The principle is that the soluble antigens and antibodies form visible sediments in the presence of electrolytes according to their abundance. PDE has various subtypes, and these subtypes have different functions, the classification of the PDE4 subtype is important in the research and development of novel drugs.
The cAMP signaling pathway and multiple activated factors are involved in regulating numerous physiological processes, including growth, reproduction, differentiation and apoptosis Previous studies have demonstrated that the disruption of the cAMP signaling pathway or the function of any factor within this pathway can contribute to the treatment of numerous human diseases 66 — For example, by targeting the interruption of the cAMP pathway, a variety of inhibitors of various factors have been identified, and as a result, associated drugs for treating various diseases have been developed.
Alzheimer's disease has been reported to be associated with an alteration in the activity of AC; therefore, it is suggested that forskolin may be used as a targeted drug to treat Alzheimer's disease Cholera toxin CTX has an effect similar to that of forskolin. H89 is a commonly used PKA inhibitor. Previous studies have indicated that H89 blocks LPS-, prostaglandin E 2 PGE 2 -, and phospho-ceramide analogueinduced cellular secretion of cyclooxygenase 2 3 , nitric oxide NO 7 and additional inflammatory factors such as interleukin 6 26 , Cho et al 80 demonstrated that LPS stimulates the production of inflammatory factors and the amplification of the immune response via the mitogen-activated protein kinase MAPK pathway.
In the process of gastrointestinal inflammation, muscularis macrophages produce NO to induce resident intestinal macrophage dysfunction. Therefore, inhibitors of PKA can be used in the treatment of gastrointestinal inflammation. The intracellular cAMP concentration is regulated via the stimulation of adenyl and guanyl cyclases in response to extracellular signaling The PDEs are a superfamily of enzymes; there are a minimum of different PDE enzymes, which degrade cyclic nucleotides 1.
PDE inhibitors cause an increase in the intracellular concentration of cAMP and have an impact on a variety of cells PDE inhibitors have become a research focus. PDE inhibitors have the potential to treat incontinence, regulate heart rate disorders, prevent heart failure 62 , and antagonize malignant tumors in myeloid and lymphoid tissue and in the prostate 83 , In addition, PDE isozymes participate in several pathological processes in kidney cells.
Therefore, it is suggested that PDE inhibitors can be used for the treatment of nephritis and renal failure PDE4 inhibitors have been most extensively applied; for example, these inhibitors are used to treat chronic obstructive pulmonary disease 86 , inflammation 87 , asthma 61 , autoimmune diseases 88 , and depression 64 , in addition to learning and memory disorders The most well known PDE4 inhibitor is rolipram.
Rolipram has been demonstrated to significantly increase cAMP levels 79 , 89 , strengthen arginine enzyme activity 90 , treat depression 91 , ameliorate memory and intelligence 22 and suppress several types of inflammation Due to the fact that rolipram is not highly selective for the PDE4 subtype, this drug has strong side effects, such as inducing vomiting. Another commonly used inhibitor is 3-isobutylmeth-ylxanthine In addition, a variety of other inhibitors have been developed based on S-adenosylmethionine SAM.
SAM functions as an anti-inflammatory drug and has been demonstrated to act as an effective PDE4B inhibitor for the treatment of chronic inflammatory diseases Another PDE inhibitor, pentoxifylline, increases intracellular cAMP, acts as an immunosuppressant, has anti-fibrotic activity, and improves hemodynamics.
In recent years, pentoxifylline and rolipram have been increasingly used in clinical settings These drugs have been observed to be able to increase bone mass in mice and are thus used in the treatment of osteoporosis. Pentoxifylline and rolipram can block macrophage activation and the production of NO in vivo and in vitro 5. Furthermore, pyrazolopyridines 96 , as novel PDE4 inhibitors, have the capacity to treat chronic obstructive pulmonary disease, chronic bronchitis and emphysema.
Cilostazol, an inhibitor of PDE3, not only has strong anti-inflammatory effects but also inhibits platelet aggregation and leads to vasodilation It has been demonstrated that PDE8 serves a decisive role in modulating the concentration of steroids, T-cell adhesion and heart rhythm 2. In addition, Dong et al 98 demonstrated that dipyridamole, an inhibitor of PDE8, strongly inhibited the migration of unstimulated and stimulated splenocytes.
The physiological processes that occur in vivo and in vitro frequently involve a signaling network, rather than one pathway alone.
A high concentration of one type of nucleotide, cAMP or cGMP, will prevent the generation, metabolism or degradation of the other cyclic nucleotide ; there is an antagonistic association between the physiological effects.
For example, isoproterenol promotes myocardial contraction and increases the concentration of cAMP, while the concentration of cGMP is simultaneously reduced.
