. Effect of fluoxetine on the inhibition of adenylate cyclase activity in foskolin-stimulated MLTC-1 leydig cells

Fluoxetine (FLX), a widely used antidepressant primarily acting as a selective serotonin reuptake inhibitor, has been shown to exhibit other mechanisms of action in various cell types. Cyclic adenosine monophosphate (cAMP) is a second messenger used for intracellular signal induction. Cyclic AMP is a nucleotide synthesized within the cell from adenosine triphosphate by the adenylyl cyclase enzyme, and is inactivated enzymatically to 5′AMP by hydroxylation with a group of enzymes called phosphodiesterase. The aim of this study was to determine the effects of FLX on MLTC-1 Leydig cells on intracellular cyclic AMP response to forskolin (FSK). MLTC-1 cells were incubated at 37°C in media supplemented with or without different doses of FLX (0, 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 µM). We then looked for how the concentration of FLX for a short-time (2 hours) and a long-time (24 hours) affects the concentration of intracellular cyclic AMP response to FSK and ATP levels on MLTC-1 cells. Our results show that FLX decreased the intracellular cAMP response to FSK depending on FLX concentration. FLX decreased significantly cAMP levels only at 10 µM after 2 hours of incubation but after 24 hours of incubation FLX caused an effect on cAMP levels at 5 µM and at 10 µM. Moreover, as expected, FLX also caused a decline of steroidogenesis, which is under the control of cAMP and ATP levels in the cells. Taken together, these findings demonstrate that the inhibition of cAMP synthesis by FLX is dose-dependent, and that FLX also inhibited hormone-induced steroidogenesis in MLTC-1 cells.

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Anti-Allergic and Anti-Inflammatory Effects of Undecane on Mast Cells and Keratinocytes

Molecules ◽

10.3390/molecules25071554 ◽

2020 ◽

Vol 25 (7) ◽

pp. 1554

Author(s):

Dabin Choi ◽

Wesuk Kang ◽

Taesun Park

Keyword(s):

Mast Cells ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Allergic Diseases ◽

Intracellular Camp ◽

Tnf Α ◽

Anti Inflammatory ◽

Skin Conditions ◽

Factor Α ◽

Camp Levels

The critical roles of keratinocytes and resident mast cells in skin allergy and inflammation have been highlighted in many studies. Cyclic adenosine monophosphate (cAMP), the intracellular second messenger, has also recently emerged as a target molecule in the immune reaction underlying inflammatory skin conditions. Here, we investigated whether undecane, a naturally occurring plant compound, has anti-allergic and anti-inflammatory activities on sensitized rat basophilic leukemia (RBL-2H3) mast cells and HaCaT keratinocytes and we further explored the potential involvement of the cAMP as a molecular target for undecane. We confirmed that undecane increased intracellular cAMP levels in mast cells and keratinocytes. In sensitized mast cells, undecane inhibited degranulation and the secretion of histamine and tumor necrosis factor α (TNF-α). In addition, in sensitized keratinocytes, undecane reversed the increased levels of p38 phosphorylation, nuclear factor kappaB (NF-κB) transcriptional activity and target cytokine/chemokine genes, including thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and interleukin-8 (IL-8). These results suggest that undecane may be useful for the prevention or treatment of skin inflammatory disorders, such as atopic dermatitis, and other allergic diseases.

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Genetic Polymorphisms of the ß2-Adrenergic Receptor Relate to Guanosine Protein-coupled Stimulator Receptor Dysfunction in Fibromyalgia Syndrome

The Journal of Rheumatology ◽

10.3899/jrheum.101104 ◽

2011 ◽

Vol 38 (6) ◽

pp. 1095-1103 ◽

Cited By ~ 17

Author(s):

YANGMING XIAO ◽

WEIJING HE ◽

I. JON RUSSELL

Keyword(s):

Adrenergic Receptor ◽

Fibromyalgia Syndrome ◽

Mononuclear Cells ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Intracellular Camp ◽

