Cyclic Nucleotide Concentrations Research Articles

Dynamics of Changes in the cAMP/cGMP Concentration Ratio in the Thymus and Spleen of Laboratory Mice during Vaccination against Plague and Tularemia against the Background of Immunomodulation.

Vaccine strains Yersinia pestis EV NIIEG at a dose of 103 CFU and Francisella tularensis 15 NIIEG at a dose of 102 CFU induced changes in the concentration of cyclic nucleotides in the thymus and spleen of white mice. Antigen-induced changes in the cAMP/cGMP ratio in immunocompetent organs had a phase or oscillatory character, which seems to be related to the regulation of postvaccination immunoreactivity in the body. Synthetic organoselenium compound 974zh stimulated an increase in the amplitude of cAMP/cGMP oscillations, indicating its stimulating effect on the immunogenic properties of vaccine strains at doses an order of magnitude below the standard doses.

Role of alpha-7 nicotinic acetylcholine receptor down-regulation in cyclic nucleotide reduction in memory-related brain regions of Alzheimer’s rats

In this study, we investigated the effect of the downregulation of α7 nicotinic acetylcholine receptors (α7nAChRs) by Streptozotocin (STZ) on the level of cyclic nucleotides. Three groups of six rats each—Control, Sham (received an Intracerebroventricular injection of artificial cerebrospinal fluid, and STZ were formed. Except for the control group, all rats were anesthetized with 45 mg/kg of pentobarbitone. The STZ was administered intracerebroventricularly at a dose of 3 mg/kg to STZ group rats on Day 1 as well as Day 3 of an experimental period of 18 days. By using the Morris Water Maze (MWM) along with the Y-maze test, behavioral parameters were assessed, and biochemical analysis was assessed within the hippocampus, prefrontal cortex as well as amygdala of the rat brain. Statistical analysis was performed using repeated measures with one-way and two-way analysis of variance. Bonferroni and Student Newman–Keuls post hoc test was used to determine significance between experimental groups, with a significance threshold of p < 0.05. STZ administration resulted in a notable decline in the cognitive function of rats, as evidenced by longer transfer latency, reduced duration spent, and decreased overall distance covered within the designated quadrant (in percentage) in the MWM assessment. Moreover, STZ lowered the spontaneous change in behavior observed in the rodents during the Y-maze evaluation and decreased Cyclic Adenosine Monophosphate and Cyclic Guanosine Monophosphate (cyclic nucleotides) concentrations within the HIP, PFC, and Amygdale of rat brain regions. The α7nAChR downregulation may form a basis that could serve as a foundation for cognitive impairments alongside reduced cyclic nucleotides’ concentration in specific areas of the rat brain. Thus, cyclic nucleotides could be a probable target to stimulate the α7nAChR mediated mechanism in STZ-mediated conditions.

The effect of γ-irradiation at a dose of 1 Gy on the adenylate- and guanylate cyclase systems of rat platelets at different times of the post-radiation period

Objective. To study the effect of total γ-irradiation of the organism of rats at a dose of 1 Gy on the adenylate- and guanylate cyclase systems and their relationship with Ca2+ levels in the platelet cytoplasm.Materials and methods. The studies were carried out on outbred white male rats of mature age (6-7 months) weighing 250±30 g. Animals were irradiated (once and evenly) on the IGUR unit with 137Сs γ-quanta at a dose of 1 Gy (dose rate 0.62 Gy/min, for 1.61 min). Animals of the corresponding age served as controls. To determine the content of cyclic nucleotides, RIO cAMP/cGMP-iodine125-M-IBOX reagent kits were used. The amount of calcium in platelets was determined using a Fura-2/AM fluorescent probe using spectrofluorimeter SM 2203 “SOLAR” (Minsk, Belarus).Results. In the short period after irradiation, an increase in the activity of the adenylate cyclase system is observed. The cAMP level rises 1.8-1.5 times during 3-30 days of the post-radiation period. However, the increase in [Ca2+]cyt on the 3rd day after irradiation leads to its predominance by 1.8 times in relation to cAMP, which explains the appearance of an increased aggregation activity of the blood platelets in the immediate post-radiation period. An increase in the activity of the guanylate cyclase system and an increase in the intracellular content of cGMP are observed on the 90th day, which leads to a decrease in the Ca2+/cGMP ratio by 1.83 times and corresponds to a reduced platelet aggregation activity and the probability of bleeding in the long term of the rehabilitation period.Conclusion. The effects of radiation on the platelets of rats irradiated at a dose of 1 Gy are due to systemic disorders of the main intracellular regulatory mechanisms: Ca2+, cAMP, cGMP. Based on the data obtained, it can be concluded that pharmacological correction in the short-term after irradiation should be aimed at inhibiting the effects of [Ca2+]cyt and adenylate cyclase system activity, and in the long-term, at inhibition of guanylate cyclase system activity.

