cAMP Degradation Research Articles

Highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonadotropin allowing their measurement in plasma.

In previous studies, we had shown the synergistic effect of 10−5 M forskolin (FSK) on the detection threshold of the cyclic AMP response to luteinizing hormones (LH) and chorionic gonadotropins (CG) from various species in the mouse Leydig tumor cell (mLTC) cell line. Independently, we started to study the effect of 10−12–10−6 M oxytocin (OXT) also on the cyclic AMP response to LH and CG preparations on these same cells and found an amplifying effect on the luminescence response caused by gonadotropins. The aim was then to explore the effects of 10−12–10−6 M OXT on the gonadotropin-induced cAMP response, in the presence or absence of 10 µM FSK to optimize the assay down to a sensitivity compatible with the detection of the circulating concentrations of these hormones in various species. Finally, the optimization relies on three independent phenomena: (1) the inhibition of nucleotide phosphodiesterase by IBMX (3-isobutyl-1-methylxanthine) to avoid cAMP degradation; (2) the strong synergy of 10 µM forskolin with low concentrations of LH or CG during the 1-h luminescence measurement; (3) the stimulatory effect of 10−8M OXT on the amplitude of transfected cAMP-sensitive luciferase response. By doing this, the detectable concentrations are at the 1–10 pg/well (pM range) for the LHs and CGs from various species. The bioactivities of circulating LHs and CGs in blood or urine are therefore expected to be measurable in 10 µL-plasma samples from mammalian species and maybe others. Indeed, a preliminary study with equine and donkey plasma samples shows that the measured bioactivity was fully inhibited by a specific MAB against the receptor-binding region of equine LH (eLH) and equine CG (eCG), thus eliminating a possible response due to interfering substances other than eLH or eCG. From these data, it is expected that the bioactivity profiles of these hormones will be measurable in the blood of human, equine, and ovine species and very likely in rodents, ruminants, and hopefully in most other mammalian species.Lay summaryLuteinizing hormone (LH) plays a central role in controlling ovary and testicle functions in many animals, including humans. The highly sensitive method, known as an assay, described in this paper, measures the biological activity of LH in the blood of mammals. The assay is performed in culture of cells derived from mouse testicles in the presence of factors that diminish the detection threshold for LH. The knowledge of the bioactive LH concentration dynamics in the blood is very informative about the reproductive status of male and female mammals. This new in vitro bioassay provides a powerful tool to get this information.

A three-dimensional finite element model of cAMP signals

This paper presents a three-dimensional finite element model for cyclic adenosine monophosphate (cAMP) signaling. Governing equations for the synthesis, diffusion, and degradation of cAMP were numerically implemented using the finite element method. Simulated results were displayed as time course plots of cAMP concentrations at selected nodes within the discretized geometry. The validity of the finite element model was assessed by comparing simulated results against analytical or other numerical solutions of cAMP concentration distribution for a spherical cellular volume. An endothelial cell was also simulated using its discretized geometry obtained from microscopic cellular cross-sectional images. Simulated solutions using the spherical cellular volume produced near identical cAMP concentration plots to the analytical solutions and were in good agreements with numerical results obtained from VCell, an existing software package for modeling cell biological systems. The validated 3-D finite element model was then employed to simulate the cAMP signaling pathway within a pulmonary microvascular endothelial cell geometry.

Abstract MP241: Roflumilast-mediated Phosphodiesterase 4d Inhibition Reverses Diabetes-associated Cardiac Dysfunction And Remodeling: Effects Beyond Glucose Lowering

Introduction: Patients with type 2 diabetes (T2DM) have a substantial risk of developing cardiovascular disease. Our recent studies reveal that hyperinsulinemia attenuates cardiac contractility by inducing expression of phosphodiesterase 4D (PDE4D) that increases cAMP degradation. Furthermore, there is growing evidence that PDE4 dysregulation is of pathophysiological importance in metabolic disorders. Hypothesis: We propose that inhibition of PDE4D might ameliorate diabetes-associated cardiac dysfunction, in addition to lowering glucose. Methods: Male C57BL/6J mice fed with high-fat diet (HFD) were treated with PDE4 inhibitor roflumilast (currently used to treat chronic obstructive pulmonary disease, COPD). Myocardial structure, contractile function and remodeling were evaluated. For mechanistic studies, specific silencing of cardiac PDE4D and overexpression of miR-1 were administrated in mice undergoing high-fat feeding. These studies were complemented by in vitro analysis in primary cultured rat cardiomyocytes and cardiac fibroblasts. Results: Mice on HFD developed greater body weight compared to normal chow diet-fed mice, manifesting hyperglycemia, hyperinsulinemia, cardiac contractile dysfunction and remodeling, and cardiac PDE4D5 upregulation by 24 weeks. The expression of PDE4D5 was also elevated in human hearts with diabetes. In comparison with vehicle-treated HFD controls, PDE4 inhibitor roflumilast (1mg/kg/day) can prevent and even reverse hyperglycemia and cardiac dysfunction, accompanied by the decrease of cardiac PDE4D expression. In addition, cardiac miR-1 level was reduced in HFD mice, which was restored by PDE4 inhibitor roflumilast treatment. Either cardiac specific PDE4D5 knockdown or miR-1 overexpression significantly reversed cardiac dysfunction in HFD-mice, despite persistence of hyperglycemia and glucose intolerance. Gain- and loss-of-function studies of PDE4D in cardiomyocytes implicated that suppression of PDE4D protected cardiac hypertrophy via SERCA2a-mediated miR-1 restoration. Moreover, inhibition of PDE4D prevented insulin-activated TGF-β1 signaling which promotes miR-1 reduction in cardiac fibroblasts and subsequent fibrosis. Conclusions: These studies elucidate a novel mechanism by which PDE4D contributes to HFD-induced heart failure through reducing miR-1 expression in both cardiomyocytes and cardiac fibroblasts and suggest a therapeutic potential of PDE4 inhibitor roflumilast in preventing or treating cardiac dysfunction associated with diabetes.

