Increase In Ciliary Beat Frequency Research Articles

Effects of Benzalkonium Chloride and Potassium Sorbate on Airway Ciliary Activity

Objectives: Preservatives are indispensable components of aqueous multidose topical formulations. The purpose of our study was to investigate the effects of two representative preservatives, benzalkonium chloride (BKC) and potassium sorbate (PS), on rabbit tracheal ciliary beat frequency (CBF). Methods: Rabbit tracheal ciliated cell culture was established and CBF was determined using high-speed digital imaging methods. The effects of preservatives at different concentrations on CBF were observed over a 10-min exposure period. Results: BKC induced inhibition of CBF in a concentration-dependent manner. Ciliary beating was stopped by 0.01% BKC after 5 min of exposure. A low concentration of PS (0.12%) only resulted in a mild decrease in CBF during a 10-min exposure. The CBF decreased by 13.0% from baseline after 10 min. However, there was no statistically significant difference compared with the corresponding control condition. Application of 0.24, 0.48 and 0.96% of PS to rabbit tracheal cells resulted in an increase in CBF, with an increase of 105 ± 9.8, 107.6 ± 4.0, and 117.1 ± 9.5% relative to baseline CBF after 10 min of exposure, respectively. Conclusions: PS could be considered as a safer and more promising preservative than BKC for use in topical formulations.

Activation of ciliary beating by a mucolytic agent, ambroxol, in bronchioles of mice

Ambroxol has been used as a mucolytic agent in pulmonary diseases. In this study, we studied the effects of ambroxol on ciliary beat frequency (CBF) and ciliary bend amplitude (CBA) of the bronchioles. Mice were anestetized by pentobarbital sodium (70mg/kg, ip) and bronchiolar ciliary cells were isolated from lungs by an elastase treatment. In a HCO3−‐containing solution, ambroxol increased CBA and CBF in a concentration dependent manner: ambroxol (10μM) increased CBA and CBF by 30 % within 15 min. However, in a HCO3−‐free solution, it did not induce any increase in CBA and CBF, although the switch from the HCO3−‐containing solution to the HCO3−‐free solution increased them. Measurement of intracellular pH (pHi) demonstrated that ambroxol (10 μM) increased pHi gradually in bronchiolar ciliary cells. These suggest that an increase in pHi induced by ambroxol increases CBA and CBF. To confirm the effects of pHi on CBA and CBF, an NH4+ pulse was applied. An increase in pHi increased CBA and CBF, whereas a decrease in pHi decreased them. Diisothiocyanostilbene‐disulfonic acid (DIDS) abolished the increases in CBA and CBF induced by ambroxol. In bronchiolar ciliary cells, ambroxol increases pHi via activation of HCO3−/Cl− exchanges, which increases CBA and CBF.

Conservation of aTritoniaPedal peptides network in gastropods

Adults of the nudibranch mollusc Tritonia diomedea crawl using mucociliary locomotion. Crawling is controlled in part by the large Pedal 5 (Pd5) and Pedal 6 (Pd6) neurons that produce Tritonia Pedal peptides (TPeps). TPeps elicit an increase in ciliary beat frequency, thereby increasing crawling speed. In adults of T. diomedea, an extensive network of TPep-containing neurites adjacent to the basement membrane of the pedal epithelium delivers TPeps to the ciliated cells. In this study, we show that diverse nudibranchs all have a pattern of TPep-like immunoreactivity similar to that of T. diomedea, with thin tracts of TPep-like immunoreactive (TPep-LIR) neurites projecting to the epithelial layer. We also show that members of two non-nudibranch gastropod species have a pattern of TPep-innervation similar to that of the nudibranchs. In addition, we characterized two pairs of motor neurons in adults of the nudibranch Armina californica that are possible homologues of the Pd5 and Pd6 cells in T. diomedea. Activity in one of these pairs, the Pedal Peptidergic Dorsal 1 (PPD1) cells, was correlated with mucociliary locomotion. The second pair, the Pedal Peptidergic Ventral 1 cells, shared synchronous synaptic input with the PPD1 cells, a pattern consistent with the shared synaptic input of the T. diomedea Pd5 and Pd6 cells. These findings suggest that the roles of the Pd5 and Pd6 cells as mucociliary motor neurons in nudibranchs are conserved evolutionarily. Additionally, the extensive network of TPep-LIR neurites seen in the foot of T. diomedea appears likely to be a common feature among gastropods.