Peripheral Blood Mononuclear ◽

Genotype Frequencies ◽

Camp Levels

Objective.To determine the genotype frequencies of ß2-adrenergic receptor (ß2AR) gene polymorphisms (Gly16Arg, Glu27Gln) in patients with fibromyalgia syndrome (FM) by comparison with unrelated healthy controls. We sought any clinical association with these polymorphisms and determined whether the polymorphisms would associate with a biologic guanosine protein-coupled stimulator receptor (Gs) dysfunction in FM.Methods.Study subjects included 97 clinically characterized patients with FM and 59 controls. The ß2AR polymorphisms at codons 16 and 27 were determined using polymerase chain reaction-restriction fragment length polymorphism. The Gs functions of peripheral blood mononuclear cells (PBMC) were tested using isoproterenol (ISO) as the adrenergic Gs ligand and measuring intracellular cyclic adenosine monophosphate (cAMP) levels.Results.The frequency of the ß2AR gene polymorphism Gly16Arg in FM (43.5%) was significantly lower than in controls (63.2%), suggesting that this genotype might have some effect on the risk of developing FM. The only clinical association in FM was with sleep dysfunction. Patients with FM who carried the ß2AR polymorphism Arg16Arg also exhibited significantly lower PBMC basal cAMP levels (p < 0.05) and lower ISO-stimulated cAMP levels (p < 0.05) than FM carrying Gly16Gly or Gly16Arg.Conclusion.This confirms a relationship between ß2AR polymorphism and FM. It is the first study to demonstrate ß2AR polymorphism-related differences in intracellular cAMP responses of FM PBMC after ß2AR stimulationin vitro. These findings may explain some of the differences in responsiveness of FM subgroups to the adrenergic agonist medications currently approved for FM treatment.

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Primary Cilia Mediate Intracellular cAMP Responses in Bone Cells Exposed to Dynamic Fluid Flow

ASME 2008 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2008-192511 ◽

2008 ◽

Cited By ~ 1

Author(s):

Ronald Y. Kwon ◽

Sara Temiyasathit ◽

Padmaja Tummala ◽

Clarence Quah ◽

Christopher R. Jacobs

Keyword(s):

Fluid Flow ◽

Primary Cilia ◽

Bone Structure ◽

Bone Cells ◽

Cyclic Adenosine Monophosphate ◽

Second Messenger ◽

Adenosine Monophosphate ◽

Cell Types ◽

Intracellular Camp ◽

And Function

It is well accepted that fluid flow is an important mechanical signal in regulating bone structure and function. Primary cilia, which are non-motile, microtubule based organelles that extend from the centrosome and project into extracellular space in many cell types, have recently been shown to mediate fluid flow-induced osteogenic responses in MLO-Y4 osteocyte-like cells [1]. However, primary cilia did not mediate increases in intracellular Ca2+ concentration, and the second messenger system(s) involved in primary cilia-mediated mechanosensing has yet to be elucidated. In this study, our goals were to (1) determine whether exposing bone cells to oscillatory fluid flow modulates intracellular levels of cyclic adenosine monophosphate (cAMP), another ubiquitous second messenger molecule, and (2) investigate whether this modulation may be mediated by primary cilia.

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Shockwaves Suppress Adipocyte Differentiation via Decrease in PPARγ

Cells ◽

10.3390/cells9010166 ◽

2020 ◽

Vol 9 (1) ◽

pp. 166

Author(s):

Wonkyoung Cho ◽

SeoYeon Kim ◽

Myeongsook Jeong ◽

Young Mi Park

Keyword(s):

Adipocyte Differentiation ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Intracellular Camp ◽

Peroxisome Proliferator ◽

Mechanical Stimuli ◽

Western Blots ◽

Peroxisome Proliferator Activated Receptor ◽

Pparγ Expression ◽

Camp Levels

Adipogenesis is a crucial cellular process that contributes to the expansion of adipose tissue in obesity. Shockwaves are mechanical stimuli that transmit signals to cause biological responses. The purpose of this study is to evaluate the effects of shockwaves on adipogenesis. We treated 3T3L-1 cells and human primary preadipocytes for differentiation with or without shockwaves. Western blots and quantitative real-time reverse transcriptase PCR (qRT-PCR) for adipocyte markers including peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding proteins (C/EBPα) were performed. Extracellular adenosine triphosphate (ATP) and intracellular cyclic adenosine monophosphate (cAMP) levels, which are known to affect adipocyte differentiation, were measured. Shockwave treatment decreased intracellular lipid droplet accumulation in primary human preadipocytes and 3T3-L1 cells after 11–12 days of differentiation. Levels of key adipogenic transcriptional factors PPARγ and/or C/EBPα were lower in shockwave-treated human primary preadipocytes and 3T3L-1 cells after 12–13 days of differentiation than in shockwave-untreated cells. Shockwave treatment induced release of extracellular ATP from preadipocytes and decreased intracellular cAMP levels. Shockwave-treated preadipocytes showed a higher level of β-catenin and less PPARγ expression than shockwave-untreated cells. Supplementation with 8-bromo-cAMP analog after shockwave treatment rescued adipocyte differentiation by preventing the effect of shockwaves on β-catenin, Wnt10b mRNA, and PPARγ expression. Low-energy shockwaves suppressed adipocyte differentiation by decreasing PPARγ. Our study suggests an insight into potential uses of shockwave-treatment for obesity.