Dual Activation of Phosphodiesterase 3 and 4 Regulates Basal Cardiac Pacemaker Function and Beyond.

The sinoatrial (SA) node is the physiological pacemaker of the heart, and resting heart rate in humans is a well-known risk factor for cardiovascular disease and mortality. Consequently, the mechanisms of initiating and regulating the normal spontaneous SA node beating rate are of vital importance. Spontaneous firing of the SA node is generated within sinoatrial nodal cells (SANC), which is regulated by the coupled-clock pacemaker system. Normal spontaneous beating of SANC is driven by a high level of cAMP-mediated PKA-dependent protein phosphorylation, which rely on the balance between high basal cAMP production by adenylyl cyclases and high basal cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). This diverse class of enzymes includes 11 families and PDE3 and PDE4 families dominate in both the SA node and cardiac myocardium, degrading cAMP and, consequently, regulating basal cardiac pacemaker function and excitation-contraction coupling. In this review, we will demonstrate similarities between expression, distribution, and colocalization of various PDE subtypes in SANC and cardiac myocytes of different species, including humans, focusing on PDE3 and PDE4. Here, we will describe specific targets of the coupled-clock pacemaker system modulated by dual PDE3 + PDE4 activation and provide evidence that concurrent activation of PDE3 + PDE4, operating in a synergistic manner, regulates the basal cardiac pacemaker function and provides control over normal spontaneous beating of SANCs through (PDE3 + PDE4)-dependent modulation of local subsarcolemmal Ca2+ releases (LCRs).

CAMP Compartmentalization in Cerebrovascular Endothelial Cells: New Therapeutic Opportunities in Alzheimer's Disease.

The vascular hypothesis used to explain the pathophysiology of Alzheimer’s disease (AD) suggests that a dysfunction of the cerebral microvasculature could be the beginning of alterations that ultimately leads to neuronal damage, and an abnormal increase of the blood–brain barrier (BBB) permeability plays a prominent role in this process. It is generally accepted that, in physiological conditions, cyclic AMP (cAMP) plays a key role in maintaining BBB permeability by regulating the formation of tight junctions between endothelial cells of the brain microvasculature. It is also known that intracellular cAMP signaling is highly compartmentalized into small nanodomains and localized cAMP changes are sufficient at modifying the permeability of the endothelial barrier. This spatial and temporal distribution is maintained by the enzymes involved in cAMP synthesis and degradation, by the location of its effectors, and by the existence of anchor proteins, as well as by buffers or different cytoplasm viscosities and intracellular structures limiting its diffusion. This review compiles current knowledge on the influence of cAMP compartmentalization on the endothelial barrier and, more specifically, on the BBB, laying the foundation for a new therapeutic approach in the treatment of AD.

Physiological Temperature Changes Fine-Tune β 2- Adrenergic Receptor-Induced Cytosolic cAMP Accumulation.

