ABSTRACT
The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined.
Subject(s)
Autocrine Communication , Blood Pressure , Cyclic AMP , Oligopeptides , Signal Transduction , Animals , Humans , Mice , Cyclic AMP/metabolism , Oligopeptides/pharmacology , Oligopeptides/metabolism , Receptors, Neuropeptide/metabolism , Kidney Tubules, Proximal/metabolism , Male , Kidney/metabolism , Mice, Inbred C57BL , Receptors, Dopamine D1/metabolismABSTRACT
Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6ßrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.
Subject(s)
Disease Models, Animal , Drug Repositioning , Retinitis Pigmentosa , Animals , Mice , Dogs , Retinitis Pigmentosa/drug therapy , Retinitis Pigmentosa/genetics , Mutation , Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Mice, Knockout , Leber Congenital Amaurosis/drug therapy , Leber Congenital Amaurosis/genetics , Bromocriptine/pharmacology , Bromocriptine/therapeutic use , cis-trans-Isomerases/genetics , cis-trans-Isomerases/metabolism , Humans , Drug Therapy, Combination , Mice, Inbred C57BL , Retinal Cone Photoreceptor Cells/drug effects , Retinal Cone Photoreceptor Cells/metabolism , Retinal Cone Photoreceptor Cells/pathology , Female , Cyclic AMP/metabolism , Retinal Degeneration/drug therapy , Retinal Degeneration/genetics , Male , Calcium/metabolismABSTRACT
Platelet activation is critical for haemostasis, but if unregulated can lead to pathological thrombosis. Endogenous platelet inhibitory mechanisms are mediated by prostacyclin (PGI2)-stimulated cAMP signalling, which is regulated by phosphodiesterase 3A (PDE3A). However, spatiotemporal regulation of PDE3A activity in platelets is unknown. Here, we report that platelets possess multiple PDE3A isoforms with seemingly identical molecular weights (100 kDa). One isoform contained a unique N-terminal sequence that corresponded to PDE3A1 in nucleated cells but with negligible contribution to overall PDE3A activity. The predominant cytosolic PDE3A isoform did not possess the unique N-terminal sequence and accounted for >99% of basal PDE3A activity. PGI2 treatment induced a dose and time-dependent increase in PDE3A phosphorylation which was PKA-dependent and associated with an increase in phosphodiesterase enzymatic activity. The effects of PGI2 on PDE3A were modulated by A-kinase anchoring protein (AKAP) disruptor peptides, suggesting an AKAP-mediated PDE3A signalosome. We identified AKAP7, AKAP9, AKAP12, AKAP13, and moesin expressed in platelets but focussed on AKAP7 as a potential PDE3A binding partner. Using a combination of immunoprecipitation, proximity ligation techniques, and activity assays, we identified a novel PDE3A/PKA RII/AKAP7 signalosome in platelets that integrates propagation and termination of cAMP signalling through coupling of PKA and PDE3A.
Subject(s)
A Kinase Anchor Proteins , Blood Platelets , Cyclic AMP-Dependent Protein Kinases , Cyclic Nucleotide Phosphodiesterases, Type 3 , Epoprostenol , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 3/genetics , Blood Platelets/metabolism , Blood Platelets/drug effects , Humans , A Kinase Anchor Proteins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Epoprostenol/metabolism , Epoprostenol/pharmacology , Phosphorylation , Cyclic AMP/metabolism , Signal TransductionABSTRACT
The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.
