Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP

Increases in the second messenger cAMP are associated with receptor-mediated ATP release from erythrocytes. In other signaling pathways, cAMP-specific phosphodiesterases (PDEs) hydrolyze this second messenger and thereby limit its biological actions. Although rabbit and human erythrocytes possess adenylyl cyclase and synthesize cAMP, their PDE activity is poorly characterized. It was reported previously that the prostacyclin analog iloprost stimulated receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the PDEs that hydrolyze erythrocyte cAMP synthesized in response to iloprost were not identified. PDE3 inhibitors were reported to augment increases in cAMP stimulated by prostacyclin analogs in platelets and pulmonary artery smooth muscle cells. Additionally, PDE3 activity was identified in embryonic avian erythrocytes, but the presence of this PDE in mammalian erythrocytes has not been investigated. Here, using Western blot analysis, we determined that PDE3B is a component of rabbit and human erythrocyte membranes. In addition, we report that the preincubation of rabbit and human erythrocytes with the PDE3 inhibitors milrinone and cilostazol potentiates iloprost-induced increases in cAMP. In addition, cilostamide, the parent compound of cilostazol, potentiated iloprost-induced increases in cAMP in human erythrocytes. These findings demonstrate that PDE3B is present in rabbit and human erythrocytes and are consistent with the hypothesis that PDE3 activity regulates cAMP levels associated with a signaling pathway activated by iloprost in these cells.     

Expression and activity of cAMP phosphodiesterase isoforms in pulmonary artery smooth muscle cells from patients with pulmonary hypertension: role for PDE1

Pulmonary hypertension (PHT) is associated with increased vascular resistance due to sustained contraction and enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC); the abnormal tone and remodeling in the pulmonary vasculature may relate, at least in part, to decreased cyclic nucleotide levels. Cyclic nucleotide phosphodiesterases (PDEs), of which 11 families have been identified, catalyze the hydrolysis of cAMP and cGMP. We tested the hypothesis that PASMC isolated from patients with PHT, either idiopathic pulmonary arterial hypertension (IPAH) or secondary pulmonary hypertension (SPH), have increased expression and activity of PDE isoforms that reduce the responsiveness of agents that raise cellular cAMP. Real-time PCR and immunoblotting demonstrated that the expression of PDE1A, PDE1C, PDE3B, and PDE5A was enhanced in PASMC from both IPAH and SPH patients compared with control PASMC. Consistent with this enhanced expression of PDEs, agonist-stimulated cAMP levels were significantly reduced in IPAH and SPH PASMC unless a PDE inhibitor was present. The use of specific PDE inhibitors revealed that an increase in PDE1 and PDE3 activity largely accounted for reduced agonist-induced cAMP levels and increased proliferation in IPAH and SPH PASMC. Treatment with PDE1C-targeted small interference RNA enhanced cAMP accumulation and inhibited cellular proliferation to a greater extent in PHT PASMC than controls. The results imply that an increase in PDE isoforms, in particular PDE1C, contributes to decreased cAMP and increased proliferation of PASMC in patients with PHT. PDE1 isoforms may provide novel targets for the treatment of both primary and secondary forms of the disease.     

cAMP analogs and selective inhibitors used to study low Km Mucor rouxii cAMP phosphodiesterase.

1. The sensitivity of partially purified low Km phosphodiesterase (PDE) from Mucor rouxii to pharmacological agents and cAMP analogs was studied. The IC50 obtained were compared with those reported for PDEs from higher eukaryotes. 2. The best inhibitors of the hydrolysis of 1 microM cAMP were SQ 65.442 (IC50 c 10 microM), dipyridamol and CI 930. cGMP was not an inhibitor (IC50 greater than 1000 microM). 3. The cAMP analogs were tested as inhibitors of the hydrolysis of 0.1 microM cAMP. 8-Aminohexylamino cAMP was the best inhibitor with an IC50 of c 1 microM. 4. A sedimentation profile of Mucor PDE was assayed in the presence of several pharmacological inhibitors and cAMP analogs. No isoforms with different sensitivity towards the inhibitors were detected. Forms with slightly different behaviour towards some cAMP analogs were observed.     