Norepinephrine (NE) controls many vital body functions by activating adrenergic receptors (ARs). Average core body temperature (CBT) in mice is 37°C. Of note, CBT fluctuates between 36 and 38°C within 24 hours, but little is known about the effects of CBT changes on the pharmacodynamics of NE. Here, we used Peltier element-controlled incubators and challenged murine hypothalamic mHypoA -2/10 cells with temperature changes of ±1°C. We observed enhanced NE-induced activation of a cAMP-dependent luciferase reporter at 36 compared with 38°C. mRNA analysis and subtype specific antagonists revealed that NE activates β 2- and β 3-AR in mHypoA-2/10 cells. Agonist binding to the β 2-AR was temperature insensitive, but measurements of cytosolic cAMP accumulation revealed an increase in efficacy of 45% ± 27% for NE and of 62% ± 33% for the β 2-AR-selective agonist salmeterol at 36°C. When monitoring NE-promoted cAMP efflux, we observed an increase in the absolute efflux at 36°C. However, the ratio of exported to cytosolic accumulated cAMP is higher at 38°C. We also stimulated cells with NE at 37°C and measured cAMP degradation at 36 and 38°C afterward. We observed increased cAMP degradation at 38°C, indicating enhanced phosphodiesterase activity at higher temperatures. In line with these data, NE-induced activation of the thyreoliberin promoter was found to be enhanced at 36°C. Overall, we show that physiologic temperature changes fine-tune NE-induced cAMP signaling in hypothalamic cells via β 2-AR by modulating cAMP degradation and the ratio of intra- and extracellular cAMP. SIGNIFICANCE STATEMENT: Increasing cytosolic cAMP levels by activation of G protein-coupled receptors (GPCR) such as the β 2-adrenergic receptor (AR) is essential for many body functions. Changes in core body temperature are fundamental and universal factors of mammalian life. This study provides the first data linking physiologically relevant temperature fluctuations to β 2-AR-induced cAMP signaling, highlighting a so far unappreciated role of body temperature as a modulator of the prototypic class A GPCR.

Cautionary Notes on the Use of Arabinose- and Rhamnose-Inducible Expression Vectors in Pseudomonas aeruginosa.

The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of vfr transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed vfr transcription under the control of the rhaBp rhamnose-inducible promoter system (designated PRha) and found that PRha promoter activity was highly dependent upon vfr. Vfr dependence was also observed for the araBp arabinose-inducible promoter (designated PBAD). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the PRha and PBAD promoters in vitro. Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in PRha and PBAD promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution. IMPORTANCE Heterologous gene expression and complementation constitute a valuable and widely utilized tool in bacterial genetics. The arabinose-inducible ParaBAD (PBAD) and rhamnose-inducible PrhaBAD (PRha) promoter systems are commonly used in P. aeruginosa genetics and prized for the tight control and dynamic expression ranges that can be achieved. In this study, we demonstrate that the activity of both promoters is dependent upon the cAMP-dependent transcription factor Vfr. While this poses an obvious problem for use in a vfr mutant background, the issue is more pervasive, considering that vfr transcription/synthesis and cAMP homeostasis are highly integrated into the cellular physiology of the organism and influenced by numerous global regulatory systems. Fortunately, the synthetic PTac promoter is not subject to Vfr regulatory control.

Selective Inhibition of Phosphodiesterase 7 Enzymes Reduces Motivation for Nicotine Use through Modulation of Mesolimbic Dopaminergic Transmission.

Approximately 5 million people die from diseases related to nicotine addiction and tobacco use each year. The nicotine-induced increase of corticomesolimbic dopaminergic (DAergic) transmission and hypodopaminergic conditions occurring during abstinence are important for maintaining drug-use habits. We examined the notion of reequilibrating DAergic transmission by inhibiting phosphodiesterase 7 (PDE7), an intracellular enzyme highly expressed in the corticomesolimbic circuitry and responsible for the degradation of cyclic adenosine monophosphate (cAMP), the main second messenger modulated by DA receptor activation. Using selective PDE7 inhibitors, we demonstrated in male rats that systemic PDE7 enzyme inhibition reduced nicotine self-administration and prevented reinstatement to nicotine seeking evoked by cues or by the pharmacological stressor yohimbine. The effect was also observed by direct application of the PDE7 inhibitors into the nucleus accumbens (NAc) shell but not into the core. Inhibition of PDE7 resulted in increased DA- and cAMP-regulated neuronal phosphoprotein and cAMP response element-binding protein and their phosphorylated forms in the NAc. It also enhanced the DA D1 receptor agonism-mediated effects, indicating potentiation of protein kinase A–dependent transmission downstream of D1 receptor activation. In electrophysiological recordings from DA neurons in the lateral posterior ventral tegmental area, the PDE7 inhibitors attenuated the spontaneous activity of DA neurons. This effect was exerted through the potentiation of D1 receptor signaling and the subsequent facilitation of γ-aminobutyric acid transmission. The PDE7 inhibitors did not elicit conditioned place preference and did not induce intravenous self-administration, indicating lack of reinforcing properties. Thus, PDE7 inhibitors have the potential to treat nicotine abuse.SIGNIFICANCE STATEMENT The World Health Organization estimates that there are 1.25 billion smokers worldwide, representing one-third of the global population over the age of 15. Nicotine-induced increase of corticomesolimbic DAergic transmission and hypodopaminergic conditions occurring during abstinence are critical for maintaining drug-use habits. Here, we demonstrate that nicotine consumption and relapse to nicotine seeking are attenuated by reequilibrating DAergic transmission through inhibition of PDE7, an intracellular enzyme responsible for the degradation of cAMP, the main second messenger modulated by DA receptor activation. PDE7 inhibition may represent a novel treatment approach to aid smoking cessation.