Subject(s)
5'-Nucleotidase , Adenosine , Cell Proliferation , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , Phosphoric Diester Hydrolases , Pyrophosphatases , Signal Transduction , Phosphoric Diester Hydrolases/metabolism , Phosphoric Diester Hydrolases/genetics , Pyrophosphatases/metabolism , Pyrophosphatases/genetics , 5'-Nucleotidase/metabolism , 5'-Nucleotidase/genetics , Animals , Cell Proliferation/drug effects , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Adenosine/metabolism , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Myocytes, Smooth Muscle/drug effects , Mice , Humans , Adenosine Monophosphate/metabolism , Mice, Inbred C57BL , Cyclic AMP/metabolism , Male , Vascular Calcification/metabolism , Vascular Calcification/pathology , Vascular Calcification/geneticsABSTRACT
Introduction: Biphasic insulin secretion is an intrinsic characteristic of the pancreatic islet and has clinical relevance due to the loss of first-phase in patients with Type 2 diabetes. As it has long been shown that first-phase insulin secretion only occurs in response to rapid changes in glucose, we tested the hypothesis that islet response to an increase in glucose is a combination of metabolism plus an osmotic effect where hypertonicity is driving first-phase insulin secretion. Methods: Experiments were performed using perifusion analysis of rat, mouse, and human islets. Insulin secretion rate (ISR) and other parameters associated with its regulation were measured in response to combinations of D-glucose and membrane-impermeable carbohydrates (L-glucose or mannitol) designed to dissect the effect of hypertonicity from that of glucose metabolism. Results: Remarkably, the appearance of first-phase responses was wholly dependent on changes in tonicity: no first-phase in NAD(P)H, cytosolic calcium, cAMP secretion rate (cAMP SR), or ISR was observed when increased D-glucose concentration was counterbalanced by decreases in membrane-impermeable carbohydrates. When D-glucose was greater than 8 mM, rapid increases in L-glucose without any change in D-glucose resulted in first-phase responses in all measured parameters that were kinetically similar to D-glucose. First-phase ISR was completely abolished by H89 (a non-specific inhibitor of protein kinases) without affecting first-phase calcium response. Defining first-phase ISR as the difference between glucose-stimulated ISR with and without a change in hypertonicity, the peak of first-phase ISR occurred after second-phase ISR had reached steady state, consistent with the well-established glucose-dependency of mechanisms that potentiate glucose-stimulated ISR. Discussion: The data collected in this study suggests a new model of glucose-stimulated biphasic ISR where first-phase ISR derives from (and after) a transitory amplification of second-phase ISR and driven by hypertonicity-induced rise in H89-inhibitable kinases likely driven by first-phase responses in cAMP, calcium, or a combination of both.
Subject(s)
Glucose , Insulin Secretion , Insulin , Animals , Insulin Secretion/drug effects , Glucose/metabolism , Rats , Humans , Insulin/metabolism , Mice , Male , Islets of Langerhans/metabolism , Islets of Langerhans/drug effects , Cyclic AMP/metabolism , Calcium/metabolismABSTRACT
Cyclic nucleotide phosphodiesterases (PDEs) modulate neurohormonal regulation of cardiac function by degrading cAMP and cGMP. In cardiomyocytes, multiple isoforms of PDEs with different enzymatic properties and subcellular locally regulate cyclic nucleotide levels and associated cellular functions. This organisation is severely disrupted during hypertrophy and heart failure (HF), which may contribute to disease progression. Clinically, PDE inhibition has been seen as a promising approach to compensate for the catecholamine desensitisation that accompanies heart failure. Although PDE3 inhibitors such as milrinone or enoximone can be used clinically to improve systolic function and relieve the symptoms of acute CHF, their chronic use has proved detrimental. Other PDEs, such as PDE1, PDE2, PDE4, PDE5, PDE9 and PDE10, have emerged as potential new targets for the treatment of HF, each with a unique role in local cyclic nucleotide signalling pathways. In this review, we describe cAMP and cGMP signalling in cardiomyocytes and present the different families of PDEs expressed in the heart and their modifications in pathological cardiac hypertrophy and HF. We also review results from preclinical models and clinical data indicating the use of specific PDE inhibitors or activators that may have therapeutic potential in CI.