Safety evaluation of sinus surfactant solution on respiratory cilia function

A common complaint of chronic rhinosinusitis (CRS) is thickened inspissated nasal secretions and concomitant postnasal drip. Recently, the use of diluted, baby shampoo demonstrated modest improvement in these complaints. Removal of hair-specific ingredients, such as thickeners and fragrances, and addition of a humectant and optimization of mucoactive ingredients generated a topical surfactant solution designed for sinonasal use. This study evaluates the safety of this solution on respiratory cilia function. Murine nasal explants as well as murine nasal air-liquid interface cultures were tested. Using high-speed video microscopy, baseline ciliary beat frequency (CBF) was established, followed by addition of the surfactant solution. CBF was recorded every 30 seconds for 15 minutes. Sinonasal surfactant solution diluted in bicarbonate buffered saline resulted in a transient increase of CBF in murine explants and mature epithelial cultures with no evidence of toxicity on respiratory cilia over the 15 minutes. Respiratory mucosal explants and air-liquid interface cultures demonstrate robust ciliary beating and represent 2 model systems for screening topical agents for ciliotoxicity. In both systems, application of a novel sinonasal surfactant solution diluted in bicarbonate buffered saline was not ciliotoxic to respiratory epithelium over a 15-minute period.

Effect of macrolide antibiotics on ciliary motility in rabbit airway epithelium in-vitro.

We have studied ciliary beat frequency (CBF) of rabbit cultured tracheal epithelium by a photoelectric method in-vitro. Addition of erythromycin and roxithromycin increased CBF in a dose-dependent fashion, whereas clarithromycin was without effect. The rank order potency of macrolide was roxithromycin > erythromycin >> clarithromycin. The roxithromycin-induced increase in CBF was not altered by propranolol, AA-861, or verapamil, but partially attenuated by indomethacin. Roxithromycin increased intracellular cAMP concentrations. These results suggest that certain macrolides can stimulate airway ciliary motility probably via prostaglandin- and cAMP-dependent regulatory pathways, which may affect mucociliary transport function in the respiratory tract.

Effects of Lactoferrin and Lysozyme on Ciliary Motility in Rabbit Tracheal Epithelium

We studied the effects of lactoferrin and lysozyme on ciliary beat frequency (CBF) of rabbit cultured tracheal epithelium by a photoelectric method in-vitro. Lactoferrin (50 mg mL−1) and lysozyme (100 mg mL−1) elicited rapid increases in CBF from 13·3 ± 0·3 to 15·9 ± 0·4 Hz (mean ± s.e., P<0·01) and 13·0 ± 0·4 to 17·2 ± 0·5 Hz (P<0·01), respectively, followed by sustained increases which were maintained at least for 30 min. These effects were dose dependent. The maximal increases from the baseline values (ΔCBFmax) and the concentrations required to produce a half-maximal responses (EC50) were 32·4 ± 5·3% and 14·9 ± 2·8 mg mL−1, respectively, for lactoferrin, and 38·0 ± 4·4% and 2·3 ± 0·3 mg mL−1 for lysozyme. The increases in CBF produced by lactoferrin and lysozyme were not affected by pretreatment of cells with propranolol or Ca2+-free medium. Intracellular cAMP levels remained unchanged after the addition of lactoferrin and lysozyme. These results suggest that lactoferrin and lysozyme stimulate airway epithelial ciliary motility, thereby playing a role in the regulation of mucociliary transport in the respiratory tract.

