Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification.

Cyclic AMP (cAMP) signaling is essential to Mycobacterium tuberculosis (Mtb) pathogenesis. However, the roles of phosphodiesterases (PDEs) Rv0805, and the recently identified Rv1339, in cAMP homeostasis and Mtb biology are unclear. We found that Rv0805 modulates Mtb growth within mice, macrophages and on host-associated carbon sources. Mycobacterium bovis BCG grown on a combination of propionate and glycerol as carbon sources showed high levels of cAMP and had a strict requirement for Rv0805 cNMP hydrolytic activity. Supplementation with vitamin B12 or spontaneous genetic mutations in the pta-ackA operon restored the growth of BCGΔRv0805 and eliminated propionate-associated cAMP increases. Surprisingly, reduction of total cAMP levels by ectopic expression of Rv1339 restored only 20% of growth, while Rv0805 complementation fully restored growth despite a smaller effect on total cAMP levels. Deletion of an Rv0805 localization domain also reduced BCG growth in the presence of propionate and glycerol. We propose that localized Rv0805 cAMP hydrolysis modulates activity of a specialized pathway associated with propionate metabolism, while Rv1339 has a broader role in cAMP homeostasis. Future studies will address the biological roles of Rv0805 and Rv1339, including their impacts on metabolism, cAMP signaling and Mtb pathogenesis.

Gastrodin promotes hippocampal neurogenesis via PDE9-cGMP-PKG pathway in mice following cerebral ischemia.

Gastrodin, which is extracted from the Chinese herbal medicine Gastrodia elata Blume, can ameliorate neurogenesis after cerebral ischemia. However, it's possible underlying mechanisms remain still elusive. PDE9-cGMP-PKG signaling pathway is involved in the proliferation of neural stem cells (NSCs) after cerebral ischemia. In this study, we investigated whether the beneficial effect of gastrodin on hippocampal neurogenesis after cerebral ischemia is correlated with the PDE9-cGMP-PKG signaling pathway. Bilateral common carotid artery occlusion (BCCAO) in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in primary cultured hippocampal NSCs were used to mimic brain ischemic injury. The Morris water maze (MWM) test was executed to detect spatial learning and memory. Proliferation, differentiation, and mature neurons were examined using immunofluorescence. The survival and proliferation of NSCs were assessed by CCK-8 assay and BrdU immunofluorescence staining, respectively. ELISA and western blot were used to detect the level of the PDE9-cGMP-PKG signaling pathway. In BCCAO mice, administering gastrodin (50 and 100 mg/kg) for 14 d restored cognitive behaviors; meanwhile, neurogenesis in hippocampus was stimulated, and PDE9 was inhibited and cGMP-PKG was activated by gastrodin. Consistent with the results, administering gastrodin (from 0.01-1 µmol/L) for 48 h dose-dependently ameliorated the cell viability and promoted greatly the proliferation in primary hippocampal NSCs exposed to OGD/R. Gastrodin further decreased PDE9 activity and up-regulated cGMP-PKG level. KT5823, a PKG inhibitor, markedly abrogated the protective effects of gastrodin on OGD/R-injured NSCs, accompanied by the down-regulation of PKG protein expression, but had no effects on PDE9 activity and cGMP level. Gastrodin could accelerate hippocampal neurogenesis after cerebral ischemia, which is mediated, at least partly, by PDE9-cGMP-PKG signaling pathway.

Development of Phosphodiesterase-Protein-Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity.

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems-cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG, and dual-specificity RegA-RD complexes-and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

A Novel, Pan-PDE Inhibitor Exerts Anti-Fibrotic Effects in Human Lung Fibroblasts via Inhibition of TGF-ß Signaling and Activation of cAMP/PKA Signaling.