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Cyclic AMP-binding protein Epac1 acts as a metabolic sensor to promote cardiomyocyte lipotoxicity

Cell Death and Disease ◽

10.1038/s41419-021-04113-9 ◽

2021 ◽

Vol 12 (9) ◽

Author(s):

Marion Laudette ◽

Yannis Sainte-Marie ◽

Grégoire Cousin ◽

Dorian Bergonnier ◽

Ismahane Belhabib ◽

...

Keyword(s):

Cyclic Amp ◽

Mitochondrial Dysfunction ◽

Cyclic Adenosine Monophosphate ◽

Molecular Complexes ◽

Adenosine Monophosphate ◽

Intracellular Camp ◽

Mitochondrial Enzymes ◽

Amino Acid Residues ◽

Α Subunit ◽

Key Enzyme

AbstractCyclic adenosine monophosphate (cAMP) is a master regulator of mitochondrial metabolism but its precise mechanism of action yet remains unclear. Here, we found that a dietary saturated fatty acid (FA), palmitate increased intracellular cAMP synthesis through the palmitoylation of soluble adenylyl cyclase in cardiomyocytes. cAMP further induced exchange protein directly activated by cyclic AMP 1 (Epac1) activation, which was upregulated in the myocardium of obese patients. Epac1 enhanced the activity of a key enzyme regulating mitochondrial FA uptake, carnitine palmitoyltransferase 1. Consistently, pharmacological or genetic Epac1 inhibition prevented lipid overload, increased FA oxidation (FAO), and protected against mitochondrial dysfunction in cardiomyocytes. In addition, analysis of Epac1 phosphoproteome led us to identify two key mitochondrial enzymes of the the β-oxidation cycle as targets of Epac1, the long-chain FA acyl-CoA dehydrogenase (ACADL) and the 3-ketoacyl-CoA thiolase (3-KAT). Epac1 formed molecular complexes with the Ca2+/calmodulin-dependent protein kinase II (CaMKII), which phosphorylated ACADL and 3-KAT at specific amino acid residues to decrease lipid oxidation. The Epac1-CaMKII axis also interacted with the α subunit of ATP synthase, thereby further impairing mitochondrial energetics. Altogether, these findings indicate that Epac1 disrupts the balance between mitochondrial FA uptake and oxidation leading to lipid accumulation and mitochondrial dysfunction, and ultimately cardiomyocyte death.

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PACmn for improved optogenetic control of intracellular cAMP

BMC Biology ◽

10.1186/s12915-021-01151-9 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Shang Yang ◽

Oana M. Constantin ◽

Divya Sachidanandan ◽

Hannes Hofmann ◽

Tobias C. Kunz ◽

...

Keyword(s):

Plasma Membrane ◽

Signaling Pathways ◽

Hippocampal Neurons ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Cell Types ◽

Camp Signaling ◽

Intracellular Camp ◽

Extracellular Signals ◽

Starting Point

Abstract Background Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that transduces extracellular signals in virtually all eukaryotic cells. The soluble Beggiatoa photoactivatable adenylyl cyclase (bPAC) rapidly raises cAMP in blue light and has been used to study cAMP signaling pathways cell-autonomously. But low activity in the dark might raise resting cAMP in cells expressing bPAC, and most eukaryotic cyclases are membrane-targeted rather than soluble. Our aim was to engineer a plasma membrane-anchored PAC with no dark activity (i.e., no cAMP accumulation in the dark) that rapidly increases cAMP when illuminated. Results Using a streamlined method based on expression in Xenopus oocytes, we compared natural PACs and confirmed bPAC as the best starting point for protein engineering efforts. We identified several modifications that reduce bPAC dark activity. Mutating a phenylalanine to tyrosine at residue 198 substantially decreased dark cyclase activity, which increased 7000-fold when illuminated. Whereas Drosophila larvae expressing bPAC in mechanosensory neurons show nocifensive-like behavior even in the dark, larvae expressing improved soluble (e.g., bPAC(R278A)) and membrane-anchored PACs exhibited nocifensive responses only when illuminated. The plasma membrane-anchored PAC (PACmn) had an undetectable dark activity which increased >4000-fold in the light. PACmn does not raise resting cAMP nor, when expressed in hippocampal neurons, affect cAMP-dependent kinase (PKA) activity in the dark, but rapidly and reversibly increases cAMP and PKA activity in the soma and dendrites upon illumination. The peak responses to brief (2 s) light flashes exceed the responses to forskolin-induced activation of endogenous cyclases and return to baseline within seconds (cAMP) or ~10 min (PKA). Conclusions PACmn is a valuable optogenetic tool for precise cell-autonomous and transient stimulation of cAMP signaling pathways in diverse cell types.