The regulation of ciliary beat frequency by ovarian steroids in the guinea pig Fallopian tube: interactions between oestradiol and progesterone

Ciliary beat frequency (CBF) was measured in slice preparations of the Fallopian tube fimbria, using videomicroscopy with a high-speed (500 Hz) camera in guinea pigs that were treated with β-oestradiol benzoate (βE2B) and medroxy progesterone (mPRG). In non-ovulating guinea pigs at 4 weeks of age, the CBF of the fimbria was high (17.8 Hz). In sexually mature guinea pigs (12-16 weeks of age) with constant ovulation, the CBF varied from 12 Hz to 16 Hz. The in vivo administration of both ICI-182,780 (a blocker of βE2 receptors) and mifepristone (a blocker of PRG receptors) induced high CBF (17.4 Hz). The administration of βE2B at a low (3.2 mg/kg/day) or high (32 mg/kg/day) dose decreased the CBF to 14.5 Hz or 11 Hz, respectively. ICI-182,780 abolished the βE2B-induced changes in CBF and decreased CBF to 12 Hz. The administration of mPRG (6.4 mg/kg/day) decreased CBF to 12.5 Hz. Mifepristone abolished this mPRG-induced decrease in CBF and maintained the CBF at 15 Hz. However, administering both βE2B and mPRG increased CBF to 17.5 Hz, suggesting that βE2B inhibits mPRG actions and vice versa. To confirm the interactions between βE2B and mPRG, we administered both βE2B and mPRG to guinea pigs that were pretreated for 1.5 days with either mPRG (6.4 mg/kg/day) or βE2B (3.2 mg/kg/day). Prior treatment with βE2B or mPRG prevented the increase in CBF that was otherwise by βE2B plus mPRG, and maintained the CBF at 14.5 Hz or 13 Hz, respectively. The administration of βE2B plus mPRG still induced the expression of PRG receptors, indicating that the highest CBF is not the result of no expression of the receptors. In the beating cilia of the fimbria, the signals that are activated by the βE2 and PRG receptors are proposed to antagonize each other in regulating the frequency.

Effect of cyclic AMP-elevating agents on airway ciliary beat frequency in central and lateral airways in rat precision-cut lung slices

In patients with chronic obstructive pulmonary disease (COPD), mucociliary clearance of the respiratory tract is impaired due to enhanced mucus secretion and deterioration of normal ciliary activity. We investigated the effects of cyclic AMP-elevating agents with a different mode of action on ciliary beat frequency (CBF) in rat large central and small lateral airways by comparing the phosphodiesterase-4 (PDE4) inhibitors rolipram and roflumilast to the β 2-adrenoceptor agonist terbutaline and the adenylyl cyclase activator forskolin. Rat precision-cut lung slices were prepared and effects of cyclic AMP-elevating agents on CBF were assessed for up to 4 h. In central airways a time- and concentration-dependent increase in CBF was seen for roflumilast (59 ± 4%, 1 µM, 60 min), rolipram (55 ± 4%, 1 µM, 60 min), terbutaline (64 ± 8%, 10 µM, 60 min) and forskolin (55 ± 8%, 100 µM, 60 min). Only roflumilast and rolipram increased CBF in lateral airways, with a similar time course and maximum efficacy (roflumilast 48 ± 5%, rolipram 54 ± 2%). Incubation of lateral airways with terbutaline (10 µM, + 11%) or forskolin (100 µM, + 1%) had negligible effects. As a major novel finding this study reveals that PDE4 inhibitors increased CBF in central as well as in lateral airways, while terbutaline and forskolin affected CBF in proximal airways only.

