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Electron micrograph showing a highly enriched preparation of cilia isolated from porcine airway epithelium. Thin section of the ciliary pellet demonstrates the features of intact axonemes (nine outer doublet microtubules and an inner pair). Bar, 1 μm.
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![Effect of extracellular ATP on [Ca2+]i and CBF enhancement....](publication/8239376/figure/fig2/AS:203162318315524@1425449079430/Effect-of-extracellular-ATP-on-Ca2-i-and-CBF-enhancement-Representative-experiment-of_Q320.jpg)
The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the abse...
Citations
... cGMP may activate cGMP-dependent kinase (PKG) and, potentially, cAMP-dependent protein kinase (PKA) (Lorenz et al, 2017), which in turn may phosphorylate dyneins or kinesins of the axoneme, altering the cilia beating. PKG has been detected in the axonemes of the tracheal cilia; moreover, in isolated cilia preparations submicromolar concentrations of cGMP induced phosphorylation of the ciliary proteins (Gertsberg et al, 2004;Wyatt, 2015). ...
Clearance of the airway is dependent on directional mucus flow across the mucociliary epithelium, and deficient flow is implicated in a range of human disorders. Efficient flow relies on proper polarization of the multiciliated cells and sufficient ciliary beat frequency. We show that NO, produced by nNOS in the multiciliated cells of the mouse trachea, controls both the planar polarity and the ciliary beat frequency and is thereby necessary for the generation of the robust flow. The effect of nNOS on the polarity of ciliated cells relies on its interactions with the apical networks of actin and microtubules and involves RhoA activation. The action of nNOS on the beat frequency is mediated by guanylate cyclase; both NO donors and cGMP can augment fluid flow in the trachea and rescue the deficient flow in nNOS mutants. Our results link insufficient availability of NO in ciliated cells to defects in flow and ciliary activity and may thereby explain the low levels of exhaled NO in ciliopathies.
... The molecular structure and biochemical function of these enzymes are evolutionarily conserved from unicellular algae to plants and animals (130). Taken together, homeostatic control of extracellular ATP and its intracellular signaling pathways in plants could utilize molecular components similar to those observed in mammalian cells (40,100,115). ...
Danger signals trigger immune responses upon perception by a complex surveillance system. Such signals can originate from the infectious nonself or the damaged self, the latter termed damage-associated molecular patterns (DAMPs). Here, we apply Matzinger’s danger model to plant innate immunity to discuss the adaptive advantages of DAMPs and their integration into preexisting signaling pathways. Constitutive DAMPs (cDAMPs), e.g., extracellular ATP, histones, and self-DNA, fulfill primary, conserved functions and adopt a signaling role only when cellular damage causes their fragmentation or localization to aberrant compartments. By contrast, immunomodulatory peptides (also known as phytocytokines) exclusively function as signals and, upon damage, are activated as inducible DAMPs (iDAMPs). Dynamic coevolutionary processes between the signals and their emerging receptors and shared co-receptors have likely linked danger recognition to preexisting, conserved downstream pathways.
... Accumulating evidence shows that mammalian CBF changes in response to the phosphorylation state of ciliary targets and to the levels of [Ca 2+ ] i , both events mainly occurring through variation of cAMP levels inside the cells [22]. So, for the regulation of airway cilia, cAMP needs to be made available to the axonemes from within the cell and beta-adrenergic agonists or phosphodiesterase -4 inhibitors are used to increase CBF in a cAMP-dependent manner via beta 2-receptors expressed at the apical membrane [23]. Herein, we found that thyme extract is able to increase c-AMP levels at comparable levels with salmeterol or isoproterenol (beta-adrenergic agonists) or YM 976 (phosphodiesterase-4 inhibitor) suggesting its possible involvement in increasing CBF in a cAMP-dependent manner in COPD cell lines. ...
Chronic obstructive pulmonary disease (COPD) is a respiratory disorder characterized by a progressive and irreversible airflow limitation. COPD is associated to a chronic inflammatory response with infiltration of in-flammatory cells in the surface epithelium of large airways and abnormalities in structure and functions of cilia. Thyme (Thymus vulgaris L.) is a traditional medicinal plant of the Mediterranean area used to treat respiratory disorders. We previously evidenced that thyme extract reduce IL-1beta and IL-8, by downregulating the activated NF-κB levels, suggesting its potential therapeutically use in COPD. Cilia beating frequency (CBF) is dramatically impaired in COPD and different pharmacological agents can modulate cilia function. Herein we evaluated the effect of a commercial thyme extract in modulating CBF by measuring its activity in stimulating cAMP, Ca 2+ levels and CBF in a MucilAir 3D human COPD airway epithelia reconstituted in vitro system using salmeterol, YM976, isoproterenol and GSK1016790 A as positive controls. Results showed that thyme extract increased cAMP levels starting from 12 h post-treatment, decreased extracellular Ca 2+ levels and increased the CBF in airway epithelia from COPD donors. Overall, this work demonstrated that thyme extract is effective in stimulating CBF by inducing an increase of cAMP and Ca 2+ levels, thus supporting its therapeutical use in the treatment of COPD.
