Decrease In Ciliary Beat Frequency Research Articles

The effects of benomyl and its breakdown products carbendazim and butyl isocyanate on the structure and function of tracheal ciliated cells.

The effects of the fungicide benomyl and its breakdown products, carbendazim and butyl isocyanate, were examined on canine tracheal epithelial tissue in primary culture. Changes in ciliary frequencies were monitored with an optical spectrum analysis system. Serial dilutions of the test compounds were prepared in 100% corn oil and applied to the cell cultures for intervals up to 6 hours and frequencies measured at intervals of 15 minutes to 1 hour. Benomyl and butyl isocyanate caused concentration-dependent decreases in ciliary beat frequency. Benomyl at 300 micrograms/ml (3 mM) caused ciliostasis within 75 minutes of exposure. Butyl isocyanate at a molar concentration three times lower than benomyl (1 mM) caused a similar response, although within 30 minutes. The IBC50 for benomyl was 0.75 mM, while for butyl isocyanate it was 0.52 mM. Carbendazim caused a moderate decrease in frequency over a 6 hour exposure period. Benomyl caused moderate to severe swelling of the mitochondria of ciliated epithelial cells with other cell organelles appearing normal. Butyl isocyanate did not cause any noticeable effect on cell ultrastructure and the apparently low rate of penetration of carbendazim into cells made it impossible to obtain an effect which justified ultrastructural analysis. It appears, at least for benomyl and butyl isocyanate, that while the physiological effect of these two compounds (inhibition of ciliary beat) is the same, the sites of action in the cell may be different.

Polymorphonuclear leukocyte-generated oxygen metabolites decrease beat frequency of human respiratory cilia.

We investigated the effect of polymorphonuclear leukocyte (PMN)-generated oxygen metabolites on the ciliary beat frequency. PMNs were incubated with human respiratory cilia obtained by nasal brushing. The oxidative metabolism was stimulated by opsonized zymosan, and ciliary beat frequency was evaluated before and after activation of PMNs. Ciliary beat frequency was studied using video microscopy. Our results demonstrate a significant decrease in ciliary beat frequency after activation of PMNs. This effect was reduced by catalase. These data suggest that the PMN-generated oxygen metabolites, particularly H2O2, decrease beat frequency of human respiratory cilia.

Effect of sulfur dioxide on mucociliary activity and ciliary beat frequency in guinea pig trachea.

The effects of 30 min exposure to sulfur dioxide on mucociliary activity (MCA) and ciliary beat frequency (CBF) were studied in 31 guinea pig tracheas. MCA was measured by recording the light reflected from ciliated mucous membranes using an infrared bar code reader. CBF of single ciliated cells obtained by brushing was measured with phase-contrast microscopy. Each tracheal sample was exposed to SO2 at concentrations ranging from 2.5 to 12.5 ppm, or to air for control purposes. MCA and CBF were measured before and immediately after gas exposure. A reduction in mean MCA of 63% (P = 0.0007) and statistically insignificant changes in CBF (P > 0.05) were recorded at concentrations of 2.5 ppm SO2. Higher SO2 concentrations caused a further impairment of MCA as well as a dose-dependent decrease in CBF (P = 0.002). A concentration of 12.5 ppm SO2 induced a decrease from baseline values of approximately 80% in mean MCA and of roughly 70% in mean CBF. This study demonstrates a dose-dependent SO2-induced decrease in MCA of guinea pig tracheas. The decrease in MCA was associated with an impairment of CBF only at SO2 concentrations higher than 5.0 ppm.

An experimental model for the exposure of human ciliated cells to sulfur dioxide at different concentrations

Mucociliary transport is an important nonimmunological defense mechanism of the respiratory tract. The aim of this study was to investigate the effect of sulfur dioxide (SO2) at different concentrations on ciliary beat frequency (CBF). Ciliated cells were obtained from 12 volunteers by nose brush. CBF was quantified using video-interference microscopy. The cells were placed on a polycarbonate membrane in contact with the surface of a reservoir filled with RPMI 1640 (bicarbonate buffered) or Ringer's (electrolyte) solution, allowing the cells to be supplied by capillarity. In an exposure chamber the cells were exposed for 30 min to SO2 2.5-12.5 ppm at 37 degrees C and 100% air humidity. SO2 induced a dose-dependent decrease in CBF of the cells cultured in Ringer's solution. SO2 at 2.5 ppm caused a 42.8% decrease and at 12.5 ppm a 96.5% decrease (8.1 +/- 0.24 versus 0.28 +/- 0.20 Hz). CBF of cells cultured in RPMI 1640 was reduced only moderately after 12.5 ppm SO2 exposure (7.9 +/- 0.26 versus 6.70 +/- 0.30 Hz). In Ringer's solution a decrease in pH was observed after 30 min of SO2 exposure to 12.5 ppm to a minimum value of 3.6. By contrast, the pH of RPMI 1640 remained constant at 7.5 under identical conditions. After adding RPMI 1640 to Ringer's solution, CBF increased in parallel to the pH to control values (5.0 ppm: 4.64 +/- 0.45 to 8.51 +/- 0.60 Hz). These data suggest that the highly water-soluble SO2 reversibly eliminates CBF in correlation with a decrease in pH.