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm.

Na+/H+ exchangers (NHEs) are a family of ion transporters that regulate the pH of various cell compartments across an array of cell types. In eukaryotes, NHEs are encoded by the SLC9 gene family comprising 13 genes. SLC9C2, which encodes the NHE11 protein, is the only one of the SLC9 genes that is essentially uncharacterized. Here, we show that SLC9C2 exhibits testis/sperm-restricted expression in rats and humans, akin to its paralog SLC9C1 (NHE10). Similar to NHE10, NHE11 is predicted to contain an NHE domain, a voltage sensing domain, and finally an intracellular cyclic nucleotide binding domain. An immunofluorescence analysis of testis sections reveals that NHE11 localizes with developing acrosomal granules in spermiogenic cells in both rat and human testes. Most interestingly, NHE11 localizes to the sperm head, likely the plasma membrane overlaying the acrosome, in mature sperm from rats and humans. Therefore, NHE11 is the only known NHE to localize to the acrosomal region of the head in mature sperm cells. The physiological role of NHE11 has yet to be demonstrated but its predicted functional domains and unique localization suggests that it could modulate intracellular pH of the sperm head in response to changes in membrane potential and cyclic nucleotide concentrations that are a result of sperm capacitation events. If NHE11 is shown to be important for male fertility, it will be an attractive target for male contraceptive drugs due to its exclusive testis/sperm-specific expression.

PDE2A Inhibition Enhances Axonal Sprouting, Functional Connectivity, and Recovery after Stroke.

Phosphodiesterase (PDE) inhibitors have been safely and effectively used in the clinic and increase the concentration of intracellular cyclic nucleotides (cAMP/cGMP). These molecules activate downstream mediators, including the cAMP response element-binding protein (CREB), which controls neuronal excitability and growth responses. CREB gain of function enhances learning and allocates neurons into memory engrams. CREB also controls recovery after stroke. PDE inhibitors are linked to recovery from neural damage and to stroke recovery in specific sites within the brain. PDE2A is enriched in cortex. In the present study, we use a mouse cortical stroke model in young adult and aged male mice to test the effect of PDE2A inhibition on functional recovery, and on downstream mechanisms of axonal sprouting, tissue repair, and the functional connectivity of neurons in recovering cortex. Stroke causes deficits in use of the contralateral forelimb, loss of axonal projections in cortex adjacent to the infarct, and functional disconnection of neuronal networks. PDE2A inhibition enhances functional recovery, increases axonal projections in peri-infarct cortex, and, through two-photon in vivo imaging, enhances the functional connectivity of motor system excitatory neurons. PDE2A inhibition after stroke does not have an effect on other aspects of tissue repair, such as angiogenesis, gliogenesis, neurogenesis, and inflammatory responses. These data suggest that PDE2A inhibition is an effective therapeutic approach for stroke recovery in the rodent and that it simultaneously enhances connectivity in peri-infarct neuronal populations.SIGNIFICANCE STATEMENT Inhibition of PDE2A enhances motor recovery, axonal projections, and functional connectivity of neurons in peri-infarct tissue. This represents an avenue for a pharmacological therapy for stroke recovery.