Title: Les phosphodiestérases des nucléotides cycliques - Cibles thérapeutiques dans l'hypertrophie et l'insuffisance cardiaques. Abstract: Les phosphodiestérases des nucléotides cycliques (PDE) modulent la régulation neuro-hormonale de la fonction cardiaque en dégradant l'AMPc et le GMPc. Dans les cardiomyocytes, de multiples isoformes de PDE, aux propriétés enzymatiques et aux localisations subcellulaires différentes, régulent localement les niveaux de nucléotides cycliques et les fonctions cellulaires associées. Cette organisation est fortement perturbée au cours de l'hypertrophie et de l'insuffisance cardiaque à fraction d'éjection réduite (IC), ce qui peut contribuer à la progression de la maladie. Sur le plan clinique, l'inhibition des PDE a été considérée comme une approche prometteuse pour compenser la désensibilisation aux catécholamines qui accompagne l'IC. Bien que des inhibiteurs de la PDE3, tels que la milrinone ou l'énoximone, puissent être utilisés cliniquement pour améliorer la fonction systolique et soulager les symptômes de l'IC aiguë, leur utilisation chronique s'est avérée préjudiciable. D'autres PDE, telles que les PDE1, PDE2, PDE4, PDE5, PDE9 et PDE10, sont apparues comme de nouvelles cibles potentielles pour le traitement de l'IC, chacune ayant un rôle unique dans les voies de signalisation locales des nucléotides cycliques. Dans cette revue, nous décrivons la signalisation de l'AMPc et du GMPc dans les cardiomyocytes et présentons les différentes familles de PDE exprimées dans le cÅur ainsi que leurs modifications dans l'hypertrophie cardiaque pathologique et dans l'IC. Nous évaluons également les résultats issus de modèles précliniques ainsi que les données cliniques indiquant l'utilisation d'inhibiteurs ou d'activateurs de PDE spécifiques qui pourraient avoir un potentiel thérapeutique dans l'IC.
Subject(s)
Cardiomegaly , Heart Failure , Phosphodiesterase Inhibitors , Humans , Cardiomegaly/drug therapy , Heart Failure/drug therapy , Animals , Phosphodiesterase Inhibitors/therapeutic use , Phosphodiesterase Inhibitors/pharmacology , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , 3',5'-Cyclic-AMP Phosphodiesterases/physiology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Molecular Targeted Therapy/methods , Cyclic GMP/metabolism , Cyclic GMP/physiology , Signal Transduction/drug effects , Signal Transduction/physiology , Cyclic AMP/metabolism , Cyclic AMP/physiology , Phosphoric Diester Hydrolases/metabolism , Phosphoric Diester Hydrolases/physiologyABSTRACT
INTRODUCTION: Creating induced pluripotent stem cells (iPSCs) from somatic cells of patients with genetic diseases offers a pathway to generate disease-specific iPSCs carrying genetic markers. Differentiating these iPSCs into renal tubular cells can aid in understanding the pathophysiology of rare inherited renal tubular diseases through cellular experiments. MATERIALS AND METHODS: Two Japanese patients with Pseudohypoparathyroidism (PHP), a 49-year-old woman and a 71-year-old man, were studied. iPSC-derived tubular cells were established from their peripheral blood mononuclear cells (PBMCs). We examined changes in intracellular and extracellular cyclic adenosine monophosphate (cAMP) levels in these cells in response to parathyroid hormone (PTH) stimulation. RESULTS: Renal tubular cells, differentiated from iPSCs of a healthy control (648A1), showed a PTH-dependent increase in both intracellular and extracellular cAMP levels. However, the renal tubular cells derived from the PHP patients' iPSCs showed inconsistent changes in cAMP levels upon PTH exposure. CONCLUSION: We successfully created disease-specific iPSCs from PHP patients' PBMCs, differentiated them into tubular cells, and replicated the distinctive response of the disease to PTH in vitro. This approach could enhance our understanding of the pathophysiology of inherited renal tubular diseases and contribute to developing effective treatments.