Protein kinase C-dependent phosphorylation of a ciliary membrane protein and inhibition of ciliary beating.

The present study examined whether protein kinase C phosphorylated a ciliary protein and whether this phosphorylation event was temporally correlated with a decrease in ciliary beat frequency. Activation of protein kinase C decreased ciliary beat frequency of sheep tracheal epithelium, an effect fully blockable by pretreatment of the tissue pieces with H-7, a protein kinase inhibitor. Using cilia removed from these epithelial surfaces and incubated in solutions containing stimulators of protein kinase C along with [gamma-32P]ATP or [gamma-35S]ATP, a single protein target of ciliary protein kinase C activity was identified. The protein is a polypeptide of molecular mass 37 kDa (p37) as estimated by SDS-polyacrylamide gel electrophoresis. Protein kinase C dependency of p37 phosphorylation was proven by showing that Calphostin C, a specific protein kinase C inhibitor, blocked label incorporation into p37 completely, and by demonstrating that purified protein kinase C phosphorylated p37. Inhibitors of cAMP-dependent kinase and calcium/calmodulin-dependent kinase did not change the phosphorylation of p37 in the presence of protein kinase C activators. p37 was recovered in a Triton X-100-extractable fraction of this ciliary preparation, suggesting that p37 is membrane associated. This hypothesis was further supported by the fact that p37 was present in a pellet representing reconstituted membranes. Thin-layer electrophoresis revealed that p37 was phosphorylated on serine and tyrosine residues, suggesting that the activation of protein kinase C also stimulated tyrosine kinase activity. p37 did not precipitate with annexin I or II antibodies. These results show that sheep tracheal cilia contain protein kinase C activity and that activated protein kinase C phosphorylates a membrane-associated ovine ciliary target, an effect temporally related to a protein kinase C-mediated decrease in ciliary beat frequency.

Mechanism of hydrogen peroxide-induced inhibition of sheep airway cilia.

To study the effect of the inflammatory mediator hydrogen peroxide (H2O2) on airway ciliary activity, we measured ciliary beat frequency (CBF) in cultured tracheal explants from sheep. Addition of H2O2 (10(-8) to 10(-4) M) produced a concentration-dependent mean (+/- SEM) decrease in CBF between 11.1 +/- 0.4% (P less than 0.01) and 100 +/- 0% (P less than 0.001); at each concentration, the maximal effect was reached by 20 to 25 min. Between 10(-8) and 10(-6) M H2O2, the decrease in CBF was reversible, lactate dehydrogenase (LDH) release was not significantly increased, and major morphologic lesions were not seen. At higher concentrations of H2O2, incomplete recovery of CBF (10(-5) M) or irreversible ciliostasis (10(-4) M) developed, and a significant increase in LDH and morphologic lesions were present. Catalase (2,000 U/ml) and H-7 (10(-5) M), a protein kinase inhibitor, abolished cilioinhibition produced by H2O2 at 10(-6) M and lower concentrations but not at 10(-5) M and higher concentrations. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, caused a dose-dependent (10(-11) to 10(-5) M), reversible decrease in CBF; this effect was abolished by H-7. We suggest that at nonlethal concentrations, H2O2 inhibits the beat frequency of airway epithelial cilia reversibly, through the activation of second messengers, including protein kinase C. This mechanism might contribute to the previously demonstrated impairment of mucociliary clearance in airway inflammation.

Influence of levocabastine suspension on ciliary beat frequency and mucociliary clearance.

