cGMP-specific Phosphodiesterase Research Articles

Differential relaxant responses of guinea-pig lung strips and bronchial rings to sodium nitroprusside: a mechanism independent of cGMP formation.

The biochemical mechanism subserving smooth muscle relaxant effects of sodium nitroprusside was examined on U46619, 9,11-dideoxy-9 alpha,11 alpha-methanoepoxy PGF2 alpha, precontracted guinea-pig lung strips and hilar bronchial rings. Lung strips were resistant to the relaxant action of sodium nitroprusside or sodium nitrite (NaNO2), whereas they markedly relaxed to 8-bromo-cyclic GMP (8-Br-cGMP), a membrane permeable analogue of cGMP. Precontracted bronchial rings completely relaxed to sodium nitroprusside, NaNO2, or 8-Br-cGMP in a concentration-dependent manner. Sodium nitroprusside (10 microM) substantially raised tissue cGMP level in lung strips. Conversely, sodium nitroprusside had no detectable effect on cGMP levels in bronchial rings. In the presence of 10 microM dipyridamole, an agent which preferentially inhibits cGMP-specific phosphodiesterase, cGMP levels in lung strips treated with sodium nitroprusside was significantly enhanced, but sodium nitroprusside demonstrated no relaxant effect on the preparations. However, dipyridamole potentiated sodium nitroprusside-induced precontracted bronchial ring relaxation without affecting the bronchial tissue cGMP level. In the presence of 10 microM LY83583 (6-anilino-5,8-quinoline-dione), a specific cGMP concentration-lowering agent, sodium nitroprusside-mediated elevation of cGMP level in lung strips was significantly reduced with no effect on the functional response. LY83583 demonstrated no inhibitory effect on either relaxation or cGMP level in bronchial rings treated with sodium nitroprusside. Our results suggest that precontracted smooth muscle in lung strips and in hilar bronchi respond distinctly to sodium nitroprusside. Furthermore, sodium nitroprusside mediates bronchial smooth muscle relaxation by mechanisms unrelated to cGMP.

2-(2-Fluorobenzamido)benzoate ethyl ester (EFB-1) inhibits superoxide production by human neutrophils and attenuates hemorrhagic shock-induced organ dysfunction in rats

Neutrophil activation after trauma–hemorrhagic shock (T/H) has been implicated in the development of multiple organ dysfunction (MOD). In this study, we report that a small chemical compound, 2-(2-fluorobenzamido)benzoic acid ethyl ester (EFB-1), exhibited a potent inhibitory effect on the formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-induced superoxide anion (O2•−) release and CD11b expression by human neutrophils. Additionally, administration of EFB-1 in rats subjected to T/H caused a significant improvement in MOD. EFB-1 treatment induced an increase in cAMP formation and protein kinase (PK) A activity in FMLP-activated neutrophils, which occurred through the selective inhibition of cAMP-specific phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function or cGMP-specific PDE activity. FMLP-induced phosphorylation of protein kinase B (AKT), but not calcium mobilization, was reduced by EFB-1. The inhibitory effects of EFB-1 on O2•− production, CD11b expression, and AKT phosphorylation were reversed by PKA inhibitors (H89 and KT5720). Significantly, administration of EFB-1 (1mg/kg body wt) attenuated the myeloperoxidase activity of the intestines, lungs, and liver and reduced the wet/dry weight ratio of the intestines and lungs and plasma alanine aminotransferase and aspartate aminotransferase levels in Sprague–Dawley rats after T/H. Therefore, EFB-1 is a new inhibitor of cAMP-specific PDE that potently suppresses O2•− release and CD11b expression by human neutrophils and attenuates T/H-induced MOD in rats.

Distinct Allostery Induced in the Cyclic GMP-binding, Cyclic GMP-specific Phosphodiesterase (PDE5) by Cyclic GMP, Sildenafil, and Metal Ions

