Procaterol-stimulated increases in ciliary bend amplitude and ciliary beat frequency in mouse bronchioles

The beating cilia play a key role in lung mucociliary transport. The ciliary beating frequency (CBF) and ciliary bend amplitude (CBA) of isolated mouse bronchiolar ciliary cells were measured using a light microscope equipped with a high-speed camera (500 Hz). Procaterol (aβ(2)-agonist) increased CBA and CBF in a dose dependent manner via cAMP. The time course of CBA increase is distinct from that of CBF increase: procaterol at 10 nM first increased CBA and then CBF. Moreover, 10 pM procaterol increased CBA, not CBF, whereas 10 nM procaterol increased both CBA and CBF. Concentration-response studies of procaterol demonstrated that the CBA curve was shifted to a lower concentration than the CBF curve, which suggests that CBA regulation is different from CBF regulation. Measurements of microbead movements on the bronchiole of lung slices revealed that 10 pM procaterol increased the rate of ciliary transport by 37% and 10 nM procaterol increased it by 70%. In conclusion, we have shown that increased CBA is of particular importance for increasing the bronchiolar ciliary transport rate, although CBF also plays a role in increasing it.     

Automated measurement of ciliary beat frequency

Measurements of ciliary beat frequency using video images are dependent on observer interpretation. To obtain objective estimates of ciliary beat frequency from video-image sequences, a computer-based method was developed. Regions of interest of video-image sequences were selected and digitized. Variations in numerical values representing light intensity resulting from cilia beating were extracted and analyzed using autocorrelation techniques. The ciliary beat frequencies obtained for 14 in vitro experiments on ciliated cells or epithelium from the frog palate (Rana catesbeiana) over the range of frequencies 2-25 Hz correlated well with independent observer measurements (r = 0.979). The addition of such computer-based methods to video observer-based systems allows more objective and efficient determinations of ciliary beat frequency.     

The control of ciliary beat frequency

Ciliary movement is powered by axonemal dynein. This article considers how a signal transduction cascade initiated at the cell membrane may activate outer dynein arms to change the velocity of microtubule sliding and the swimming speed of ciliated cells. For Paramecium, a critical event in the cascade is the cAMP-dependent phosphorylation of a 29 kDa polypeptide that is associated with the outer dynein arm.     

Stimulation of tracheal ciliary beat frequency by capsaicin

To determine the possible involvement of neural and cyclooxygenase pathways whereby irritants might affect cilia activity in vivo, the temporal response of canine tracheal ciliary beat frequency (CBF) to the inhaled surrogate irritant capsaicin was studied. CBF was measured on the ventral midtracheal surface of barbiturate-anesthetized eucapnically ventilated beagle dogs by heterodyne-mode laser light scattering. After base-line CBF was established, hexamethonium bromide (2 mg/kg iv), ipratropium bromide (0.5 microgram/kg iv), indomethacin (2 mg/kg iv), or intravenous 0.9% saline was administered. Aerosolized 3 Z 10(-9) M capsaicin in 0.9% saline was delivered for 2 min, and CBF was measured for the following 60 min. Control experiments used 0.9% saline sham aerosol with a 0.9% saline sham block. Aerosolized capsaicin stimulated CBF from a base line of 6.2 +/- 1.4 (SD) Hz (n = 230) to a mean maximum of 17.7 +/- 7.3 Hz (n = 16) 23 min after aerosol delivery, and CBF returned to base line within 60 min. Neither hexamethonium bromide, ipratropium bromide, nor indomethacin changed CBF from base-line values. The episodic CBF stimulatory response to capsaicin after commencement of aerosol was completely inhibited by hexamethonium bromide. Ipratropium bromide partially inhibited the first 15 min and totally inhibited the following 45 min of stimulatory response. Indomethacin inhibited the initial 15 min but had less effect on the following 45 min of stimulatory response. These data indicate that multiple stimulatory mechanisms function over a prolonged period of time to affect the removal of irritants from the airways and that these mechanisms differ from those involved in the maintenance of basal CBF.     

Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells

Objectives

Our purpose was to investigate the role of the nitric oxide (NO) signaling pathway in the regulation of ciliary beat frequency (CBF) in mouse nasal and tracheal epithelial cells.

Methods

We studied the effects of the NO donor l-arginine (L-Arg) and specific inhibitors of the NO signaling pathway on CBF of both nasal and tracheal epithelial cells by using high-speed digital microscopy. We also examined eNOS, sGC β, PKG I and acetylated α tubulin expression in native mouse nasal and tracheal epithelium using immunohistochemical methods.

