2+/calmodulin-dependent phosphodiesterases in human myocardium, and we therefore measured cAMP hydrolytic activity in the absence of Ca2+ (i.e. at 2.0 mm EGTA) or in the presence of 200 μm CaCl2 and 50 nm calmodulin. We also measured cAMP hydrolytic activity in the presence of 0.1 and 1.0 μm cAMP, two concentrations that are likely to be physiologically relevant based on the published Km values for the cAMP-dependent protein kinase (27Taskén K. Solberg R. Foss K. Skålhegg B. Hansson V. Jahnsen T. Hardie D.G. Hanks S.K. Facts Book on Protein Kinases. Academic Press, London1995: 58-63Google Scholar), in order to examine the possible effects of increases in [cAMP] that might result from increased β-adrenergic receptor stimulation. Such increases would be expected to increase the contribution of other cAMP phosphodiesterases whose affinity for cAMP is lower than that of PDE3. We first quantified the effect of [cAMP] and Ca2+/calmodulin on the activity of PDE3 itself. As expected (Fig. 3), the activity of rtPDE3A-136 increased when [cAMP] was raised from 0.1 to 1.0 μm. Ca2+/calmodulin affected neither the activity of rtPDE3A nor its sensitivity to inhibition by cilostazol or cGMP. We proceeded to measure cAMP hydrolytic activities in subcellular fractions of human myocardium. At 0.1 μm cAMP, PDE3 comprised the majority of cAMP hydrolytic activity in microsomal fractions in the absence and in the presence of Ca2+/calmodulin (Fig. 4). At 1.0 μm cAMP, PDE3 activity increased, but its relative contribution to total cAMP activity was reduced to a small degree by a slightly greater increase in the activity of other (cilostazol-insensitive) cAMP phosphodiesterases. Results in cytosolic fractions were quite different. At 0.1 μm cAMP, PDE3 comprised the majority of cAMP hydrolytic activity in the absence of Ca2+/calmodulin (Fig. 4). In the presence of Ca2+/calmodulin, however, the relative contribution of PDE3 to cAMP hydrolytic activity was markedly reduced by a large increase in the activity of cilostazol-insensitive, Ca2+/calmodulin-activated cAMP phosphodiesterases (presumably PDE1). As in microsomal fractions, raising the cAMP concentration from 0.1 to 1.0 μm increased the activity of PDE3 in cytosolic fractions, but its relative contribution to total cAMP hydrolytic activity declined because of a larger increase in the activity of cilostazol-insensitive cAMP phosphodiesterases. The decrease in the relative contribution of PDE3 to total cAMP hydrolysis was decreased even further by the addition of Ca2+/calmodulin and the consequent increase in the activity of Ca2+/calmodulin-activated cAMP phosphodiesterases. At 1.0 μm cAMP, in the presence of Ca2+/calmodulin, PDE3 consisted of <20% of the total cAMP hydrolytic activity in cytosolic fractions. Effects of cGMP on cAMP Hydrolytic Activity—Our third objective was to examine the effect of cGMP on cAMP hydrolysis in subcellular fractions. Because cGMP is a competitive inhibitor of PDE3 activity, and because cGMP analogues and agents that raise cGMP content can raise intracellular cAMP content in cardiac myocytes (21Patel K.N. Yan L. Gandhi A. Scholz P.M. Weiss H.R. Basic Res. Cardiol. 2001; 96: 34-41Crossref PubMed Scopus (6) Google Scholar, 28Wen J.F. Cui X. Jin J.Y. Kim S.M. Kim S.Z. Kim S.H. Lee H.S. Cho K.W. Circ. Res. 2004; 94: 936-943Crossref PubMed Scopus (56) Google Scholar, 29Musialek P. Rigg L. Terrar D.A. Paterson D.J. Casadei B. J. Mol. Cell. Cardiol. 2000; 32: 1831-1840Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 30Weiss H.R. Lazar M.J. Punjabi K. Tse J. Scholz P.M. Eur. J. Pharmacol. 2003; 481: 25-31Crossref PubMed Scopus (5) Google Scholar), our expectation was that cGMP would inhibit cAMP hydrolysis under different conditions to an extent that would reflect the contribution of PDE3 to total cAMP hydrolytic activity. We first examined the effects of 1.0 μm cGMP on rtPDE3A activity at different concentrations of cAMP in the absence or presence of Ca2+/calmodulin. The sensitivity of PDE3A cAMP hydrolytic activity to inhibition by cGMP was not affected by Ca2+/calmodulin at cAMP concentrations of 0.1 and 1.0 μm (Fig. 5). We proceeded to quantify the effect of 1.0 μm cGMP on the cAMP hydrolytic activity in subcellular fractions. In microsomal fractions, at both 0.1 and 1.0 μm cAMP, 1.0 μm cGMP inhibited cAMP phosphodiesterase activity (Fig. 6). This inhibition was unaffected by