Inhibition of cAMP‐Phosphodiesterase 4 (PDE4) Induces Salivation in Mice

While exploring potential anti-inflammatory benefits of PDE4 inhibition in a mouse model of bacterial lung infection, we noticed that treatment with the PDE4 inhibitor Piclamilast visibly induced salivation in the animals. Given the predominant role of autonomic nervous system regulation in the control of salivation, and the potential impact of altered autonomics triggered by PDE4 inhibition on our infection model, we have further explored this observation. InKetamine/Xylazine-anesthetized mice, in which saliva production was tracked by the weight increase of thin filter paper strips placed into the mouths of the animals, treatment with PDE4 inhibitors did not induce saliva production per se, but synergized with low doses of the β-adrenoceptor agonist Isoprenaline to induce substantial salivation that was ablated by treatment with the β-blocker Propranolol, but not the mACh-blocker Atropine, and was absent in CFTRΔF508/ΔF508 mice that lack functional CFTR. Conversely, in awake mice, visual scoring (elevated/abnormal salivation=1; no elevated/abnormal salivation=0) indicated that treatment with PDE4 inhibitors was by itself sufficient to induce substantial salivation, which was ablated by treatment with either Propranolol or Atropine, and was also absent in ΔF508-CFTR-mutant mice. Our findings suggest that in anesthetized mice, in which autonomic nervous system regulations are suppressed, inhibition of cAMP degradation by PDE4 within salivary gland cells potentiates the effects of increased cAMP production resulting from β-adrenoceptor activation, and thereby promote the cAMP/PKA-dependent activation of CFTR that promotes saliva secretion. However, in awake mice, PDE4 inhibition acts via an additional mechanism on the autonomic, muscarinic regulation of salivation, likely by affecting neurotransmitter release. In both awake and anesthetized mice, salivation was induced by several structurally distinct PDE4 inhibitors, but not by a PDE3 inhibitor, suggesting that salivation is a class effect of PAN-PDE4 inhibitors. The PDE4 family comprises four PDE4 subtypes, PDE4A to D. Genetic ablation of any individual PDE4 subtype in the respective KO mouse strains did not induce salivation in either awake or anesthetized mice, indicating that salivation must result from the concurrent inactivation of multiple (at least two) PDE4 subtypes. Taken together, our data identify PDE4 as a critical regulator of CFTR function in an in vivo model and suggests a therapeutic potential of PDE4 inhibition to alleviate CFTR hypofunction in cystic fibrosis. In addition, our data reveal a therapeutic potential for PDE4 inhibition in the treatment of dry mouth, a condition frequently associated with drug-, radiation-, or ageing-induced salivary gland dysfunction.

Phosphodiesterase-4 Inhibition in Psoriasis.

Psoriasis is a chronic immune-mediated inflammatory disorder. Phosphodiesterase-4 (PDE-4) is an enzyme that mediates inflammatory responses and plays a role in psoriasis pathogenesis. PDE-4 degrades its substrate cyclic adenosine monophosphate (cAMP) to adenosine monophosphate (AMP), which subsequently leads to the production of pro-inflammatory mediators. Inhibitors of PDE-4 work by blocking the degradation of cAMP, which leads to a reduction in inflammation. Apremilast is the only approved oral PDE-4 inhibitor for the treatment of psoriasis. While it is effective for some patients, it may be limited by adverse effects in others. A topical PDE-4 inhibitor, roflumilast, is being investigated in psoriasis and showing promising results. Crisaborole, a topical PDE-4 inhibitor approved for use in atopic dermatitis, has also been investigated in psoriasis. This is an updated comprehensive review to summarize the currently available evidence for the PDE-4 inhibitors apremilast, roflumilast and crisaborole in the treatment of psoriasis, with a focus on data from randomized clinical trials.

Role of phosphodiesterases in the development of takotsubo syndrome

Abstract Background/Purpose Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. We identified a higher sensitivity to catecholamine-induced stress toxicity as mechanism associated with the TTS phenotype in our former study, but the pathogenesis of TTS is still not completely understood. In this study our aim was to prove the hypothesis of an altered phosphodiesterase (PDE)-dependent 3',5'-cyclic adenosine monophosphate (cAMP)-signaling in TTS in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Methods and results We generated functional TTS-iPSC-CMs and treated them with catecholamines to mimic a TTS-phenotype. To directly address the hypothesis that local cAMP dynamics might be altered in TTS, we used Förster resonance energy transfer (FRET) based cAMP sensors, which are specifically located in the cytosol or at the sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA) micro domain. We demonstrated that β-adrenergic receptor (β-AR) stimulations resulted in stronger cytosolic FRET responses in TTS-CMs compared to controls. In contrast, no differences of cAMP level were observed in the SERCA-PLN micro domain between TTS- and control-iPSC-CMs. To analyze the interplay of β-AR signaling and specific PDE contribution to the cAMP signaling in TTS, specific PDE-inhibitors were used. We were able to show in the cytosol that after β-AR stimulation, the strong effects of the PDE4 family of control cells were significantly decreased in diseased TTS CMs, which is in line with previously described reduced PDE4 activity in failing mouse hearts. In contrast, the contribution of PDE3 to cytoplasmic cAMP degradation was increased in TTS (Figure 1 A). This is in line with increased PDE3A and down-regulated PDE4D protein expression in TTS-iPSC-CMs compared to control cells. Analysis of PDE-dependent cAMP level in the SERCA micro domain show also a significantly reduced PDE4 activity. But the dynamic cytosolic PDE contribution of PDE2 and PDE3 after catecholamine treatment in TTS is lost in SERCA micro domain (Figure1B). Conclusion Our data showed for the first time alterations of local cAMP signaling in healthy and diseased TTS-iPSC-CMs. We demonstrated an isozym shift from PDE4 in control to PDE3 and PDE2 in TTS and identified PDE4 as an important player in the β-adrenergic cAMP signaling in TTS. Therefore, PDE4 activators may be a possible new therapeutic target option in the treatment of TTS. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DZHK