The activity of many proteins orchestrating different biological processes is regulated by allostery, where ligand binding at one site alters the function of another site. Allosteric changes can be brought about by either a change in the dynamics of a protein, or alteration in its mean structure. We have investigated the mechanisms of allostery induced by chemically distinct ligands in the cGMP-binding, cGMP-specific phosphodiesterase, PDE5. PDE5 is the target for catalytic site inhibitors, such as sildenafil, that are used for the treatment of erectile dysfunction and pulmonary hypertension. PDE5 is a multidomain protein and contains two N-terminal cGMP-specific phosphodiesterase, bacterial adenylyl cyclase, FhLA transcriptional regulator (GAF) domains, and a C-terminal catalytic domain. Cyclic GMP binding to the GAFa domain and sildenafil binding to the catalytic domain result in conformational changes, which to date have been studied either with individual domains or with purified enzyme. Employing intramolecular bioluminescence resonance energy transfer, which can monitor conformational changes both in vitro and in intact cells, we show that binding of cGMP and sildenafil to PDE5 results in distinct conformations of the protein. Metal ions bound to the catalytic site also allosterically modulated cGMP- and sildenafil-induced conformational changes. The sildenafil-induced conformational change was temperature-sensitive, whereas cGMP-induced conformational change was independent of temperature. This indicates that different allosteric ligands can regulate the conformation of a multidomain protein by distinct mechanisms. Importantly, this novel PDE5 sensor has general physiological and clinical relevance because it allows the identification of regulators that can modulate PDE5 conformation in vivo.

Drosophila gustatory preference behaviors require the atypical soluble guanylyl cyclases

The intracellular messenger cGMP has been suggested to play a role in taste signal transduction in both vertebrates and invertebrates. In the present study, we have examined the role of the Drosophila atypical soluble guanylyl cyclases (sGCs), Gyc-89Da and Gyc-89Db, in larval and adult gustatory preference behaviors. We showed that in larvae, sucrose attraction requires Gyc-89Db and caffeine avoidance requires Gyc-89Da. In adult flies, sucrose attraction is unaffected by mutations in either gene whereas avoidance of low concentrations of caffeine is eliminated by loss of either gene. Similar defective behaviors were observed when cGMP increases were prevented by the expression of a cGMP-specific phosphodiesterase. We also showed that both genes were expressed in gustatory receptor neurons (GRNs) in larval and adult gustatory organs, primarily in a non-overlapping pattern, with the exception of a small group of cells in the adult labellum. In addition, in adults, several cells co-expressed the bitter taste receptor, Gr66a, with either Gyc-89Da or Gyc-89Db. We also showed that the electrophysiological responses of a GRN to caffeine were significantly reduced in flies mutant for the atypical sGCs, suggesting that at least part of the adult behavioral defects were due to a reduced ability to detect caffeine.

Oncogenic BRAF Induces Melanoma Cell Invasion by Downregulating the cGMP-Specific Phosphodiesterase PDE5A

We show that in melanoma cells oncogenic BRAF, acting through MEK and the transcription factor BRN2, downregulates the cGMP-specific phosphodiesterase PDE5A. Although PDE5A downregulation causesa small decrease in proliferation, its major impact is to stimulate a dramatic increase in melanoma cell invasion. This is because PDE5A downregulation leads to an increase in cGMP, which induces an increase in cytosolic Ca(2+), stimulating increased contractility and inducing invasion. PDE5A downregulation also this leads to an increase in short-term and long-term colonization of the lungs by melanoma cells. We do not observe this pathway in NRAS mutant melanoma or BRAF mutant colorectal cells. Thus, we show that in melanoma cells oncogenic BRAF induces invasion through downregulation of PDE5A.

Involvement of phosphodiesterase type 6 and retinopathy

磷酸二酯酶6(PDE6)是对环鸟苷酸(cGMP)特异的PDE,广泛分布于视网膜杆状光感受器细胞中,是脊椎动物光转录过程中的关键酶.它通过调节细胞内特定区域的cGMP水平,参与脊椎动物视觉传导级联反应.PDE蛋白质亚基的结构域通过与转导蛋白的直接作用来调节PDE的活性,从而调节光子的转导过程.视网膜色素变性(RP)等疾病的发生与PDE蛋白质亚基的突变密切相关.近年来PDE同工酶在细胞中的定位及定量研究为选择性调节药物的开发提供了研究基础.就PDE6的结构、功能、视功能调控机制及应用前景进行综述。

Effects of a cGMP-Specific Phosphodiesterase Inhibitor on Expression of Endothelial Nitric Oxide Synthase and Vascular Endothelial Growth Factor in Rats with Cyclosporine-Induced Nephrotoxicity