Time-dependent changes in nasal ciliary beat frequency

As the ex vivo lifetime of nasal ciliary cells is limited, these cells have to be transferred to a culture medium for analysis with vital cytology immediately. Although the ciliary beat frequency (CBF) is likely to change over time, sufficient data regarding changes in the ex vivo CBF has not been published to date. In the present study, nasal epithelial cells were harvested from the mucosa of the inferior turbinate of 19 healthy volunteers with a cytology brush. Beating cilia were visualized with phase-contrast microscopy. Over a 12-h timeframe, 2 s epochs of video were captured every 5 min from the identical group of cells using a high-speed digital camera with a sampling rate of 100 fps. Temperature was maintained at about 22 degrees C and controlled by an infrared pyrometer. The CBF rapidly increased by 47 +/- 53% during the first 3 h of measurement. A relative plateau followed this increase from 3 to 9 h. After 9 h, CBF reduced linearly. After 12 h, the mean frequency reduced to 20 +/- 69% of baseline. However, there was considerable variance between the samples. The initial increase in CBF has not been reported previously. This interval seems to be unsuitable for meaningful measurements. Measurements of CBF are most reliable during the plateau phase between 3 and 9 h. After 9 h, there is clearly a significant decrease in CBF, together with a considerable interindividual variance.

TRPV4 channel participates in receptor-operated calcium entry and ciliary beat frequency regulation in mouse airway epithelial cells

The rate of mucociliary clearance in the airways is a function of ciliary beat frequency (CBF), and this, in turn, is increased by increases in intracellular calcium. The TRPV4 cation channel mediates Ca(2+) influx in response to mechanical and osmotic stimuli in ciliated epithelia. With the use of a TRPV4-deficient mouse, we now show that TRPV4 is involved in the airways' response to physiologically relevant physical and chemical stimuli. Ciliary TRPV4 expression in tracheal epithelial cells was confirmed with immunofluorescence in TRPV4(+/+) mice. Ciliated tracheal cells from TRPV4(-/-) mice showed no increases in intracellular Ca(2+) and CBF in response to the synthetic activator 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and reduced responses to mild temperature, another TRPV4-activating stimulus. Autoregulation of CBF in response to high viscosity solutions is preserved in TRPV4(-/-) despite a reduced Ca(2+) signal. More interestingly, TRPV4 contributed to an ATP-induced increase in CBF, providing a pathway for receptor-operated Ca(2+) entry but not store-operated Ca(2+) entry as the former mechanism is lost in TRPV4(-/-) cells. Collectively, these results suggest that TRPV4 is predominantly located in the cilia of tracheal epithelial cells and plays a key role in the transduction of physical and chemical stimuli into a Ca(2+) signal that regulates CBF and mucociliary transport. Moreover, these studies implicate the participation of TRPV4 in receptor-operated Ca(2+) entry.

Effects of Ephedrine on Human Nasal Ciliary Beat Frequency

The purpose of this study was to investigate the effects of ephedrine, an α,β-adrenoceptor agonist, on the regulation of human nasal ciliary beat frequency (CBF). Changes in (cultured) human nasal CBF in response to 0.25, 0.5, 1.0 and 2.0% ephedrine were quantified by using high-speed (240 frames per second) digital microscopy combined with a beat-by-beat CBF analysis. At ∼25°C, we found that ephedrine induces an instant and moderate increase in human nasal CBF followed by an inhibitory response. With concentrations ranging from 0.25 to 2.0%, while the increment is independent of ephedrine concentration, the inhibitory effect is dependent on the concentration of ephedrine. Based on these results we propose that the clinically used concentration of ephedrine, 0.5%, has maximum stimulatory effect without obvious inhibitory effect on (cultured) human nasal CBF.

