Correlation between rheologic properties and in vitro ciliary transport of rat nasal mucus.

The relationship between mucus rheologic variables and in vitro ciliary transport was investigated in mucus samples collected from the upper airways of 30 Wistar rats. In vitro mucus transportability was determined by means of the frog palate preparation. Rheologic evaluation was done by measuring the rigidity modulus (log G*, representing the vectorial sum of viscosity and elasticity) and the loss tangent (tan delta, i.e. the ratio between viscosity and elasticity) at 1 and 100 radian/s using a magnetic microrheometer. The correlation between the rheologic variables and in vitro mucus transportability was made by stepwise multiple linear regression analysis, with frog palate transport rate considered as the dependent variable. A significant relationship was obtained between the rheologic parameters (log G* and tan delta) measured at 1 radian/s and the frog palate transport ratio. The relative speed of mucus samples was related to rheology according to the following relationship: rat/frog speed ratio = 1.666-0.434 log G*-0.331 tan delta, for G* and delta determined at 1 radian/s (multiple r = 0.666, p < 0.001). Transport rates predicted from the above formula gave a satisfactory fit to those observed in a second set of 30 rats. The present results indicate that the overall mucus impedance, as well as the ratio between viscosity and elasticity, are important in determining the efficiency of clearance. In addition, it was shown that measurements performed by applying relatively low frequency deformations are preferable for predicting ciliary transport.     

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.     

Rheological properties controlling mucociliary frequency and respiratory mucus transport

Respiratory mucus and mucosa possess highly hydrophilic structures which are difficult to preserve using standard fixative methods. The close interaction between cilia and mucus can be observed after instantaneously interrupting the ciliary movement using ultra rapid and cryosubstitution fixation methods. Mucus possess several rheological properties such as pseudoplasticity, elastothixotropy, spinability and adhesiveness. Rheological properties of mucus may control, per se, the ciliary beating frequency. By measuring the mucociliary frequency on the excised mucus-depleted frog palate of native mucus and xanthan gum using a simulant of mucus, we observed that beyond an optimal value of viscosity (close to 12 Pa.s) the mucociliary frequency and transport rate decrease in parallel. Other rheological factors such as adhesion and spinability of mucus can also be implicated in the regulation of the mucociliary transport rate.     

The role of the periciliary fluid in mucociliary flows: flow velocity profiles in frog palate mucus

Observations of flow velocity profiles over frog mucociliated palate are used to estimate viscosity, shear rate and shear stress in the periciliary flow field. The ability of cilia to generate significant shear stress at long distances and their utility as rhoeometers are examined. It is proposed that the depth of significant ciliary shear penetration into the periciliary fluid is sufficient to move mucus masses well beyond the ciliary tips, obviating the need for tip penetration where anchoring phenomena are sufficiently reduced.     

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.     

Effects of hormones and nucleotides on ciliary beating in frog esophagus and guinea pig trachea

The $\text{in vivo}$ effects of various hormones, nucleotides and related substances on the rate of ciliary beating in frog esophagus and guinea pig trachea were studied using both particle transport and photoelectric methods. Frog esophageal ciliary beating $\text{in vitro}$ was greatly accelerated by acetycholine, eserine, prostaglandin A₁ and E₂, N⁶- 2′-0-dibutyryl-adenosine--3′,5′-cyclic monophosphate, all tri- and di-phosphonucleotides (ATP, ADP, UTP, UDP, etc.), EDTA, EGTA, and calcium-free medium. Adenosine monophosphate, epinephrine, serotonin at low concentrations, 3,3′5-L-triiodothyronine, creative phosphate, and phosphoenolpyruvate were inactive or only minimally stimulatory in the frog. All substances tested, including those trachea. Furthermore, guinea pig ciliary activity was unaffected by hyperthyroidism or hypothyroidism induced in the intact animal before testing.     

Extracellular ATP induces hyperpolarization and motility stimulation of ciliary cells.