... Recently, a novel localization of nNOS at the proximal portion of cilia in airway epithelium has been shown, suggesting that the independent and local regulation of NO levels is crucial for normal cilia function (16). In addition, authors pointed out that [Ca 2+ ]i levels regulate the phosphorylation and the dephosphorylation of ciliary proteins, via the NO pathway, modulating the cilia movement (17). ...
In Paramecium cilia beating is correlated to intracellular calcium concentration ([Ca2+]i) and nitric oxide (NO) synthesis. Recent findings affirm that photobiomodulation (PBM) can transiently increase the [Ca2+]i in mammalian cells. In the present study we investigated the effect of both 808nm and 980nm diode laser irradiated with flat-top hand-piece on [Ca2+]i and NO production of Paramecium primaurelia, to provide basic information for the development of new therapeutic approaches. In the experiments, the laser power in CW varied (0.1W; 0.5W; 1W; 1.5W) to generate the following respective fluences: 6.4J/cm2; 32J/cm2; 64J/cm2; 96J/cm2. The 6.4J/cm2 didn't induce PBM if irradiated by both 808nm and 980nm diode laser. Conversely, the 32J/cm2 fluence had no effect on Paramecium cells if irradiated by the 808nm laser, while if irradiated by the 980 nm laser induced increment in swimming speed (suggesting an effect on the [Ca2+]i, NO production, similar to the 64J/cm2 with the 808nm wavelength). The more evident discordance occurred with the 96J/cm2fluence, which had the more efficient effect on PBM among the parameters if irradiated with the 808nm laser and killed the Paramecium cells if irradiated by the 980nm laser. Lastly, the 980nm and 64J/cm2 or 96J/cm2 were the only parameters to induce a release of stored calcium. This article is protected by copyright. All rights reserved.
... Western blots of porcine cilia axonemes probed for PKG I-alpha demonstrated the presence of PKG on the ciliary axoneme [60]. Numerous cilia substrates were also identified to be phosphorylation targets of PKG. ...
Motile cilia of the lungs respond to environmental challenges by increasing their ciliary beat frequency in order to enhance mucociliary clearance as a fundamental tenant of innate defense. One important second messenger in transducing the regulable nature of motile cilia is cyclic guanosine 3',5'-monophosphate (cGMP). In this review, the history of cGMP action is presented and a survey of the existing data addressing cGMP action in ciliary motility is presented. Nitric oxide (NO)-mediated regulation of cGMP in ciliated cells is presented in the context of alcohol-induced cilia function and dysfunction.
... Since both acetylcholine and serotonin seem to generate independently an increase in ciliary activity we observed the independent effects of both on cilia function. Among a cholinergic stimulation of cGMP, which constitutes equal to cAMP, an important messenger to activate a phosphorylation and mediate an increase in cilliary beat frequency, NO induces the synthesis of cGMP based on activation of soluble guanylyl cyclase [32] [33] [34] [4]. But neither inhibition of NO generation in the present study using L-NMMA or L-NAME did prevent the TNF-a induced increase in PTV, nor a muscarinic receptor blockade using atropine was able to reduce or inhibit the response to TNFa. ...
The tracheal epithelium prevents via its highly effective clearance mechanism the contamination of the lower airways by pathogens. This mechanism is driven by ciliary bearing cells which are not only in contact with the gas phase; in addition they are also influenced by inflammatory mediators. These mediators can alter the protective function of the epithelium. Since the pro-inflammatoric cytokine tumor necrosis factor-α (TNF-α) plays a pivotal role within the inflammatory cascade, we investigated its effect onto the tracheal epithelium measured by its ciliary beat frequency and the particle transport velocity. In organ explant experiments the ciliary beat frequency and the particle transport velocity were measured under the application of TNF-α using tracheae from male C57BL6J mice. We observed a dose dependent TNF-α induced increase of both particle transport velocity and ciliary beat frequency. Knock out mice experiments made evident that the increase was depended on the expression of tumor necrosis factor receptor 1 (TNF-R1). The increases in ciliary beat frequency as well as the accelerated particle transport velocity were either inhibited by the unspecific serotonin antagonist methysergide or by cyproheptadine a specific 5-HT2 receptor antagonist. Thus, acetylcholine antagonists or nitric oxide synthase (NOS) inhibitors failed to inhibit the TNF-α induced activation. In conclusion, TNF-α may play a pivotal role in the protection of lower airways by inducing ciliary activity and increase in particle transport velocity via TNF-R1 and 5-HT2 receptor.
... This enzyme catalyzes the conversion of guanosine triphosphate (GTP) to 3´-5´cyclic guanosine monophosphate (cGMP), an important intracellular second messenger molecule (Schaap 2005). Increased cGMP synthesis regulates cGMP-dependent protein kinases (PKGs), phosphodiesterases, and ion channels, thus modulating the phosphorylation state of several proteins and affecting cellular ion homeostasis Gertsberg et al. 2004). In the musculoskeletal system the NO/cGMP/PKG signaling pathway has been identified in osteoblasts, osteoclasts, motor nerve endings, and vascular smooth muscle cells of arterioles supplying muscles. ...