Background The mechanism of cyclosporine (CsA)–induced nephrotoxicity has been suggested to be vasoconstriction due to reduced nitric oxide (NO), providing tissue fibrosis by elevation of transforming growth factor beta and vascular endothelial growth factor (VEGF). In this study using a rat model of CsA-induced nephrotoxicity, we administered a phosphodiesterase-5 inhibitor to ameliorate the renal injury and alter the expression of endothelial No synthase (eNOS) and VEGF. Methods A right nephrectomy was performed in Sprague-Dawley rats (n = 30; 200–250 g, all male). The Ischemia group (n = 6) underwent ligation of the left renal artery for 45 minutes (IR) before observation for 28 days. After IR, the udenafil group (n = 6) was treated with 10 mg/kg drug orally, the CsA group (n = 6) received 15 mg/kg CsA injected subcutaneously and the CsA plus udenafil group (n = 6) received 15 mg/kg CsA injected subcutaneously together with the oral administration of 10 mg/kg udenafil. Results Administration of udenafil significantly decreased serum creatinine either alone (0.21 ± 0.04 mg/dL) or in combination with CsA (1.86 ± 0.35 mg/dL) versus the ischemia (0.85 ± 0.22 mg/dL) and the CsA alone (3. 10 ± 0.77 mg/dL) group. ( P = .002; P = .002). Comparing the Hematoxylin-eosin staining of the ischemia (0.41 ± 0.09) and CsA (0.44 ± 0.08) groups showed a significantly decreased loss of nuclei in proximal tubules after the administration of udenafil (0.27 ± 0.05 [ P = .004] and 0.26 ± 0.02 [ P = .002] respectively). Immunohistochemical staining showed strong eNOS staining in the udenafil and CsA plus udenafil groups. Western blots for eNOS showed decreased expression in the CsA group and increased expression in the udenafil group. Western blots for VEGF revealed reduced expression only in the CsA plus udenafil group. eNOS mRNA was decreased in the CsA (0.017 ± 0.010) compared with the ischemia group (0.048 ± 0.015; P = .000). VEGF mRNA which was decreased in the CsA group (2.026 ± 1.109), showed greater tendency after administration of udenafil (0.440 ± 0.449) ( P = .003). Conclusion The phosphodiesterase inhibitor ameliorated renal injury in a rat model of CsA-induced nephrotoxicity, possibly related to increased eNOS and reduced VEGF expression.

Use of a Schizosaccharomyces pombe PKA-repressible reporter to study cGMP metabolising phosphodiesterases

The Schizosaccharomyces pombe fbp1 gene is transcriptionally repressed by protein kinase A (PKA) that is activated by extracellular glucose via a cAMP-signaling pathway. We previously used an fbp1–ura4 reporter that places uracil biosynthesis under the control of the glucose-sensing pathway to identify mutations in genes of the cAMP pathway. More recently, this reporter has been used in high throughput screens for small molecule inhibitors of heterologously-expressed cyclic nucleotide phosphodiesterases (PDEs) that hydrolyse cAMP to 5′ AMP. Here we show that strains lacking the adenylyl cyclase gene respond to either exogenous cAMP or cGMP to activate PKA, thus regulating fbp1–ura4 expression and other PKA-regulated processes such as conjugation and the nuclear export of an Rst2–GFP fusion protein. Expression of cGMP-specific PDEs or ones that hydrolyse both cAMP and cGMP increases the amount of exogenous cGMP required to activate PKA in order to repress fbp1–ura4 expression, creating conditions that allow detection of inhibitors of these PDEs. As proof of this concept, we screened a collection of compounds previously identified as inhibitors of cAMP-specific PDE4 or PDE7 enzymes for their ability to inhibit the mammalian cGMP-specific PDE5A enzyme. We identified compound BC76, which inhibits PDE5A in an in vitro enzyme assay with an IC50 of 232nM. Further yeast-based assays show that BC76 inhibits PDE1, PDE4, PDE5, PDE8, PDE10 and PDE11, thus demonstrating the utility of this system for detecting and characterising inhibitors of either cAMP- or cGMP-metabolising PDEs.