Signaling role of PDE isozymes in pathobiology of glomerular mesangial cells

Mesangial cells (MC) of renal glomeruli respond to immune-inflammatory injury by accelerated proliferation and generation of reactive oxygen metabolites (ROM). We studied in vivo and in vitro roles of cAMP-protein kinase A (PKA) signaling in modulation of these pathobiologic processes with focus on PDE isozymes. Mitogenic synthesis of DNA in mesangial cells grown in primary culture was blocked by forskolin and dibutyryl cyAMP. Incubation of MC with PDE-3 inhibitors, cilostamide and lixazione, inhibited (>50%) mitogenesis, whereas inhibitors of PDE-4, rolipram and denbufylline, caused little or no inhibition. Conversely, inhibitors of PDE-4 suppressed generation of ROM in MC, whereas inhibitors of PDE-3 had no effect. Incubation of mesangial cells with cilostamide or with rolipram increasedin situ activity of PKA, and effects of the two inhibitors were additive. PDE inhibitors also decreased activity of mitogen-activated protein kinase. The efficacy of PDE isozyme inhibitors (IC₅₀) to suppress mitogenesis or ROM generation paralleled IC₅₀ for inhibition of cAMP hydrolysis by extracts from mesangial cells. Administration of lixazinone or lixazione in combination with rolipram to rats with mesangial proliferative glomerulonephritis induced by antithymic serum suppressed proliferation of mesangial cells and also reduced other histopathologic manifestations of the disease. Based on these observations, we propose that in MC, a cAMP pool that is hydrolyzed by PDE-3 inhibits by negative crosstalk via activation of PKA, mitogen-activated protein kinase (MAPK) pathway, and mitogenesis; whereas cAMP pool linked to PDE-4 inhibits, also via activation of PKA, ROM generation in mesangial cells. Results also suggest that PDE isozyme inhibitors, in particular inhibitors of PDE-3, should be investigated for potential use for “signal transduction pharmacotherapy” of glomerulonephritis.     

Cyclic nucleotide-mediated regulation of vascular smooth muscle cell cyclic nucleotide phosphodiesterase activity

Cultured rat aortic vascular smooth muscle cells (VSMC) express both cGMP- inhibited cAMP phosphodiesterase (PDE-3) and Ro,20-1724-inhibited cAMP phosphodiesterase (PDE-4) activities. Utilizing a PDE-3-selective inhibitor (cilostamide) and a PDE-4-selective inhibitor (Ro,20-1724), PDE-3 and PDE-4 activities were shown to account for 15 and 55% of total VSMC cAMP phosphodiesterase (PDE) activity. Incubations of VSMC with either forskolin or 8-bromo-cAMP caused a concentration- and time-dependent increase in total cellular cAMP PDE activity. In these cells, both PDE-3 and PDE-4 activities were increased, with a relatively larger effect observed on PDE-3 activity. Similar incubations with an activator of soluble guanylyl cyclase (sodium nitroprusside) or with 8-bromo-cGMP did not increase cAMP PDE activity. cAMP-induced increases in cAMP PDE activity were inhibited with actinomycin D or cycloheximide, demonstrating that new mRNA and protein synthesis were required. We conclude that VSMC cAMP PDE activity is elevated following long-term elevation of cAMP, and that increases in PDE-3 and PDE-4 activities account for more than 70% of this increase. These results may have implications for long-term use of cAMP PDE inhibitors as therapeutic agents.     