The effects of levocabastine, a new fast-acting, highly potent H1-antagonist, on nasal ciliary epithelial function were investigated in an in vitro and in vivo study. In the in vitro study, a suspension of levocabastine in Locke-Ringer solution was applied to 10 bioptic specimens of ciliated human adenoid tissue. Each specimen was exposed to the test solution for 60 min. Ciliary beat frequency (CBF) was recorded with a photoelectric recording device at 10-min intervals. There were small, insignificant decreases in CBF, which were minimal compared to that observed with ciliotoxic agents. In the in vivo study, 8 healthy volunteers were given, intranasally, one droplet of the levocabastine suspension. Mucociliary transit time (MTT) was measured by placing a saccharin particle drenched in indigo carmine in the nose just below the top of the concha and measuring the time until appearance of the dye in the pharyngeal cavity. No statistically significant differences were found in the MTT before and after application of the levocabastine suspension. The studies thus indicate that nasally administered levocabastine does not interfere with ciliary beat frequency and mucociliary function.

Nasal Ciliary Beat Frequency after Nasal Allergen Challenge

The aim of this study was to estimate nasal ciliary beat frequency (CBF) before and after nasal allergen challenge. Patients were challenged with Pharmalgen D Pteronyssinus or grass pollen. The nasal reaction was measured with active anterior rhinomanometry and visual analog scale. CBF was measured by a photometric technique, on nasal specimens obtained with a brush from the inferior turbinate before and 20 minutes after challenge. A positive response to nasal challenge was seen in 12 patients and associated with a decrease in CBF in all but one patient. The CBF mean value for this group fell from 13.1 (±1.9) Hz to 11.5 (±1.8) Hz (p < .01). In another 11 patients where the challenge was negative, there was a tendency to increased CBF after challenge, 14.1 (±2.1) Hz before and 14.8 (±2.1) Hz after (p = .07). Thus, in this study CBF was decreased when an allergic reaction was provoked and, in contrast, moderately increased after a negative challenge. In a nasal allergic reaction it is possible that decreased mucociliary function contributes to nasal obstruction and decreased ventilation of the sinuses.

Leukotriene C4 decreases the activity of respiratory cilia in vitro

In the present study we assessed the effect of leukotriene C4 on ciliary beat frequency in vitro. Chicken tracheas were sliced into thin rings and ciliary activity was viewed microscopically. Ciliary beat frequency was measured through an optical fibre by a fast Fourier transformer analyser and recorded on an oscilloscope. Ciliary beat frequency was measured at one fixed point during 30 min at room temperature in RPMI-HEPES medium alone and in medium containing leukotriene C4 at a range of concentrations (10(-6)-10(-9) M). Our results demonstrate that leukotriene C4 (10(-7) and 10(-8) M) caused a significant decrease in ciliary beat frequency in all tracheal rings tested (mean decrease of 24%). The specificity and Ca2(+)-dependency of leukotriene C4 activity was elucidated by the ability of FPL 55712 (SRS-A specific antagonist), Ca2(+)-free medium, calcium channel blockers (nifedipine and verapamil) and the calmodulin inhibitor trifluroperazine to abrogate its effect on ciliary beating.

Inhibition of ciliary activity by phorbol esters in rabbit tracheal epithelial cells

To study the effect of protein kinase C activation on respiratory ciliary activity, we measured ciliary beat frequency (CBF) by a photoelectric technique in response to phorbol esters and cell-permeable diglyceride in cultured tracheal epithelial cells from rabbits. Phorbol 12-myristate 13-acetate (PMA) resulted in a concentration- and time-dependent inhibition of CBF (half maximum inhibitory concentration (IC50) = 3 x 10(-10) M) with the maximal decrease being 21.0 +/- 1.4% (mean +/- SE, p less than 0.001) observed at 10(-6) M. L-alpha-dioctanoylglycerol (DiC8), another known activator of protein kinase C, likewise reduced CBF in a dose-dependent fashion. In contrast, phorbol 12,13-didecanoate, a non-tumor-promoting phorbol ester that does not stimulate protein kinase C, produced no significant changes in CBF. The decrease in CBF induced by PMA was not affected by blockade of arachidonic acid metabolism with indomethacin and nordihydroguaiaretic acid, but was antagonized by pretreatment with H-7, a specific inhibitor of protein kinase C (p less than 0.01). Maximal ciliary inhibition with either PMA or DiC8 was not accompanied by a decrease in intracellular concentration of cyclic AMP. These results indicate that activation of protein kinase C has a significant depressive effect on ciliary activity, and hence the airway mucociliary transport function, presumably through a regulatory pathway that is not dependent on cyclic AMP or arachidonic acid metabolites.