Endogenous sulfur dioxide is a novel inhibitor of hypoxia-induced mast cell degranulation

IntroductionMast cell (MC) degranulation is an important step in the pathogenesis of inflammatory reactions and allergies; however, the mechanism of stabilizing MC membranes to reduce their degranulation is unclear. MethodsSO2 content in MC culture supernatant was measured by HPLC-FD. The protein and mRNA expressions of the key enzymes aspartate aminotransferase 1 (AAT1) and AAT2 and intracellular AAT activity were detected. The cAMP level in MCs was detected by immunofluorescence and ELISA. The release rate of MC degranulation marker β-hexosaminidase was measured. The expression of AAT1 and cAMP, the MC accumulation and degranulation in lung tissues were detected. ObjectivesTo exam whether an endogenous sulfur dioxide (SO2) pathway exists in MCs and if it serves as a novel endogenous MC stabilizer. ResultsWe firstly show the existence of the endogenous SO2/AAT pathway in MCs. Moreover, when AAT1 was knocked down in MCs, MC degranulation was significantly increased, and could be rescued by a SO2 donor. Mechanistically, AAT1 knockdown decreased the cyclic adenosine monophosphate (cAMP) content in MCs, while SO2 prevented this reduction in a dose-independent manner. Pretreatment with the cAMP-synthesizing agonist forskolin or the cAMP degradation inhibitor IBMX significantly blocked the increase in AAT1 knockdown-induced MC degranulation. Furthermore, in hypoxia-stimulated MCs, AAT1 protein expression and SO2 production were markedly down regulated, and MC degranulation was activated, which were blunted by AAT1 overexpression. The cAMP synthesis inhibitor SQ22536 disrupted the suppressive effect of AAT1 overexpression on hypoxia-induced MC degranulation. In a hypoxic environment, mRNA and protein expression of AAT1 was significantly reduced in lung tissues of rats. Supplementation of SO2 elevated the cAMP level and reduced perivascular MC accumulation and degranulation in lung tissues of rats exposed to a hypoxic environment in vivo. ConclusionSO2 serves as an endogenous MC stabilizer via upregulating the cAMP pathway under hypoxic circumstance.

PKA functions in metabolism and resistance to obesity: lessons from mouse and human studies.

Both direct and indirect evidence demonstrate a central role for the cAMP-dependent protein kinase (PKA) signaling pathway in the regulation of energy balance and metabolism across multiple systems. However, the ubiquitous pattern of PKA expression across cell types poses a challenge in pinpointing its tissue-specific regulatory functions and further characterizing its many downstream effects in certain organs or cells. Mouse models of PKA deficiency and over-expression and studies in living cells have helped clarify PKA function in adipose tissue (AT), liver, adrenal, pancreas, and specific brain nuclei, as they pertain to energy balance and metabolic dysregulation. Limited studies in humans suggest differential regulation of PKA in AT of obese compared to lean individuals and an overall dysregulation of PKA signaling in obesity. Despite its complexity, under normal physiologic conditions, the PKA system is tightly regulated by changes in cAMP concentrations upstream via adenylate cyclase and downstream by phosphodiesterase-mediated cAMP degradation to AMP and by changes in PKA holoenzyme stability. Adjustments in the PKA system appear to be important to the development and maintenance of the obese state and its associated metabolic perturbations. In this review we discuss the important role of PKA in obesity and its involvement in resistance to obesity, through studies in humans and in mouse models, with a focus on the regulation of PKA in energy expenditure, intake behavior, and lipid and glucose metabolism.

Cyclic Nucleotides Signaling and Phosphodiesterase Inhibition: Defying Alzheimer's Disease.

