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

2008 ◽  
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.

Adenylyl Cyclase 6 Mediates Primary Cilia-Dependent Changes in Cyclic Adenosine Monophosphate in Response to Dynamic Fluid Flow

2009 ◽  
Author(s):  
Sara Temiyasathit ◽  
Ronald Y. Kwon ◽  
Padmaja Tummala ◽  
Clarence C. Quah ◽  
Christopher R. Jacobs
Keyword(s):  
Fluid Flow ◽  
Adenylyl Cyclase ◽  
Primary Cilium ◽  
Primary Cilia ◽  
Bone Structure ◽  
Bone Cells ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cell Types ◽  
Dependent Flow

It is well accepted that fluid flow is an important mechanical signal in regulating bone structure and function. Primary cilia, which are solitary, microtubule-based organelles that extend from the centrosome into extracellular space in many cell types, have 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 [1]. Recently, we identified cAMP as a novel early signaling molecule in primary cilia-dependent mechanotransduction of fluid flow in osteocytes. Specifically, we show that MLO-Y4 osteocyte-like cells respond to oscillatory flow with a rapid decrease in intracellular levels of cAMP that is dependent on the primary cilium [2]. Adenylyl cyclase 6 (AC6) is an enzyme responsible for the synthesis of cAMP from ATP. We found that AC 6 localizes to the primary cilium of bone cells (Fig. 1). In this study, our goal was to determine whether AC6 mediates the primary cilia-dependent, flow-induced decrease in cAMP.

Secondary Messenger Systems in PTSD

2016 ◽  
Author(s):  
Ulrike Schmidt
Keyword(s):  
Second Messengers ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Brain Regions ◽  
Guanosine Monophosphate ◽  
Intracellular Camp ◽  
Post Traumatic Stress ◽  
Secondary Messenger

Second messengers such as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositoltriphosphate, and diacylglycerol (DAG) are a prerequisite for the signal transduction of extracellular receptors. The latter are central for cellular function and thus are implicated in the pathobiology of a variety of disorders, such as schizophrenia, bipolar disorder, major depression, and post-traumatic stress disorder (PTSD). This chapter focuses on the involvement of second messenger molecules and their regulators as direct targets in human and animal PTSD and aims to stimulate the underdeveloped research in this field. The synthesis of literature reveals that second messengers clearly play a central role in PTSD-associated brain regions and processes. In particular, pituitary adenylate cyclase-activating polypeptide (PACAP), an important regulator of intracellular cAMP levels, as well as protein kinase c, the major target of DAG, belong to the hitherto most promising PTSD candidate molecules directly involved in second messenger signaling.

scholarly journals Extracellular vesicles: another compartment for the second messenger, cyclic adenosine monophosphate

2019 ◽  
Vol 316 (4) ◽  
pp. L691-L700 ◽  
Author(s):  
Sarah L. Sayner ◽  
Chung-Sik Choi ◽  
Marcy E. Maulucci ◽  
K. C. Ramila ◽  
Chun Zhou ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Cell Communication ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Cell Types ◽  
Signaling Specificity ◽  
Phosphodiesterase 4 ◽  
Pulmonary Endothelial Cells

The second messenger, cAMP, is highly compartmentalized to facilitate signaling specificity. Extracellular vesicles (EVs) are submicron, intact vesicles released from many cell types that can act as biomarkers or be involved in cell-to-cell communication. Although it is well recognized that EVs encapsulate functional proteins and RNAs/miRNAs, currently it is unclear whether cyclic nucleotides are encapsulated within EVs to provide an additional second messenger compartment. Using ultracentrifugation, EVs were isolated from the culture medium of unstimulated systemic and pulmonary endothelial cells. EVs were also isolated from pulmonary microvascular endothelial cells (PMVECs) following stimulation of transmembrane adenylyl cyclase (AC) in the presence or absence of the phosphodiesterase 4 inhibitor rolipram over time. Whereas cAMP was detected in EVs isolated from endothelial cells derived from different vascular beds, it was highest in EVs isolated from PMVECs. Treatment of PMVECs with agents that increase near-membrane cAMP led to an increase in cAMP within corresponding EVs, yet there was no increase in EV number. Elevated cell cAMP, measured by whole cell measurements, peaked 15 min after treatment, yet in EVs the peak increase in cAMP was delayed until 60 min after cell stimulation. Cyclic AMP was also increased in EVs collected from the perfusate of isolated rat lungs stimulated with isoproterenol and rolipram, thus corroborating cell culture findings. When added to unperturbed confluent PMVECs, EVs containing elevated cAMP were not barrier disruptive like cytosolic cAMP but maintained monolayer resistance. In conclusion, PMVECs release EVs containing cAMP, providing an additional compartment to cAMP signaling.

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

2020 ◽  
Vol 17 (4) ◽  
pp. 595-602
Author(s):  
Nguyen Thi Mong Diep ◽  
Nguyen Thi Bich Hang ◽  
Nguyen Le Cong Minh ◽  
Tran Thanh Son ◽  
Nguyen Thuy Duong
Keyword(s):  
Cyclic Amp ◽  
Leydig Cells ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cell Types ◽  
Intracellular Camp ◽  
Atp Levels ◽  
Long Time ◽  
Short Time ◽  
Camp Levels

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.

scholarly journals PACmn for improved optogenetic control of intracellular cAMP

BMC Biology ◽  
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.

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

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

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

Molecules ◽  
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.

scholarly journals Increased intracellular cyclic adenosine monophosphate inhibits T lymphocyte-mediated cytolysis by two distinct mechanisms.

1988 ◽  
Vol 167 (6) ◽  
pp. 1963-1968 ◽  
Author(s):  
L S Gray ◽  
J Gnarra ◽  
E L Hewlett ◽  
V H Engelhard
Keyword(s):  
Cholera Toxin ◽  
T Lymphocyte ◽  
Pertussis Toxin ◽  
Target Cell ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Stimulation Of

Cholera toxin (CT), but not pertussis toxin (PT), treatment of cloned murine CTL inhibited target cell lysis in a dose-dependent fashion. The effects of CT were mimicked by forskolin and cyclic adenosine monophosphate (cAMP) analogues. Inhibition of cytotoxicity by CT and cAMP analogs was mediated in part by attenuation of conjugate formation. Additionally, both CT and cAMP analogs blocked the increase in intracellular Ca2+ induced by stimulation of the TCR complex by mAbs. These findings indicate that cAMP inhibits the activity of CTL by two distinct mechanisms and suggests a role for this second messenger in CTL-mediated cytolysis.

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

2015 ◽  
Vol 4 (1) ◽  
pp. 14-19 ◽  
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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