Phosphodiesterase inhibition with tadalafil provides longer and sustained protection of stem cells

We hypothesized that inhibition of the cGMP-specific enzyme phosphodiesterase 5A (PDE5A) promoted cGMP/protein kinase G (PKG) activity to condition stem cells for enhanced survival and proliferation. One-time tadalafil treatment (1 μM for 30 min) of mesenchymal stem cells ((Tada)MSCs) provided sustained protection of cells for 36 h. Higher cGMP activity with concomitantly increased PKG1 activity was observed in (Tada)MSCs, which peaked within 12 h after tadalafil treatment. Pretreatment with PKG1 blockers (1 μM KT-5823 or 20 nM K-252a) or transduction with adenoviral PKG1-short-hairpin RNA abolished tadalafil-induced cytoprotection of the cells. A higher proliferation rate was observed in (Tada)MSCs compared with nontreated MSCs ((Cont)MSCs). In a rat model of acute myocardial infarction, (Tada)MSCs transplanted 0 and 24 h after tadalafil treatment showed higher survival compared with (Cont)MSCs on day 2 and day 4 after engraftment. (Tada)MSCs transplanted 48 h after tadalafil treatment lost their protection on both day 2 and day 4 after engraftment, and their rate of survival was similar to (Cont)MSCs. Reduced terminal dUTP nick end-labeling positivity (P < 0.01 vs. (Cont)MSCs) and higher proliferation of (Tada)MSCs (P < 0.01 vs. (Cont)MSCs) was observed in the infarcted heart. Fluorescence immunostaining revealed neomyogenesis in both the infarct and peri-infarct areas. Blood vessel density was significantly increased in group 2 compared with group 1. Transthoracic echocardiographic heart function revealed significant preservation of the indexes of left ventricle contractility and attenuation of remodeling in (Tada)MSC-engrafted animal hearts (group 2) compared with (Cont)MSCs (group 1). PDE5A inhibition using long-acting tadalafil is an innovative approach to promote stem cell survival and proliferation in the infarcted heart.

Co-incubation of oocytes with the selective phosphodiesterase 9 (PDE9) inhibitor BAY 73-6691 (BAY) and low levels of cGMP blocks spontaneous resumption of meiosis in vitro

OBJECTIVE: A drop in cAMP triggered by PDE 3 is a key step in meiotic resumption of the oocyte. cGMP is an endogenous inhibitor of PDE 3, and is known to prevent oocyte maturation at high levels. The objective for this study was to determine if a PDE9 (cGMP-specific phosphodiesterase expressed in the oocyte) inhibitor could inhibit spontaneous resumption of meiosis in oocytes. DESIGN: Controlled rodent dose response experiments. MATERIALS AND METHODS: Germinal vesicle (GV) intact oocytes collected from PMG-stimulated BDF-1 mice and stripped of cumulus cells, were incubated in KSOM media containing either BAY (10 μM, 100 μM, or 1 mM), 8-Br-cGMP, [the membrane-permeable analogue of cGMP, (500 μM, 1 mM, or 10 mM)], a combination of both treatments [8-Br-cGMP (1 mM) and either 100 μM or 1 mM BAY], or no additional treatment (control). Oocytes were evaluated for GV-breakdown (GVBD) as an indicator of meiotic resumption after 20-22 hours. Results were analyzed using two-tailed Fisher's exact test. RESULTS: In total, 46/49 (94%) control oocytes progressed to GVBD, compared with 40/48 (83%, P = 0.76), 20/60 (33%, P < 0.001) and 0/54 (0%, P < 0.001) in oocytes incubated with 8-Br-cGMP at 500 μM, 1 mM, or 10 mM respectively. While culture of oocytes in any concentration of BAY alone did not significantly affect meiosis, co-culture with either 1mM or 100 μM BAY and 1 mM 8-Br-cGMP prevented GVBD in the majority of oocytes (92 & 93% respectively; p<0.001). Interestingly, none of the oocytes which resumed meiosis in co-culture matured to metaphase II, but rather arrested at metaphase I, whereas 23% of the oocytes cultured in only 1mM 8-Br-cGMP progressed to MII. CONCLUSION: While a PDE9 inhibitor alone is not sufficient to inhibit oocyte maturation in vitro, a powerful effect is seen in combination with added cGMP. Therefore a combined approach that increases cGMP transport to the oocyte from granulosa cells and blocks degradation with a PDE9 inhibitor may have contraceptive potential.

