Phosphodiesterase Assay Research Articles

PDE-5 inhibitors in selected herbal supplements from the Ghanaian market for better erectile function as tested by a bioassay

Herbal supplements sold as ‘all natural’ on various markets in Accra (Ghana) and advertised as highly efficacious in treating erectile dysfunction (ED) were bought and analysed by a PDE-5 enzyme inhibition assay. The claimed efficacy of these products could be the result of inherent plant constituents, but also of intentionally added pharmaceuticals. Medically, ED is treated with potent inhibitors of the phosphodiesterase-5 (PDE-5) enzyme, as in the case of sildenafil. To test the efficacy of the Ghanaian supplements, extracts were made and tested using a PDE-Glo phosphodiesterase assay, a luminescent high-throughput screening (HTS) method.Results revealed that about 90% of the selected samples were able to inhibit PDE-5 activity to a high extent. Estimated concentrations in sildenafil equivalents ranged from traces to very high, with 25 samples (62.5%) pointing at daily doses higher than 25 mg sildenafil equivalents and 9 (22.5%) of these at doses higher than the maximal recommended daily intake of 100 mg sildenafil equivalents. Further investigations are needed to confirm if the observed effects are due to inherent plant constituents or merely the result of added synthetic PDE-5 enzyme inhibitors, especially because doses above 100 mg sildenafil equivalents per day may result in severe health risks.

An NMR based phosphodiesterase assay.

We propose a phosphodiesterase assay based on 1D 1H NMR to monitor the hydrolysis of cyclic nucleotides directly, without requiring tags or the addition of exogenous reagents. The method is suitable to measure phosphodiesterase KM and kcat parameters and to identify phosphodiesterase inhibitors.

In Vitro and In Silico Studies of Secang Wood (Caesalpinia sappan L.) Extracts and Brazilin as Natural Phosphodiesterase-1 (PDE1) Inhibitor for Herbal Cognitive Enhancer Development

Secang (Caesalpinia sappan L.) (CS) is one of the plants widely found in Indonesia and known to have a various biological activities. However, there is no scientific evidence regarding its cognitive enhancing activity through the inhibition of Phosphodisesterase-1 (PDE1). PDE1 is recently discovered as a promising molecular target for the development of cognitive enhancer agents to combat cognitive diseases. This study aimed to identify in vitro PDE1-inhibition activity of n-hexane, chloroform and ethanol extracts of CS wood and its major constituent, brazilin, with Cyclic Nucleotide Phosphodiesterase Assay Kit and in silico interaction of brazilin with PDE1 used Molecular Operating Environment (MOE) ver. 2016.01. This study demonstrates that the ethanol extract was the most active PDE1-inhibition activity compared to the other extracts with the inhibition of 52.7% at concentration of 100μg/mL. Interestingly, brazilin at concentration of 11.45μg/mL inhibited PDE1 activity (30.6%) equal to the positive control, vinpocetine (14.02 μg/mL). In conclusion, the ethanol extract of CS wood and its major constituent, brazilin, inhibited PDE1 activity and could be develop as cognitive enhancer.

Prokaryotic Expression, Purification and Identification of Human PDE9A Protein

Cerebral aggregation of beta amyloid plaques (Aβ) and neurofibrillary tangles is responsible for the onset of Alzheimer’s disease (AD), and PDE9A inhibition rescues Aβ-induced deficits in synaptic plasticity and cognition. This study was aimed to express active PDE9A protein for subsequent inhibitor screening. The PDE9A gene was cloned from human cDNA by real-time polymerase chain reaction, and then the gene sequence and its amino acid sequence were analyzed on Lasergene. An inducible expression vector was constructed by enzyme digestion-seamless cloning and transformed into Escherichia coli BL21 (DE3) for PDE9A expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. The recombinant protein was purified by Ni-NTA affinity chromatography and its activity was determined by a phosphodiesterase assay kit. It was found the open reading frame of PED9A was 1035 bp long, the deduced protein was composed of 345 amino acids, and its predicted isoelectric point was about 4.84. The E. coli vector ST6-PDE9A successfully expressed the recombinant PDE9A protein in the supernatant of bacterial lysate. The optimal culture conditions were that the bacterium ST6-PDE9A was grown first in a lysogeny broth at 37 ºC to an OD600 of 0.6-0.8 and then at 16 ºC for 40 h with the addition of 1 M IPTG. Activity test showed PDE9A significantly hydrolyzed the substrate cyclic guanosine monophosphate. In conclusion, we constructed a prokaryotic expression vector and expressed active proteins, laying a solid foundation for screening PDE9 inhibitors.