Influence of Essential and Fatty Oils on Ciliary Beat Frequency of Human Nasal Epithelial Cells

In alternative and complementary medicine, the use of essential and fatty oils has become more and more popular. In addition to conventional medical therapies, self-medication is showing increasing popularity, using agents with unclear compounds and poorly controlled dosages. Among other disorders, these alternative treatments are used in bronchitis and rhinitis, including some topical applications. Thus, the influence on ciliated epithelia should be evaluated, because a disturbance of the ciliary function can lead to recurrent sinusitis and chronic rhinosinusitis. The aim of this study was to test the influence of fatty and essential oils on the ciliary beat frequency (CBF) of nasal mucosa in vivo. The influence of sesame oil, soy oil, peanut oil, Miglyol 840, thyme oil, lavender oil, eucalyptus oil, and menthol on the ciliary activity of nasal brushings was evaluated by digital high-speed imaging. The presence of most fatty oils resulted in an increase in CBF, the effect being highest for peanut oil. Miglyol 840 had no significant influence on CBF. The essential oils were tested at a concentration of 0.2 and 2%. Thyme oil did not affect CBF, whereas the presence of all other essentials oils resulted in an increase in CBF; the effect was higher at 0.2% than at 2%. Except thyme oil and Miglyol 840, all tested oils caused an increase in CBF. Interestingly, the 0.2% concentrations of essential oils resulted in stronger effects when compared with the 2% concentrations.

Maternal alcohol ingestion reduces surfactant protein A expression by preterm fetal lung epithelia

In addition to neurodevelopmental effects, alcohol consumption at high levels during pregnancy is associated with immunomodulation and premature birth. Premature birth, in turn, is associated with increased susceptibility to various infectious agents such as respiratory syncytial virus (RSV). The initial line of pulmonary innate defense includes the mucociliary apparatus, which expels microorganisms trapped within the airway secretions. Surfactant proteins A and D (SP-A and SP-D, respectively) are additional components of pulmonary innate immunity and have an important role in pulmonary defense against inhaled pathogens. The purpose of this study was to determine if chronic alcohol consumption during the third trimester of pregnancy alters the function of the mucociliary apparatus and expression of SP-A and SP-D of fetal lung epithelia. Sixteen, date-mated ewes were assigned to two different groups; an ethanol-exposed group in which ewes received ethanol through surgically implanted intra-abomasal cannula during the third trimester of pregnancy, and a control group in which ewes received the equivalent amount of water instead of ethanol. Within these two groups, ewes were further randomly assigned to a full-term group in which the lambs were naturally delivered, and a preterm group in which the lambs were delivered prematurely via an abdominal incision and uterotomy. Ethanol was administered five times a week as a 40% solution at 1 g/kg of body weight. The mean maternal serum alcohol concentration measured 6 h postadministration was 16.3 ± 4.36 mg/dl. Tracheas from six full-term lambs were collected to assess ciliary beat frequency (CBF). The lung tissue from all (24) lambs was collected for immunohistochemistry analysis of SP-A and SP-D protein production and fluorogenic real-time quantitative polymerase chain reaction analysis of SP-A and SP-D mRNA levels. Exposure to ethanol during pregnancy significantly blocked stimulated increase in CBF through ethanol-mediated desensitization of cAMP-dependent protein kinase. In addition, preterm born/ethanol-exposed lambs showed significantly decreased SP-A mRNA expression when compared with the preterm born/control group ( P = .004); no significant changes were seen with SP-D. The full-term/ethanol-exposed lambs had no significant alterations in mRNA levels, but had significantly less detectable SP-A protein when compared with the full-term/control lambs ( P = .02). These findings suggest that chronic maternal ethanol consumption during the third trimester of pregnancy alters innate immune gene expression in fetal lung. These alterations may underlie increased susceptibility of preterm infants, exposed to ethanol in utero, to RSV and other microbial agents.