Subject(s)
Cell Differentiation , Cyclic AMP , Induced Pluripotent Stem Cells , Kidney Tubules , Leukocytes, Mononuclear , Parathyroid Hormone , Pseudohypoparathyroidism , Humans , Parathyroid Hormone/pharmacology , Parathyroid Hormone/metabolism , Induced Pluripotent Stem Cells/metabolism , Pseudohypoparathyroidism/genetics , Pseudohypoparathyroidism/metabolism , Female , Cell Differentiation/drug effects , Male , Cyclic AMP/metabolism , Kidney Tubules/metabolism , Kidney Tubules/pathology , Middle Aged , Aged , Leukocytes, Mononuclear/metabolism , Cells, CulturedABSTRACT
Estrogen excess in females has been linked to a diverse array of chronic and acute diseases. Emerging research shows that exposure to estrogen-like compounds such as bisphenol S leads to increases in 17ß-estradiol levels, but the mechanism of action is unclear. The aim of this study was to reveal the underlying signaling pathway-mediated mechanisms, target site and target molecule of action of bisphenol S causing excessive estrogen synthesis. Human ovarian granulosa cells SVOG were exposed to bisphenol S at environmentally relevant concentrations (1 µg/L, 10 µg/L, and 100 µg/L) for 48 h. The results confirms that bisphenol S accumulates mainly on the cell membrane, binds to follicle stimulating hormone receptor (FSHR) located on the cell membrane, and subsequently activates the downstream cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway, leading to enhanced conversion of testosterone to 17ß-estradiol. This study deepens our knowledge of the mechanisms of environmental factors in pathogenesis of hyperestrogenism.
Subject(s)
Cyclic AMP-Dependent Protein Kinases , Cyclic AMP , Estrogens , Phenols , Receptors, FSH , Signal Transduction , Sulfones , Phenols/toxicity , Cyclic AMP-Dependent Protein Kinases/metabolism , Humans , Cyclic AMP/metabolism , Signal Transduction/drug effects , Female , Estrogens/metabolism , Receptors, FSH/metabolism , Receptors, FSH/genetics , Sulfones/pharmacology , Estradiol/metabolism , Granulosa Cells/metabolism , Granulosa Cells/drug effectsABSTRACT
Neurotransmitters are key modulators in neuro-immune circuits and have been linked to tumor progression. Medullary thyroid cancer (MTC), an aggressive neuroendocrine tumor, expresses neurotransmitter calcitonin gene-related peptide (CGRP), is insensitive to chemo- and radiotherapies, and the effectiveness of immunotherapies remains unknown. Thus, a comprehensive analysis of the tumor microenvironment would facilitate effective therapies and provide evidence on CGRP's function outside the nervous system. Here, we compare the single-cell landscape of MTC and papillary thyroid cancer (PTC) and find that expression of CGRP in MTC is associated with dendritic cell (DC) abnormal development characterized by activation of cAMP related pathways and high levels of Kruppel Like Factor 2 (KLF2), correlated with an impaired activity of tumor infiltrating T cells. A CGRP receptor antagonist could offset CGRP detrimental impact on DC development in vitro. Our study provides insights of the MTC immunosuppressive microenvironment, and proposes CGRP receptor as a potential therapeutic target.
Subject(s)
Calcitonin Gene-Related Peptide , Carcinoma, Neuroendocrine , Dendritic Cells , Thyroid Neoplasms , Tumor Microenvironment , Tumor Microenvironment/immunology , Humans , Thyroid Neoplasms/genetics , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/immunology , Thyroid Neoplasms/pathology , Calcitonin Gene-Related Peptide/metabolism , Carcinoma, Neuroendocrine/genetics , Carcinoma, Neuroendocrine/metabolism , Carcinoma, Neuroendocrine/pathology , Carcinoma, Neuroendocrine/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Thyroid Cancer, Papillary/metabolism , Thyroid Cancer, Papillary/immunology , Thyroid Cancer, Papillary/genetics , Thyroid Cancer, Papillary/pathology , Receptors, Calcitonin Gene-Related Peptide/metabolism , Cyclic AMP/metabolism , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Neurotransmitter Agents/metabolism , Gene Expression Regulation, Neoplastic , Cell Line, Tumor , Calcitonin Gene-Related Peptide Receptor Antagonists/pharmacology , Single-Cell AnalysisABSTRACT
BACKGROUND: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 µg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h. RESULTS: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria. CONCLUSIONS: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.