Structural characterization of dimethyldithiodenafil and dimethylthiocarbodenafil, analogs of sildenafil

Two analogs of sildenafil (compounds 1 and 2) were detected in three powder products acquired from online drug markets during an LC–UV–MS analysis of psychotropic drugs. Their structures were established by high-resolution mass spectrometry and NMR spectroscopy. Compound 1 was identified as 5-(5-(3,5-dimethylpiperazine-1-carbonothioyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-thione and named dimethyldithiodenafil. Compound 2 was identified as 5-(5-(3,5-dimethylpiperazine-1-carbonothioyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one and named dimethylthiocarbodenafil. Compound 1 was subjected to the phosphodiesterase assay (IC50=0.20nM). The three powder products were found to contain 12–19mg of dimethyldithiodenafil and 1.4–3.9mg of dimethylthiocarbodenafil per tube.

Effect of hydrogen sulfide on vasorelaxation and content of guanosine 3', 5'-cyclic phosphate in vascular tissue of rats

Objective To observe the effect of hydrogen sulfide (H2S) on vasorelaxation and expression of guanosine 3', 5'-cyclic phosphate (cGMP) and activity of cyclic nucleotide phosphodiesterase (PDE) in vascular tissue. Methods H2S donor was provided by sodium bisulfide sodium hydrosulfide.The isolated perfused rat thoracic aorta rings were used to test the relaxation responses to H2S, which recorded by Power Lab system, and the enzyme-linked immuno assay was used to detect intracellular cGMP.The activity of PDE was evaluated by using cyclic nucleotide PDE assay kit. Results (1)H2S relaxed the thoracic aorta rings, and the half maximal effective concentration (EC50) for the H2S relaxation curve, represented by the corresponding concentration of H2S that achieved 50% of the maximum relaxation effect, was (1.79±0.31)×10-5 mol/L.(2)The cGMP content in vascular tissue increased from(22.29±1.59) pmol/L to(41.45±7.49) pmol/L and (31.35±2.56) pmol/L after incubation with 50 μmol/L and 300 μmol/L H2S, respectively (t= -3.09, t=-2.88; all P<0.05, n=7-8). (3)cGMP could be lysed into 5'-guanylicacid(5'-GMP) by PDE, which was an important pathway for cGMP degradation.This study showed that PDE activity was decreased in vascular tissue, the 5'-GMP decreased from (0.52±0.06) mol/L to (0.25±0.06) mol/L and (0.27±0.07) mol/L after incubation with 50 μmol/L and 300 μmol/L H2S, respectively (t=3.21, t=2.58; all P<0.05, n=7-8). Conclusion The vasorelaxant effects of H2S might be related to the inhibited activity of PDE and elevated content of cGMP. Key words: Hydrogen sulfide; Guanosine 3', 5'-cyclic phosphate; Cyclic nucleotide phosphodiesterase; Vasorelaxation

The inotropic effect of the active metabolite of levosimendan, OR-1896, is mediated through inhibition of PDE3 in rat ventricular myocardium.

AimsWe recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca2+ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors.MethodsContractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.ResultsOR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca2+. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α1-AR response.ConclusionThe observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca2+ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.

The Resveratrol Attenuates Ethanol‐Induced Hepatocyte Apoptosis Via Inhibiting ER‐Related Caspase‐12 Activation and PDE Activity In Vitro