Cellular membrane potential and ciliary motility were examined in tissues cultures prepared from frog palate and esophagus epithelia. Addition of micromolar concentrations of extracellular ATP caused membrane hyperpolarization and enhanced the beat frequency. These two effects of ATP were 1) dose dependent, reaching a maximum at 10 microM ATP; 2) dependent on the presence of extracellular Ca2+ or Mg2+; 3) insensitive to inhibitors of voltage-gated calcium channels; 4) abolished after depleting the intracellular Ca2+ stores with thapsigargin; 5) attenuated by quinidine (1 mM), Cs+ (5-20 mM), and replacement of extracellular Na+ by K+; 6) insensitive to charybdotoxin (5-20 nM), TEA (1-20 microM), and apamin (0.1-1 microM); 7) independent of initial membrane potential; and 8) unaffected by amiloride. In addition, extracellular ATP induced an appreciable rise in intracellular Ca2+. Addition of thapsigargin caused an initial enhancement of the ciliary beat frequency and membrane hyperpolarization. These results strongly suggest the involvement of calcium-dependent potassium channels in the response to ATP. The results show that moderate hyperpolarization is closely associated with a sustained enhancement of ciliary beating by extracellular ATP.     

Cholinergic nerves stimulate mucociliary transport,ciliary activity,and mucus secretion in the frog palate

Summary Mucociliary transport, ciliary activity, and mucus secretion were studied in the palate of the frog Rana pipiens by direct observation, stroboscopic synchronization of ciliary beating, and histochemistry. Excised palates were studied in vitro, and intact palates were studied in vivo. Electrical stimulation of the glossopharyngeal nerve in vivo or of the palatine nerve in vitro stimulated all three activities. The effect was mimicked by acetylcholine and pilocarpine, enhanced by physostigmine, and blocked by atropine but unaffected by d-tubocurarine. Stimulation increased the number of cilia beating and their rate of beating, the number of goblet cells secreting and, for small acidic cells, the amount of mucus secreted, and the rate and extent of particle transport. The response to tactile stimulation was locally restricted in vitro but widespread in vivo. It was concluded that, although there is a low basal rate of mucus secretion and ciliary activity that is independent of nervous control, stimulation of these activities in the intact animal is mediated through the central nervous system and cholinergic nerves to the palate.Supported in part by Grant HL-16730 from the U.S. Public Health Service     

Spectral characterization of ciliary beating. Temperature dependence on spectral parameters

Ciliary beating was optically examined in tissue cultures from frog palate epithelium. Consecutive segments of the analog signal were Fast-Fourier transformed. The three main parameters which define the spectrum, position of the peak maximum (f), width of the spectral line (S.D.), and area (A) under the spectrum, were all measured as a function of temperature. These measurements were also examined as a function of the number of cilia by varying the examined area from 1.2 to 122 microns2. It was found that: all the parameters were exponentially temperature dependent; and the average frequency was independent of the number of cilia examined, while S.D. was dependent on it. On a physiological level, we demonstrated that the ciliary fluctuation in frequency is temperature dependent, increasing with increase in temperature. Moreover, it was shown that where a relatively small number of cilia were measured (d = 1.24 micron), the area A under the observed spectrum was directly proportional to the amplitude of ciliary beating. Increasing the temperature decreases the amplitude and vice versa. According to our suggested model the dependence of A on f was predicted and verified experimentally. A mathematical model which simulates the S.D. as a function of examined area and temperature is suggested. The calculated results from the model are in a good agreement with our experimental findings.     

Intensification of ciliary motility by extracellular ATP

Ciliary metachronism and motility were examined optically in tissue cultures from frog palate epithelium as a function of extracellular ATP concentration in the range of 10(-7)-10(-3) M. The main findings were: a) upon addition of ATP the metachronal wavelength increased by a factor of up to 2. b) the velocity of the metachronal wave increased by a factor of up to 5. c) the frequency of ciliary beating increased by a factor of up to 2-3, the increase being temperature insensitive in the range of 15 degrees C-25 degrees C. d) the area under the 1-second FFT spectrum decreased by a factor of up to 2.5. e) the energy of the metachronal wave is increased by a factor of up to 9.5. f) all the spectrum parameters are subject to influence by ATP, as also by ADP and AMP. However, there are pronounced differences in the various responses to them. Based on these findings, physical aspects of the rate increase of particle transport caused by addition of extracellular ATP are explained. A plausible overall chemical mechanism causing pronounced changes in ciliary motility is discussed.     

Expression of endothelial nitric oxide synthase in ciliated epithelia of rats.