Defects in brain functions associated with aging and neurodegenerative diseases benefit insignificantly from existing options, suggesting that there is a lack of understanding of pathological mechanisms. Alzheimer's disease (AD) is such a nearly untreatable, allied to age neurological deterioration for which only the symptomatic cure is available and the agents able to mould progression of the disease, is still far away. The altered expression of phosphodiesterases (PDE) and deregulated cyclic nucleotide signaling in AD has provoked a new thought of targeting cyclic nucleotide signaling in AD. Targeting cyclic nucleotides as an intracellular messenger seems to be a viable approach for certain biological processes in the brain and controlling substantial. Whereas, the synthesis, execution, and/or degradation of cyclic nucleotides has been closely linked to cognitive deficits. In relation to cognition, the cyclic nucleotides (cAMP and cGMP) have an imperative execution in different phases of memory, including gene transcription, neurogenesis, neuronal circuitry, synaptic plasticity and neuronal survival, etc. AD is witnessed by impairments of these basic processes underlying cognition, suggesting a crucial role of cAMP/cGMP signaling in AD populations. Phosphodiesterase inhibitors are the exclusive set of enzymes to facilitate hydrolysis and degradation of cAMP and cGMP thereby, maintains their optimum levels initiating it as an interesting target to explore. The present work reviews a neuroprotective and substantial influence of PDE inhibition on physiological status, pathological progression and neurobiological markers of AD in consonance with the intensities of cAMP and cGMP.

The phosphodiesterase-4 and glycine transporter-1 inhibitors enhance in vivo hippocampal theta network connectivity and synaptic plasticity, whereas D-serine does not

Dysfunctional N-methyl-D-aspartate receptors (NMDARs) and cyclic adenosine monophosphate (cAMP) have been associated with deficits in synaptic plasticity and cognition found in neurodegenerative and neuropsychiatric disorders such as Alzheimer’s disease (AD) and schizophrenia. Therapeutic approaches that indirectly enhance NMDAR function through increases in glycine and/or D-serine levels as well as inhibition of phosphodiesterases that reduces degradation of cAMP, are expected to enhance synaptic strength, connectivity and to potentially impact cognition processes. The present in vivo study investigated effects of subcutaneous administration of D-serine, the glycine transporter 1 (GlyT1) inhibitor SSR504734 and the PDE4 inhibitor rolipram, on network oscillations, connectivity and long-term potentiation (LTP) at the hippocampi circuits in Sprague-Dawley rats. In conscious animals, multichannel EEG recordings assessed network oscillations and connectivity at frontal and hippocampal CA1–CA3 circuits. Under urethane anaesthesia, field excitatory postsynaptic potentials (fEPSPs) were measured in the CA1 subfield of the hippocampus after high-frequency stimulation (HFS) of the Schaffer collateral-CA1 (SC) pathway. SSR504734 and rolipram significantly increased slow theta oscillations (4–6.5 Hz) at the CA1–CA3, slow gamma oscillations (30–50 Hz) in the frontal areas and enhanced coherence in the CA1–CA3 network, which were dissociated from motor behaviour. SSR504734 enhanced short-term potentiation (STP) and fEPSP responses were extended into LTP response, whereas the potentiation of EPSP slope was short-lived to STP with rolipram. Unlike glycine, increased levels of D-serine had no effect on network oscillations and limits the LTP induction and expression. The present data support a facilitating role of glycine and cAMP on network oscillations and synaptic efficacy at the CA3–CA1 circuit in rats, whereas raising endogenous D-serine levels had no such beneficial effects.

MRP4/ABCC4 expression is regulated by histamine in acute myeloid leukemia cells, determining cAMP efflux.

Intracellular cAMP (i-cAMP) levels play an important role in acute myeloid leukemia (AML) cell proliferation and differentiation. Its levels are the result of cAMP production, degradation, and exclusion. We have previously described histamine H2 receptors and MRP4/ABCC4 as two potential targets for AML therapy. Acting through histamine H2 receptors, histamine increases cAMP production/synthesis, while MRP4/ABCC4 is responsible for the exclusion of this cyclic nucleotide. In this study, we show that histamine treatment induces MRP4/ABCC4 expression, augmenting cAMP efflux, and that histamine, in combination with MRP inhibitors, is able to reduce AML cell proliferation. Histamine, through histamine H2 receptor, increases i-cAMP levels and induces MRP4 transcript and protein levels in U937, KG1a, and HL-60 cells. Moreover, histamine induces MRP4 promoter activity in HEK293T cells transfected with histamine H2 receptor (HEK293T-H2 R). Our results support that the cAMP/Epac-PKA pathway, and not MEK/ERK nor PI3K/AKT signaling cascades, is involved in histamine-mediated upregulation of MRP4 levels. Finally, the addition of histamine potentiates the inhibition of U937, KG1a, and HL-60 cell proliferation induced by MRP4 inhibitors. Our data highlight that the use of a poly-pharmacological approach aimed at different molecular targets would be beneficial in AML treatment.