Subject(s)
Acrosome Reaction , Acrosome , Calcium , Lead , Sperm Motility , Spermatozoa , Male , Spermatozoa/drug effects , Calcium/metabolism , Sperm Motility/drug effects , Animals , Acrosome/drug effects , Lead/toxicity , Acrosome Reaction/drug effects , Cyclic AMP/metabolism , Cattle , Membrane Potential, Mitochondrial/drug effects , Signal Transduction/drug effects , Semen Analysis , DNA Damage/drug effects , Organometallic Compounds/toxicity , Organometallic Compounds/pharmacologyABSTRACT
The presence of cyclic adenosine monophosphate (cAMP) has been considered to be a fundamental factor in ensuring meiotic arrest prior to ovulation. cAMP is regarded as a key molecule in the regulation of oocyte maturation. However, it has been reported that increased levels of intracellular cAMP can result in abnormal cytokinesis, with some MI oocytes leading to symmetrically cleaved 2-cell MII oocytes. Consequently, we aimed to investigate the effects of elevated intracellular cAMP levels on abnormal cytokinesis and oocyte maturation during the meiosis of mouse oocytes. This study found that a high concentration of isobutylmethylxanthine (IBMX) also caused chromatin/chromosomes aggregation (AC) after the first meiosis. The rates of AC increased the greater the concentration of IBMX. In addition, AC formation was found to be reversible, showing that the re-formation of the spindle chromosome complex was possible after the IBMX was removed. In human oocytes, the chromosomes aggregate after the germinal vesicle breakdown and following the first and second polar body extrusions (the AC phase), while mouse oocytes do not have this AC phase. The results of our current study may indicate that the AC phase in human oocytes could be related to elevated levels of intracytoplasmic cAMP.
Subject(s)
1-Methyl-3-isobutylxanthine , Chromatin , Oocytes , Animals , Oocytes/metabolism , Female , Chromatin/metabolism , 1-Methyl-3-isobutylxanthine/pharmacology , Mice , Humans , Meiosis/drug effects , Cyclic AMP/metabolism , Phosphodiesterase Inhibitors/pharmacologyABSTRACT
BACKGROUND: Bile acids (BAs) concentration can affect metabolic improvement caused by bariatric surgery and BA concentrations increase in patients after sleeve gastrectomy (SG). Here, how BAs after SG affect metabolism in nonalcoholic fatty liver disease (NAFLD) was studied. METHODS: Mice were given high-fat diet (HFD) to induce NAFLD and received SG surgery. Hepatic and fecal BA concentrations in mice were detected by liquid chromatography-tandem mass spectrometry method. BA-related genes were detected by quantitative real-time polymerase chain reaction. G protein BA receptor 1 (GPBAR1) expression was identified using western blot analysis. NAFLD mice after SG received GPBAR1 inhibitor Triamterene. The weight of mice and mice liver was detected. Mouse liver tissue was observed by hematoxylin-eosin and Oil Red O staining. Triglyceride (TG), nonesterified fatty acid (NEFA), and cyclic adenosine monophosphate (cAMP) levels in mouse liver tissue were analyzed by metabolic assay and enzyme-linked immune sorbent assay. RESULTS: SG boosted increase in hepatic total/conjugated BAs and related genes and GPBAR1 expression, and attenuated increase in fecal total BAs/muricholic acid in HFD-induced mice and increased fecal taurine-BAs in HFD-induced mice. Triamterene (72 mg/kg) reversed the inhibitory role of SG in HFD-induced increase of body weight, lipid accumulation, inflammatory cell infiltration, and increase of hepatic weight and TG/NEFA content, and counteracted the positive role of SG in HFD-induced increase of hepatic cAMP concentration in mice. CONCLUSIONS: BAs improve metabolism via activating GPBAR1 to increase cAMP in NAFLD mice after SG.
Subject(s)
Bile Acids and Salts , Cyclic AMP , Gastrectomy , Non-alcoholic Fatty Liver Disease , Receptors, G-Protein-Coupled , Animals , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/genetics , Mice , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/surgery , Non-alcoholic Fatty Liver Disease/pathology , Bile Acids and Salts/metabolism , Cyclic AMP/metabolism , Male , Diet, High-Fat/adverse effects , Mice, Inbred C57BL , Liver/metabolism , Liver/surgery , Liver/pathology , Disease Models, AnimalABSTRACT
Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.