Phosphodiesterase (PDE) inhibitors are currently a widespread and extensively studied group of anti-inflammatory and anti-fibrotic compounds which may find use in the treatment of numerous lung diseases, including asthma and chronic obstructive pulmonary disease. Several PDE inhibitors are currently in clinical development, and some of them, e.g., roflumilast, are already recommended for clinical use. Due to numerous reports indicating that elevated intracellular cAMP levels may contribute to the alleviation of inflammation and airway fibrosis, new and effective PDE inhibitors are constantly being sought. Recently, a group of 7,8-disubstituted purine-2,6-dione derivatives, representing a novel and prominent pan-PDE inhibitors has been synthesized. Some of them were reported to modulate transient receptor potential ankyrin 1 (TRPA1) ion channels as well. In this study, we investigated the effect of selected derivatives (832-a pan-PDE inhibitor, 869-a TRPA1 modulator, and 145-a pan-PDE inhibitor and a weak TRPA1 modulator) on cellular responses related to airway remodeling using MRC-5 human lung fibroblasts. Compound 145 exerted the most considerable effect in limiting fibroblast to myofibroblasts transition (FMT) as well as proliferation, migration, and contraction. The effect of this compound appeared to depend mainly on its strong PDE inhibitory properties, and not on its effects on TRPA1 modulation. The strong anti-remodeling effects of 145 required activation of the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway leading to inhibition of transforming growth factor type ß1 (TGF-ß1) and Smad-dependent signaling in MRC-5 cells. These data suggest that the TGF-ß pathway is a major target for PDE inhibitors leading to inhibitory effects on cell responses involved in airway remodeling. These potent, pan-PDE inhibitors from the group of 7,8-disubstituted purine-2,6-dione derivatives, thus represent promising anti-remodeling drug candidates for further research.

Discovery of Potent, Selective, and Orally Bioavailable Inhibitors against Phosphodiesterase-9, a Novel Target for the Treatment of Vascular Dementia.

To identify phosphodiesterase-9 (PDE9) as a novel target for the treatment of vascular dementia (VaD), a series of pyrazolopyrimidinone analogues were discovered based on a hit 1. Hit-to-lead optimization resulted in a potent inhibitor 2 with excellent selectivity and physicochemical properties to enable in vivo studies. Oral administration of 2 (5.0 mg/kg) caused notable therapeutic effects in the VaD mouse model, providing a promising lead or chemical probe for investigating the biological functions of PDE9 inhibition.

The Phosphodiesterase 9 Inhibitor PF-04449613 Promotes Dendritic Spine Formation and Performance Improvement after Motor Learning.

The cyclic nucleotide cGMP is an intracellular second messenger with important roles in neuronal functions and animals' behaviors. The phosphodiesterases (PDEs) are a family of enzymes that hydrolyze the second messengers cGMP and cAMP. Inhibition of phosphodiesterase 9 (PDE9), a main isoform of PDEs hydrolyzing cGMP, has been shown to improve learning and memory as well as cognitive function in rodents. However, the role of PDE9 in regulating neuronal structure and function in vivo remains unclear. Here we used in vivo two-photon microscopy to investigate the effect of a selective PDE9 inhibitor PF-04449613 on the activity and plasticity of dendritic spines of layer V pyramidal neurons in the mouse primary motor cortex. We found that administration of PF-04449613 increased calcium activity of dendrites and dendritic spines of layer V pyramidal neurons in mice under resting and running conditions. Chronic treatment of PF-04449613 over weeks increased dendritic spine formation and elimination under basal conditions. Furthermore, PF-04449613 treatment over 1-7 days increased the formation and survival of new spines as well as performance improvement after rotarod motor training. Taken together, our studies suggest that elevating the level of cGMP with the PDE9 inhibitor PF-04449613 increases synaptic calcium activity and learning-dependent synaptic plasticity, thereby contributing to performance improvement after learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000-000, 2018.

Hydroxyurea and a cGMP-amplifying agent have immediate benefits on acute vaso-occlusive events in sickle cell disease mice.

Inhibition of leukocyte adhesion to the vascular endothelium represents a novel and important approach for decreasing sickle cell disease (SCD) vaso-occlusion. Using a humanized SCD-mouse-model of tumor necrosis factor-α-induced acute vaso-occlusion, we herein present data demonstrating that short-term administration of either hydroxyurea or the phosphodiesterase 9 (PDE9) inhibitor, BAY73-6691, significantly altered leukocyte recruitment to the microvasculature. Notably, the administration of both agents led to marked improvements in leukocyte rolling and adhesion and decreased heterotypic red blood cell-leukocyte interactions, coupled with prolonged animal survival. Mechanistically, these rheologic benefits were associated with decreased endothelial adhesion molecule expression, as well as diminished leukocyte Mac-1-integrin activation and cyclic guanosine monophosphate (cGMP)-signaling, leading to reduced leukocyte recruitment. Our findings indicate that hydroxyurea has immediate beneficial effects on the microvasculature in acute sickle-cell crises that are independent of the drug's fetal hemoglobin-elevating properties and probably involve the formation of intravascular nitric oxide. In addition, inhibition of PDE9, an enzyme highly expressed in hematopoietic cells, amplified the cGMP-elevating effects of hydroxyurea and may represent a promising and more tissue-specific adjuvant therapy for this disease.