Phosphodiesterase inhibition by a gastroprotective agent irsogladine: preferential blockade of cAMP hydrolysis

The effect of irsogladine [2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate], an antiulcer drug, on contents of cyclic nucleotides including cAMP and cGMP was investigated in rat stomachs. Irsogladine concentration-dependently increased cAMP content in rat glandula stomach. However, irsogladine at higher concentration (10(-5) M) was unable to further increase cAMP level in the presence of non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine, although 3-isobutyl-1-methylxanthine by itself increased cAMP level. On the other hand, irsogladine had no effect on the glandula cGMP content. Subsequently, the effect of irsogladine on the cyclic nucleotide degradation by purified bovine brain and heart PDEs was investigated. The cAMP degradation by purified bovine brain PDE was partially suppressed by PDE1 inhibitor vinpocetin, PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride and PDE4 inhibitor rolipram but not by PDE3 inhibitor cilostamide, and completely inhibited by 3-isobutyl-1-methylxanthine, suggesting that is attributed almost exclusively to PDE1, PDE2 and PDE4. Meanwhile, cGMP degradation by purified bovine brain PDE was partially suppressed by erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride. Irsogladine preferentially inhibited the response to cAMP degradation compared with cGMP degradation by this brain PDE. The cAMP degradation by bovine heart PDE was almost completely inhibited by the combination with vinpocetine and cilostamide, indicating that is mediated almost exclusively by PDE1 and PDE3. Irsogladine suppressed this cAMP degradation measured in the presence of vinpocetine to almost the same extent as that determined in the presence of cilostamide. These results indicate that irsogladine produces the increase of intracellular cAMP content via non-selective inhibition of PDE isozymes, which may be a key mechanism involved in its gastroprotective actions.     

Phosphodiesterase 5 inhibitors augment UT-15C-stimulated ATP release from erythrocytes of humans with pulmonary arterial hypertension

Both prostacyclin analogs and phosphodiesterase 5 (PDE5) inhibitors are effective treatments for pulmonary arterial hypertension (PAH). In addition to direct effects on vascular smooth muscle, prostacyclin analogs increase cAMP levels and ATP release from healthy human erythrocytes. We hypothesized that UT-15C, an orally available form of the prostacyclin analog, treprostinil, would stimulate ATP release from erythrocytes of humans with PAH and that this release would be augmented by PDE5 inhibitors. Erythrocytes were isolated and the effect of UT-15C on cAMP levels and ATP release were measured in the presence and absence of the PDE5 inhibitors, zaprinast or tadalafil. In addition, the ability of a soluble guanylyl cyclase inhibitor to prevent the effects of tadalafil was determined. Erythrocytes of healthy humans and humans with PAH respond to UT-15C with increases in cAMP levels and ATP release. In both groups, UT-15C-induced ATP release was potentiated by zaprinast and tadalafil. The effect of tadalafil was prevented by pre-treatment with an inhibitor of soluble guanylyl cyclase in healthy human erythrocytes. Importantly, UT-15C-induced ATP release was greater in PAH erythrocytes than in healthy human erythrocytes in both the presence and the absence of PDE5 inhibitors. The finding that prostacyclin analogs and PDE5 inhibitors work synergistically to enhance release of the potent vasodilator ATP from PAH erythrocytes provides a new rationale for the co-administration of these drugs in this disease. Moreover, these results suggest that the erythrocyte is a novel target for future drug development for the treatment of PAH.     

Phosphodiesterase 10A upregulation contributes to pulmonary vascular remodeling

Phosphodiesterases (PDEs) modulate the cellular proliferation involved in the pathophysiology of pulmonary hypertension (PH) by hydrolyzing cAMP and cGMP. The present study was designed to determine whether any of the recently identified PDEs (PDE7-PDE11) contribute to progressive pulmonary vascular remodeling in PH. All in vitro experiments were performed with lung tissue or pulmonary arterial smooth muscle cells (PASMCs) obtained from control rats or monocrotaline (MCT)-induced pulmonary hypertensive (MCT-PH) rats, and we examined the effects of the PDE10 inhibitor papaverine (Pap) and specific small interfering RNA (siRNA). In addition, papaverine was administrated to MCT-induced PH rats from day 21 to day 35 by continuous intravenous infusion to examine the in vivo effects of PDE10A inhibition. We found that PDE10A was predominantly present in the lung vasculature, and the mRNA, protein, and activity levels of PDE10A were all significantly increased in MCT PASMCs compared with control PASMCs. Papaverine and PDE10A siRNA induced an accumulation of intracellular cAMP, activated cAMP response element binding protein and attenuated PASMC proliferation. Intravenous infusion of papaverine in MCT-PH rats resulted in a 40%-50% attenuation of the effects on pulmonary hypertensive hemodynamic parameters and pulmonary vascular remodeling. The present study is the first to demonstrate a central role of PDE10A in progressive pulmonary vascular remodeling, and the results suggest a novel therapeutic approach for the treatment of PH.     