From a previous study focussing upon the antidepressant medications fluoxetine and amitriptyline , it is known that the cGMP and cAMP signaling pathways are able to function simultaneously.
These two second messengers regulate a variety of cellular functions, including protein synthesis, protein phosphorylation, the regulation of enzymatic activity and gene expression.
The efficiency of the research methods commonly used for elucidating the cAMP signaling pathway must be improved. The high-throughput and high-content screening technologies developed in recent years may be applied to increase the speed of screening for inhibitors and agonists of the cAMP signaling pathway, and may also improve the efficiency of novel drug research and development , For instance, chIP was shown to markedly shorten the early drug discovery process , and recent innovations in flow cytometry have allowed up to fold faster serial processing of samples The methods of disease treatment described in the present review predominantly focus on blocking or reducing signaling messengers using pathway inhibitors.
Conversely, few drugs exert curative effects by increasing the concentration of cAMP. Therefore, further elucidating the role of the cAMP signaling pathway in diseases associated with signal dysfunction and interruption may aid in the development of a therapeutic strategy based on pathway activation. Conti M and Beavo J: Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem. FEBS Lett. Biochem J.
Genes Dev. Beshay E, Croze F and Prud'homme GJ: The phosphodiesterase inhibitors pentoxifylline and rolipram suppress macrophage activation and nitric oxide production in vitro and in vivo. Clin Immunol. J Leukoc Biol. J Cell Biochem. View Article : Google Scholar. Biochem Pharmacol. View Article : Google Scholar :. Biochim Biophys Acta.
Nat Rev Drug Discov. Prog Neuropsychopharmacol Biol Psychiatry. Annu Rev Pharmacol Toxicol. Mol Pharmacol. Neurobiol Learn Mem.
Am J Physiol Renal Physiol. Physiol Rev. Mol Endocrinol. Front Behav Neurosci. J Biol Chem. Cell Signal. Mol Immunol. Methods Mol Biol.
Mol Cell. Curr Biol. EMBO J. Anhui Med Pharm J. Mol Cell Proteomics. Lei H, Venkatakrishnan A, Yu S and Kazlauskas A: Protein kinase A-dependent translocation of Hsp90 alpha impairs endothelial nitric-oxide synthase activity in high glucose and diabetes. Methods Mol Med. Neurosci Lett. This method combined the high separation capability of chromatography with the high selectivity and high sensitivity of mass spectrometry; therefore, HPLC-MS has the advantages of rapid analysis and amenability to automation.
CREB is a nuclear transcription factor. Non-phosphorylated CREB is predominantly located in the nucleus. PKA that is activated by cAMP translocates to the nucleus and activates CREB through the phosphorylation of the amino terminal kinase inducible domain, in turn regulating target gene transcription 41 — The N-terminus is the transcription activation site, which contains multiple phosphorylation sites, including serine residue Ser , Ser and Ser, which can be phosphorylated by a variety of protein kinases.
Ser serves an important role in the transcriptional activity of CREB 44 — The phosphorylation of these sites is associated with downstream protein expression and function. Therefore, the detection of these phosphorylated sites is crucial to the study of signal transduction pathways. A variety of methods have been developed to detect CREB. The most widely used is western blot analysis and transfection together with the luciferase assay.
In western blotting method, the protein samples are separated by PAGE; the proteins are then transferred to membranes and subsequently probed with a specific antibody. Due to the importance of CREB and its phosphorylation, it has become a focus in research for targets of novel drug research and development. The ELISA method, which is suitable for high throughput screening, is well established and is widely used in drug discovery.
Therefore, the cell-based ELISA method based on the double fluorescent labeling technique has been widely used previously. In this method, an immobilized capture antibody specific for CREB binds to phosphorylated and unphosphorylated proteins. Subsequent to washing the unbound antibodies away, a biotinylated detection antibody that recognizes p-CREB Ser is used to detect only the phosphorylated protein, utilizing a standard streptavidin-HRP format.
The traditional detection method is an electrophoretic mobility shift assay EMSA. However, due to safety concerns regarding the use of radioactive isotopes, radioactive labeling has been replaced by non-radioactive visualization techniques.
The luciferase reporter gene assay 54 involves the transfection of the reporter gene plasmid CREB-Luc into cells. The cells undergo appropriate stimulation and are then lysed, followed by treatment to detect luciferase activity. This method can gauge the expression of a reporter gene easily and effectively. The construction of a reporter gene plasmid is accomplished by cloning the gene transcription regulatory elements upstream of, or at other appropriate locations relative to, the luciferase gene.