The effect of phosphodiesterase inhibitors on the extinction of cocaine-induced conditioned place preference in mice.

Several phosphodiesterase inhibitors (PDEis) improve cognition, suggesting that an increase in brain cAMP and cGMP facilitates learning and memory. Since extinction of drug-seeking behavior requires associative learning, consolidation and formation of new memory, the present study investigated the efficacy of three different PDEis in the extinction of cocaine-induced conditioned place preference (CPP) in B6129S mice. Mice were conditioned by escalating doses of cocaine which was resistant to extinction by free exploration. Immediately following each extinction session mice received (a) saline/vehicle, (b) rolipram (PDE4 inhibitor), (c) BAY-73-6691 (PDE9 inhibitor) or (d) papaverine (PDE10A inhibitor). Mice that received saline/vehicle during extinction training showed no reduction in CPP for >10 days. BAY-73-6691 (a) dose-dependently increased cGMP in hippocampus and amygdala, (b) significantly facilitated extinction and (c) diminished the reinstatement of cocaine CPP. Rolipram, which selectively increased brain cAMP levels, and papaverine which caused increases in both cAMP and cGMP levels, had no significant effect on the extinction of cocaine CPP. The results suggest that increase in hippocampal and amygdalar cGMP levels via blockade of PDE9 has a prominent role in the consolidation of extinction learning.

Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases.

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKß-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.

Betulinic acid has an inhibitory effect on pancreatic lipase and induces adipocyte lipolysis.

Betulinic acid is a pentacyclic triterpenic acid that exists naturally in many kinds of food and has many biological functions. The present study investigated the antiobesity properties of betulinic acid and possible mechanisms by which betulinic acid functions. To examine the antilipase function of betulinic acid, the ability of betulinic acid to inhibit pancreatic lipase activity in vitro and to prevent the elevation of plasma triacylglycerol levels was tested after oral administration of a lipid emulsion in rats. In addition, the lipolytic effects of betulinic acid were assayed in rat adipose tissues. The activity of cAMP-dependent phosphodiesterase was also measured in vitro. Betulinic acid inhibited pancreatic lipase activity in a dose-dependent manner at concentrations of 1.5-100 µM (IC50 value of 21.10 µM) and prevented the elevation of plasma triacylglycerol levels 2 h after oral administration of the lipid emulsion at a dose of 100 mg/kg. In addition, betulinic acid had a strong lipolytic effect, which was mediated by cAMP-dependent phosphodiesterase inhibition. In conclusion, betulinic acid may exert antiobesity effects by directly inhibiting pancreatic lipase, which would prevent the absorption of lipid from the small intestine. In addition, it was found that betulinic acid may further accelerate fat mobilization by enhancing the levels of lipolysis in adipose tissues.

Influence of Paeonia lactiflora roots extract on cAMP-phosphodiesterase activity and related anti-inflammatory action.

BACKGROUND: Paeonia lactiflora root (baishao in Chinese) is a commonly used herb in TCM. Research has shown baishao to have positive pharmacological actions, including, particularly, anti-inflammatory properties. In this paper we studied the influence of baishao extract on cAMP-phosphodiesterase (PDE) activity and related anti-inflammatory action to identify new pharmacologic action for its clinically widespread use. METHODS: PDE activity was calculated by cAMP change examined with HPLC, respiratory burst of neutrophils was detected with method of cytochrome C reduction, elastase release was indicated with the substrate reduction, rat arthritis model was caused by complete Freund's adjuvant, mouse capillary permeability model was made by acetic acid, and chemical constituents of baishao extract was identified by HPLC, mass spectroscopy and NMR spectrum. RESULTS: Baishao extract had significant inhibition on cAMP-PDE activity (p<0.01), had dose dependent restraint on neutrophils respiratory burst (p<0.001), had inhibition at low concentration and promotion at high concentration on elastase release (p<0.05), and had obvious restraint on local inflammation of animal model (p<0.01). Analysis of HPLC, mass spectroscopy and NMR spectrum showed baishao extract mainly had five components (identified as gallic acid, paeoniflorin sulfonate, albiflorin, paeoniflorin and benzoic acid), among which gallic acid had the largest inhibition on cAMP-PDE activity. CONCLUSION: The anti-inflammatory effects of baishao may be mediated, at least in part, through its gallic acid content, and this effect may be regulated in part by an inhibition on cAMP-PDE.