Structural complexity of the testis and PKG I / StAR interaction regulate the Leydig cell adaptive response to repeated immobilization stress

The role of the structural complexity of the testis and the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway was analysed in adult male rats exposed to acute and repeated immobilization stress (IMO). In whole testis preparations, exposure to acute and repeated IMO caused an increase in NO production. In contrast, NO production was inhibited in interstitial cell preparations after exposure to all types of stress. In purified Leydig cell preparations, NO production was inhibited only after exposure to prolonged IMO. These findings indicate that biologically active compounds released from various testicular compartments exert both stimulatory and inhibitory effects on NO production. TaqMan Low Density Array of rat phosphodiesterases revealed a decrease in the expression of cGMP-specific phosphodiesterase 5 (PDE5) in Leydig cells of animals exposed to repeated IMO. In contrast, the expression of cGMP-dependent protein kinase type I (PKG I), total and phosphorylated steroidogenic acute regulatory protein (StAR), and PKG I/StAR immunoprecipitated complex was increased during repeated exposure to IMO. The increase in both total and phosphorylated StAR formation was effectively blocked by inhibition of PKG I in vitro. Thus, increased expressions of PKG I and StAR complex, accompanied by decreased PDE5 activity, suggest that the NO-cGMP signalling pathway and consequent activation of the StAR protein regulate the adaptive response of Leydig cells to repeated IMO stress.

Dendritic spine density deficits in the hippocampal CA1 region of young Tg2576 mice are ameliorated with the PDE9A inhibitor PF-04447943

Many forms of synaptic plasticity are critically dependent upon production of cGMP to trigger activity-dependent increases in synaptic size and strength. Phosphodiesterase 9A (PDE9A) is a high affinity, cGMP-specific phosphodiesterase with widespread distribution in the central nervous system. Inhibition of PDE9A results in significant accumulation of cGMP in brain tissue and CSF of rodents and also increases CSF cGMP in human volunteers (Schmidt et al., 2009 ICAD; Nicholas et al 2009 ICAD). We hypothesize that chronic exposure to a PDE9 inhibitor, and the associated elevations in brain cGMP could provide a therapeutic benefit to vulnerable synapses chronically exposed to Abeta in transgenic amyloid overexpressing mice. A total of N = 20 animals per group of 4 month old Tg2576+ and non-transgenic animals were implanted with Alzet osmotic minipumps to deliver vehicle or the PDE9A inhibitor PF-4447943, currently in clinical trials for the treatment of mild to moderate Alzheimer's Disease. Neurobehavioral outcomes were measured as conditioned fear response after 30 days of treatment and subsequently the brains were harvested for measurement of Abeta, gene expression profiling or synaptic density as assessed by Golgi staining of dendrites. Dendritic spine density on apical dendrites of CA1 neurons exhibited a small but significant deficit in the density of dendritic spines in vehicle treated transgenic animals as compared to non-transgenic animals. This deficit was ameliorated by 30 days of exposure to PF-04447943. No significant drug effect was observed in the non-transgenic animals. No significant effects of drug treatment were observed on Abeta levels in Tg2576 mice. Behavioral analysis of transgenic animals showed deficits in fear conditioning as early as 2 months of age, and therefore were considered unlikely to be due to the accumulation of oligomeric Abeta. These deficits were not affected by drug treatment. Transcriptional profiles of animals treated with drug compared to vehicle showed evidence of regulation of pathways related to synaptic plasticity and remodeling of the dendritic cytoskeleton, consistent with stabilization of vulnerable spine structure. This data supports the hypothesis that PDE9 inhibition can stabilize vulnerable synapses early in the Alzheimer's disease process.

Testosterone-Induced Modulation of Nitric Oxide-cGMP Signaling Pathway and Androgenesis in the Rat Leydig Cells1

Testosterone, acting as a systemic and local factor, is one of the major regulatory molecules that initiate and maintain testicular function. In the present study, different experimental approaches were used to evaluate the role of testosterone in regulation of the nitric oxide (NO)-cGMP pathway in Leydig cells derived from normal and hypogonadotropic male rats treated with testosterone for 24 h and 2 wk. Real-time quantitative PCR and Western blot analysis revealed increased inducible NO synthase (NOS2) expression followed by increased NO secretion from Leydig cells ex vivo after continuous treatment with testosterone for 2 wk in vivo. The cGMP-specific phosphodiesterases Pde5, Pde6, and Pde9 were up-regulated, whereas PRKG1 protein was decreased after a 2-wk testosterone treatment. Induction of Nos2 and Pde5 in Leydig cells was blocked by androgen receptor antagonist. In experimental hypogonadotropic hypogonadism, expression of NOS2 was significantly reduced, and treatment with testosterone increased NOS2 expression above control levels. PDE5 protein level was unchanged in hypogonadal rats, whereas treatment of hypogonadal rats with testosterone significantly increased it. In contrast, hypogonadism and testosterone replacement reduced PRKG1 protein in Leydig cells. In vitro treatment with testosterone caused gradually increased Nos2 gene expression followed by increased nitrite and cGMP production by purified Leydig cells. In summary, testosterone up-regulated NO signaling via increased NOS2 expression and contributed to down-regulation of cGMP signaling in Leydig cells. Thus, testosterone-induced modulation of NO-cGMP signaling may serve as a potent autocrine regulator of testicular steroidogenesis.