the presence of Ca2+/calmodulin, and the magnitude of inhibition at 0.1 μm cAMP was comparable with the relative contribution of PDE3 to total activity (Fig. 4). These findings are consistent with the notion that cGMP inhibits cAMP hydrolysis in microsomal fractions by inhibition of PDE3. The somewhat lower magnitude of inhibition seen at 1.0 μm cAMP is consistent with the lower inhibitor:substrate ratio at this concentration. In contrast, in cytosolic fractions the inhibition of cAMP hydrolysis by cGMP was greater than could be explained on the basis of the contribution of PDE3 to cAMP hydrolytic activity, particularly in the presence of Ca2+/calmodulin (Figs. 4 and 6). This suggested that cGMP was also inhibiting non-PDE3 cAMP hydrolytic activities in these fractions. To test this, we examined the inhibition of cAMP hydrolysis by cGMP in cytosolic fractions from which PDE3 had been removed by immunoprecipitation with anti-PDE3 antibodies. cGMP had a significant inhibitory effect on non-PDE3 (cilostazol-insensitive) cAMP hydrolytic activity in these fractions (Fig. 7). The fact that a magnitude of this effect was greatly increased in the presence of Ca2+/calmodulin suggests it reflected inhibition of isoforms in the PDE1 family, which also hydrolyze cAMP and cGMP in a mutually competitive manner (31Sharma R.K. Hickie R.A. Schudt C. Dent G. Rabe K.F. Phosphodiesterase Inhibitors. Academic Press, San Diego1996: 65-80Crossref Google Scholar). In previous studies, we identified three isoforms of PDE3 cyclic nucleotide phosphodiesterase localized to different intracellular compartments of cardiac myocytes. Our intent in this study was to gain further insight into the possible roles of these isoforms in the compartment-selective regulation of cAMP-mediated signaling in human myocardium. Our first consideration was whether these isoforms would differ with regard to their catalytic activity and inhibitor sensitivity. In the PDE4 family, as in the PDE3 family, alternative transcriptional and post-transcriptional processing yields isoforms with conserved C-terminal catalytic domains but different N-terminal domains. Differences in the N-terminal domains of PDE4 isoforms result in major differences in their catalytic activity and inhibitor sensitivity, suggesting that these N-terminal domains have an important role in regulating enzyme activity (13Bolger G.B. Erdogan S. Jones R.E. Loughney K. Scotland G. Hoffmann R. Wilkinson I. Farrell C. Houslay M.D. Biochem. J. 1997; 328: 539-548Crossref PubMed Scopus (173) Google Scholar, 14Huston E. Lumb S. Russell A. Catterall C. Ross A.H. Steele M.R. Bolger G.B. Perry M.J. Owens R.J. Houslay M.D. Biochem. J. 1997; 328: 549-558Crossref PubMed Scopus (90) Google Scholar, 32Saldou N. Obernolte R. Huber A. Baecker P.A. Wilhelm R. Alvarez R. Li B. Xia L. Callan O. Su C. Jarnagin K. Shelton E.R. Cell. Signal. 1998; 10: 427-440Crossref PubMed Scopus (52) Google Scholar, 33Hoffmann R. Wilkinson I.R. McCallum J.F. Engels P. Houslay M.D. Biochem. J. 1998; 333: 139-149Crossref PubMed Scopus (142) Google Scholar, 34McPhee I. Yarwood S.J. Scotland G. Huston E. Beard M.B. Ross A.H. Houslay E.S. Houslay M.D. J. Biol. Chem. 1999; 274: 11796-11810Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 35Lim J. Pahlke G. Conti M. J. Biol. Chem. 1999; 274: 19677-19685Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 36MacKenzie S.J. Houslay M.D. Biochem. J. 2000; 347: 571-578Crossref PubMed Scopus (128) Google Scholar, 37Huston E. Beard M. McCallum F. Pyne N.J. Vandenabeele P. Scotland G. Houslay M.D. J. Biol. Chem. 2000; 275: 28063-28074Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We therefore expected to find significant differences in catalytic activity and inhibitor sensitivity among the three PDE3 isoforms attributable to differences in their N-terminal sequences. Instead, we found that the three PDE3 isoforms are comparable with respect to both Km and kcat values for cAMP, as well as with respect to their sensitivity to competitive inhibitors of cAMP hydrolysis. (To our knowledge, this is the first comparison of kcat values among PDE3A isoforms.) These observations suggest that the role of the N terminus in PDE3 isoforms is likely to have more to do with intracellular localization than with the allosteric effects on the C-terminal catalytic region. Interactions between the N terminus of PDE4 and other proteins