Levels of phosphodiesterase 4A and 4B are altered by chronic treatment with psychotropic medications in rat frontal cortex.

Our laboratory has recently demonstrated altered expression of phosphodiesterase (PDE) 4A and 4B in subjects with autism, bipolar disorder, and schizophrenia, suggesting disrupted cAMP signaling in these diagnostic groups. In the current study, we measured expression of PDEs in rat frontal cortex (FC) following chronic treatment with clozapine, fluoxetine, haloperidol, lithium, olanzapine, valproic acid (VPA), or sterile saline for 21 days. Western blotting experiments showed decreased expression of PDE4A subtypes in FC following treatment with clozapine, haloperidol, lithium, and VPA. PDE4B subtypes were similarly reduced in FC following treatment with clozapine, fluoxetine, and lithium. We also measured levels of nine PDE subtypes via qRT-PCR in FC and found significant upregulation of PDE1A and PDE8B following treatment with olanzapine, while treatment with lithium reduced expression of mRNA for PDE8B. Our results demonstrate altered expression of PDE4A and PDE4B in response to a variety of psychotropic medications suggesting potentially new therapeutic avenues for treatment of neuropsychiatric diseases.

Development and validation of a transcreener assay for detection of AMP- and GMP-producing enzymes.

Screening of AMP- and GMP-producing enzymes such as phosphodiesterases (PDEs), ligases, and synthetases would be simplified by the ability to directly detect unmodified nucleoside monophosphates. To address this need, we developed polyclonal and monoclonal antibodies that recognize AMP and GMP with nanomolar sensitivity and high selectivity vs. the corresponding triphosphate and 3',5'-cyclic monophosphate nucleotides that serve as substrates for many enzymes in these classes. One of these antibodies was used to develop a Transcreener AMP/GMP assay with a far red fluorescence polarization (FP) readout. This polyclonal antibody exhibited extremely high selectivity, with IC(50) ratios of 6,000 for ATP/AMP, 3,810 for cAMP/AMP, and 6,970 for cGMP/GMP. Standard curves mimicking enzymatic conversion of cAMP, cGMP, and ATP to the corresponding monophosphates yielded Z' values of >0.85 at 10% conversion. The assay reagents were shown to be stable for 24 h at room temperature, both before and after dispensing. The Transcreener AMP/GMP FP assay was used for enzymatic detection of cGMP- and cAMP-dependent PDEs 4A1A, 3A, and 9A2 and ATP-dependent ligases, acetyl CoA synthetase, and ubiquitin- activating enzyme (UBE1). Shifts of >100 mP were observed in the linear part of the progress curves for all enzymes tested, and the PDE isoforms exhibited the expected substrate and inhibitor selectivity. These studies demonstrate that direct immunodetection of AMP and GMP is a flexible, robust enzyme assay method for diverse AMP- and GMP-producing enzymes. Moreover, it eliminates many of the shortcomings of other methods including the need for fluorescently labeled substrates, the low signal:background inherent in substrate depletion assays, and the potential for interference with coupling enzymes.

Action of a Novel PDE4 inhibitor ZL-n-91 on lipopolysaccharide-induced acute lung injury.