Effect of type 3 and type 4 phosphodiesterase inhibitors on the maintenance of bovine oocytes in meiotic arrest.

The use of broad-spectrum inhibitors first suggested that phosphodiesterases (PDEs) are involved in the maturation of bovine oocytes. Modulation of individual PDE families is now possible with the use of newly developed type-specific PDE inhibitors. This study evaluated the role of type 3- and type 4-specific PDE inhibitors on the meiotic arrest of bovine cumulus-oocyte complexes (COCs) and denuded oocytes (DOs). It also evaluated the role of these specific inhibitors on meiotic arrest when COCs are incubated in the presence or absence of theca cell monolayers. Bovine COCs were aspirated from ovaries collected at the abattoir. Denuded oocytes and COCs were incubated for 12 h in culture medium alone or culture medium containing the type 3 PDE inhibitors cilostamide (10 and 20 microM) or milrinone (10 and 50 microM) or the type 4 PDE inhibitor rolipram (10 and 50 microM). Oocytes were then fixed and classified according to the status of nuclear maturation. Cumulus-oocyte complexes were coincubated with untreated theca cell monolayers or theca cell monolayers treated with the different specific PDE inhibitors. Bovine COCs or DOs incubated in culture medium resumed meiosis, but supplementation of the culture medium with the PDE3 inhibitors cilostamide or milrinone resulted in meiotic arrest. On the other hand, supplementation of the culture medium with rolipram did not prevent oocyte maturation. Furthermore, PDE3 inhibitors, but not PDE 4 inhibitors, had an additive effect on the inhibitory action of theca cell monolayers on oocyte maturation. These data support the hypothesis that inhibition of PDE3 prevents the meiotic resumption of bovine oocytes, whereas inhibition of PDE4 does not block oocyte maturation even under normally inhibitory conditions. The additive effect of PDE3 inhibitors on the ability of theca cells to maintain bovine oocytes in meiotic arrest suggests that type 3 PDE has an important role in meiotic resumption of bovine oocytes.     

The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway

Recent studies of cyclooxygenase-2 (COX-2) inhibitors suggest that the balance between thromboxane and prostacyclin is a critical factor in cardiovascular homeostasis. Disruption of prostacyclin signaling by genetic deletion of the receptor or by pharmacological inhibition of COX-2 is associated with increased atherosclerosis and restenosis after injury in animal models and adverse cardiovascular events in clinical trials (Vioxx). Human vascular smooth muscle cells (VSMC) in culture exhibit a dedifferentiated, migratory, proliferative phenotype, similar to what occurs after arterial injury. We report that the prostacyclin analog iloprost induces differentiation of VSMC from this synthetic, proliferative phenotype to a quiescent, contractile phenotype. Iloprost induced expression of smooth muscle (SM)-specific differentiation markers, including SM-myosin heavy chain, calponin, h-caldesmon, and SM alpha-actin, as determined by Western blotting and RT-PCR analysis. Iloprost activated cAMP/protein kinase A (PKA) signaling in human VSMC, and the cell-permeable cAMP analog 8-bromo-cAMP mimicked the iloprost-induced differentiation. Both myristoylated PKA inhibitor amide-(14-22) (PKI, specific PKA inhibitor), as well as ablation of the catalytic subunits of PKA by small interfering RNA, opposed the upregulation of contractile markers induced by iloprost. These data suggest that iloprost modulates VSMC phenotype via G(s) activation of the cAMP/PKA pathway. These studies reveal regulation of VSMC differentiation as a potential mechanism for the cardiovascular protective effects of prostacyclin. This provides important mechanistic insights into the induction of cardiovascular events with the use of selective COX-2 inhibitors.     