Subject(s)
Cyclic AMP , Guanine Nucleotide Exchange Factors , Sumoylation , Ubiquitin-Conjugating Enzymes , rap1 GTP-Binding Proteins , Guanine Nucleotide Exchange Factors/metabolism , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/chemistry , Humans , Cyclic AMP/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/genetics , rap1 GTP-Binding Proteins/metabolism , rap1 GTP-Binding Proteins/genetics , HEK293 Cells , Molecular Dynamics Simulation , Shelterin Complex/metabolism , Signal Transduction , Telomere-Binding Proteins/metabolism , rap GTP-Binding Proteins/metabolism , rap GTP-Binding Proteins/genetics , Heat-Shock Response , Amino Acid Sequence , Protein BindingABSTRACT
Most α2-AR agonists derived from dexmedetomidine have few structural differences between them and have no selectivity for α2A/2B-AR or Gi/Gs, which can lead to side effects in drugs. To obtain novel and potent α2A-AR agonists, we performed virtual screening for human α2A-AR and α2B-AR to find α2A-AR agonists with higher selectivity. Compound P300-2342 and its three analogs significantly decreased the locomotor activity of mice (p < 0.05). Furthermore, P300-2342 and its three analogs inhibited the binding of [3H] Rauwolscine with IC50 values of 7.72 ± 0.76 and 12.23 ± 0.11 µM, respectively, to α2A-AR and α2B-AR. In α2A-AR-HEK293 cells, P300-2342 decreased forskolin-stimulated cAMP production without increasing cAMP production, which indicated that P300-2342 activated α2A-AR with coupling to the Gαi/o pathway but without Gαs coupling. P300-2342 exhibited no agonist but slight antagonist activities in α2B-AR. Similar results were obtained for the analogs of P300-2342. The docking results showed that P300-2342 formed π-hydrogen bonds with Y394, V114 in α2A-AR, and V93 in α2B-AR. Three analogs of P300-2342 formed several π-hydrogen bonds with V114, Y196, F390 in α2A-AR, and V93 in α2B-AR. We believe that these molecules can serve as leads for the further optimization of α2A-AR agonists with potentially few side effects.
Subject(s)
Adrenergic alpha-2 Receptor Agonists , Molecular Docking Simulation , Receptors, Adrenergic, alpha-2 , Humans , Animals , HEK293 Cells , Receptors, Adrenergic, alpha-2/metabolism , Adrenergic alpha-2 Receptor Agonists/pharmacology , Mice , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Cyclic AMP/metabolism , Male , Protein BindingABSTRACT
Protein kinase A (PKA) is a ubiquitous cAMP-dependent enzyme in mammalian tissues. The inactive PKA holoenzyme disassociates into a homodimer of regulatory (R) subunits and two active catalytic (C) subunits upon cAMP binding to two tandem domains (termed CBD-A and CBD-B) in R subunits. The release of cAMP facilitates reassociation of R and C subunits, resetting PKA to its basal state. The cAMP-mediated structural changes in the activation-termination cycle remain partially understood. The multimeric states of PKA complicate the issue and are particularly less studied. Therefore, we computationally investigated the conformational dynamics of the PKA RIα homodimer in different cAMP-bound states. The absence of cAMP in two CBDs differently affects the conformational dynamics of protomers. Moreover, such disparate responses are extended to the dimer interface constituted by the N-terminal helical sub-domains termed N3A motifs. The removal of cAMP from CBD-A induces large-scale structural changes of individual R subunits towards the holoenzyme state, consistent with previous simulations of a single R subunit. Meanwhile it keeps the structural heterogeneity of the N3A-N3A' dimer interface observed in the fully bound state. By contrast, the removal of cAMP from CBD-B does not affect individual R subunits but alters the conformational space of the N3A-N3A' dimer interface. The cAMP-coupled structural changes of each protomer and conserved conformational space of the N3A-N3A' dimer interface are essential for the transition between the fully cAMP-bound R2 homodimer and the R2C2 holoenzyme as suggested by their crystal structures. Our work provides structural insights into the regulatory mechanism of cAMP in PKA signaling.