Kinetic control of signaling: role of RGS proteins and GRKs

G protein‐coupled receptors interact with the GDP‐bound heterotrimeric G‐protein, G protein‐coupled receptor kinases (GRKs), and arrestins through their cytosolic surface. Previous studies suggest that the interaction of GRKs and the heterotrimer with the receptor occur in the same space; therefore, there is likely steric hindrance allowing only one pathway to operate at a time. Supporting this claim, experimentally induced competition for the intracellular heterotrimeric G‐protein binding site results in a reduction in GRK and Gα function. We created a Gαs chimera, called sis, containing most of the helical domain of Gαi1. In initial studies, sis prevented GRK2, 4, 5, and 6‐mediated reductions in isoproterenol‐mediated cAMP accumulation. Therefore, we tested the hypothesis that sis disrupts GRK/arrestin function due to an alteration in the turnover of the sis protein. To exclude any background effects of endogenous Gαs, we used GnasE2−/E2− MEFs re‐expressing Gαs (control) or sis (experimental) as well as the β2‐adrenergic receptor. Experimentally, cAMP accumulation in the presence and absence of receptor and GRK inhibitors, receptor‐Gs kinetics, and receptor internalization were measured. The introduction of the Gαi1 helical domain into Gαs did not significantly affect the biochemistry of the chimeric sis protein; however, GRK function and cAMP production altered. GRK2 failed to attenuate isoproterenol‐mediated cAMP accumulation in sis‐expressing cells, but attenuated cAMP accumulation in Gs‐expressing cells. Accordingly, the rate of endocytosis decreased in sis‐expressing cells compared to Gαs‐expressing cells at 10 μM, but not 0.1 and 1 μM, isoproterenol (0.77 ± 0.10 versus 1.00 ± 0.14 min−1). The kinetics of 10 μM isoproterenol‐mediated cAMP production demonstrates that the rate of accumulation of cAMP is similar, yet sis‐expressing cells have an increase in maximal response, and the rate of degradation of cAMP fits two different models. Degradation of cAMP in Gαs‐expressing cells is fit to a classical first‐order equation with a rate constant of 0.068 ± 0.012 luminescence/min, whereas, in sis‐expressing cells, the degradation of cAMP fit a linear zero‐order model with a slope of −0.012 ± 0.0012 luminescence/min. As there is 10 μM of isoproterenol present, we hypothesized that the receptor is nearly always active, and sis has altered kinetics and GRK activity due to rapid cycling between the cyclase and receptor. To test this second hypothesis, exploratory experiments are being conducted to determine if the alterations observed can be explained through a chimerically‐derived interaction with RGS proteins. The collected preliminary data suggest that altered kinetics of signaling alter GRK function and receptor internalization, and consequently, RGS proteins may indirectly inhibit GRK function.Support or Funding InformationWesternU MSPS program funds and NIH grants DK073911 (MB), DK079307 (EKJ), and HL109002 (EKJ) supported this work.

Role of Impaired CFTR Function in Pathogenesis of Pneumonia Among Alcohol Users

INTRODUCTIONImpairment of mucociliary clearance (MCC) increases the risk of respiratory infections among excessive alcohol users. The mechanisms underlying this impact of alcohol on MCC are not currently understood. Our previous in vitro studies indicated that alcohol decreases ion transport via CFTR channels, the defective protein that causes cystic fibrosis. Here, we have attempted to validate these findings in vivo and test the hypothesis that alcohol‐induced CFTR dysfunction reduces airway surface hydration and mucus transport thereby impairing airway defense against inhaled bacterial pathogens, in particular Klebsiella pneumoniae, to which alcohol users are disproportionately susceptible. Since CFTR dysfunction correlates with reduced PKA‐dependent CFTR phosphorylation and channel gating due to increased cAMP degradation by phosphodiesterase‐4B (PDE4B) enzymes, we also tested the effects of PDE4 inhibitor, roflumilast, on CFTR function and bacterial clearance in alcohol‐fed rats.METHODSAge and gender‐matched rats (CFTR+/+) were pair‐fed with Lieber‐DeCarli alcohol diet or isocaloric control diet for 4 weeks and with K. pneumoniae (KP, ATCC 43816). Subsets of rats were randomly treated with vehicle/roflumilast (5mg/kg/day) by oral gavage starting 7 days before the end of the alcohol diet. CFTR activity was measured with nasal potential difference (NPD) and MCC was assayed with radiographic clearance of Tc99. Airway surface liquid (ASL), periciliary layer (PCL), and ciliary beat frequency (CBF) of excised trachea were quantified with micro‐optical coherence tomography (μOCT).RESULTSAlcohol administration reduced CFTR ion transport by NPD. Clearance of Tc99 label in 30 min indicated delayed MCC in alcohol‐treated rats. Trachea excised from alcohol‐treated rats exhibited significantly decreased PCL depth, but no meaningful differences in ASL or CBF. Roflumilast treatment significantly increased CFTR activity in both control and alcohol‐administered rats. Increased CFTR activation by roflumilast resulted in augmented PCL depth, reduced mucus viscosity and significantly accelerated MCT rate. Compared to controls, histopathological examination of lung sections from alcohol‐treated animals exhibited more severe evidence of multifocal pneumonia with edema, fibrin deposition and numerous bacteria in alveolar spaces. Ongoing studies are determining the impact of roflumilast treatment on bacterial clearance and severity of pneumonia by K. pneumoniae.CONCLUSIONSAlcohol‐induced defects in CFTR ion transport resulted in reduced airway surface hydration and delayed mucus clearance in rat airways that was associated with diminished ability to clear bacterial pathogens. Acquired (non‐genetic) CFTR dysfunction may be a suitable pharmacologic target to improve mucus clearance impaired by chronic alcohol use. Administration of roflumilast may boost lung host defense against bacterial pathogens and improve pneumonia outcomes in individuals affected by excessive alcohol use.Support or Funding InformationNIH 1R01AA027528