磷酸二酯酶的心血管功能调节作用

磷酸二酯酶(phosphodiesterase,PDE)是细胞内第二信使cAMP和cGMP降解的关键酶,作为药物研发的靶点受到广泛关注。近年研究发现,PDE在心肌细胞中能与β-肾上腺素受体及一些与兴奋收缩相关的蛋白形成复合物而使细胞内信号传递区室化分布,该现象可能为PDE抑制剂治疗慢性心力衰竭提供新的启示。血管平滑肌功能调节主要为血管张力和表型的调控,PDE5抑制剂舒张血管的作用已成功应用到勃起障碍的治疗。PDE4和PDE1C等在增殖的平滑肌细胞中表达量增高,单独抑制PDE的某一亚型将为治疗与平滑肌增殖有关的疾病(如肺动脉高压、血管成行术后再狭窄)提供新的途径。本文将重点阐述近年来PDE在心血管系统功能调节研究中的主要进展,以及PDE抑制剂在心血管系统疾病治疗中的应用。     

The role of cyclic nucleotide phosphodiesterases in the regulation of adipocyte lipolysis

This study assessed the effects of selective inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) on adipocyte lipolysis. IC224, a selective inhibitor of type 1 phosphodiesterase (PDE1), suppressed lipolysis in murine 3T3-L1 adipocytes (69.6 +/- 5.4% of vehicle control) but had no effect in human adipocytes. IC933, a selective inhibitor of PDE2, had no effect on lipolysis in either cultured murine 3T3-L1 adipocytes or human adipocytes. Inhibition of PDE3 with cilostamide moderately stimulated lipolysis in murine 3T3-L1 and rat adipocytes (397 +/- 25% and 235 +/- 26% of control, respectively) and markedly stimulated lipolysis in human adipocytes (932 +/- 7.6% of control). Inhibition of PDE4 with rolipram moderately stimulated lipolysis in murine 3T3-L1 adipocytes (291 +/- 13% of control) and weakly stimulated lipolysis in rat adipocytes (149 +/- 7.0% of control) but had no effect on lipolysis in human adipocytes. Cultured adipocytes also responded differently to a combination of PDE3 and PDE4 inhibitors. Simultaneous exposure to cilostamide and rolipram had a synergistic effect on lipolysis in murine 3T3-L1 and rat adipocytes but not in human adipocytes. Hence, the relative importance of PDE3 and PDE4 in regulating lipolysis differed in cultured murine, rat, and human adipocytes.     

Cyclic nucleotide phosphodiesterase (PDE) inhibitors: novel therapeutic agents for progressive renal disease

Cyclic nucleotides are recognized as critical mediators of many renal functions, including solute transport, regulation of vascular tone, proliferation of parenchymal cells, and inflammation. Although most studies have linked elevated cAMP levels to activation of protein kinase A, cAMP can also directly activate cyclic nucleotide gated ion channels and can signal through activation of GTP exchange factors. Cyclic AMP signaling is highly compartmentalized through plasma membrane localization of adenylyl cyclase and expression of scaffolding proteins that anchor protein kinase A to specific intracellular locations. Cyclic nucleotide levels are largely regulated through catabolic processes directed by phosphodiesterases (PDEs). The PDE superfamily is large and complex, with over 60 distinct isoforms that preferentially hydrolyze cAMP, cGMP, or both. PDEs contribute to compartmentalized cyclic nucleotide signaling. The unique cell- and tissue-specific distribution of PDEs has prompted the development of highly specific PDE inhibitors to treat a variety of inflammatory conditions. In experimental systems, PDE inhibitors have been employed to demonstrate functional compartmentalization of cyclic nucleotide signaling in the kidney. For example, mitogenesis in glomerular mesangial cells and normal tubular epithelial cells is negatively regulated by an intracellular pool of cAMP that is metabolized by PDE3, but not by other PDEs. In Madin-Darby canine kidney cells, an in vitro model of polycystic kidney disease, an intracellular pool of cAMP directed by PDE3 stimulates mitogenesis. In mesangial cells, an intracellular pool of cAMP directed by PDE4 inhibits reactive oxygen species and expression of the potent proin-flammatory cytokine monocyte chemoattractant protein 1. An intracellular pool of cGMP directed by PDE5 regulates solute transport. PDE5 inhibitors ameliorate renal injury in a chronic renal disease model. In this overview, we highlight recent studies to define relationships between PDE expression and renal function and to provide evidence that PDE inhibitors may be effective agents in treating chronic renal disease.     