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ID: 1039 The antidepressant fluoxetine inhibits adenylate cyclase stimulation by FSH or Forskolin in the COV434 human ovarian granulosa tumor cell line

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.315 ◽

2017 ◽

Vol 4 (S) ◽

pp. 117

Author(s):

Thi Mong Diep Nguyen ◽

Danièle Klett ◽

Minh Thu Vo ◽

Yves Combarnous

Keyword(s):

Signaling Pathways ◽

Granulosa Cells ◽

Molecular Mechanisms ◽

Follicular Development ◽

Tumor Cell Line ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Cell Types ◽

Intracellular Camp ◽

Human Granulosa Cells

Fluoxetine (Prozac), a selective Serotonin Reuptake Inhibitor antidepressant, exhibits other mechanisms of action in various cell types and has been shown to induce cell death in cancer cells, paving the way for its potential use in cancer therapy. The ovary is a complex endocrine organ responsible for steroidogenesis and folliculogenesis, and human granulosa cells are essential for scientific research to improve the understanding of these two processes. However, little is known about fundamental signaling pathways in human granulosa cells. In this study, we investigated the dynamics of intracellular cyclic adenosine monophosphate AMP, a conserved signaling messenger that can regulate virtually every physiological process. We show that incubating COV434 human ovarian granulosa cells with fluoxetine induces a decrease in intracellular cAMP response to Follicle-stimulating hormone (FSH) and forskolin (FSK). In order to study the intracellular cAMP kinetic responses of COV434 cells to FSH or FSK, we used COV434 cells transiently expressing a chimeric cAMP-responsive luciferase so that real-time variations of intracellular cAMP concentration could be monitored, by using oxiluciferin luminescence produced from catalyzed luciferin oxidation. Our data show that fluoxetine induces an increase in the extracellular Ca2+ entry and reduces ATP concentration as well as cell viability. Targeting these signaling pathways with fluoxetine could permit to get better knowledge in the molecular mechanisms involved in ovarian follicular development

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Caffeine in the Treatment of Atopic Dermatitis and Psoriasis: A Review

SKIN The Journal of Cutaneous Medicine ◽

10.25251/skin.3.2.38 ◽

2019 ◽

Vol 3 (2) ◽

pp. 59-71 ◽

Cited By ~ 1

Author(s):

Mais Bassam Alashqar

Keyword(s):

Atopic Dermatitis ◽

Skin Diseases ◽

Kinase Inhibitor ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Intracellular Camp ◽

Ataxia Telangiectasia Mutated ◽

Inflammatory Skin Diseases ◽

Camp Levels ◽

Effective Adjunct

Atopic dermatitis (AD) and psoriasis are inflammatory skin diseases. AD is characterized by immune dysregulation and barrier impairment, while psoriasis is by immune dysfunction and resultant keratinocyte hyper-proliferation. Caffeine has shown effective in ameliorating the symptoms of both diseases, but it is not conclusive through which pathways. The aim of this study was to provide a detailed discussion of available work on this topic, as well as known modes of action of caffeine that are relevant to these two conditions. After an extensive review of the literature, we found that both diseases have decreased intracellular cyclic adenosine monophosphate (cAMP) levels in cutaneous leukocytes, so it is very likely that being a methylxanthine, and hence a phosphodiesterase (PDE) inhibitor, caffeine raises intracellular cAMP levels, which suppresses inflammatory pathways and potentiates anti-inflammatory ones. Moreover, caffeine is known to be an ATR (ataxia-telangiectasia mutated) kinase and an ATM (ATM- and Rad3-Related) kinase inhibitor, which promotes prompt apoptosis of damaged cells. It was also found to have anti-necrotic effects in reactive oxygen species (ROS)-damaged cells. These pro-apoptotic and anti-necrotic properties may also be reducing the inflammation. Finally, caffeine's metabolites have shown antioxidising effects against ROS, which certainly would reduce inflammation caused by lipid peroxidation, DNA damage and organelle destruction. We find that caffeine acts in a number of ways to improve symptoms of inflammation and that it is an effective adjunct to therapy in AD and psoriasis.