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Goal. The study of the content of cyclic nucleotides in the eye tissues both in normal and in experimental conditions of toxic-allergic uveitis and the assessment of their possible impact on the development of the pathological process. Material and methods. The concentration of cAMP and cGMP in the eye tissues of experimental animals (rabbits) was determined using standard Amerscham kits by radioimmune method. The samples were counted on a Mark III counter. The results were processed according to the Student's method. Results. A significant decrease in the concentration of cAMP in the cornea, retina and vasculature of the rabbit's eye was found, which significantly affects the processes of catabolism and energy production. The content of another messenger cGMP in the same tissues is also reduced, and this suppresses the processes of cell proliferation. Conclusion. The results obtained add significant new elements to the picture of uveitis development, which should be considered when developing sound approaches to its effective treatment. Key words: cyclic nucleotides, uveitis, regulation of metabolism, cAMP, cGMP, eye tissues.

Alteration in Cngb1 Expression upon Maternal Immune Activation in a Mouse Model and Its Possible Association with Schizophrenia Susceptibility.

ObjectiveThe cyclic nucleotide-gated channel (Cng) regulates synaptic efficacy in brain neurons by modulating Ca2＋ levels in response to changes in cyclic nucleotide concentrations. This study investigated whether the expression of Cng channel, cyclic nucleotide-gated channel subunit beta 1 (Cngb1) exhibited any relationship with the pathophysiology of schizophrenia in an animal model and whether genetic polymorphisms of the human gene were associated with the progression of schizophrenia in a Korean population.MethodsWe investigated whether Cngb1 expression was related to psychiatric disorders in a mouse model of schizophrenia induced by maternal immune activation. A case-control study was conducted of 275 schizophrenia patients and 410 controls with single-nucleotide polymorphisms (SNPs) in the 5′-near region of CNGB1.ResultsCngb1 expression was decreased in the prefrontal cortex in the mouse model. Furthermore, the genotype frequency of a SNP (rs3756314) of CNGB1 was associated with the risk of schizophrenia.ConclusionOur results suggest that CNGB1 might be associated with schizophrenia susceptibility and maternal immune activation. Consequently, it is hypothesized that CNGB1 may be involved in the pathophysiology of schizophrenia.

CNG channel structure, function, and gating: a tale of conformational flexibility.

Cyclic nucleotide-gated (CNG) channels are key to the signal transduction machinery of certain sensory modalities both in vertebrate and invertebrate organisms. They translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through sensory cells. Despite CNG and voltage-gated potassium channels sharing a remarkable amino acid sequence homology and basic architectural plan, their functional properties are dramatically different. While voltage-gated potassium channels are highly selective and require membrane depolarization to open, CNG channels have low ion selectivity and are not very sensitive to voltage. In the last few years, many high-resolution structures of intact CNG channels have been released. This wealth of new structural information has provided enormous progress toward the understanding of the molecular mechanisms and driving forces underpinning CNG channel activation. In this review, we report on the current understanding and controversies surrounding the gating mechanism in CNG channels, as well as the deep intertwining existing between gating, the ion permeation process, and its modulation by membrane voltage. While the existence of this powerful coupling was recognized many decades ago, its direct structural demonstration, and ties to the CNG channel inherent pore flexibility, is a recent achievement.

Phosphodiesterase-2 inhibitor reverses post-traumatic stress induced fear memory deficits and behavioral changes via cAMP/cGMP pathway

Phosphodiesterase 2 is one of the phosphodiesterase (PDEs) family members that regulate cyclic nucleotide (namely cAMP and cGMP) concentrations. The present study determined whether PDE2 inhibition could rescue post-traumatic stress disorder (PTSD)-like symptoms. Mice were subjected to single prolonged stress (SPS) and treated with selective PDE2 inhibitor Bay 60–7550 (0.3, 1, or 3 mg/kg, i.p.). The behavioral tests such as forced swimming, sucrose preference test, open field, elevated plus maze, and contextual fear paradigm were conducted to determine the effects of Bay 60–7550 on SPS-induced depression- and anxiety-like behavior and fear memory deficits. The results suggested that Bay 60–7550 reversed SPS-induced depression- and anxiety-like behavior and fear memory deficits. Moreover, Bay 60–7550 prevented SPS-induced changes in the adrenal gland index, synaptic proteins synaptophysin and PSD95 expression, PKA, PKG, pCREB, and BDNF levels in the hippocampus and amygdala. These effects were completely prevented by PKG inhibitor KT5823. While PKA inhibitor H89 also prevented Bay 60-7550-induced pCREB and BDNF expression, but only partially prevented the effects on PSD95 expression in the hippocampus. These findings suggest that Bay 60–7550 protects mice against PTSD-like stress induced traumatic injury by activation of cGMP- or cAMP-related neuroprotective molecules, such as synaptic proteins, pCREB and BDNF.