Apical Oxidative Hyaluronan Degradation Stimulates Airway Ciliary Beating via RHAMM and RON

Hyaluronan (HA) is synthesized in high-molecular-weight form at the apical pole of airway epithelial cells, covering the luminal surface. When human airway epithelial cells grown and redifferentiated at the air-liquid interface (ALI) were exposed to xanthine/xanthine oxidase (X/XO), ciliary beat frequency (CBF) increased. This effect was blocked by superoxide dismutase (SOD) and catalase. Inhibition of hyaluronan synthesis inhibited the CBF response to X/XO, while addition of exogenous HA amplified it. A functionally blocking antibody to the receptor for hyaluronic acid-mediated motility (RHAMM) reduced the CBF response to X/XO. Since RHAMM has no transmembrane domain and thus cannot signal on its own, the association of RHAMM with recepteur d'origine nantais (RON), a member of the hepatocyte growth factor receptor family, was explored. Immunohistochemistry of human airway epithelium showed co-localization of RHAMM and RON at the apex of ciliated cells. Physical association of RHAMM and RON was confirmed with co-immunoprecipitations. Macrophage-stimulating protein (MSP), an agonist of RON, stimulated CBF. Genistein, a nonspecific tyrosine kinase inhibitor, and MSP beta chain (beta-MSP), a specific RON inhibitor, blocked the X/XO-induced CBF increase. HA present in the apical secretions of human airway epithelial cells was shown to degrade upon exposure to X/XO, a process inhibited by SOD. Low-molecular-weight HA fragments stimulated CBF, an effect blocked by anti-RHAMM antibody and genistein. These data suggest that high molecular form HA is broken down by reactive oxygen species to form low-molecular-weight fragments that signal via RHAMM and RON to stimulate CBF.

TUMOR NECROSIS FACTOR ALPHA MODIFIES THE CILIARY BEAT FREQUENCY (CBF) OF OVIDUCTAL CILIATED, THROUGH THE PROSTAGLANDINS AND THE NITRIC OXIDE PATHWAY

Mucociliary transport (MCT) is a mechanism that is responsible for the movement of gametes and embryos through the oviduct and serves as physical barrier in the epithelium, to prevent microorganisms invasion. The rate of MCT depends on the ciliary beat frequency (CBF) of ciliated cells of the epithelium. The CBF is regulated by several molecules that are secreted by gametes, embryos or even the epithelium itself, such as ATP, prostaglandins (PG) and nitric oxide. ATP and PG increased the CBF, while NO antagonized this effects, in this system. Other molecule that is secreted by the oviductal epithelium is Tumor Necrosis factor-a (TNFα). High concentrations of this molecule are produced in pathological conditions, causing sloughing of ciliated cells. By the other hand, in physiological conditions TNF is present at low concentrations in the oviductal fluid. Although, in the oviduct there is no evidence about control of CBF by TNFα and the impact on the MTC. In others ciliated epitheliums, TNFα modifies the CBF, effects that are mediated by the nitric oxide pathway. In the oviduct, however there is no evidence about NO production in response to TNFα, but it was established that this molecule induce the synthesis of PG by the epithelial cells. The main goal of this work was to determine the concentration-dependent effect of TNFα, over the CBF in the rat oviduct, and the involvement of prostaglandins and nitric oxide pathways in this effect. Methods: Using microphotodensitometric analysis of primary cultured rat oviductal ciliated cells, we determined changes in CBF in response to TNFα, (0,01-100 ng/ml), in the presence or absence of an inhibitor of arachidonic acid release (AACOCF3 50 μM), an inhibitor of prostaglandins (Indometacin 100 μM), and nitric oxide (L-NAME 100 μM) production. Results: TNFα, in a concentration range of 0,1–10 ng/ml increased the CBF, while concentrations of 50 and 100 ng/ml did not affect the CBF. The concentration-response curve fit to a biphasic curve, with an ascending EC50 of 6,123 × 10−12 M (0,1 ng/ml) and a descending EC50 of 2,03 × 10−9 M (36 ng/ml). The increase in CBF observed with TNFα, 1 ng/ml is inhibited in the presence of AACOCF3 and Indometacin, while TNFα 50 ng/ml in the presence of L-NAME, increased CBF in a similar way as TNFα, 1 ng/ml. Conclusion: TNFa show a biphasic effect on the CBF that is concentration-dependent. The ascending phase of the CBF, observed at low concentration of TNFα, seems to be PG–dependent, while the nitric oxide pathway seems to be involve in the descending phase of CBF, observed at higher concentrations of TNFα, suggesting that nitric oxide pathway, is a negative regulator of the CBF, at this conditions. Our results suggest that TNFα, may have dual effects on the CBF and the MCT in the oviduct. FONDECYT 1040804. (poster)