In the present study, we investigated the effect of classic PDE4 inhibitor rolipram and novel PDE4 inhibitor ZL-n-91 on LPS-induced acute lung injury (ALI) in mice and its mechanism. ALI was induced in ICR mice by instilling intratracheally with LPS, and mice were divided into seven groups: control (Saline), LPS group, ZL-n-91 (3 microg, 10 microg, and 30 microg kg(-1), ip), Rolipram (1.0 mg kg(-1), ip) and dexamethasone (0.5 mg kg(-1), ip). After the 6h of instilling intratracheally with LPS in mice, total leukocyte number, neutrophil number and protein content in BALF increased rapidly, a large number of neutrophil infiltration around the pulmonary vessel and airway, the lung wet weight/dry weight (w/d)ratio raised significantly. MPO activity, TNF-alpha level and cAMP-PDE, PDE4 activity in lung homogenate raised significantly. P(a)O(2), P(a)CO(2) and PH value in peripheral arterial blood also changed obviously, P(a)O(2) and PH value dropped slightly and P(a)CO(2) increased significantly in LPS group. ZL-n-91 (3 microg, 10 microg, 30 microg kg(-1)) dose-dependently reduced the total leukocyte number, neutrophil number and total protein content in BALF, MPO activity, TNF-alpha level and cAMP-PDE, PDE4 activity in lung homogenate, but the effect of ZL-n-91 in pathological changes and lung wet w/d ratio is slight; Rol and Dex significantly reduced lung wet w/d ratio and improved pathological changes, neutrophil around the pulmonary vessel and airway significantly reduced, symptoms of lung edema relieved; The PH value, P(a)O(2) and P(a)CO(2) in ZL-n-91 high dosage group and Rol group had changes, but there was no significant difference compared with LPS group or saline group; After the administration, the righting reflex recovery time significantly shorten in every group of ZL-n-91. the righting reflex recovery time of Rol group was similar with ZL-n-91 30 microg kg(-1) group, while Dex group was similar with saline group. The present study confirms that the inhibitory effect of ZL-n-91(30 microg kg(-1)) on the inflammatory reactivity, including inhibition of inflammatory cell and protein exudation, MPO and PDE4 activity, improvement of the blood gas, those effects were equivalent with rolipram 1 mg kg(-1), and suggested that ZL-n-91 was stronger than rolipram in PDE4 inhibition. So we speculated that ZL-n-91 may have stronger therapeutic potential for treatment of inflammatory disease than rolipram, meantime have stronger nervous system effect than rolipram.

Inhibition of human cAMP-phosphodiesterase as a mechanism of the spasmolytic effect of Matricaria recutita L.

Mechanisms underlying the spasmolytic activity of chamomile still remain unclear. Inhibition of cAMP- and cGMP-phosphodiesterases (PDE) is one of the mechanisms operated by spasmolytic drugs. In this study, the effect of chamomile on PDE was investigated. Human platelet cAMP-PDE and recombinant PDE5A1 were assayed in the presence of infusions prepared from sifted flowers and capitula. LC-ESI-MS/MS analysis showed different compositions in infusions made with sifted flowers and capitula. Chamomile inhibited cAMP-PDE activity (IC50 = 17.9-40.5 microg/mL), while cGMP-PDE5 was less affected (-15% at 50 microg/mL). Among the individual compounds tested, only flavonoids showed an inhibitory effect (IC50 = 1.3-14.9 microM), contributing to around 39% of the infusion inhibition; other compounds responsible for cAMP-PDE inhibition still remain unknown. Although experimental evidence supporting the use of chamomile for gastrointestinal minor spasms dates back to the fifties, cAMP-PDE inhibition as a likely mechanism underlying the spasmolytic activity is reported for the first time.

Development of a fission yeast-based high-throughput screen to identify chemical regulators of cAMP phosphodiesterases.

Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. The authors describe a fission yeast-based high-throughput screen to detect inhibitors of heterologously expressed adenosine 3',5'-cyclic monophosphate (cAMP) PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High-throughput screens of 2 bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. The authors verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens.

Difference in preventive effects between the phosphodiesterase iv inhibitor rolipram and anti-arthritic drugs on antigen-induced arthritis in mice.

The efficacy of the phosphodiesterase (PDE) IV inhibitor rolipram on antigen-induced arthritis (AIA) in mice was evaluated in comparison with clinically used anti-arthritic drugs. To induce AIA, DBA/1 mice were immunized with ovalbumin (OVA) emulsified with CFA (day 0) followed by intra-articular injection of OVA on day 21. Rolipram and clinically used anti-arthritic drugs including indomethacin (IND), dexamethasone (DEX), methotrexate (MTX), auranofin (AUR), and D-penicillamine (D-PA) were orally administered daily from days 0 to 20. On day 22, anti-OVA IgG in serum, proliferative responses of spleen cells to the OVA, and anti-OVA IgG2a and interferon (IFN)-gamma as indicators of Th1 responses, as well as anti-OVA IgG1 and interleukin (IL)-10 as those of Th2 reactions, were measured. Treatment with rolipram was followed by inhibition of the early phase of AIA associated with downregulation of both OVA-specific splenocyte proliferation and decreases of IFN-gamma released from the spleen cells but no decreases of the amount of IL-10, or levels of anti-OVA IgG, IgG2a, and IgG1. All clinically used anti-arthritic drugs were more effective in suppressing the late phase of AIA compared with the early phase of joint inflammation. The suppression of AIA by clinically used anti-arthritic drugs was associated with down-regulation of not only Th1 but also Th2 responses. These results suggest that PDE IV inhibitors such as rolipram may exert their suppressive effects on AIA with relatively selective downregulation of antigen-specific Th1 responses compared with anti-arthritic drugs.