Endothelial nitric oxide synthase (eNOS), originally found in the endothelium of vascular tissue, also exists in other cell types, including ciliated epithelia of airways. The eNOS is ultrastructurally localized to the basal body of the microtubules of the cilia, and nitric oxide (NO) stimulates ciliary beat frequency (CBF). We examined whether the expression of eNOS is present in ciliated cells of other organs. Western blotting analysis revealed that eNOS was expressed in the rat cerebrum, lung, trachea, testis, and oviduct. Immunohistochemical staining showed that eNOS was localized in the ciliated epithelia of airways, oviduct, testis, and ependymal cells of brain in addition to the endothelium and smooth muscle of the vasculature. To confirm the activation of eNOS in the ciliated epithelia, we examined the effect of L-arginine (L-Arg), the substrate of NOS, on the production of nitrite and nitrate (NOx) in the cultured explants of rat trachea. L-Arg (100 microM) increased NOx levels significantly (p<0.05). In explants exposed to inhibitors of NOS, the effect of l-Arg on the production of NOx was blocked. These findings suggest that epithelial NO plays an important role in signal transduction associated with ciliary functions.     

The distribution of cationized ferritin receptors on ciliated epithelial cells of rat trachea

The interaction of tracheal cilia with the biphasic mucus layer covering the surface of the mammalian respiratory tract may be influenced by many cell surface coat components including those having an overall negative charge. In order to assess the distribution of ciliary anionic sites, cationized ferritin (CF) was used to label the surface of rat tracheal epithelium. If pieces of trachea were fixed with 3% glutaraldehyde and treated with CF at low (L) (0.08 mg/ml), medium (M) (0.32 mg/ml PBS), or high (H) (0.64 mg/ml PBS) concentrations, the label was distributed evenly over the entire external surface of the ciliary membrane at all concentrations. Unfixed tracheal tissue was also treated with L, M, and H CF for 1 or 5 min at 4 degrees C in order to minimize lateral redistribution of CF receptors. To ensure accessibility of the cell surface to CF the samples were agitated thoroughly during exposure. Exposure for 1 min to L, M, and H CF resulted in a light binding of ferritin particles on all portions of the ciliary membrane with occasional areas of multilayered binding distributed randomly on the ciliary shaft. When unfixed trachea was treated with CF for 5 min at 4 degrees C, CF binding was similar except heavier and more uniform. In no instance was there any preferential binding of CF to the ciliary tips at any of the concentrations used. Moreover, as indicated by the CF binding pattern at L concentrations, high density negative charges are present over almost the entire surface of the cilium. These results suggest that, unlike the ciliary membrane of other organs such as oviduct, negatively charged cell surface coat molecules are present on all areas of the ciliary membrane of rat tracheal epithelia.     

Purinergically induced membrane fluidization in ciliary cells: characterization and control by calcium and membrane potential.

To examine the role of membrane dynamics in transmembrane signal transduction, we studied changes in membrane fluidity in mucociliary tissues from frog palate and esophagus epithelia stimulated by extracellular ATP. Micromolar concentrations of ATP induced strong changes in fluorescence polarization, possibly indicating membrane fluidization. This effect was dosage dependent, reaching a maximum at 10-microM ATP. It was dependent on the presence of extracellular Ca2+ (or Mg2+), though it was insensitive to inhibitors of voltage-gated calcium channels. It was inhibited by thapsigargin and by ionomycin (at low extracellular Ca2+ concentration), both of which deplete Ca2+ stores. It was inhibited by the calcium-activated potassium channel inhibitors quinidine, charybdotoxin, and apamine and was reduced considerably by replacement of extracellular Na+ with K+. Hyperpolarization, or depolarization, of the mucociliary membrane induced membrane fluidization. The degree of membrane fluidization depended on the degree of hyperpolarization or depolarization of the ciliary membrane potential and was considerably lower than the effect induced by extracellular ATP. These results indicate that appreciable membrane fluidization induced by extracellular ATP depends both on an increase in intracellular Ca2+, mainly from its internal stores, and on hyperpolarization of the membrane. Calcium-dependent potassium channels couple the two effects. In light of recent results on the enhancement of ciliary beat frequency, it would appear that extracellular ATP-induced changes both in ciliary beat frequency and in membrane fluidity are triggered by similar signal transduction pathways.     