Calcium-mediated, purinergic stimulation and polarized localization of calcium-sensitive adenylyl cyclase isoforms in human airway epithelia

Purinergic stimulation of human airway epithelia results in a prolonged increase in ciliary beat frequency that depends on calcium-mediated cAMP production [Lieb, T., Wijkstrom Frei, C., Frohock, J.I., Bookman, R.J. and Salathe, M. (2002) Prolonged increase in ciliary beat frequency after short-term purinergic stimulation in human airway epithelial cells. J. Physiol. (Lond.) 538, 633–646]. Here, fully differentiated human airway epithelial cells in culture are shown to express calcium-stimulated transmembrane adenylyl cyclase (tmAC) isoforms (types 1, 3, and 8) by reverse transcription polymerase chain reaction. Immunohistochemistry of tracheal sections and fully differentiated airway epithelial cell cultures revealed polarized expression of these tmACs, with types 1 and 8 localized to the apical membrane and thus at the position required for ciliary regulation. Real-time, ciliated-cell specific cAMP production by tmACs upon apical, purinergic stimulation with UTP was confirmed using fluorescent energy resonance transfer between fluorescently tagged PKA subunits.

Effects of perfluorooctane sulfonate on tracheal ciliary beating frequency in mice

Perfluorooctane sulfonate (PFOS) is one of the emerging persistent organic pollutants, ubiquitously found in the global environment, even in human serum. PFOS has been reported to perturb Ca 2+ homeostasis in Paramecium, cardiomyocytes and neurons. Since ciliary beat frequency (CBF) in the trachea is known to be increased by cytoplasmic Ca 2+ elevation, the effects of PFOS on CBF were evaluated in a slice preparation using video-enhanced contrast microscopy. PFOS increased CBF by 11% ( P < 0.05) at 100 μM, while it did not do so at 30 μM. At 100 μM, it increased intracellular Ca 2+ concentration ([Ca 2+] i) in mouse tracheal ciliary cells. In Ca 2+-free solution, PFOS at 100 μM failed to increase CBF (0.96-fold of vehicle control). The addition of Gd 3+ (1 μM), a store-operated Ca 2+ channel blocker, did not prevent the increase in CBF (1.09-fold ( P < 0.01) of vehicle control). High extracellular K + concentration (50 mM), which causes depolarization of the plasma membrane potential and a transient increase in [Ca 2+] i, increased CBF by 20% ( P < 0.05). This observation indicates involvement of voltage-dependent Ca 2+ channels (VDCCs) in stimulation of CBF. Nifedipine (30 μM), a selective VDCC blocker, antagonized the effects of high K + (0.92-fold of high K + solution) and PFOS (0.96-fold of vehicle control) on CBF. In cells from peroxisome proliferator-activated receptor α (PPARα)-null mice, PFOS still increased CBF (1.12-fold ( P < 0.05) of vehicle control), indicating that the actions of PFOS are not mediated via PPARα. These findings collectively suggest that PFOS stimulates CBF by increasing cytoplasmic Ca 2+ through VDCC.

Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells

Airway ciliary beat frequency regulation is complex but in part influenced by cyclic adenosine monophosphate (cAMP)-mediated changes in cAMP-dependent kinase activity, yet the cAMP concentration required for increases in ciliary beat frequency and the temporal relationship between ciliary beat frequency and cAMP changes are unknown. A lentiviral gene transfer system was developed to express a fluorescence resonance energy transfer (FRET)-based cAMP sensor in ciliated cells. Expression of fluorescently tagged cAMP-dependent kinase subunits from the ciliated-cell-specific foxj1 promoter enhanced expression in fully differentiated ciliated human airway epithelial cells, and permitted simultaneous measurements of ciliary beat frequency and cAMP (represented by the FRET ratio). Apical application of forskolin (1 microM, 10 microM, 20 microM) and, in permeabilized cells, basolateral cAMP (20 microM, 50 microM, 100 microM) caused dose-dependent, albeit similar and simultaneous-increases in cAMP and ciliary beat frequency. However, decreases in cAMP preceded decreases in ciliary beat frequency, suggesting that either cellular cAMP decreases before ciliary cAMP or the dephosphorylation of target proteins by phosphatases occur at a rate slower than the rate of cAMP hydrolysis.

Roles of Ca2+and protein kinase C in the excitatory response to serotonin in embryonic molluscan ciliary cells

We examined the roles of Ca2+ and protein kinase C (PKC) in the cilio-excitatory response to serotonin in pedal ciliary cells from Helisoma trivolvis embryos. Serotonin (5-hydroxytryptamine; 5-HT; 100 micromol/L) induced an increase in ciliary beat frequency (CBF) was abolished by microinjected BAPTA (50 mmol/L), but was only partially inhibited by the phospholipase C inhibitor U-73122 (10 micromol/L). The diacylglycerol analogs 1-oleoyl-2-acetyl-sn-glycerol (100 micromol/L) and 1,2-dioctanoyl-sn-glycerol (100 micromol/L) caused increases in [Ca2+]i that were smaller than those induced by serotonin. In the absence of extracellular Ca2+, 1,2-dioctanoyl-sn-glycerol (100 micromol/L) failed to elicit an increase in both CBF and [Ca2+]i. In contrast, the serotonin-induced increase in CBF persisted in the absence of extracellular Ca2+, although the increase in [Ca2+]i was abolished. PKC inhibitors bisindolylmaleimide (10 and 100 nmol/L) and calphostin C (10 nmol/L) partially inhibited the serotonin-induced increase in CBF, but didn't affect the serotonin-induced change in [Ca2+]i. These findings suggest that an intracellular store-dependent increase in [Ca2+]i mediates the cilio-excitatory response to serotonin. Furthermore, although PKC is able to cause an increase in [Ca2+]i through calcium influx, it contributes to the cilio-excitatory response to 5-HT through a different mechanism.

Altered Sinonasal Ciliary Dynamics in Chronic Rhinosinusitis

Although multiple etiologies contribute to the development of rhinosinusitis, a common pathophysiological sequelae is ineffective sinonasal mucociliary clearance, resulting in stasis of sinonasal secretions, with subsequent infection, and persistent inflammation. The respiratory cilia beat continually at a basal rate, while during times of stress, such as exercise or infection, ciliary beat frequency (CBF) increases, accelerating mucus clearance. Previous investigations have led to conflicting results with some authors reporting decreased CBF while others have found normal values of CBF in patients with chronic rhinosinusitis (CRS). Additionally, these studies have only analyzed basal CBF. The goal of this study was to compare the basal as well as the stimulated sinonasal CBF in patients with CRS versus controls. A dual temperature controlled perfusion chamber, differential interference contrast microscopy, and high-speed digital video were used to analyze both basal and adenosine triphosphate (100 microM)-stimulated CBF in human sinonasal mucosal explants. Although no difference in basal CBF was detected between control and CRS patients, a marked difference in stimulated CBF was noted. Exogenously applied adenosine triphosphate resulted in a 50-70% increase of CBF in control tissue with a minimally observed CBF increase in explants from CRS patients. Dynamic regulation of respiratory ciliary activity is critical for the respiratory epithelium to adapt to varying environmental situations. Thus, diminished or absent adaptation could predispose the sinonasal cavity to accumulation of inhaled infectious and noxious particulate matter resulting in infection/inflammation. Our findings suggest that CRS patients have decreased sinonasal ciliary adaptation to environmental stimuli.