Results

L-Arg significantly increased CBF of cultured nasal and tracheal epithelial cells, and the effects were blocked by pretreatment with N^G-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, with LY-83583, a sGC inhibitor, or with KT-5823, a PKG inhibitor. Positive immunostaining for NO signaling molecules including eNOS, sGC β and PKG I was observed in either nasal or tracheal ciliated epithelium.

Conclusion

NO plays a role in regulating CBF of mouse respiratory epithelial cells via a eNOS–NO–sGC β–cGMP–PKG I pathway.     

Factors affecting ciliary beat frequency in the intrapulmonary airways of rats.

The influence of drugs on ciliary beat frequency in the intrapulmonary airways of rats was studied in vitro. Sympathomimetic drugs significantly increased the ciliary beat frequency. Acetylcholine, pilocarpine, aminophylline, prednisolone, potassium iodide, and ammonium chloride also caused cilioexcitation, though to a lesser degree. Phentolamine, 5-hydroxytryptamine, histamine, and acetylcysteine caused an apparent cilioinhibition. However, histamine in high doses also caused an incoordination of ciliary beat. Codeine and phenobarbital were slightly cilioexcitatory in low concentrations and ciliodepressive at higher concentrations.     

Influence of rheological properties of human bronchial secretions on the ciliary beat frequency

We studied the relations between the mucociliary beat frequency (MF) measured photometrically on the depleted frog palate and the rheological properties of sputum collected in patients with chronic obstructive pulmonary disease. MF was lower (p less than 0.001) with sputa (11.3 +/- 3.3 Hz) than with frog mucus (16.9 +/- 3.3 Hz) used as controls. The relative transport rate (Tr) of sputa was closely correlated (r = 0.81, p less than 0.001) to the relative MF. Significant correlations were observed between MF on the one hand and viscosity (r = -0.68, p less than 0.01), elastic modulus (r = -0.70, p less than 0.01) and spinability (r = +0.49, p less than 0.05) on the other. These results suggest that abnormalities in the rheological properties of bronchial secretions may impair the mucociliary transport rate by first decreasing the ciliary beat frequency.     

Stimulation of ciliary beat frequency by autonomic agonists: in vivo

beta 2-Adrenergic bronchodilator and muscarinic cholinergic bronchoconstrictor agonists both stimulate ciliary activity in vitro. To test the hypothesis that increases in autonomic activity would result in increases in ciliary beat frequency (CBF) in vivo, a correlation analysis heterodyne laser light-scattering system was developed and validated to measure the stimulating effects of sympathomimetic and parasympathomimetic agonists on tracheal CBF in intact, anesthetized beagles. The mean baseline CBF from 42 studies of 274 measurements in 9 (5 male and 4 female) adult beagles was 6.6 +/- 1.1 Hz. The stimulating effects of a beta 2-adrenergic agonist, fenoterol, and a muscarinic cholinergic agonist, methacholine, on CBF were studied on four and eight beagles, respectively. The studies were randomized and blinded. Aerosolized 10(-5) M fenoterol stimulated the CBF from the base line of 6.8 +/- 2.5 to 32.0 +/- 17.9 Hz in four dogs. Aerosolized methacholine stimulated the CBF from the base line of 5.8 +/- 0.7 to 9.4 +/- 3.0 Hz for 10(-8) M, and to 12.6 +/- 3.1 Hz for 10(-6) M in eight dogs. These are the first data obtained in intact animals that demonstrate CBF in the lower respiratory tract is regulated by autonomic agonists.     

Stimulation of tracheal ciliary beat frequency by localized tissue incision

Since measurements of basal ciliary beat frequency (CBF) were significantly lower in our intact canine experiments than reports of ciliary activity in rabbits involving surgical intervention, we hypothesized that local tissue trauma stimulates CBF. The effects of minor neck surgery on tracheal CBF in eight barbiturate-anesthetized eucapnically ventilated beagles were investigated. Each dog underwent two studies. Measurements of CBF were made at 1-min intervals on the right lateral midtracheal surface by means of heterodyne mode correlation analysis laser light scattering. In the control study, CBF was measured in each dog for at least 160 min. In the incision study, base-line CBF was measured for at least 40 min. The overlying sternohyoidus muscles were then separated, and a longitudinal 2- to 3-cm incision was made in the trachea caudally from the fourth to the fifth cartilage ring. CBF was measured at least 5 cm distally from the site of tracheal injury for an additional 120 min. Electrocardiogram, rectal temperature, tracheal pressure, exhaled CO2, and arterial blood pressure, PO2, PCO2, and pH remained stable throughout both studies. The mean base-line CBF was 4.7 +/- 0.4 Hz. It increased to 19.5 +/- 2.9 Hz (P less than 0.0001) 100 min after the incision and remained elevated until the end of the study period (P less than 0.0001). The mechanism(s) causing this stimulation may also be responsible for the high "basal" CBF observed in other studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of ciliary beat frequency by autonomic mechanisms: in vitro