Preclinical assessment of curcumin as a potential therapy for B-CLL.

Curcumin, the principle component of the spice turmeric, has been used as an anti-inflammatory medication in India and China for centuries. Recent studies, predominantly using actively dividing cell lines, have suggested that this compound could be used as a chemopreventative or therapeutic agent for epithelial tumors. As curcumin has been reported to inhibit the NIK/IKK complex, an activity that would be expected to induce apoptosis in B cell malignancies, we sought to determine whether curcumin induces apoptosis in vitro in primary chronic lymphocytic leukemia (B-CLL) cells. Primary leukemic cells were incubated with varying dosages of curcumin, followed by assessment for apoptosis. The role of PPARgamma or NF-kappaB signaling in curcumin-induced apoptosis was examined by cotreatment with a PPARgamma antagonist or EMSA of nuclear NFkappaB complexes. We also examined whether a clinically achievable concentration of curcumin (1 microM) would augment the apoptotic effects of fludarabine, dexamethasone, vincristine or the PDE4 inhibitor rolipram. In B-CLL cells from 14 patients, curcumin-induced apoptosis with a mean EC(50) of 5.5 microM. In contrast, the EC(50) for whole mononuclear cells from a healthy donor was 21.8 microM. In a 48 hr wash-out time course, curcumin-induced apoptosis was time-dependent, with a substantial reduction in apoptosis observed when curcumin was removed after 5 hr. Curcumin treatment reduced basal nuclear NF-kappaB levels and 1 microM curcumin augmented both vinca alkaloid and PDE4 inhibitor-induced apoptosis in B-CLL cells. Our studies suggest that curcumin may augment the efficacy of established or experimental therapies for B-CLL.

Gene expression and protein localization of calmodulin-dependent phosphodiesterase in adult rat retina.

Calcium calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) was identified in crude extract and immunolabeled sections of rat retina. Both cAMP and cGMP PDE activities were stimulated by calcium-calmodulin (4.7-fold and 2.3-fold, respectively). To characterize PDE1 isoforms in retinal cells further, we used antibodies that specifically recognize PDE1 gene products. PDE1B antibody stained a band at molecular mass of 63 kDa whereas PDE1C antibody recognized two bands at 74- and 70-kDa molecular masses. Two PDE1A antibodies (against N-terminal and C-terminal peptides) detected a band at 79 kDa never described before. Immunohistochemical analysis showed a distribution of PDE1A in the outer retina with a bright fluorescence in the outer segments of photoreceptors. PDE1B is uniformly distributed across the retina. PDE1C is confined mainly to the inner retina, with a precise localization in the inner nuclear layer. Immunostaining with choline acetyltransferase antibody indicates localization in cholinergic amacrine cell. The present data provide evidence of expression of PDE1 isoforms in mammalian retina with a complementary distribution of PDE1A and PDE1C, suggesting different roles in retinal function.

Roflumilast for the treatment of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a progressive disease of the airways that is triggered primarily by smoking. It manifests clinically with dyspnea, cough and sputum production, all of which become aggravated with disease progression. The only intervention that can halt the decline in lung function in COPD is smoking cessation--other interventions and therapeutic treatments can only slow down the progression of the disease. Pharmacologic treatment of stable COPD consists primarily of bronchodilators, which are used for relieving symptoms and reducing lung function decline, and corticosteroids, which are used for minimizing the associated inflammation. Methylxanthines are non-selective phosphodiesterase (PDE) inhibitors with bronchodilatory and anti-inflammatory effects; however, their use in COPD and other respiratory conditions is limited by their narrow therapeutic index and poor safety profile. Cilomilast and roflumilast are selective PDE4 inhibitors that are currently in pre-registration and phase III clinical trials, respectively, for the treatment of COPD (cilomilast and roflumilast) and asthma (roflumilast).