Subject(s)
Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Cyclic AMP , Protein Multimerization , Cyclic AMP/metabolism , Cyclic AMP/chemistry , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/metabolism , Molecular Dynamics Simulation , Protein ConformationABSTRACT
Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through primary cilia (PC) that grow and retract with the cell cycle and in response to extracellular cues. Disruption of PC homeostasis corrupts SHH regulation, placing significant pressure on the pathway to maintain ciliary fitness. Mechanisms by which ciliary robustness is ensured in SHH-stimulated cells are not yet known. Herein, we reveal a crosstalk circuit induced by SHH activation of Phospholipase A2α that drives ciliary E-type prostanoid receptor 4 (EP4) signaling to ensure PC function and stabilize ciliary length. We demonstrate that blockade of SHH-EP4 crosstalk destabilizes PC cyclic AMP (cAMP) equilibrium, slows ciliary transport, reduces ciliary length, and attenuates SHH pathway induction. Accordingly, Ep4-/- mice display shortened neuroepithelial PC and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates coordination between distinct ciliary receptors to maintain PC function and length homeostasis for robust downstream signaling.
Subject(s)
Cilia , Hedgehog Proteins , Prostaglandins , Signal Transduction , Animals , Mice , Cilia/metabolism , Cyclic AMP/metabolism , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Mice, Knockout , Prostaglandins/metabolism , Receptors, Prostaglandin E, EP4 Subtype/metabolism , Receptors, Prostaglandin E, EP4 Subtype/geneticsABSTRACT
BACKGROUND: This study investigated the relaxation effect of PGE2 on the ureter and its role in promoting calculi expulsion following calculi development. METHODS: By using immunofluorescence and Western blot, we were able to locate EP receptors in the ureter. In vitro experiments assessed the impact of PGE2, receptor antagonists, and agonists on ureteral relaxation rate. We constructed a model of ureteral calculi with flowable resin and collected ureteral tissue from postoperative side of the ureter after obstruction surgery. Western blot analysis was used to determine the protein expression levels of EP receptors and the PGE2 terminal synthase mPGES-1. Additionally, PGE2 was added to smooth muscle cells to observe downstream cAMP and PKA changes. RESULTS: The expression of EP2 and EP4 proteins in ureteral smooth muscle was verified by Western blot analysis. According to immunofluorescence, EP2 was primarily found on the cell membrane, while EP4 was found in the nucleus. In vitro, PGE2 induced concentration-dependent ureteral relaxation. Maximum diastolic rate was 70.94 ± 4.57% at a concentration of 30µM. EP2 antagonists hindered this effect, while EP4 antagonists did not. Obstructed ureters exhibited elevated mPGES-1 and EP2 protein expression (P < 0.01). Smooth muscle cells treated with PGE2 displayed increased cAMP and phosphorylated PKA. CONCLUSIONS: PGE2 binding to EP2 induces ureteral relaxation through the cAMP-PKA pathway. This will provide a new theoretical basis for the development of new therapeutic approaches for the use of PGE2 in the treatment of ureteral stones.
Subject(s)
Cyclic AMP-Dependent Protein Kinases , Cyclic AMP , Dinoprostone , Receptors, Prostaglandin E, EP2 Subtype , Ureter , Ureteral Calculi , Receptors, Prostaglandin E, EP2 Subtype/metabolism , Cyclic AMP/metabolism , Dinoprostone/metabolism , Dinoprostone/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Animals , Ureter/metabolism , Signal Transduction/physiology , Male , Muscle Relaxation/drug effects , Muscle Relaxation/physiologyABSTRACT
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are opened in an allosteric manner by membrane hyperpolarization and cyclic nucleotides such as cAMP. Because of conflicting reports from experimental studies on whether cAMP binding to the four available binding sites in the channel tetramer operates cooperatively in gating, we employ here a computational approach as a promising route to examine ligand-induced conformational changes after binding to individual sites. By combining an elastic network model (ENM) with linear response theory (LRT) for modeling the apo-holo transition of the cyclic nucleotide-binding domain (CNBD) in HCN channels, we observe a distinct pattern of cooperativity matching the "positive-negative-positive" cooperativity reported from functional studies. This cooperativity pattern is highly conserved among HCN subtypes (HCN4, HCN1), but only to a lesser extent visible in structurally related channels, which are only gated by voltage (KAT1) or cyclic nucleotides (TAX4). This suggests an inherent cooperativity between subunits in HCN channels as part of a ligand-triggered gating mechanism in these channels.