The ciliated epithelium of the mammalian trachea separates the neurohumoral milieu of the tissue from that of the environment of the airway lumen. To determine whether specific autonomic receptors regulating ciliary beat frequency (CBF) were located on mucosal or serosal sides, we measured CBF by heterodyne mode correlation analysis laser light scattering in bovine tracheal tissues mounted in a two-sided chamber. A beta 2-adrenergic agonist, fenoterol, at 10(-7) M, stimulated serosal CBF from 7.9 +/- 1.3 to 20.2 +/- 5.8 Hz (P less than 0.01) and mucosal CBF from 6.6 +/- 0.9 to 14.7 +/- 4.6 Hz (P less than 0.01). A muscarinic cholinergic agonist, methacholine, at 10(-7) M, increased mucosal CBF from 8.4 +/- 1.0 to 19.5 +/- 5.5 Hz (P less than 0.01) and serosal CBF from 8.0 +/- 0.9 to 15.4 +/- 5.0 Hz (P less than 0.01). The differences in stimulation of CBF on the mucosal and serosal sides between fenoterol and methacholine were significant (P less than 0.01). Studies in which these autonomic agonist stimulating effects were inhibited by their respective antagonists, propranolol and atropine sulfate, demonstrated that CBF can be regulated independently by mediators both in the submucosa and within the mucus lining.     

The effect of cryopreservation on ciliary beat frequency of human respiratory epithelium

The effect of cryopreservation on ciliary activity of human nasal respiratory epithelium was evaluated. Samples were cryopreserved in a solution containing nutrient medium, 10% fetal calf serum, and two different concentrations (10 or 20%) of dimethyl sulfoxide and stored in liquid nitrogen at -196 degrees C for 2 weeks. Ciliary beat frequencies (CBF) of the samples before and after cryopreservation were compared. Mean CBF values did not differ significantly with both concentrations of dimethyl sulfoxide. The mean intrasample coefficient of variation of the CBF decreased significantly after cryopreservation. After thawing, CBF remained unchanged for at least 4 hr. It is concluded that normal ciliated epithelial cells can be frozen and stored in liquid nitrogen at 196 degrees C while maintaining their CBF.     

Hysteresis modeling for calcium-mediated ciliary beat frequency in airway epithelial cells

A hysteresis model is proposed to describe calcium-mediated Ciliary beat frequency (CBF) in airway epithelial cells. In this dynamic model, the kinetics of coupling between calcium and CBF is posited as a two-step configuration. First, Ca²⁺ directly binds to or indirectly acts on the axonemal proteins to modulate the activity of axonemal proteins. This step can be modeled by a Hill function in biochemistry. In the second step, the activity of axonemal proteins interacts with the sliding velocity of axonemal microtubules, the equivalent to regulating the CBF. The well-known Bouc-Wen model for hysteresis in mechanical engineering, which can only generate the stable clockwise hysteresis loops, is modified to describe the counter clockwise hysteresis loops commonly observed in the biological experiments. Based upon this new hysteresis model, the dynamic behavior of calcium-regulated CBF in epithelial airway cells is investigated through simulation studies. The numerical results demonstrate that the CBF dynamics in airway epithelial cells predicted by the hysteresis model is more consistent with the experimental observations than that predicted by previous static model in the literature.     

Intracellular calcium oscillations regulate ciliary beat frequency of airway epithelial cells

The effect of ATP-induced Ca2+ oscillations on ciliary activity was examined in airway epithelial cells by simultaneously measuring the ciliary beat frequency (CBF) and the intracellular Ca2+ concentration ([Ca2+]i) near the base of the cilia. Exposure to extracellular ATP (ATPo) induces a rapid and large increase in both [Ca2+]i and CBF, followed by oscillations in [Ca2+]i and a sustained elevation in CBF. After each Ca2+ oscillation, the [Ca2+]i returned to near basal values. By contrast, the CBF remained elevated during these Ca2+ oscillations, although each Ca2+ oscillation induced small variations in CBF. During Ca2+ oscillations, increases in CBF closely followed the rising phase of increases in [Ca2+]i, but declines in CBF lagged behind declines in [Ca2+]i. Higher frequency Ca2+ oscillations reduced variations in CBF, producing a stable and sustained elevation in CBF. The maximal CBF was induced by Ca2+ oscillations and was 15% greater than the CBF induced by the substantially larger initial [Ca2+]i increase. These data demonstrate that the rate of CBF is not directly dependent on the absolute [Ca2+]i, but is dependent on the differential changes in [Ca2+]i and suggest that CBF in airway epithelial cells is regulated by frequency-modulated Ca2+ signaling.     