Structural insights into the mechanism of rhodopsin phosphodiesterase

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

NCS 613, a Potent PDE4 Inhibitor, Displays Anti-Inflammatory and Anti-Proliferative Properties on A549 Lung Epithelial Cells and Human Lung Adenocarcinoma Explants.

Chronic inflammation is a deleterious process occurring in several pulmonary diseases; it is a driving force promoting tumorigenesis. By regulating local cyclic nucleotide concentration, cyclic nucleotide phosphodiesterases (PDE) govern important biological processes, including inflammation and proliferation. The aim of this study was to investigate the anti-inflammatory and anti-proliferative effects of NCS 613, a specific PDE4 inhibitor, on TNFα-treated human lung adenocarcinoma cell line (A549) and on human lung adenocarcinoma explants. PDE4 isoforms and inflammatory pathways mediated by p38 MAPK, ERK1/2, and IκBα were analyzed by Western blot and immunostainings. Proliferation were performed using [3H]-thymidine incorporation under different experimental conditions. TNFα-stimulation increased p38 MAPK phosphorylation and NF-κB translocation into the nucleus, which was abolished by NCS 613 treatment. Concomitantly, NCS 613 restores IκBα detection level in human adenocarcinoma. An IC50 value of 8.5 μM was determined for NCS 613 on anti-proliferative properties while ERK1/2 signaling was down-regulated in A549 cells and lung adenocarcinoma explants. These findings shed light on PDE4 signaling as a key regulator of chronic inflammation and cancer epithelial cell proliferation. It suggests that PDE4 inhibition by NCS 613 represent potential and interesting strategy for therapeutic intervention in tackling chronic inflammation and cell proliferation.

The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness.

Introduction Cold storage of platelets is considered to contribute to lower risk of bacterial growth and to more efficient hemostatic capacity. For the optimization of storage strategies, it is required to further elucidate the influence of refrigeration on platelet integrity. This study focused on adenosine diphosphate (ADP)-related platelet responsiveness. Materials and Methods Platelets were prepared from apheresis-derived platelet concentrates or from peripheral whole blood, stored either at room temperature or at 4°C. ADP-induced aggregation was tested with light transmission. Activation markers, purinergic receptor expression, and P2Y12 receptor function were determined by flow cytometry. P2Y1 and P2X1 function was assessed by fluorescence assays, cyclic nucleotide concentrations by immunoassays, and vasodilator-stimulated phosphoprotein (VASP)-phosphorylation levels by Western blot analysis. Results In contrast to room temperature, ADP-induced aggregation was maintained under cold storage for 6 days, associated with elevated activation markers like fibrinogen binding or CD62P expression. Purinergic receptor expression was not essentially different, whereas P2Y1 function deteriorated rapidly at cold storage, but not P2Y12 activity. Inhibitory pathways of cold-stored platelets were characterized by reduced responses to nitric oxide and prostaglandin E1. Refrigeration of citrated whole blood also led to the attenuation of induced inhibition of platelet aggregation, detectable within 24 hours. Conclusion ADP responsiveness is preserved under cold storage for 6 days due to stable P2Y12 activity and concomitant disintegration of inhibitory pathways enabling a higher reactivity of stored platelets. The ideal storage time at cold temperature for the highest hemostatic effect of platelets should be evaluated in further studies.