Nitric oxide induces airway smooth muscle cell relaxation by decreasing the frequency of agonist-induced Ca2+ oscillations

Nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, but the underlying mechanism is not well understood. Consequently, we investigated the effects of NO on airway SMC contraction, Ca2+ signaling, and Ca2+ sensitivity in mouse lung slices with phase-contrast and confocal microscopy. Airways that were contracted in response to the agonist 5-hydroxytryptamine (5-HT) transiently relaxed in response to the NO donor, NOC-5. This NO-induced relaxation was enhanced by zaprinast or vardenafil, two selective inhibitors of cGMP-specific phosphodiesterase-5, but blocked by ODQ, an inhibitor of soluble guanylyl cyclase, and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Simultaneous measurements of airway caliber and SMC [Ca2+]i revealed that airway contraction induced by 5-HT correlated with the occurrence of Ca2+ oscillations in the airway SMCs. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca2+ oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced airway relaxation and decreased the frequency of the Ca2+ oscillations. NOC-5 inhibited the increase of [Ca2+]i and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP3) in airway SMCs. The effect of NO on the Ca2+ sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca2+ by treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca2+-permeabilized airways. Consequently, we conclude that NO, acting via the cGMP–PKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca2+ via the IP3 receptor to decrease the frequency of agonist-induced Ca2+ oscillations.

Multiple Affinity States of cGMP-Specific Phosphodiesterase for Sildenafil Inhibition Defined by cGMP-Dependent and cGMP-Independent Mechanisms

cGMP-specific phosphodiesterase (PDE5) has become a target for drug development for the treatment of a number of physiological dysfunctions, affected by changes in the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway. PDE5 has two highly homologous regulatory domains, GAF-A and GAF-B. We showed previously that PDE5 could be converted from a low-activity (nonactivated) state to a high-activity state upon cGMP binding to the GAF-A domain with higher sensitivities toward sildenafil (EMBO J 22:469-478, 2003). Here we investigated whether sildenafil sensitivity of PDE5 could be modified by cGMP-independent mechanisms. Individually expressed recombinant GAF-A and GAF-B proteins were tested for their ability to modulate full-length recombinant PDE5 affinity to sildenafil. The GAF-A domain protein had the most dramatic effect on the affinity of the nonactivated recombinant PDE5 for sildenafil, revealing much higher sensitivity to sildenafil inhibition. The apparent affinity for sildenafil increased from the nanomolar range to the picomolar range, providing evidence for the presence of a "super-high" sensitivity state of PDE5 for sildenafil inhibition. In human platelet, higher sensitivity of PDE5 for sildenafil inhibition has been detected after blocking cGMP-binding sites of the GAF-A domain. Thus, our data demonstrate that high sensitivity of PDE5 for sildenafil can be obtained not only through cGMP-induced activation of PDE, but also through cGMP-independent modulation of PDE5 in the nonactivated state, possibly through protein-protein interaction. Furthermore, data suggest that nonactivated PDE5 with "super-high" affinities for sildenafil inhibition may be responsible for therapeutic effects of long-term treatments with low doses of PDE5 inhibitors.

A single dose of Sildenafil does not enhance FeNO: A randomised, cross-over and placebo-controlled study

Monitoring of asthma control can be performed with different means including measurement of the concentration of nitric oxide (NO) in exhaled air. Due to its action on the NO-metabolism; we hypothesized that the intake of Sildenafil might augment and falsify the NO-values in exhaled air of subjects taking the drug to treat erectile dysfunction. This randomised, placebo-controlled cross-over study including 10 male non-asthmatic volunteers taking a single dose of 50 mg Sildenafil did not confirm this assumption in non-asthmatic subjects. We cannot think of any reason why asthmatics should behave differently. On the basis of these results, it does not seem necessary to ask asthma patients with elevated NO-values if they had taken any selective inhibitor of the cGMP-specific phosphodiesterase Type 5 as Sildenafil prior to the test.

Cloning and Sequence Analysis of Red/Blue Light Chimeric Photoreceptor Genes from Three Fern Species (Coniogramme intermedia var. glabra, Plagiogyria distinctissima and Pronephrium lakhimpurnense)

Abstract Neochrome (NEO) is a particular chimeric photoreceptor that can respond to red/far red as well as blue light. Using this photoreceptor, some cryptogams may enhance light sensitivity at low light environment. However, the NEO sequence information is extremely limited. Using inverse PCR and single oligonucleotide nested PCR genome-walking approaches, the full-length genomic sequences of NEO genes were determined for three fern species Coniogramme intermedia var. glabra, Plagiogyria distinctissima and Pronephrium lakhimpurnense. Sequence and structure analysis results indicate that they possess a conserved structure and organization. Introns are absent in three genes. They each contain an 4332, 4308 and 4317-bp ORF (Open reading frame), encoding a deduced protein with 1443, 1435 and 1438 amino acid residues. Their N-terminus consists of a putative functional phytochrome sensory module including PAS (Per / Arnt / Sim), GAF (cGMP specific phosphodiesterase / adenylate cyclases / formate hydrogen lyase...

Cardiac Arrest Caused By Sildenafil Overdosage, A Case Report

Sildenafil citrate (Viagra, Revatio) is a drug used to treat erectile dysfunction and pulmonary arterial hypertension. It acts by inhibiting cGMP specific phosphodiesterase type 5, an enzyme that regulates blood flow in the penis. Since becoming available in 1998, sildenafil has been the prime treatment for erectile dysfunction, rare but serious side effect found after post marketing surveillance includes severe hypotension, myocardial infarction, ventricular arrhythmias, and stroke. We report a case of sildenafil over dosage where the patient developed severe hypotension, and had a cardiac arrest twice.

Cyclic Nucleotide Binding GAF Domains from Phosphodiesterases: Structural and Mechanistic Insights

GAF domains regulate the catalytic activity of certain vertebrate cyclic nucleotide phosphodiesterases (PDEs) by allosteric, noncatalytic binding of cyclic nucleotides. GAF domains arranged in tandem are found in PDE2, -5, -6, -10, and -11, all of which regulate the cellular concentrations of the second messengers cAMP and/or cGMP. Nucleotide binding to GAF domains affects the overall conformation and the catalytic activity of full-length PDEs. The cyclic nucleotide-bound GAF domains from PDE2, -5, -6, and -10 all adopt a conserved fold but show subtle differences within the binding pocket architecture that account for a large range of nucleotide affinities and selectivity. NMR data and details from the structure of full-length nucleotide-free PDE2A reveal the dynamic nature and magnitude of the conformational change that accompanies nucleotide binding. The discussed GAF domain structures further reveal differences in dimerization properties and highlight the structural diversity within GAF domain-containing PDEs.

Endogenous cGMP regulates adult longevity via the insulin signaling pathway in Caenorhabditis elegans

G-proteins, including GPA-3, play an important role in regulating physiological responses in Caenorhabditis elegans. When confronted with an environmental stimulus such as dauer pheromone, or poor nutrients, C. elegans receives and integrates external signals through its nervous system (i.e. amphid neurons), which interprets and translates them into biological action. Here it is shown that a suppressed neuronal cGMP level caused by GPA-3 activation leads to a significant increase (47.3%) in the mean lifespan of adult C. elegans through forkhead transcription factor family O (FOXO)-mediated signal. A reduced neuronal cGMP level was found to be caused by an increased cGMP-specific phosphodiesterase activity at the transcriptional level. Our results using C. elegans mutants with specific deficits in TGF-beta and FOXO RNAi system suggest a mechanism in that cGMP, TGF-beta, and FOXO signaling interact to differentially produce the insulin-like molecules, ins-7 and daf-28, causing suppression of the insulin/IGF-1 pathway and promoting lifespan extension. Our findings provide not only a new mechanism of cGMP-mediated induction of longevity in adult C. elegans but also a possible therapeutic strategy for neuronal disease, which has been likened to brain diabetes.