Adenosine-mediated Cyclic AMP-dependent Inhibition of Ciliary Activity in Rabbit Tracheal Epithelium

We wished to determine whether adenosine, a purine nucleotide, modulates activity of respiratory cilia and, to this end, we studied cultured rabbit tracheal epithelium in response to adenosine and related substances in vitro. Ciliary beat frequency (CBF) as determined by a photoelectric method was depressed by adenosine (10(-3) M), the maximal decrease from the baseline value (965 +/- 29 beats/min, mean +/- SE) being 31.6 +/- 5.0% (p less than 0.001). The adenosine A2-receptor agonist N-ethylcarboxamide adenosine had only a small effect on ciliary activity, whereas other adenosine analogs elicited decreases in CBF in a dose-dependent fashion. The order of potency of cilia-inhibitory action was N-cyclohexyladenosine (an agonist for adenosine A1-receptor) greater than phenylisopropyladenosine greater than adenosine greater than N-ethylcarboxamide adenosine. Intracellular cyclic AMP (cAMP) levels were decreased by 10(-3) M adenosine from 39.2 +/- 6.5 to 25.3 +/- 4.8 pM/mg protein (p less than 0.05). The effect of adenosine on CBF was enhanced by dipyridamole, an adenosine uptake inhibitor, and by deoxycoformycin, an adenosine deaminase inhibitor. The adenosine-induced decreases in CBF and cAMP content were reversed by 8-phenyltheophylline, an adenosine receptor antagonist. These results suggest that there is an adenosine A1-receptor on rabbit tracheal epithelium that inhibits adenylate cyclase, which may result in the impairment of respiratory ciliary activity, and that adenosine-induced ciliary inhibition may be modulated by adenosine uptake and its catabolism by airway epithelial cells.

Oxidant-Mediated Ciliary Dysfunction: Possible Role in Airway Disease

The effects of reactive species of oxygen on the airway are not well known. This study examined the effects of hydrogen peroxide (H2O2) on the structure and function of the airway epithelium. Tracheal rings were prepared from 200 g male rats. Damage to the airway epithelium was assayed by monitoring the ciliary beat frequency, the release of 51Cr, and histology. H2O2 at concentrations of 1.0 mM and above caused a very rapid decrease in ciliary beat frequency. After ten minutes' exposure to 1.0 mM, the ciliary beat frequency was 72 +/- 20 percent of control. Release of 51Cr was a less sensitive measure with significant release occurring after four hours of exposure to ciliotoxic concentrations of H2O2. Histologic changes were not evident within the experimental time period. All toxic effects of H2O2 were completely blocked by catalase. This study shows that H2O2 causes a rapid decline in ciliary activity and suggests that oxidant-mediated ciliary dysfunction could play a role in the pathogenesis of airway disease. The ciliary beat frequency provides a sensitive, physiologically relevant parameter for the in vitro study of these diseases.

Decreases in Ciliary Beat Frequency Due to Intranasal Administration of Propranolol

Recently the intranasal application of 5% propranolol was proposed in order to prevent the extensive first-pass metabolism of this drug. The ciliary epithelium in the nose affects the removal of dust, allergens, and microorganisms. The decreasing effect of propranolol on the ciliary beat frequency of human adenoid tissue and chicken embryo tracheas was measured with a photoelectric registration device. After nasal application of 5% propranolol, the drop is diluted by the nasal mucus. It was found that even 0.1% propranolol had a deleterious effect on the cilia of chicken and human tissue. Ciliary movement was arrested irreversibly within 20 min.

Role of ciliary motility in acute allergic mucociliary dysfunction

The purpose of this investigation was to determine if abnormal ciliary function contributes to allergic mucociliary dysfunction. In conscious sheep with Ascaris suum hypersensitivity, ciliated cells were obtained with a cytology brush and tracheal mucous velocity (TMV) was determined before and serially for 2 h following antigen inhalation. The recovered cells (also containing mast cells) were suspended in a chamber, and ciliary activity was viewed microscopically and recorded on videotape for subsequent slow-motion analysis of ciliary beat frequency (CBF). One hour after A. suum challenge mean CBF (+/- SE) showed a slight increase from a base-line value of 630 +/- 16 to 716 +/- 30 beats/min (P less than 0.05) when mean TMV was decreased to 57% of base line (P less than 0.05). After 2 h, both mean CBF and TMV returned toward base line. Since possible in vivo actions of chemical mediators liberated by antigen challenge may have been lost after suspension of the brushed cells, we also assessed the effects of antigen on CBF in vitro. A. suum caused a dose-dependent increase in CBF that was blocked by cromolyn sodium. We conclude that 1) allergic mucociliary dysfunction is not caused by a decrease in CBF and 2) antigen-induced release of chemical mediators increases CBF.