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Upregulation of cAMP prevents antibody-mediated thrombus formation in COVID-19

Blood Advances ◽

10.1182/bloodadvances.2021005210 ◽

2022 ◽

Vol 6 (1) ◽

pp. 248-258

Author(s):

Jan Zlamal ◽

Karina Althaus ◽

Hisham Jaffal ◽

Helene Häberle ◽

Lisann Pelzl ◽

...

Keyword(s):

Time Course ◽

Thrombus Formation ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Intracellular Camp ◽

Igg Antibodies ◽

Potential Therapeutic Target ◽

Increased Risk ◽

Risk For Thrombosis ◽

Camp Levels

Abstract Thromboembolic events are frequently reported in patients infected with the SARS-CoV-2 virus. The exact mechanisms of COVID-19-associated hypercoagulopathy, however, remain elusive. Recently, we observed that platelets (PLTs) from patients with severe COVID-19 infection express high levels of procoagulant markers, which were found to be associated with increased risk for thrombosis. In the current study, we investigated the time course as well as the mechanisms leading to procoagulant PLTs in COVID-19. Our study demonstrates the presence of PLT-reactive IgG antibodies that induce marked changes in PLTs in terms of increased inner-mitochondrial transmembrane potential (Δψ) depolarization, phosphatidylserine (PS) externalization, and P-selectin expression. The IgG-induced procoagulant PLTs and increased thrombus formation were mediated by ligation of PLT Fc-γ RIIA (FcγRIIA). In addition, contents of calcium and cyclic-adenosine-monophosphate (cAMP) in PLTs were identified to play a central role in antibody-induced procoagulant PLT formation. Most importantly, antibody-induced procoagulant events, as well as increased thrombus formation in severe COVID-19, were inhibited by Iloprost, a clinically approved therapeutic agent that increases the intracellular cAMP levels in PLTs. Our data indicate that upregulation of cAMP could be a potential therapeutic target to prevent antibody-mediated coagulopathy in COVID-19 disease.

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Rapid Bioassay for Detection of Thyroid-Stimulating Antibodies Using Cyclic Adenosine Monophosphate-Gated Calcium Channel and Aequorin

European Thyroid Journal ◽

10.1159/000371740 ◽

2015 ◽

Vol 4 (1) ◽

pp. 14-19 ◽

Cited By ~ 13

Author(s):

Naohiro Araki ◽

Mitsuru Iida ◽

Nobuyuki Amino ◽

Shinji Morita ◽

Akane Ide ◽

...

Keyword(s):

Calcium Channel ◽

Hormone Receptor ◽

Chinese Hamster ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Thyroid Stimulating Hormone ◽

Ovary Cell ◽

Intracellular Camp ◽

Thyroid Stimulating Antibodies ◽

Stimulating Hormone

Background: Thyroid-stimulating antibodies (TSAb) are known to be responsible for hyperthyroidism in Graves' disease (GD). The conventional methods to measure TSAb depend on cell-based assays that require cumbersome procedures and a sterilized tissue culture technique. The aim of the present study was to develop a ready-to-use cell-based assay for measuring TSAb activity without requiring sterilized conditions. Methods: We developed a new assay kit using a frozen Chinese hamster ovary cell line expressing the thyroid-stimulating hormone receptor, cyclic adenosine monophosphate (cAMP)-gated calcium channel and aequorin, tentatively named the aequorin TSAb assay. Activated stimulatory G-protein-coupled adenylate cyclase increases intracellular cAMP, which then binds to the cyclic nucleotide-gated calcium channel. Activation of this channel allows Ca2+ to enter the cell, and the influx of Ca2+ can be measured with aequorin, which is quantified by a luminometer. Results can be obtained in only 4 h without sterilized conditions. TSAb activities were expressed by international units using the NIBSC 08/204 standard. Results: Positive results of aequorin TSAb were obtained in 197 of 199 (98.9%) of untreated patients with GD. Only 1 of 42 (2.3%) patients with painless thyroiditis had a weakly positive aequorin TSAb. All 45 patients with subacute thyroiditis and 185 normal subjects showed negative aequorin TSAb. As for chronic thyroiditis, all 52 euthyroid patients showed negative aequorin TSAb, but 8 of 50 (16.0%) hypothyroid patients had a positive reaction. However, these positive reactions were not induced by serum thyroid-stimulating hormone (TSH) and were thought to be induced by the stimulating activity of anti-TSH receptor immunoglobulins. Conventional porcine TSAb and Elecsys thyroid-stimulating hormone receptor antibodies were positive in 69.3 and 95.5% of GD, respectively. Conclusion: The aequorin TSAb assay was positive in 98.9% of GD and was more sensitive than the conventional assay. This assay can be conducted in only 4 h without sterilized conditions and is practically useful in general clinical laboratories.

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