Endoplasmic reticulum (ER) stress plays a key role in cell apoptosis pathways. Caspase-12, a proapoptotic gene induced by ER stress, is also the key molecule in ER-related apoptosis. The purpose of this study is to evaluate the protective activity and possible mechanism of resveratrol (ResV) against ethanol (EtOH)-induced apoptosis in human hepatocyte Chang cell line. The human hepatocyte Chang cell line was used to test the hypothesis that ResV may alleviate the liver cell apoptosis induced by EtOH. ER stress-inducible proteins and silent mating type information regulation 2 homolog 1 (SIRT1) were assayed by Western blot. Cell viability was studied by MTT assay and apoptosis was measured by Annexin-V and propidium iodide assay. Caspase-12 activation was examined by immunofluorescence staining. Alcohol dehydrogenase-2 (ADH-2) and aldehyde dehydrogenase-2 (ALDH-2) were measured by polymerase chain reaction amplified product length polymorphism. Phosphodiesterase (PDE) activity was assayed in cell lysates using a cyclic nucleotide PDE assay. EtOH exposure significantly increased the expression of ER stress markers and activated signaling pathways associated with ER stress. These include GRP78, p-IRE1α, p-eIF2α, p-PERK, ATF4 as well as cleaved caspase-3/12, CHOP/GADD153, and Bax in human hepatocyte Chang cell line. The expression of these proteins were significantly down-regulated by ResV (10μM) in a SIRT1-dependent manner. ResV can inhibit EtOH-, tunicamycin-, thapsigargin-induced caspase-12 activation. ADH-2 and ALDH-2 activities are lower in this cell line. PDE activity increased by EtOH was inhibited by ResV (10μM). The results indicate that (i) EtOH-induced activation of caspase-12 could be one of the underlying mechanisms of hepatocyte apoptosis; (ii) EtOH-induced cell apoptosis was alleviated via ResV (10μM) by inhibiting ER stress and caspase-12 activation in a SIRT1-dependent manner; and (iii) SIRT1 activated indirectly by ResV (10μM) attenuates EtOH-induced hepatocyte apoptosis partly through inhibiting PDE activity.

Inhibition of phosphodiestrase 9 induces cGMP accumulation and apoptosis in human breast cancer cell lines, MCF‐7 and MDA‐MB‐468

Phosphodiesterase 9 (PDE9) is a major isoform of phosphodiesterase hydrolysing cGMP and plays a key role in proliferation of cells, their differentiation and apoptosis, via intracellular cGMP signalling. The study described here was designed to investigate expression, activity and apoptotic effect of PDE9 on human breast cancer cell lines, MCF-7 and MDA-MB-468. Activity and expression of PDE9 were examined using colorimetric cyclic nucleotide phosphodiesterase assay and real-time RT-PCR methods respectively; cGMP concentration was also measured. MTT viability test, annexin V-FITC staining, Hoechst 33258 staining and caspase3 activity assay were used to detect apoptosis. Treatment of both cell lines with BAY 73-6691 lead to reduction in PDE9 mRNA expression, PDE9 cGMP-hydrolytic activity and elevation of the intracellular cGMP response. BAY 73-6691 significantly reduced cell proliferation in a dose- and time-dependent manner and caused marked increase in apoptosis through caspase3 activation. Our results revealed that BAY 73-6691 induced apoptosis in these breast cancer cell lines through the cGMP pathway. These data suggest that BAY 73-6691 could be utilized as an agent in treatment of breast cancer.

Potential therapeutic applications of phosphodiesterase inhibition in prostate cancer

Phosphodiesterases (PDEs) play a role in controlling cyclic nucleotide action, including cyclic guanosine monophosphate (cGMP). Previous studies have ascribed a protective role of cGMP signaling on hypoxia-mediated cancer progression. Herein, we determine their potential role in hypoxia-mediated chemoresistance and immune escape. Phosphodiesterase assays were used to measure PDE activity in prostate cancer cell lines (DU145, PC3). Immunoblots were performed to determine the presence of PDEs in human prostate tissue samples. The effect of PDE inhibition on hypoxia-induced chemoresistance (compared to normoxic controls, 20% O2) was determined using clonogenic assays. Flow cytometry was used to determine the effects of PDE inhibition on surface MHC class I-related chain A (MICA), a natural killer (NK) cell-activating ligand. A mouse model was used to evaluate the in vivo effects of PDE inhibition on the growth of human prostate cancer cells. PDE5 and PDE11 were the most prominent PDEs in the cell lines, representing between 86 and 95% of the total cGMP-specific PDE activity. Treatment of DU-145 cells with a PDE inhibitor significantly reduced the hypoxia-associated acquisition of resistance to doxorubicin, with a mean 51% reduction in surviving fraction compared to controls (p < 0.001, ANOVA). As well, PDE inhibition completely reversed (p = 0.02, ANOVA) hypoxia-induced shedding of the immune stimulatory molecule, MICA, and attenuated the growth of human prostate tumor xenografts in an NK cell-competent murine model (p = 0.03, Wilcoxon, Mann-Whitney). These results suggest a rationale for future studies on the potential therapeutic applications of PDE inhibitors in men with prostate cancer.