Cells are transfected with the construct and luciferase activity is detected following treatment or proper stimulation. The influences of different treatments on the targeted regulatory elements, or the differences prior to and subsequent to stimulation, are quantified using the luciferase activity level. ChIP is an important method for investigating the interactions between specific proteins or modified forms of proteins and a genomic DNA region ChIP is based on the development of an in vivo analysis method.
The basic principle is to selectively enrich a chromosomal fragment chromatin , which contains a specific antigen. An antibody that can identify a protein or modified protein is used to determine the relative abundance of the antigen at one or more locations in the genome. A complex PDE gene organization and a great number of PDE splicing variants fine-tune cyclic nucleotide signals and make PDEs conducive to specificity in the signaling pathways 1. Inhibitors of PDE lead to the elevation of cAMP and cGMP levels, which in turn lead to multifarious cellular effects, including airway smooth muscle relaxation, inhibitory effects on cellular inflammation and immune responses The PDE4 inhibitors roflumilast 59 , 60 and cilomilast 61 have indicated the potential of the development of PDE inhibitors into novel drugs.
Previous studies have used a luminescence-based, high-throughput screening method in place of the enzyme kinetic method for measuring cyclic nucleotide PDE activity. The level of remaining ATP is then determined. As a result, luminescence increases. Thus, luminescence is directly proportional to the remaining ATP level, which is directly proportional to the PDE activity Peter et al 63 used immunoprecipitation and subsequent activity assays to determine total PDE activity.
Immunoprecipitation is predominantly used for the qualitative detection of antibodies or antigens. The principle is that the soluble antigens and antibodies form visible sediments in the presence of electrolytes according to their abundance. PDE has various subtypes, and these subtypes have different functions, the classification of the PDE4 subtype is important in the research and development of novel drugs.
The cAMP signaling pathway and multiple activated factors are involved in regulating numerous physiological processes, including growth, reproduction, differentiation and apoptosis Previous studies have demonstrated that the disruption of the cAMP signaling pathway or the function of any factor within this pathway can contribute to the treatment of numerous human diseases 66 — For example, by targeting the interruption of the cAMP pathway, a variety of inhibitors of various factors have been identified, and as a result, associated drugs for treating various diseases have been developed.
Alzheimer's disease has been reported to be associated with an alteration in the activity of AC; therefore, it is suggested that forskolin may be used as a targeted drug to treat Alzheimer's disease Cholera toxin CTX has an effect similar to that of forskolin.
H89 is a commonly used PKA inhibitor. Previous studies have indicated that H89 blocks LPS-, prostaglandin E 2 PGE 2 -, and phospho-ceramide analogueinduced cellular secretion of cyclooxygenase 2 3 , nitric oxide NO 7 and additional inflammatory factors such as interleukin 6 26 , Cho et al 80 demonstrated that LPS stimulates the production of inflammatory factors and the amplification of the immune response via the mitogen-activated protein kinase MAPK pathway.
In the process of gastrointestinal inflammation, muscularis macrophages produce NO to induce resident intestinal macrophage dysfunction.
Therefore, inhibitors of PKA can be used in the treatment of gastrointestinal inflammation. The intracellular cAMP concentration is regulated via the stimulation of adenyl and guanyl cyclases in response to extracellular signaling The PDEs are a superfamily of enzymes; there are a minimum of different PDE enzymes, which degrade cyclic nucleotides 1.
PDE inhibitors cause an increase in the intracellular concentration of cAMP and have an impact on a variety of cells PDE inhibitors have become a research focus. PDE inhibitors have the potential to treat incontinence, regulate heart rate disorders, prevent heart failure 62 , and antagonize malignant tumors in myeloid and lymphoid tissue and in the prostate 83 , In addition, PDE isozymes participate in several pathological processes in kidney cells.
Therefore, it is suggested that PDE inhibitors can be used for the treatment of nephritis and renal failure PDE4 inhibitors have been most extensively applied; for example, these inhibitors are used to treat chronic obstructive pulmonary disease 86 , inflammation 87 , asthma 61 , autoimmune diseases 88 , and depression 64 , in addition to learning and memory disorders The most well known PDE4 inhibitor is rolipram.
Rolipram has been demonstrated to significantly increase cAMP levels 79 , 89 , strengthen arginine enzyme activity 90 , treat depression 91 , ameliorate memory and intelligence 22 and suppress several types of inflammation Due to the fact that rolipram is not highly selective for the PDE4 subtype, this drug has strong side effects, such as inducing vomiting.
Another commonly used inhibitor is 3-isobutylmeth-ylxanthine In addition, a variety of other inhibitors have been developed based on S-adenosylmethionine SAM. SAM functions as an anti-inflammatory drug and has been demonstrated to act as an effective PDE4B inhibitor for the treatment of chronic inflammatory diseases Another PDE inhibitor, pentoxifylline, increases intracellular cAMP, acts as an immunosuppressant, has anti-fibrotic activity, and improves hemodynamics.