The structure of the tips of mammalian respiratory cilia

Summary The ciliary crown and the relationship of the ciliary crown to the underlying axoneme were studied by electron microscopy in cilia from hamster and rat trachea and bronchioles, and rabbit trachea. The ciliary crown is a cluster of 4 to 6 fibrils 35 nm long protruding beyond the plasma membrane at the tips of the cilia. The fibrils are well preserved after tannic acidglutaraldehyde-osmium tetroxide fixation and have high contrast with a periodic density of 4.5 nm. They stain relatively weakly with phosphotungstic acid. The surface of the fibrils stains with ruthenium red.The microtubules of the axoneme end in a plate of electron dense amorphous material. A five layered disc occupies the space between the membrane and the amorphous plate at the tip of the axoneme. The plasma membrane can be dissolved with the detergent triton X-100 without loss of the ciliary crown. This indicates that the ciliary crown is composed of transmembranous filaments which are bound to the disc at the tip of the axoneme.Supported by U.S.P.H.S. Research Grant number HL-12650     

Ciliary function of the frog oro-pharyngeal epithelium

Summary The palate epithelium of the frog was examined by scanning electron microscopy, light microscopy and high speed cine micrography. The cilia remain stationary for much of the time in the end-of-effective stroke position. Each beat cycle begins with a forwardly-directed recovery stroke lasting about 60 ms, followed by an effective stroke towards the oesophagus lasting about 12 ms. Activity can often be correlated with the presence of mucus, which is carried as strands on the tips of the ciliary effective strokes whilst the recovery strokes move beneath the mucus. Coordination of ciliary activity was very variable; local antiplectic metachrony of the recovery strokes could almost always be seen, and on very active epithelia effective strokes were associated with approximately diaplectic waves (either to left or right), but any particular pattern of coordinated activity was transient and quickly transformed to another pattern. Beating and coordination of these short cilia were compared with those of cilia propelling water.     

Calcium regulation of ciliary activity in rabbit tracheal epithelial explants and outgrowth

Ciliated epithelial cells from rabbit trachea were employed to examine the role of Ca2+ in the regulation of ciliary motility. Tracheal explants and outgrowths were maintained in culture, and ciliary frequency was determined using a photomultiplier interfaced with a spectrum analyzer capable of Fast Fourier transform analysis. Relative cellular Ca2+ levels were determined by measuring 45Ca2+ uptake and efflux. Elevated cellular Ca2+, from exposure to 10(-5) M calcium ionophore A23187, led to an increase in ciliary frequency followed by inhibition of motility after prolonged treatment. A decrease in ciliary frequency was observed upon lowering intracellular Ca2+ by exposing the epithelium to 1 mM EGTA. Exposure of ciliated cells to 10(-4) M trifluoperazine resulted in inhibition of ciliary motility, a result suggesting a possible role for calmodulin- or phospholipid-sensitive Ca2+-dependent protein kinases in ciliary function. These results support the hypothesis that intracellular Ca2+ is actively involved in modulating the frequency of ciliary beat.     

Effects of ethanol on in vitro ciliary motility

Consumption of ethanol can impair lung function and slow total lung clearance. High concentrations of ethanol have been shown to slow or arrest ciliary beating. This study examined the effects of concentrations of alcohol comparable to blood levels achieved from social drinking on ciliary beat frequency. We obtained ciliated cells by brushing the trachea of unanesthetized sheep during fiber-optic bronchoscopy. The cells were suspended in a perfusion chamber and physiological conditions were maintained in vitro. Ciliary beat frequency and synchrony were determined by slow-motion analysis of video images obtained by interference contrast microscopy. Metachronal ciliary coordination was observed in all preparations. The ciliary beat frequency was stimulated at ethanol concentrations from 0.01 up to but not including 0.1%, unchanged at 0.5 and 1%, and slowed at 2%. While confirming inhibition of ciliary motility at very high ethanol levels, we observed no acute impairment of ciliary function at ethanol concentrations comparable to those achieved from social drinking. Indeed, we found an unexpected stimulation of ciliary beating at low levels of ethanol. How this alteration in ciliary beating would affect pulmonary clearance remains unknown at this time.     

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.     

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.