Prospects for the Use of Thermal Extracts of Brucella abortusI-206 in S-and L-Forms in the Diagnosis and Prevention of Brucellosis

Currently, one of the topical areas of research is the development of new antigen preparations for the specific diagnosis and prevention of brucellosis, since indication of the pathogen and prevention of the disease is complicated by the ability of brucella to dissociate, and live vaccines used for specific prevention of brucellosis have residual virulence. Thermal extracts (TE) obtained from Brucella abortus I-206 in the L- and S-form can be used as such promising antigens. It is known that TE in the L- and S-forms have immunogenic properties, as well as a modulating effect on the proliferation of immunocompetent cells, morphological changes in the immunocompetent organs of experimental animals.The aimof the work is to study the effect of Brucella abortus thermal extracts in L- and S-forms on the functional state of the cells of experimental animals.Materials and methods. The study was performed on 100 certified white mice. As objects of study, we used the B. abortus I-206 TE in L- and S-forms. Evaluation of the effect of antigenic drugs on the functional state of phagocytes of laboratory animals in vitro was performed on peritoneal macrophages. The total activity of the respiratory chain enzymes in the NBT-test and superoxide dismutase was determined. Cells of intact animals served as controls. As a positive control, a commercial antigenic LPS preparation Escherichia coli was used. The content of cyclic nucleotides in homogenates of immunocompetent organs was determined using ELISA.Results.This study presents materials on the study of the effect of TE on the bactericidal activity of phagocytes and the level of cyclic nucleotides in immunocompetent organs. It has been established that TEs activate oxygen-dependent bactericidal systems of phagocytes. When studying the effect of TE on the content of cyclic nucleotides in immunocompetent organs of white mice, an increase in their concentration was revealed, indicating an increase in the functional activity of the cells.Conclusion.The obtained data make it possible to substantiate the need for a further detailed study of the immunogenic properties of B. abortus TE in the L- or S-form on the organism of experimental animals.

Phosphodiesterase Inhibitors: A Therapeutic Augmentation for Treatment of Cognitive Impairment

Cognitive dysfunction is a loss of intellectual skills. This can affect the thoughts, memories and abilities of reasoning of a person. Dementia is considered to be one of the cognitive dysfunction neuropsychological hallmarks. It is estimated to be prevalent in around 81.1 million people worldwide by 2040. Significant progress has been made in understanding the pathological basis of these disorders over the last few decades. In experimental models of neurological disease, the transmutation of intracellular signalling pathways involving cognitive deficits, synaptic dysfunction, loading of β-amyloid plaques or presynaptic density in hippocampus has demonstrated a therapeutic benefit. Cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP) are two such targets for intracellular signalling, which produced beneficial cellular effects in CNS pathologies after elevation. These second messengers strengthen signals at cell surface receptors and within the cell, making them an essential element in signal transduction and necessary for cellular signalling in various cell functions such as the release of neurotransmitters. Although the concentration of cyclic nucleotides depends on the rate of their synthesis by adenylate or guanylate cyclase’s, however, their breakdown is regulated through phosphodiesterase’s (PDEs) which are capable of employ tight, selective control over cyclic nucleotide signaling through the hydrolysis of cAMP/cGMP. PDE inhibition is therefore thought to be a new strategy for building cyclic nucleotide levels in the brain. This review evaluated recent studies and development with a focus on cognitive decline, but due to adverse effects, there are still very few PDE inhibitors for clinical use in neurology. Therefore, more specific inhibitors must be looked for at the level of PDE subclasses and their isoforms.

Why fibrosis after exposure to low frequency noise?