Effects of albuterol enantiomers on ciliary beat frequency in ovine tracheal epithelial cells.

beta(2)-Adrenergic agonists stimulate ciliary beat frequency (CBF), an integral part of mucociliary clearance. To evaluate the differential effects of albuterol enantiomers and their racemic mixture on ciliary function, CBF and intracellular calcium were measured at room temperature from single ovine airway epithelial cells with use of digital videomicroscopy. Baseline CBF was 7.2 +/- 0.2 (SE) Hz (n = 80 measurements). R-albuterol (10 microM to 1 mM) stimulated CBF in a dose-dependent manner to maximally 24.4 +/- 5.4% above baseline. Racemic albuterol stimulated CBF to maximally 12.8 +/- 3.6% above baseline, a significantly lower increase compared with R-albuterol alone, despite identical R-enantiomer amounts in both groups. Simultaneous recordings of intracellular calcium concentration and CBF from single cells indicated that the CBF increase in response to R-albuterol was mediated through beta-receptors and stimulation of protein kinase A, in a calcium-dependent and -independent fashion. S-albuterol had a negligible effect on CBF and did not change intracellular calcium. Together, these results suggest that R-albuterol is more efficacious than racemic albuterol in stimulating CBF. Thus S-albuterol may interfere with the ability of R-albuterol to increase CBF.     

Injected cyclic AMP increases ciliary beat frequency in conjunction with membrane hyperpolarization

Injections of cyclic AMP (cAMP) and 8-Br-cAMP into Paramecium and external application of isobutylmethylxanthine (IBMX), an inhibitor of cAMP breakdown, to these cells increased the frequency of ciliary beating and hyperpolarized the membrane potential. When the membrane potential was held equal to the resting potential under voltage clamp, the same experimental conditions which serve to increase intracellular cAMP did not raise the ciliary frequency. We conclude that cAMP is presumably not the direct mediator of the hyperpolarization-induced ciliary activation, although it may be associated with this motor response.     

Temperature effect on the ciliary beat frequency of human nasal and tracheal ciliated cells.

Even though all human respiratory cilia are similar in structure, they experience a wide range of temperatures between the initial part of the nasal fossae which behave as heat exchangers and the inferior part of the trachea, particularly when we inhale exceedingly cold or hot air. The ciliary beat frequency of ciliated cells from human nasal mucosa and from bronchial mucosa averages 8 Hz when measured at room temperature. In the present study we compared the ciliary beat frequency of human cells from nasal and tracheal mucosa brushings at different temperatures from 5 degrees C to 50 degrees C using two different techniques, ex vivo and in vitro: ex vivo in culture medium less than 24 h after sampling and in vitro after demembranation and reactivation according to a standard procedure developed in our laboratory. Measuring the ATP-reactivated ciliary beat frequency allowed us to check the thermal parameters of the dynein ATPase and all the axonemal machinery. No significant difference in frequency was observed between nasal fossae cilia and tracheal cilia when comparing extreme temperatures in both experimental procedures.     

Pathways of substance P stimulation of canine tracheal ciliary beat frequency.

Substance P (SP), an inflammatory neuropeptide, may be released by intraepithelial nerves in response to an irritant or inflammatory stimulus. To investigate the neural and humoral pathways mediating the response of tracheal ciliary beat frequency (CBF) to topically applied SP, CBF was measured on the ventral midtracheal surface of anesthetized beagles by using heterodyne-mode correlation analysis laser light scattering. In the first study, aerosolized SP, delivered to the lungs of eight beagle dogs, stimulated CBF in a dose-dependent manner from a baseline of 4.9 +/- 0.4 Hz to a maximum of 14.9 +/- 1.5 Hz at dose of 10(-7) M. In the second study, the tracheal lumen was isolated from the bronchial airways by inflating the cuff of an endotracheal tube near the carina. Intravenous hexamethonium bromide (2 mg/kg), ipratropium bromide (0.5 micrograms/kg), and indomethacin (2 mg/kg) were used as blocking agents to inhibit the nicotinic, muscarinic, and cyclooxygenase pathways, respectively. Aerosolized 10(-9), 10(-8), or 10(-7) M SP was delivered sequentially to the tracheal lumen for 3 min at 30-min intervals. SP caused two distinct CBF stimulatory episodes at 4 min (mean time of the maximal response) and at 18 min (mean time of the maximal response) after onset of delivery and returned to baseline after 25 min. SP stimulated CBF from the baseline of 5.1 +/- 0.4 Hz to a maximum of 14.2 +/- 2.5 Hz during the first episode (P less than 0.01) and to 10.4 +/- 0.6 Hz during the second episode (P less than 0.01) at dose of 10(-8) M. These responses were inhibited by all the blocking agents. These data suggest that SP stimulates CBF via a cyclooxygenase-dependent parasympathetic reflex.