Nitric oxide (NO) is a multifaceted signaling molecule in the musculoskeletal system. In bone, NO can act as a signal molecule and has anabolic effects by supporting bone formation and repressing bone resorption. In skeletal muscles, NO has a major role in the self-regulation of arteriolar blood flow and is also involved in the control of Ca2+ dynamics, glucose metabolism, mitochondrial oxidative phosphorylation, immune response, and muscle and tendon regeneration. Altered NO production in the skeletal system is associated with pathologies of high morbidity, such as osteoporosis, osteoarthritis, and rheumatoid arthritis and with non-curable progressive dystrophies in muscles. NO thus has medical importance; however, there are still roadblocks that do not allow effective modulation of NO homeostasis in diseases of the musculoskeletal system. This chapter provides an overview of current knowledge of the roles and possible pharmacological targeting of NO and NO derived radicals in the musculoskeletal system. © Springer-Verlag Berlin Heidelberg 2014. All rights are reserved.
... These environmental and host stimuli are transmitted via surface receptors and channels to trigger activation of second messenger cascades that regulate phosphorylation status of ciliary proteins thereby modulating the kinetics of microtubules sliding relative to each other. Recently, elegant experiments using fluo- 36,37 Decreased temperature 36,37 Ca 2ϩ concentration 86 Pyocyanin (Pseudomonas aeruginosa) 92 Mechanical shear stress 28 Lipooligosaccharide (Haemophilus influenzae) 60 Nitric oxide 31,87 Protein D (H. influenzae) 60 Bradykinin 88 Pneumolysin (Streptococcus pneumoniae) 61 TNF-␣ 31 Hydrogen peroxide (S. pneumoniae) 61 IL-1 31 Methacholine 89 Prostacyclin 90 Substance P 91 CBF ϭ ciliary beat frequency. rescence resonance energy transfer in primary ciliated cell culture showed direct evidence that activation of protein kinase A (PKA) coincides with an increase in CBF, and that the return to baseline frequency lags PKA inactivation, indicating that dephosphorylation by phosphatases is required to terminate CBF stimulation. ...
Cilia are complex and powerful cellular structures of the respiratory mucosa that play a critical role in airway defense. Respiratory epithelium is lined with cilia that perform an integrated and coordinated mechanism called mucociliary clearance. Mucociliary clearance is the process by which cilia transport the mucus blanket overlying respiratory mucosa to the gastrointestinal tract for ingestion. It is the primary means by which the airway clears pathogens, allergens, debris, and toxins. The complex structure and regulatory mechanisms that dictate the form and function of normal cilia are not entirely understood, but it is clear that ciliary dysfunction results in impaired respiratory defense.
A literature review of the current knowledge of cilia dysfunction in chronic rhinosinsusitis was conducted.
Ciliary dysfunction may be primary, the result of genetic mutations resulting in abnormal cilia structure, or, more commonly, secondary, the result of environmental, infectious, or inflammatory stimuli that disrupt normal motility or coordination. Patients with chronic rhinosinusitis (CRS) have been found to have impaired mucociliary clearance. Many biochemical, environmental, and mechanical stimuli have been shown to influence ciliary beat frequency, and common microbial pathogens of respiratory mucosa such as Pseudomonas aeruginosa and Haemophilus influenzae have developed toxins that appear to interrupt normal mucociliary function. Furthermore, inflammatory mediators known to be present in patients with CRS appear to impair secondarily mucociliary clearance.
The goal of this article is to summarize the recent developments in the understanding of cilia dysfunction and mucociliary clearance in CRS.
... However, only a few studies have focused on the kinases directly localized to the axoneme for control of motility. In most cases studied to date, the upstream regulator of phosphorylation is calcium [18, 63,113114115. The focus on kinases associated with the axoneme began with biochemical and physiological assays using isolated ciliary axonemes or detergent extracted " models " [116, 117]. ...
Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9+2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation/dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.
... Recently, ATP-induced NO production in tomato culture cells was found to occur downstream of both phospholipase C and diacylglycerol kinase activation [43]. Thus, the signaling pathways linking ATP perception to cellular response could utilize some of the same components seen in mammalian cells [44,45]. ...
Extracellular adenosine-5'-triphosphate (ATP) induces a number of cellular responses in plants and animals. Some of the molecular components for purinergic signaling in animal cells appear to be lacking in plant cells, although some cellular responses are similar in both systems [e.g. increased levels of cytosolic free calcium, nitric oxide (NO), and reactive oxygen species (ROS)]. The purpose of this review is to compare and contrast purinergic signaling mechanisms in animal and plant cells. This comparison will aid our overall understanding of plant physiology and also provide details of the general fundamentals of extracellular ATP signaling in eukaryotes.