Introduction: The cardiovascular effects of the exposure to low frequency noise (LFN) (<500 Hz) are not fully understood. Previous experimental work confirmed an increase in collagens I and III and ultrastructural changes in the intercalated discs of cardiomyocytes from rats exposed to LFN. Our work aims at characterizing the alterations in gene expression in the heart of rats exposed for 13 weeks to LFN. Materials and methods: 8 adult Wistar rats assigned randomly in equal number to continuous LFN exposure or silent conditions for control purposes were sacrificed after 13 weeks. The heart apexes were frozen and preserved at -80 ºC until RNA extraction. Gene expression was analysed using GeneChip® Rat Gene 1.0 ST (Affymetrix). The set of genes was submitted to Ingenuity Pathway Analysis (IPA) software to predict relevant outcomes. For immunohistochemistry we have used samples of heart apexes fixed in formalin and embedded in paraffin and for immunofluorescence the samples were fixed in PFA 4%. In both methodologies we have incubated the samples with rabbit Hemoglobin α polyclonal antibodies (H80). Results: Microarray analysis showed that 19,353 genes were expressed in heart, from a total of 27,342 well-annotated genes of the chip, covering the whole transcript. The LFN exposed group showed 186 genes with significant fold change above 1.5, mostly (115 genes) down-regulated. The gene with highest variation was haemoglobin alpha, adult chain 2, 6-fold down-regulated. Its expression in rat cardiomyocytes was confirmed by immunostaining and immunofluorescence. The integrated view of gene expression, performed by IPA, assigned a subset of 19 genes to fibrosis in heart. Overall their expression variation indicated an increase in fibrosis, though not significant (z-score 0.9 < 2). Analysis of Z-scores predicted several biological functions significantly altered namely transport of molecule, concentration of cyclic nucleotides, hydrolysis of acylglycerol, olfaction, cell death and permeability transition of mitochondria. Discussion and conclusions: We have found an unexpected significant change in haemoglobin alpha, adult chain 2 expression in LFN-exposed hearts and were able to confirm the gene expression results by immunohistochemistry and immunofluorescence. Although there is increasing evidence of the presence of haemoglobin in tissues other than erythrocytes, its role is not yet clarified. Despite the well-documented occurrence of tissue fibrosis in LFN-exposed cardiomyocytes, we did not found significant differences in expression of collagen-coding genes. However, the expression of a subset of genes associated with cardiac fibrosis indicated a possible increase. Our results showed both an increase in olfaction and a decrease in cell death processes, which are compatible with a response to the chronic stress to which the rats were submitted.

A unique choanoflagellate enzyme rhodopsin exhibits light-dependent cyclic nucleotide phosphodiesterase activity

Photoactivated adenylyl cyclase (PAC) and guanylyl cyclase rhodopsin increase the concentrations of intracellular cyclic nucleotides upon illumination, serving as promising second-generation tools in optogenetics. To broaden the arsenal of such tools, it is desirable to have light-activatable enzymes that can decrease cyclic nucleotide concentrations in cells. Here, we report on an unusual microbial rhodopsin that may be able to meet the demand. It is found in the choanoflagellate Salpingoeca rosetta and contains a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. We examined the enzymatic activity of the protein (named Rh-PDE) both in HEK293 membranes and whole cells. Although Rh-PDE was constitutively active in the dark, illumination increased its hydrolytic activity 1.4-fold toward cGMP and 1.6-fold toward cAMP, as measured in isolated crude membranes. Purified full-length Rh-PDE displayed maximal light absorption at 492 nm and formed the M intermediate with the deprotonated Schiff base upon illumination. The M state decayed to the parent spectral state in 7 s, producing long-lasting activation of the enzyme domain with increased activity. We discuss a possible mechanism of the Rh-PDE activation by light. Furthermore, Rh-PDE decreased cAMP concentration in HEK293 cells in a light-dependent manner and could do so repeatedly without losing activity. Thus, Rh-PDE may hold promise as a potential optogenetic tool for light control of intracellular cyclic nucleotides (e.g. to study cyclic nucleotide-associated signal transduction cascades).