Structural basis for the activity of drugs that inhibit phosphodiesterases

Phosphodiesterases (PDEs) comprise a large family of enzymes that catalyze the hydrolysis of cAMP or cGMP and are implicated in various diseases. We describe the high-resolution crystal structures of the catalytic domains of PDE4B, PDE4D, and PDE5A with ten different inhibitors, including the drug candidates cilomilast and roflumilast, for respiratory diseases. These cocrystal structures reveal a common scheme of inhibitor binding to the PDEs: (i) a hydrophobic clamp formed by highly conserved hydrophobic residues that sandwich the inhibitor in the active site; (ii) hydrogen bonding to an invariant glutamine that controls the orientation of inhibitor binding. A scaffold can be readily identified for any given inhibitor based on the formation of these two types of conserved interactions. These structural insights will enable the design of isoform-selective inhibitors with improved binding affinity and should facilitate the discovery of more potent and selective PDE inhibitors for the treatment of a variety of diseases.     

Salt-induced ANG II suppression impairs the response of cerebral artery smooth muscle cells to prostacyclin

Recent studies have demonstrated that cerebral arteries from rats fed a high-salt (HS) diet exhibit impaired vasodilation and altered electrophysiological response to reduction in PO2. The present study examined whether an increase in salt intake alters the response of vascular smooth muscle cells (VSMC) to prostacyclin, a crucial mediator of hypoxic dilation in cerebral arteries. VSMC were isolated from cerebral arteries of male Sprague-Dawley rats maintained on an HS (4% NaCl) or a low-salt diet (0.4% NaCl) for 3 days. The stable prostacyclin analog iloprost (10 ng/ml) inhibited serotonin (0.1-10 microM)-induced contractions and the increase in intracellular Ca2+ concentration ([Ca2+]i) in VSMC isolated from arteries of animals fed the low-salt diet. In contrast, iloprost had no effect on serotonin-induced contractions and increases in [Ca2+]i in VSMC isolated from arteries of rats fed the HS diet. Preventing the fall in ANG in rats fed the HS diet by infusion of a low dose of ANG II (5 ng.kg(-1).min(-1) i.v.) restored the inhibitory effect of iloprost on serotonin-induced contractions and increases in [Ca2+]i in VSMC from animals fed the HS diet. These effects were reversed by AT1 receptor blockade with losartan. These results indicate that ANG II suppression secondary to elevated dietary salt intake impairs vascular relaxation and Ca2+ regulation by prostacyclin.     

Phosphodiesterase 3B (PDE3B) antagonizes the anti-angiogenic actions of PKA in human and murine endothelial cells

Recent reports show that protein kinase A (PKA), but not exchange protein activated by cAMP (EPAC), acts in a cell autonomous manner to constitutively reduce the angiogenic sprouting capacity of murine and human endothelial cells. Specificity in the cellular actions of individual cAMP-effectors can be achieved when a cyclic nucleotide phosphodiesterase (PDE) enzyme acts locally to control the “pool” of cAMP that activates the cAMP-effector. Here, we examined whether PDEs coordinate the actions of PKA during endothelial cell sprouting. Inhibiting each of the cAMP-hydrolyzing PDEs expressed in human endothelial cells revealed that phosphodiesterase 3 (PDE3) inhibition with cilostamide reduced angiogenic sprouting in vitro, while inhibitors of PDE2 and PDE4 family enzymes had no such effect. Identifying a critical role for PDE3B in the anti-angiogenic effects of cilostamide, silencing this PDE3 variant, but not PDE3A, markedly impaired sprouting. Importantly, using both in vitro and ex vivo models of angiogenesis, we show the hypo-sprouting phenotype induced by PDE3 inhibition or PDE3B silencing was reversed by PKA inhibition. Examination of the individual cellular events required for sprouting revealed that PDE3B and PKA each regulated angiogenic sprouting by controlling the invasive capacity of endothelial cells, more specifically, by regulating podosome rosette biogenesis and matrix degradation. In support of the idea that PDE3B acts to inhibit angiogenic sprouting by limiting PKA-mediated reductions in active cdc42, the effects of PDE3B and/or PKA on angiogenic sprouting were negated in cells with reduced cdc42 expression or activity. Since PDE3B and PKA were co-localized in a perinuclear region in human ECs, could be co-immunoprecipitated from lysates of these cells, and silencing PDE3B activated the perinuclear pool of PKA in these cells, we conclude that PDE3B-mediated hydrolysis of cAMP acts to limit the anti-angiogenic potential of PKA in ECs.     