1272 POTENTIAL IMMUNOTHERAPEUTIC APPLICATIONS OF PHOSPHODIESTERASE INHIBITION FOR PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111272 POTENTIAL IMMUNOTHERAPEUTIC APPLICATIONS OF PHOSPHODIESTERASE INHIBITION FOR PROSTATE CANCER David Siemens, Tom Hamilton, Klodiana Gjoncaj, Nianping Hu, Erin Bell, and Charles Graham David SiemensDavid Siemens Kingston, Canada More articles by this author , Tom HamiltonTom Hamilton Kingston, Canada More articles by this author , Klodiana GjoncajKlodiana Gjoncaj Kingston, Canada More articles by this author , Nianping HuNianping Hu Kingston, Canada More articles by this author , Erin BellErin Bell Kingston, Canada More articles by this author , and Charles GrahamCharles Graham Kingston, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.958AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The cyclic nucleotide phosphodiesterases (PDEs) are a family of enzymes that play a central role in controlling cyclic nucleotide (cGMP) action and previous studies have ascribed a protective role of cGMP mediated signaling on hypoxia- mediated cancer progression. Herein we describe the expression levels of the known PDE variants in prostate cancer as well as explore the functional role that they play in cancer immune escape. METHODS cGMP phosphodiesterase assays were used to measure the PDE activity in human prostate cancer cell lines DU145 and PC3. Western Blot analysis was performed to determine the presence of the PDEs in human tissue samples. The effect of PDE inhibition on hypoxia-mediated immune escape mechanisms was determined after pre-incubating cells in 0.5% or 20% O2 in the absence or presence of Zaprinast (10-7–10-6 M) and subsequent fow cytometric analysis of MICA, an essential tumor-associated antigen required for innate immune responses. A NK-competent murine model was used to measure the in vivo effects of Zaprinast on the growth of human prostate cancer xenografts. RESULTS PDE activity assays indicated that the majority of cGMP PDE activity in prostate cancer cell lines is made up from a combination of PDE5 and PDE11. The same two PDE variants also predominated in human malignant prostate tissue as detected using Western blot. TUNEL assays revealed some increased apoptosis when prostate cells were treated with a PDE inhibitor in both hypoxic and standard oxygen conditions. MICA expression was reduced when prostate cancer cells were exposed to hypoxia; however, immunogenicity could be restored by incubating hypoxic samples with a PDE inhibitor (see figure, p<0.01). Finally, growth of human prostate tumor xenografts in mice was inhibited by PDE inhibition compared to controls (p<0.05). CONCLUSIONS PDE 5 and 11 are present in human prostate cancer tissue and contribute to the majority of cGMP PDE activity in prostate cancer cell lines. Inhibition of PDE activity, reestablishing cGMP cell signaling in the hypoxic tumor environment, would appear to have beneficial effects including modulating hypoxia-induced cancer immune escape. These results indicate that PDE inhibition may represent a novel therapeutic or adjuvant target for men with prostate cancer. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e509 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information David Siemens Kingston, Canada More articles by this author Tom Hamilton Kingston, Canada More articles by this author Klodiana Gjoncaj Kingston, Canada More articles by this author Nianping Hu Kingston, Canada More articles by this author Erin Bell Kingston, Canada More articles by this author Charles Graham Kingston, Canada More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Vasorelaxant effect of flavonoids through calmodulin inhibition: Ex vivo, in vitro, and in silico approaches

In our search for potential antihypertensive agents, a series of structurally-related flavonoids was screened. Ex vivo and in vitro biological evaluations indicated that compounds 1-7 displayed an important vasorelaxant effect on the endothelium-intact (E(+)) and -denuded (E(-)) aortic rings test. Their in vitro anti-calmodulin (CaM) properties were determined by means of the inhibitory effect on the activation of the calmodulin-sensitive cAMP phosphodiesterase (PDE1) assay. Molecular modeling experiments were also performed in order to explore the probable binding site of 1-7 with CaM, and the results indicated that they could bind to the protein in the same pocket as trifluoperazine (TFP), a well-known CaM inhibitor.

Cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase is functional in bovine mammary gland

Previous studies have shown that using nonspecific phosphodiesterase (PDE) inhibitors such as caffeine improved milk production, supporting the premise that modulation of intracellular concentration of cyclic nucleotides (cyclic AMP, cyclic guanosine 3′-5′-monophosphate) is involved. Intracellular cyclic nucleotides are degraded by the PDE enzyme family. The contribution of type IV PDE (PDE4) in the secretion of casein has been reported in rat mammary gland. The objective of this study was to demonstrate the functional presence of the PDE4 family in the bovine mammary gland. To understand the enzymatic expression pattern in the mammary gland, tissue samples were taken randomly from udders obtained from a local slaughterhouse. Reverse transcription PCR revealed that the PDE4D transcript was amplified, and the expected size fragment was obtained in a 1% agarose gel. Sequence analysis of the amplicon resulted in 99% homology to PDE4D. Moreover, Western blotting using a specific PDE4D antibody has confirmed that the protein of the isoenzyme PDE4D1 is present. A clear immunoreactive signal was also observed within the acini where epithelial cells are located. Assaying cyclic AMP PDE activity reported a total activity of 38.71±3.22 fmol/min per μg of total protein. Rolipram, a specific PDE4 inhibitor, showed a sensitive activity of 8.48±1.28 fmol/min per μg of total protein, indicating that PDE4 is responsible for one-fifth of the total enzymatic activity of PDE in the mammary gland. To further validate the presence of PDE4D in the bovine mammary epithelial cells, protein extracts from bovine mammary epithelial cells were separated on SDS-PAGE gels, and PDE4D protein was detected. The PDE assays reported a total activity of 30.16±4.82 fmol/min per μg of total protein. Rolipram showed a sensible activity of 11.91±5.93 fmol/min per μg of total protein. In conclusion, these results not only demonstrate the presence of PDE4D transcript and protein, but also show an active enzyme, suggesting a functional role of PDE4D in bovine mammary gland.

Characterization of an Evolutionarily Conserved Metallophosphoesterase That Is Expressed in the Fetal Brain and Associated with the WAGR Syndrome

Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2',3'-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events.

Protection against β-Amyloid-induced Apoptosis by Peptides Interacting with β-Amyloid

beta-Amyloid peptide produces apoptosis in neurons at micromolar concentrations, but the mechanism by which beta-amyloid exerts its toxic effect is unknown. The normal biological function of beta-amyloid is also unknown. We used phage display, co-precipitation, and mass spectrometry to examine the protein-protein interactions of beta-amyloid in normal rabbit brain in order to identify the biochemical receptors for beta-amyloid. beta-Amyloid was found to bind primarily to proteins involved in low density lipoprotein and cholesterol transport and metabolism, including sortilin, endoplasmic reticulum-Golgi intermediate compartment 2 (ERGIC2), ERGIC-53, steroid 5alpha-reductase, and apolipoprotein B. beta-Amyloid also bound to the C-reactive protein precursor, a protein involved in inflammation, and to 14-3-3, a protein that regulates glycogen synthase kinase-3beta, the kinase involved in tau phosphorylation. Of eight synthetic peptides identified as targets of beta-amyloid, three were found to be effective blockers of the toxic effect of beta-amyloid on cultured neuronal cells. These peptides bound to the hydrophobic region (residues 17-21) or to the nearby protein kinase C pseudo-phosphorylation site (residues 26-30) of beta-amyloid, suggesting that these may be the most critical regions for beta-amyloid effector action and for aggregation. Peptides or other small molecules that bind to this region may protect against beta-amyloid toxic effect by competitively blocking its ability to bind beta-amyloid effector proteins such as sortilin and 14-3-3.

Critical Role of the Heme Axial Ligand, Met95, in Locking Catalysis of the Phosphodiesterase from Escherichia coli (Ec DOS) toward Cyclic diGMP

Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O(2) or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg(97) in the heme distal side in Ec DOS interacts with the O(2) molecule and Met(95) serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O(2) complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O(2). In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O(2), CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met(95) coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met(95) caused by the binding of the external ligands or mutations at Met(95) releases the catalytic lock and activates catalysis.

Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133-135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domains and a catalytic domain highly homologous with that of the T. brucei TbPDE2/TbrPDEB2 family. The N-terminus+GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2 N-terminus+GAF-A domain was found to bind cAMP with an affinity of approximately 500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of approximately 1 muM. The N-terminus+GAF-A domain of TcrPDEB1 did not bind cGMP, whereas the N-terminus+GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of approximately 3 muM. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2'-O-(N-methylanthraniloyl)adenosine-3',5'-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.

PDE7A1, a cAMP-specific Phosphodiesterase, Inhibits cAMP-dependent Protein Kinase by a Direct Interaction with C

The N-terminal regulatory region of the high affinity cAMP-specific phosphodiesterase, PDE7A1, contains two copies of the cAMP-dependent kinase (PKA) pseudosubstrate site RRGAI. In betaTC3 insulinoma cells, PDE7A1 co-localizes with PKA II in the Golgi-centrosome region. The roles PDE7A1 and its regulatory region play in cAMP signaling were examined by studying interactions with PKA subunits. PDE7A1 associates with the dissociated C subunit of PKA (C), but does not bind tetrameric PKA holoenzyme. High affinity binding of C by PDE7A1 inhibits kinase activity in vitro (IC50 = 0.5 nm). The domain containing PKA pseudosubstrate sites at the N terminus of PDE7A1 mediates complex formation with C. The PDE7A1 N-terminal repeat region inhibits C activity in CHO-K1 cells and also suppresses C dependent, cAMP-independent, physiological responses in yeast. Thus, PDE7A1 possesses a non-catalytic activity that can contribute to the termination of cAMP signals via direct inhibition of C. This study identifies a novel inhibitor of PKA and a non-catalytic affect of a cyclic nucleotide phosphodiesterase.

Inhibition of Autotaxin by Lysophosphatidic Acid and Sphingosine 1-Phosphate

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki approximately 10(-7) M). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand.

Endothelium-dependent and -independent vasorelaxation by a theophylline derivative MCPT: Roles of cyclic nucleotides, potassium channel opening and phosphodiesterase inhibition

The vasorelaxation activities of MCPT, a newly synthesized xanthine derivative, were investigated in this study. In phenylephrine (PE)-precontracted rat aortic rings with intact endothelium, MCPT caused a concentration-dependent relaxation, which was inhibited by endothelium removed. This relaxation was also reduced by the presence of nitric oxide synthase inhibitor L ω-nitro-L-arginine methylester (L-NAME, 100 μM), soluble guanylyl cyclase (sGC) inhibitors methylene blue (10 μM), 1 H-[1,2,4] oxidazolol [4,3-a] quinoxalin-1-one (ODQ, 1 μM), adenylyl cyclase (AC) blocker SQ 22536 (100 μM), ATP-sensitive K + channel blocker (K ATP) glibenclamide (1 μM), a Ca 2+ activated K + channels blocker tetraethylammonium (TEA, 10 mM) and a voltage-dependent potassium channels blocker 4-aminopyridine (4-AP, 100 μM). The vasorelaxant effects of MCPT together with IBMX (0.5 μM) had an additive action. In PE-preconstricted endothelium-denuded aortic rings, the vasorelaxant effects of MCPT were attenuated by pretreatments with glibenclamide (1 μM), SQ 22536 (100 μM) or ODQ (1 μM), respectively. MCPT enhanced cAMP-dependent vasodilator isoprenaline- and NO donor/cGMP-dependent vasodilator sodium nitroprusside-induced relaxation activities in endothelium-denuded aortic rings. In A-10 cell and washed human platelets, MCPT induced a concentration-dependent increase in intracellular cyclic GMP and cyclic AMP levels. In phosphodiesterase assay, MCPT displayed inhibition effects on PDE 3, PDE 4 and PDE 5. The inhibition % were 52 ± 3.9, 32 ± 2.6 and 8 ± 1.1 respectively. The Western blot analysis on HUVEC indicated that MCPT increased the expression of eNOS. It is concluded that the vasorelaxation by MCPT may be mediated by the inhibition of phosphodiesterase, stimulation of NO/sGC/ cGMP and AC/cAMP pathways, and the opening of K + channels.