Subject(s)
Cyclic AMP , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Ion Channel Gating , Models, Molecular , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/chemistry , Cyclic AMP/metabolism , Anisotropy , Protein Subunits/metabolism , Protein Subunits/chemistry , Protein Conformation , Humans , Potassium Channels/metabolism , Potassium Channels/chemistry , Binding SitesABSTRACT
Cordycepin, or 3'-deoxyadenosine, is an adenosine analog with a broad spectrum of biological activity. The key structural difference between cordycepin and adenosine lies in the absence of a hydroxyl group at the 3' position of the ribose ring. Upon administration, cordycepin can undergo an enzymatic transformation in specific tissues, forming cordycepin triphosphate. In this study, we conducted a comprehensive analysis of the structural features of cordycepin and its derivatives, contrasting them with endogenous purine-based metabolites using chemoinformatics and bioinformatics tools in addition to molecular dynamics simulations. We tested the hypothesis that cordycepin triphosphate could bind to the active site of the adenylate cyclase enzyme. The outcomes of our molecular dynamics simulations revealed scores that are comparable to, and superior to, those of adenosine triphosphate (ATP), the endogenous ligand. This interaction could reduce the production of cyclic adenosine monophosphate (cAMP) by acting as a pseudo-ATP that lacks a hydroxyl group at the 3' position, essential to carry out nucleotide cyclization. We discuss the implications in the context of the plasticity of cancer and other cells within the tumor microenvironment, such as cancer-associated fibroblast, endothelial, and immune cells. This interaction could awaken antitumor immunity by preventing phenotypic changes in the immune cells driven by sustained cAMP signaling. The last could be an unreported molecular mechanism that helps to explain more details about cordycepin's mechanism of action.
Subject(s)
Cyclic AMP , Deoxyadenosines , Molecular Dynamics Simulation , Neoplasms , Deoxyadenosines/metabolism , Deoxyadenosines/pharmacology , Deoxyadenosines/chemistry , Humans , Neoplasms/metabolism , Neoplasms/drug therapy , Neoplasms/pathology , Cyclic AMP/metabolism , Adenosine Triphosphate/metabolism , Signal Transduction/drug effects , Computer Simulation , Adenylyl Cyclases/metabolismABSTRACT
Cytoplasmic mislocalization and aggregation of TDP-43 protein are hallmarks of amyotrophic lateral sclerosis (ALS) and are observed in the vast majority of both familial and sporadic cases. How these two interconnected processes are regulated on a molecular level, however, remains enigmatic. Genome-wide screens for modifiers of the ALS-associated genes TDP-43 and FUS have identified the phospholipase D (Pld) pathway as a key regulator of ALS-related phenotypes in the fruit fly Drosophila melanogaster [M. W. Kankel et al., Genetics 215, 747-766 (2020)]. Here, we report the results of our search for downstream targets of the enzymatic product of Pld, phosphatidic acid. We identify two conserved negative regulators of the cAMP/PKA signaling pathway, the phosphodiesterase dunce and the inhibitory subunit PKA-R2, as modifiers of pathogenic phenotypes resulting from overexpression of the Drosophila TDP-43 ortholog TBPH. We show that knockdown of either of these genes results in a mitigation of both TBPH aggregation and mislocalization in larval motor neuron cell bodies, as well as an amelioration of adult-onset motor defects and shortened lifespan induced by TBPH. We determine that PKA kinase activity is downstream of both TBPH and Pld and that overexpression of the PKA target CrebA can rescue TBPH mislocalization. These findings suggest a model whereby increasing cAMP/PKA signaling can ameliorate the molecular and functional effects of pathological TDP-43.