Inhibition of phosphodiesterase 4 amplifies cytokine-dependent induction of arginase in macrophages

Arginase is greatly elevated in asthma and is thought to play a role in the pathophysiology of this disease. As inhibitors of phosphodiesterase 4 (PDE4), the predominant PDE in macrophages, elevate cAMP levels and reduce inflammation, they have been proposed for use in treatment of asthma and chronic obstructive pulmonary disease. As cAMP is an inducer of arginase, we tested the hypothesis that a PDE4 inhibitor would enhance macrophage arginase induction by key cytokines implicated in asthma and other pulmonary diseases. RAW 264.7 cells were stimulated with IL-4 or transforming growth factor (TGF)-beta, with and without the PDE4 inhibitor rolipram. IL-4 and TGF-beta increased arginase activity 16- and 5-fold, respectively. Rolipram alone had no effect but when combined with IL-4 and TGF-beta synergistically enhanced arginase activity by an additional 15- and 5-fold, respectively. The increases in arginase I protein and mRNA levels mirrored increases in arginase activity. Induction of arginase II mRNA was also enhanced by rolipram but to a much lesser extent than arginase I. Unlike its effect in RAW 264.7 cells, IL-4 alone did not increase arginase activity in human alveolar macrophages (AM) from healthy volunteers. However, combining IL-4 with agents to induce cAMP levels induced arginase activity in human AM significantly above the level obtained with cAMP-inducing agents alone. In conclusion, agents that elevate cAMP significantly enhance induction of arginase by cytokines. Therefore, consequences of increased arginase expression should be evaluated whenever PDE inhibitors are proposed for treatment of inflammatory disorders in which IL-4 and/or TGF-beta predominate.     

NADPH oxidase 4 mediates upregulation of type 4 phosphodiesterases in human endothelial cells

The protective actions of prostacyclin (PGI(2) ) are mediated by cyclic AMP (cAMP) which is reduced by type 4 phosphodiesterases (PDE4) which hydrolyze cAMP. Superoxide (O2(-)) from NADPH oxidase (Nox) is associated with impaired PGI(2) bioactivity. The objective of this study, therefore, was to study the relationship between Nox and PDE4 expression in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with the thromboxane A(2) analog, U46619, 8-isoprostane F(2α) (8IP), or tumor necrosing factor alpha (TNFα) [±iloprost (a PGI(2) analog)] and the expression of PDE4A, B, C, and D and splice variants thereof assessed using Western blotting and qPCR and mRNA silencing of Nox4 and Nox5. Effects on cell replication and angiogenesis were also studied. U46619, 8IP, and TNFα increased the expression of Nox 4 and Nox 5 and all PDE4 isoforms as well as cell replication and tubule formation (index of angiogenesis), effects inhibited by mRNA silencing of Nox4 (but not Nox5) and iloprost and rolipram. These data demonstrate that upregulation of Nox4 leads to an upregulation of PDE4A, B, and D and increased hydrolysis of cAMP which in turn augments cell replication and angiogenesis. This mechanism may be central to vasculopathies associated with endothelial dysfunction since the PGI(2)-cAMP signaling axis plays a key role in mediating functions that include hemostasis and angiogenesis.     

Role of phosphodiesterase 2 in growth and invasion of human malignant melanoma cells

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N⁶-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2′-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines.