Cyclic Nucleotide-mediated Signaling Research Articles

Expression of nitric oxide synthase (NOS) in Anabas testudineus ovary and participation of nitric oxide-cyclic GMP cascade in maintenance of meiotic arrest

Participation of cyclic nucleotide-mediated signaling in nitric oxide/soluble guanylate cyclase (NO/sGC) regulation of oocyte maturation (OM) in perch (Anabas testudineus) follicle-enclosed oocytes has been investigated. Congruent with sharp decline in follicular cyclic GMP (cGMP) level, nitric oxide synthase (NOS)-inhibitor (L-NAME) attenuates protein kinase A (PKA) phosphorylation but promotes p-ERK1/2 and p-p34Cdc2 (Thr-161) in maturing oocytes. Conversely, NO donor (SNP) prevents OM, potentially through elevated cGMP synthesis. Expression and localization of Nos2 and Nos3 immunoreactivity in perch ovary varied considerably at progressively higher stages of folliculogenesis. While sGC inhibitor (ODQ) alone could induce OM, 8-bromo-cGMP attenuates 17,20β-P-induced OM indicating functional significance of NO/sGC/cGMP in perch ovary. Interestingly, high NO/cGMP inhibition of OM shows positive relation with elevated cAMP level. MIS induced OM is more susceptible to the oocyte-specific phosphodiesterase (PDE) 3 than PDE4 inhibition. Collectively, high NO/cGMP attenuation of OM potentially involves PDE3 inhibition, cAMP accumulation and PKA activation.

Cyclic Nucleotides Differentially Regulate Cx43 Gap Junction Function in Uterine Artery Endothelial Cells From Pregnant Ewes.

Cell-cell communication is dependent on GJ (gap junction) proteins such as Cx43 (connexin 43). We previously demonstrated the importance of Cx43 function in establishing the enhanced pregnancy vasodilatory phenotype during pregnancy in uterine artery endothelial cells from pregnant (P-UAEC) ewes. Cx43 is regulated by elevating cAMP and PKA (protein kinase A)-dependent Cx43 S365 phosphorylation-associated trafficking and GJ open gating, which is opposed by PKC (protein kinase C)-dependent S368 phosphorylation-mediated GJ turnover and closed gating. However, the role of cyclic nucleotide-mediated signaling mechanisms that control Cx43 and GJ function in P-UAECs is unknown. We hypothesize that cAMP will mediate increases in S365 phosphorylation, thereby, enhancing GJ trafficking and open gating, while cGMP will stimulate S368, but not S365, phosphorylation to enhance GJ turnover and closed gating in P-UAECs. Treatment with 8-Bromo (8-Br)-cAMP signal significantly (P<0.05) increased nonphosphorylated S365 signal and total Cx43 phosphorylation, but not S368 phosphorylation, while 8-Br-cGMP significantly (P<0.05) increased Cx43 C-terminus-S365 signal, S368, and total Cx43 phosphorylation. Inhibition of PKA, but not PKG (protein kinase G), abrogated the 8-Br-cAMP-stimulated increase in nonphosphorylated S365 and total Cx43 phosphorylation and inhibited S368 below basal levels, whereas inhibition of PKG blocked (P<0.05) the 8-bromo-cGMP-stimulated rises in nonphosphorylated S365, total Cx43, and S368 phosphorylation levels in P-UAECs. Functional studies showed that 8-Br-cAMP increased dye transfer and sustained calcium bursts, while 8-Br-cGMP decreased both. Thus, in P-UAECs, only 8-Br-cAMP and not 8-Br-cGMP effectively enhances nonphosphorylated S365 and total Cx43 expression that correspondingly reduces S368 phosphorylation, allowing increased GJ communication. This provides new insights into the regulatory mechanisms behind Cx43 function and GJ communication.

Identification of suitable genes contributes to lung adenocarcinoma clustering by multiple meta‐analysis methods

With the widespread application of high-throughput technology, numerous meta-analysis methods have been proposed for differential expression profiling across multiple studies. We identified the suitable differentially expressed (DE) genes that contributed to lung adenocarcinoma (ADC) clustering based on seven popular multiple meta-analysis methods. Seven microarray expression profiles of ADC and normal controls were extracted from the ArrayExpress database. The Bioconductor was used to perform the data preliminary preprocessing. Then, DE genes across multiple studies were identified. Hierarchical clustering was applied to compare the classification performance for microarray data samples. The classification efficiency was compared based on accuracy, sensitivity and specificity. Across seven datasets, 573 ADC cases and 222 normal controls were collected. After filtering out unexpressed and noninformative genes, 3688 genes were remained for further analysis. The classification efficiency analysis showed that DE genes identified by sum of ranks method separated ADC from normal controls with the best accuracy, sensitivity and specificity of 0.953, 0.969 and 0.932, respectively. The gene set with the highest classification accuracy mainly participated in the regulation of response to external stimulus (P = 7.97E-04), cyclic nucleotide-mediated signaling (P = 0.01), regulation of cell morphogenesis (P = 0.01) and regulation of cell proliferation (P = 0.01). Evaluation of DE genes identified by different meta-analysis methods in classification efficiency provided a new perspective to the choice of the suitable method in a given application. Varying meta-analysis methods always present varying abilities, so synthetic consideration should be taken when providing meta-analysis methods for particular research.

Therapeutic Potential of Pde Modulation in Treating Heart Disease

Altered cyclic nucleotide-mediated signaling plays a critical role in the development of cardiovascular pathology. By degrading cAMP/cGMP, the action of cyclic nucleotide PDEs is essential for controlling cyclic nucleotide-mediated signaling intensity, duration, and specificity. Altered expression, localization and action of PDEs have all been implicated in causing changes in cyclic nucleotide signaling in cardiovascular disease. Accordingly, pharmacological inhibition of PDEs has gained interest as a treatment strategy and as an area of drug development. While targeting of certain PDEs has the potential to ameliorate cardiovascular disease, inhibition of others might actually worsen it. This review will highlight recent research on the physiopathological role of cyclic nucleotide signaling, especially with regard to PDEs. While the physiological roles and biochemical properties of cardiovascular PDEs will be summarized, the primary emphasis will be pathological. Research into the potential benefits and hazards of PDE inhibition will also be discussed.

Phosphodiesterases in neurodegenerative disorders

Cyclic nucleotide phosphodiesterases (PDEs) are responsible for the breakdown of cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). As such, they are crucial regulators of levels of cyclic nucleotide-mediated signaling. cAMP signaling and cGMP signaling have been associated with neuroplasticity and protection, and influencing their levels in the cell by inhibition of PDEs has become a much studied target for treatment in a wide array of disorders, including neurodegenerative disorders. In this review, we will focus on the involvement of PDEs in neurodegenerative disorders. In comparison with preclinical work, data on human patients are scarce. Alzheimer's disease is associated with changes in PDE4, PDE7, and PDE8 expression in the brain. Altered functioning of PDE4 as well as PDE11 is associated with major depressive disorder. In multiple sclerosis, there are indications of alterations in expression of several PDE subtypes in the central nervous system; however, evidence is indirect. In Huntington's disease and Parkinson's disease, most research has focused on PDE1B and PDE10, because of their abundant presence in striatal neurons. In another rare, neurodegenerative striatal motor disorder, that is, autosomal-dominant striatal degeneration, genetic defects in PDE8B gene are thought to underlie the neurodegenerative processes. Although the latter disorder has showed a causative dysfunction of PDEs, this does not hold for the neurodegenerative disorders discussed above, in which changes in PDE levels seemingly rather represent secondary changes and compensation to prior existing dysfunction. However, normalizing cyclic nucleotide signaling via PDE inhibition remains interesting for the treatment of neurodegenerative disorders.

Nucleotidyl Cyclase Activity of Soluble Guanylyl Cyclase α1β1

Soluble guanylyl cyclase (sGC) regulates several important physiological processes by converting GTP into the second-messenger cGMP. sGC has several structural and functional properties in common with adenylyl cyclases (ACs). Recently, we reported that membranous ACs and sGC are potently inhibited by 2',3'-O-(2,4,6-trinitrophenyl)-substituted purine and pyrimidine nucleoside 5'-triphosphates. Using a highly sensitive high-performance liquid chromatography-tandem mass spectrometry method, we report that highly purified recombinant sGC of rat possesses nucleotidyl cyclase activity. As opposed to GTP, ITP, XTP and ATP, the pyrimidine nucleotides UTP and CTP were found to be sGC substrates in the presence of Mn(2+). When Mg(2+) is used, sGC generates cGMP, cAMP, cIMP, and cXMP. In conclusion, soluble "guanylyl" cyclase possesses much broader substrate specificity than previously assumed. Our data have important implications for cyclic nucleotide-mediated signal transduction.

Targeting Cyclic Nucleotide Phosphodiesterase in the Heart: Therapeutic Implications

The second messengers, cAMP and cGMP, regulate a number of physiological processes in the myocardium, from acute contraction/relaxation to chronic gene expression and cardiac structural remodeling. Emerging evidence suggests that multiple spatiotemporally distinct pools of cyclic nucleotides can discriminate specific cellular functions from a given cyclic nucleotide-mediated signal. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing intracellular cyclic AMP and/or cyclic GMP, control the amplitude, duration, and compartmentation of cyclic nucleotide signaling. To date, more than 60 different isoforms have been described and grouped into 11 broad families (PDE1-PDE11) based on differences in their structure, kinetic and regulatory properties, as well as sensitivity to chemical inhibitors. In the heart, PDE isozymes from at least six families have been investigated. Studies using selective PDE inhibitors and/or genetically manipulated animals have demonstrated that individual PDE isozymes play distinct roles in the heart by regulating unique cyclic nucleotide signaling microdomains. Alterations of PDE activity and/or expression have also been observed in various cardiac disease models, which may contribute to disease progression. Several family-selective PDE inhibitors have been used clinically or pre-clinically for the treatment of cardiac or vascular-related diseases. In this review, we will highlight both recent advances and discrepancies relevant to cardiovascular PDE expression, pathophysiological function, and regulation. In particular, we will emphasize how these properties influence current and future development of PDE inhibitors for the treatment of pathological cardiac remodeling and dysfunction.

Regulation of cGMP by phosphodiesterases (PDEs) in the central nervous system

It is becoming well established that metabolic inactivation of the cyclic nucleotides cAMP and cGMP is intimately involved in the regulation of cyclic nucleotidemediated signaling cascades. There are four families of PDEs that metabolize cGMP, PDE1, PDE2, PDE9, and PDE10, that are prominently and differentially expressed in the central nervous system. We have begun to characterize the role of these enzymes on cGMP metabolism in vivo using pharmacological and genetic approaches in mice. The effects of manipulating enzyme activity differ for each enzyme in different brain regions, corresponding to differential localization. However, the involvement of each enzyme also appears dependent on the level of activation of cGMP formation. These findings begin to define and differentiate the functions for the different PDEs in regulating cGMP signaling in the central nervous system. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007

Functional Responses of Isolated Human Seminal Vesicle Tissue to Selective Phosphodiesterase Inhibitors

To further elucidate the significance of the cyclic nucleotide-mediated signal transduction, we examined the in vitro functional responses of isolated seminal vesicle (SV) smooth muscle tissue to selective phosphodiesterase (PDE) inhibitors. Using the organ bath technique, the effects of increasing concentrations (1 nM to 10 microM) of the PDE inhibitors vinpocetine (PDE1 inhibitor), rolipram (PDE4 inhibitor), and sildenafil and vardenafil (PDE5 inhibitors) on the tension induced by 10 microM of norepinephrine on SV tissue strips were investigated. To examine the drug effects on the tissue levels of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), the SV strips were exposed to different concentrations of the compounds (0.1, 1, and 10 microM). After freezing, homogenization, and extraction of cyclic nucleotides, cAMP and cGMP were measured using radioimmunoassays. In the experiments, sodium nitroprusside and forskolin were used as reference compounds. The norepinephrine-induced tension was reversed by the drugs in a dose-dependent manner. The rank order of efficacy was rolipram greater than sildenafil greater than vardenafil greater than or equal to vinpocetine greater than sodium nitroprusside greater than forskolin. The reversion of the norepinephrine-induced tension at maximum drug concentration ranged from 79% (rolipram) to 32% (forskolin). Only rolipram and sildenafil reached a median effective concentration. The effects of the PDE inhibitors were paralleled by a 1.7-fold to 173-fold increase in tissue cGMP or cAMP. Our results have demonstrated that PDE inhibitors can reverse the adrenergic tension of human SV tissue and increase levels of cyclic nucleotides. This outlines the potential significance of cAMP and cGMP in the control of SV smooth muscle function. These findings might be of importance with regard to the pharmacologic treatment of premature ejaculation.

Regulation of Phosphodiesterase 3 and Inducible cAMP Early Repressor in the Heart

Growing evidence suggests that multiple spatially, temporally, and functionally distinct pools of cyclic nucleotides exist and regulate cardiac performance, from acute myocardial contractility to chronic gene expression and cardiac structural remodeling. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP and cyclic GMP, regulate the amplitude, duration, and compartmentation of cyclic nucleotide-mediated signaling. In particular, PDE3 enzymes play a major role in regulating cAMP metabolism in the cardiovascular system. PDE3 inhibitors, by raising cAMP content, have acute inotropic and vasodilatory effects in treating congestive heart failure but have increased mortality in long-term therapy. PDE3A expression is downregulated in human and animal failing hearts. In vitro, inhibition of PDE3A function is associated with myocyte apoptosis through sustained induction of a transcriptional repressor ICER (inducible cAMP early repressor) and thereby inhibition of antiapoptotic molecule Bcl-2 expression. Sustained induction of ICER may also cause the change of other protein expression implicated in human and animal failing hearts. These data suggest that the downregulation of PDE3A observed in failing hearts may play a causative role in the progression of heart failure, in part, by inducing ICER and promoting cardiac myocyte dysfunction. Hence, strategies that maintain PDE3A function may represent an attractive approach to circumvent myocyte apoptosis and cardiac dysfunction.

Expression of cAMP and cGMP-phosphodiesterase isoenzymes 3, 4, and 5 in the human clitoris: Immunohistochemical and molecular biology study

Objectives Only a little research has focused on the evaluation of female sexual function. With sexual stimulation, the clitoris becomes engorged with blood and tumescent. Nevertheless, only little is known about the significance of the cyclic nucleotide-mediated signal transduction in the control of this process. We sought to elucidate the presence of the phosphodiesterase (PDE) isoenzymes 3, 4, and 5 in the human clitoris using immunohistochemical and molecular biology methods. Methods Thin sections of clitoral specimens were incubated with primary antibodies directed against PDE isoenzymes 3, 4, and 5. Next, the sections were incubated with either Texas red or fluorescein isothiocyanate-labeled secondary antibodies, and visualization was done using laser microscopy. The expression of mRNA encoding for various PDE isoenzymes was evaluated using reverse transcriptase polymerase chain reaction. Results Immunofluorescence indicating the presence of PDE4 (cyclic adenosine monophosphate-PDE) was observed in the nonvascular smooth musculature of the corpus cavernosum clitoris, sinusoidal endothelial and subendothelial layers, and nerve fibers innervating the tissue. Immunoreactivity specific for PDE5 (cyclic guanosine monophosphate-PDE) was limited to the smooth muscle of the clitoral erectile tissue. The fluorescein isothiocyanate reaction indicating the expression of PDE3 (cyclic adenosine monophosphate-PDE) was registered to a certain degree only in the clitoral epidermis. In the reverse transcriptase polymerase chain reaction studies, a predominant expression of mRNA encoding for PDE1A was registered, but only small amounts of mRNA encoding for PDE4 and PDE5 were detected. Conclusions Our results have demonstrated the presence of cyclic adenosine monophosphate-PDE and cyclic guanosine monophosphate-PDE in the human clitoris and may indicate a regulatory function of these enzymes in the cyclic nucleotide-mediated control of smooth muscle tone.

In vitro functional responses of isolated human vaginal tissue to selective phosphodiesterase inhibitors

Only little is known as to the significance of the cyclic nucleotide-mediated signal transduction in the control of the function of human vaginal smooth musculature. Recently, the presence of the phosphodiesterase (PDE) isoenzymes 4 (cAMP-PDE) and 5 (cGMP-PDE) in the human vagina was reported. Thus, it was the aim of the study to elucidate the effects of some PDE inhibitors on the tension induced by endothelin 1 (ET-1), as well as on levels of cGMP and cAMP in isolated human vaginal wall tissue. Using the organ bath technique, the ability of norepinephrine (NE), carbachol, serotonin (5-HT), oxytocin and ET-1 to contract isolated vaginal wall muscle strips was evaluated. In another set-up, the effects of the PDE4 inhibitor rolipram and PDE5 inhibitors sildenafil and vardenafil (1 nM-10 microM) on the tension induced by 0.1 microM ET-1 of human vaginal wall tissue strips were investigated. In order to measure drug effects on tissue levels of cGMP and cAMP, vaginal tissue was exposed to different concentrations (0.1, 1 and 10 microM) of the compounds and the accumulation of cyclic nucleotides was determined. The adenylyl cyclase stimulating agents forskolin and nitric oxide donor sodium nitroprusside (SNP) (0.01, 0.1 and 1 microM) were used as reference compounds. While NE, carbachol and oxytocin failed to contract the vaginal tissue, ET-1 and, to a certain degree, 5-HT elicited contractile responses of the isolated strip preparations. The tension induced by 0.1 microM ET-1 was dose-dependently reversed by the drugs. The rank order of efficacy was sildenafil > forskolin > rolipram >or= vardenafil > SNP. Rmax values ranged from 24% (SNP) to 50% (sildenafil). With sildenafil being the only exception, none of the compounds reached an EC50 value. The relaxing effects of the drugs were paralleled by a fourfold to tenfold increase in tissue levels of cGMP and/or cAMP. Our results demonstrate that PDE inhibitors can relax human vaginal tissue and increase levels of cyclic nucleoside monophosphates. The findings with regard to the PDE5 inhibitors may indicate that the NO-cGMP pathway is, to a certain degree, involved in the control of vaginal smooth muscle tone. This might be of significance with regard to the pharmacological treatment of disorders connected with female sexual arousal and the ability to achieve orgasm.

Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system.

Cyclic AMP (cAMP) and cGMP regulate a myriad of cellular functions, such as metabolism, contractility, motility, and transcription in virtually all cell types, including those of the cardiovascular system. Considerable effort over the last 20 years has allowed identification of the cellular components involved in the synthesis of cyclic nucleotides, as well as effectors of cyclic nucleotide-mediated signaling. More recently, a central role for cyclic nucleotide phosphodiesterase (PDE) has also been elaborated in many cell types, including those involved in regulating the activities of the cardiovascular system. In this review, we introduce the PDE families whose members are expressed in cells of the cardiovascular system including cardiomyocytes, vascular smooth muscle cells, and vascular endothelial cells. Because cell behavior is a dynamic process influenced by numerous factors, we will attempt to emphasize how changes in the activity, expression, and targeting of PDE influence cyclic nucleotide-mediated regulation of the behavior of these cells.

Evidence of a novel transduction pathway mediating detection of polyamines by the zebrafish olfactory system.

To better understand the full extent of the odorant detection capabilities of fish, we investigated the olfactory sensitivity of zebrafish to a monoamine and several polyamines using electrophysiological and activity-dependent labeling techniques. Electro-olfactogram (EOG) recording methods established the relative stimulatory effectiveness of these odorants as: spermine >> spermidine approximately agmatine > glutamine > putrescine >or= cadaverine >or= histamine > artificial freshwater. The detection threshold for the potent polyamines was approximately 1 micromol l(-1). Cross-adaptation experiments suggested that multiple receptors are involved in polyamine detection. Three observations indicated that polyamine signaling may involve a transduction cascade distinct from those used by either amino acids or bile salts. Like bile salts and the adenylate cyclase activator forskolin, but unlike amino acid odorants, polyamines failed to stimulate activity-dependent labeling of olfactory sensory neurons with the cation channel permeant probe agmatine, suggesting a signaling pathway different from that used by amino acid stimuli. Also supporting distinct amino acid and polyamine signaling pathways is the finding that altering phospholipase C activity with the inhibitor U-73122 significantly reduced amino acid-evoked responses, but had little effect on polyamine- (or bile salt-) evoked responses. Altering cyclic nucleotide-mediated signaling by adenylate cyclase activation with forskolin, which significantly reduced responses to bile salts, failed to attenuate polyamine responses, suggesting that polyamines and bile salts do not share a common transduction cascade. Collectively, these findings suggest that polyamines are a new class of olfactory stimuli transduced by a receptor-mediated, second messenger signaling pathway that is distinct from those used by amino acids or bile salts.

CGMP Pump

Cyclic nucleotides are important second messengers. Jedlitschky et al . expressed the human multidrug resistance protein 5 (MDR5) in hamster fibroblasts and showed that these cells exhibit an enhanced high-affinity ATP-dependent transport of cyclic nucleotides into inside-out membrane vesicles. The transport of cyclic guanosine monophosphate (cGMP) was faster and occurred at lower concentrations than that of cyclic adenosine monophosphate (cAMP), indicating that MDR5 is a high-affinity cGMP-specific pump. Also consistent with MDR5's being a cGMP-specific pump, membranes from MDR5-transfected cells did not transport glutathione or glucuronate conjugates, which are substrates for other isoforms of MDR protein. These findings have important implications for signaling both in terms of mechanisms for terminating cyclic nucleotide-mediated signaling cascades and for the possibility that exported cyclic nucleotides may initiate signal transduction in receptive cells. Jedlitschky, G., Burchell, B., and Keppler, D. (2000) The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides. J. Biol. Chem. 275 : 30069-30074. [Abstract] [Full Text]

Organizational Complexity in Lobster Olfactory Receptor Cellsa

The current working model of transduction in lobster olfactory receptor cells suggests that: (1) inositol-1,4,5-triphosphate (IP3) is the excitatory olfactory second messenger in these cells; (2) activation of the cell also involves a secondary, current-carrying channel; and (3) the phosphoinositol pathway works in parallel to a second, cyclic nucleotide-mediated signaling pathway that provides input of opposite polarity into the cell. The complexity of intracellular signaling in lobster olfactory receptor cells renders the cells capable of fine tuning, and even integrating, the signal they send to the brain.

Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers.

The protease thrombin is a potent activator of various cell types. Thrombin cleaves and thereby activates its own seven-transmembrane-domain receptor which couples to G proteins. Thrombin also can inhibit neuronal differentiation, supposedly by degrading components of the extracellular matrix. Here we report that active thrombin induces immediate cell rounding and neurite retraction in differentiating N1E-115 and NG108-15 neural cells in serum-free culture. Serum (0.5-5% vol/vol) evokes similar responses, but the cell-rounding and neurite-retracting activity of serum is not attributable to thrombin. Neural cell rounding is transient, subsiding after 10-15 min, and subject to homologous desensitization, whereas retracted neurites rapidly degenerate. Thrombin action is inhibited by cytochalasin, but not colchicine. A novel 14-amino acid peptide agonist of the thrombin receptor fully mimics thrombin's morphoregulatory activity, indicating that thrombin-induced shape changes are receptor-mediated and not secondary to extracellular matrix degradation. Although thrombin receptors couple to phosphoinositide hydrolysis and Ca2+ mobilization, thrombin-induced shape changes appear to depend neither on the Ca2+/protein kinase C- nor the cyclic nucleotide-mediated signal transduction pathways; however, the morphological response to thrombin is blocked by pervanadate, an inhibitor of tyrosine phosphatases, and by broad-specificity kinase inhibitors. Our results suggest that the thrombin receptor communicates to an as-yet-uncharacterized effector to reorganize the actin cytoskeleton and to reverse the differentiated phenotype of neural cells.