In recent years, pentoxifylline and rolipram have been increasingly used in clinical settings These drugs have been observed to be able to increase bone mass in mice and are thus used in the treatment of osteoporosis.
Pentoxifylline and rolipram can block macrophage activation and the production of NO in vivo and in vitro 5. Furthermore, pyrazolopyridines 96 , as novel PDE4 inhibitors, have the capacity to treat chronic obstructive pulmonary disease, chronic bronchitis and emphysema. Cilostazol, an inhibitor of PDE3, not only has strong anti-inflammatory effects but also inhibits platelet aggregation and leads to vasodilation It has been demonstrated that PDE8 serves a decisive role in modulating the concentration of steroids, T-cell adhesion and heart rhythm 2.
In addition, Dong et al 98 demonstrated that dipyridamole, an inhibitor of PDE8, strongly inhibited the migration of unstimulated and stimulated splenocytes. The physiological processes that occur in vivo and in vitro frequently involve a signaling network, rather than one pathway alone. A high concentration of one type of nucleotide, cAMP or cGMP, will prevent the generation, metabolism or degradation of the other cyclic nucleotide ; there is an antagonistic association between the physiological effects.
For example, isoproterenol promotes myocardial contraction and increases the concentration of cAMP, while the concentration of cGMP is simultaneously reduced. From a previous study focussing upon the antidepressant medications fluoxetine and amitriptyline , it is known that the cGMP and cAMP signaling pathways are able to function simultaneously.
These two second messengers regulate a variety of cellular functions, including protein synthesis, protein phosphorylation, the regulation of enzymatic activity and gene expression.
The efficiency of the research methods commonly used for elucidating the cAMP signaling pathway must be improved. The high-throughput and high-content screening technologies developed in recent years may be applied to increase the speed of screening for inhibitors and agonists of the cAMP signaling pathway, and may also improve the efficiency of novel drug research and development , For instance, chIP was shown to markedly shorten the early drug discovery process , and recent innovations in flow cytometry have allowed up to fold faster serial processing of samples The methods of disease treatment described in the present review predominantly focus on blocking or reducing signaling messengers using pathway inhibitors.
Conversely, few drugs exert curative effects by increasing the concentration of cAMP. Therefore, further elucidating the role of the cAMP signaling pathway in diseases associated with signal dysfunction and interruption may aid in the development of a therapeutic strategy based on pathway activation. National Center for Biotechnology Information , U.
Mol Med Rep. Published online Mar Author information Article notes Copyright and License information Disclaimer. China, E-mail: nc. Received Apr 6; Accepted Feb 8. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License , which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
This article has been cited by other articles in PMC. Abstract During development of disease, complex intracellular signaling pathways regulate an intricate series of events, including resistance to external toxins, the secretion of cytokines and the production of pathological phenomena. Keywords: cyclic adenosine monophosphate, detection method, inhibitor, activator, drug discovery. Introduction Substances that convert extracellular signals received by cell surface receptors to intracellular signals are known as second messengers Fig.
Open in a separate window. Figure 1. Methods to detect cAMP cAMP, as an important messenger involved in the regulation of metabolism and biological functions in cells, transfers information regarding cellular status. Methods to detect PKA By catalyzing phosphorylation in response to hormonal stimulation, PKA is the primary mediator of cAMP function and a key regulatory enzyme in pivotal cellular processes, such as DNA replication 28 , 29 , cell growth and metabolism 30 , cell division and rearrangement of the actin cytoskeleton 31 , Inhibitors and activators involved in cAMP signaling pathway-associated diseases The cAMP signaling pathway and multiple activated factors are involved in regulating numerous physiological processes, including growth, reproduction, differentiation and apoptosis Association with the cGMP pathway A high concentration of one type of nucleotide, cAMP or cGMP, will prevent the generation, metabolism or degradation of the other cyclic nucleotide ; there is an antagonistic association between the physiological effects.
Future directions The efficiency of the research methods commonly used for elucidating the cAMP signaling pathway must be improved. References 1. Conti M, Beavo J. Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. The hormone causes an increase in blood pressure and the breakdown of glucose for energy. This helps humans in danger to engage in physical activity to meet the challenges of a situation.
The body responds with a dry mouth, rapid heartbeat, and high blood pressure. A biochemical chain of events leads to these responses. The second messenger, cyclic AMP, is made by the enzyme adenylate cyclase. Figure 1. Instead, cyclic AMP stimulates a protein kinase cascade that ultimately leads to a cellular response.
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