Cold-Inducible RNA-Binding Protein Prevents an Excessive Heart Rate Response to Stress by Targeting Phosphodiesterase.

The stress response of heart rate, which is determined by the plasticity of the sinoatrial node (SAN), is essential for cardiac function and survival in mammals. As an RNA-binding protein, CIRP (cold-inducible RNA-binding protein) can act as a stress regulator. Previously, we have documented that CIRP regulates cardiac electrophysiology at posttranscriptional level, suggesting its role in SAN plasticity, especially upon stress conditions. Our aim was to clarify the role of CIRP in SAN plasticity and heart rate regulation under stress conditions. Telemetric ECG monitoring demonstrated an excessive acceleration of heart rate under isoprenaline stimulation in conscious CIRP-KO (knockout) rats. Patch-clamp analysis and confocal microscopic Ca2+ imaging of isolated SAN cells demonstrated that isoprenaline stimulation induced a faster spontaneous firing rate in CIRP-KO SAN cells than that in WT (wild type) SAN cells. A higher concentration of cAMP-the key mediator of pacemaker activity-was detected in CIRP-KO SAN tissues than in WT SAN tissues. RNA sequencing and quantitative real-time polymerase chain reaction analyses of single cells revealed that the 4B and 4D subtypes of PDE (phosphodiesterase), which controls cAMP degradation, were significantly decreased in CIRP-KO SAN cells. A PDE4 inhibitor (rolipram) abolished the difference in beating rate resulting from CIRP deficiency. The mechanistic study showed that CIRP stabilized the mRNA of Pde4b and Pde4d by direct mRNA binding, thereby regulating the protein expression of PDE4B and PDE4D at posttranscriptional level. CIRP acts as an mRNA stabilizer of specific PDEs to control the cAMP concentration in SAN, maintaining the appropriate heart rate stress response.

Airway Ciliary Beating Affected by the Pcp4 Dose-Dependent [Ca2+]i Increase in Down Syndrome Mice, Ts1Rhr

In Ts1Rhr, a Down syndrome model mouse, the airway ciliary beatings are impaired; that is, decreases in ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of ciliary beat amplitude)). A resumption to two copies of the Pcp4 gene on the Ts1Rhr trisomic segment (Ts1Rhr:Pcp4+/+/-) rescues the decreases in CBF and CBA that occur in Ts1Rhr. In airway cilia, upon stimulation with procaterol (a β2-agonist), the CBF increase is slower over the time course than the CBA increase because of cAMP degradation by Ca2+/calmodulin-dependent phosphodiesterase 1 (PDE1) existing in the metabolon regulating CBF. In Ts1Rhr, procaterol-stimulated CBF increase was much slower over the time course than in the wild-type mouse (Wt) or Ts1Rhr:Pcp4+/+/-. However, in the presence of 8MmIBMX (8-methoxymethyl isobutylmethyl xanthine, an inhibitor of PDE1) or calmidazolium (an inhibitor of calmodulin), in both Wt and Ts1Rhr, procaterol stimulates CBF and CBA increases over a similar time course. Measurements of cAMP revealed that the cAMP contents were lower in Ts1Rhr than in Wt or in Ts1Rhr:Pcp4+/+/-, suggesting the activation of PDE1A that is present in Ts1Rhr airway cilia. Measurements of the intracellular Ca2+ concentration ([Ca2+]i) in airway ciliary cells revealed that temperature (increasing from 25 to 37 °C) or 4αPDD (a selective transient receptor potential vanilloid 4 (TRPV4) agonist) stimulates a larger [Ca2+]i increase in Ts1Rhr than in Wt or Ts1Rhr:Pcp4+/+/-. In airway ciliary cells of Ts1Rhr, Pcp4-dose dependent activation of TRPV4 appears to induce an increase in the basal [Ca2+]i. In early embryonic day mice, a basal [Ca2+]i increased by PCP4 expressed may affect axonemal regulatory complexes regulated by the Ca2+-signal in Ts1Rhr, leading to a decrease in the basal CBF and CBA of airway cilia.