appear to be involved in the intracellular localization of these isoforms (37Huston E. Beard M. McCallum F. Pyne N.J. Vandenabeele P. Scotland G. Houslay M.D. J. Biol. Chem. 2000; 275: 28063-28074Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 38Yarwood S.J. Steele M.R. Scotland G. Houslay M.D. Bolger G.B. J. Biol. Chem. 1999; 274: 14909-14917Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 39Dodge K.L. Khouangsathiene S. Kapiloff M.S. Mouton R. Hill E.V. Houslay M.D. Langeberg L.K. Scott J.D. EMBO J. 2001; 20: 1921-1930Crossref PubMed Scopus (403) Google Scholar, 40Baillie G.S. Sood A. McPhee I. Gall I. Perry S.J. Lefkowitz R.J. Houslay M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 940-945Crossref PubMed Scopus (305) Google Scholar), and previous studies have indicated that the N-terminal hydrophobic domains of PDE3 are involved in intracellular localization in transfected cells at least in part through
Phosphodiesterase 3
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Zaprinast
Thio-
Rolipram
IC50
Phosphodiesterase 3
Cyclic nucleotide phosphodiesterase
Phosphodiesterase inhibitor
PDE10A
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Chronic obstructive pulmonary disease (COPD) is a worldwide health problem resulting in significant morbidity and mortality; however, it could not be understood totally so far. Treatment options for the disease are quite limited and there is an urgent need for new treatment strategies. Among new therapeutic agents that are under development, a group of significant importance is phosphodiesterase-4 (PDE-4) inhibitors shown to have antiinflammatory actions. Phosphodiesterases are the enzymes responsible from the breakdown and inactivation of cyclic adenosine monophosphate (cAMP) which is an intracellular second messenger molecule. They are present in several structural and inflammatory cells, in these cells the inactivation of cAMP results in a proinflammatory cascade. So, in COPD which goes together with chronic inflammation, prevention of cAMP inactivation via phosphodiesterase enzyme inhibition made phosphodiesterase enzymes potential targets. Main phosphodiesterase playing a part in COPD is PDE-4 which is predominantly present in inflammatory cells and airway smooth muscle cells. The studies therefore focused on inhibitors selective to PDE-4 subtype. The two selective PDE-4 inhibitors that are at Phase III clinical trial stage are cilomilast and roflumilast. The studies have demonstrated that antiinflammatory effects of cilomilast and roflumilast positively contribute to the respiratory function, frequency of exacerbations and quality of life of COPD patients. Despite we need new studies to evaluate the influence of these agents on the natural course of COPD as well as their long-term safety; we can certainly comment that cilomilast and roflumilast are promising hope in COPD treatment by their clinical and antiinflammatory effects.
Roflumilast
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Roflumilast
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Proinflammatory cytokine
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Vascular smooth muscle cells (VSMC) in situ function to control contraction and are said to express a contractile phenotype. However, during development or in response to vascular damage, VSMC proliferate and express a more synthetic phenotype. A survey of literature values for contractile and synthetic VSMC phosphodiesterase (PDE) 3 and PDE4 activities identified a marked difference in the PDE3 and PDE4 activities of these cells. In this study, a comparison of PDE3 and PDE4 activities in contractile and synthetic VSMC demonstrates that a reduced PDE3/PDE4 activity ratio in synthetic VSMC correlates with a reduced PDE3 activity and is associated with marked reductions in PDE3A mRNA and protein levels. Because we show that similar reductions in PDE3 activity and PDE3A levels occur upon culture of human aortic VSMC and that this phenomenon associates with the phenotypic switch that occurs to VSMC in response to vascular damage, our findings are presented in the context that PDE3 inhibition might be expected to selectively alter functions of contractile VSMC.
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Phosphodiesterase 3
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IC50
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