Investigating Ligand Binding to HCN Channels by Surface Plasmon Resonance

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding (CNB) domain in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides in the CNB domain to the pore opening. Surface plasmon resonance (SPR) is a powerful biophysical tool for quantitatively investigating ligand-protein and protein-protein interactions. Here we used SPR to detect ligand binding to the isolated C-linker/CNB domain of HCN channels. The isolated C-linker/CNB domains of wild-type (WT) and L586W mutant HCN2 channels were immobilized on a NTA sensor chip and cAMP was injected over the protein coated surface. The mutant C-linker/CNB domain has been used before for ligand binding studies based on the changes in the fluorescence of the introduced tryptophan upon ligand binding. From the cyclic nucleotide concentration dependent SPR responses, we determined the binding affinity for cAMP to be 6.3 + 2.6 μM for the WT and 10.8 + 2.3μM for the mutant C-linker/CNB domains. The binding affinity determined for the mutant L586W C-linker-CNB domains determined with SPR is in agreement with the binding affinity of 13 + 2 μM determined with the fluorescence-based method. These results indicate that SPR is well suited for the detection of binding of known HCN channel ligands. Therefore, SPR can be used to identify novel HCN channel regulators for treatment of diseases associated with abnormal functions of these channels, such as epilepsy and cardiac arrhythmias.

CUMP hydrolysis by PDE3A.

As previously reported, the cardiac phosphodiesterase PDE3A hydrolyzes cUMP. Moreover, cUMP-degrading activity was detected in cow and dog hearts several decades ago. Our aim was to characterize the enzyme kinetic parameters of PDE3A-mediated cUMP hydrolysis and to investigate whether cUMP and cUMP-hydrolyzing PDEs are present in cardiomyocytes. PDE3A-mediated cUMP hydrolysis was characterized in time course, inhibitor, and Michaelis-Menten kinetics experiments. Intracellular cyclic nucleotide (cNMP) concentrations and the mRNAs of cUMP-degrading PDEs were quantitated in neonatal rat cardiomyocytes (NRCMs) and murine HL-1 cardiomyogenic cells. Moreover, we investigated cUMP degradation in HL-1 cell homogenates and intact cells. Educts (cNMPs) and products (NMPs) of the PDE reactions were detected by HPLC-coupled tandem mass spectrometry. PDE3A degraded cUMP (measurement of UMP formation) with a K M value of ~143μM and a V max value of ~42μmol/min/mg. PDE3A hydrolyzed cAMP with a K M value of ~0.7μM and a V max of ~1.2μmol/min/mg (determination of AMP formation). The PDE3 inhibitor milrinone inhibited cUMP hydrolysis (determination of UMP formation) by PDE3A (K i=57nM). Significant amounts of cUMP as well as of PDE3A mRNA (in addition to PDE3B and PDE9A transcripts) were detected in HL-1 cells and NRCMs. Although HL-1 cell homogenates contain a milrinone-sensitive cUMP-hydrolyzing activity, intact HL-1 cells may use additional PDE3-independent mechanisms for cUMP disposal. PDE3A is a low-affinity and high-velocity PDE for cUMP. Future studies should investigate biological effects of cUMP in cardiomyocytes and the role of PDE3A in detoxifying high intracellular cUMP concentrations under pathophysiological conditions.

A novel thermoregulatory role for PDE10A in mouse and human adipocytes.

Phosphodiesterase type 10A (PDE10A) is highly enriched in striatum and is under evaluation as a drug target for several psychiatric/neurodegenerative diseases. Preclinical studies implicate PDE10A in the regulation of energy homeostasis, but the mechanisms remain unclear. By utilizing small‐animal PET/MRI and the novel radioligand [18F]‐AQ28A, we found marked levels of PDE10A in interscapular brown adipose tissue (BAT) of mice. Pharmacological inactivation of PDE10A with the highly selective inhibitor MP‐10 recruited BAT and potentiated thermogenesis in vivo. In diet‐induced obese mice, chronic administration of MP‐10 caused weight loss associated with increased energy expenditure, browning of white adipose tissue, and improved insulin sensitivity. Analysis of human PET data further revealed marked levels of PDE10A in the supraclavicular region where brown/beige adipocytes are clustered in adults. Finally, the inhibition of PDE10A with MP‐10 stimulated thermogenic gene expression in human brown adipocytes and induced browning of human white adipocytes. Collectively, our findings highlight a novel thermoregulatory role for PDE10A in mouse and human adipocytes and promote PDE10A inhibitors as promising candidates for the treatment of obesity and diabetes.

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.