Ciliary Beat Frequency In Response Research Articles

Influenza A virus enhances ciliary activity and mucociliary clearance via TLR3 in airway epithelium

BackgroundViral respiratory tract infections, such as influenza A virus (IAV), are common and life-threatening illnesses worldwide. The mechanisms by which viruses are removed from the respiratory tract are indispensable for airway host defense. Mucociliary clearance is an airway defense mechanism that removes pathogens from the respiratory tract. The coordination and modulation of the ciliary beating of airway epithelial cells play key roles in maintaining effective mucociliary clearance. However, the impact of respiratory virus infection on ciliary activity and mucociliary clearance remains unclear.MethodsTracheal samples were taken from wild-type (WT) and Toll-like receptor 3 (TLR3)-knockout (KO) mice. Transient organ culture of murine trachea was performed in the presence or absence of IAV, polyI:C, a synthetic TLR3 ligand, and/or reagents. Subsequently, cilia-driven flow and ciliary motility were analyzed. To evaluate cilia-driven flow, red fluorescent beads were loaded into culture media and movements of the beads onto the tracheal surface were observed using a fluorescence microscope. To evaluate ciliary motility, cilia tips were labeled with Indian ink diluted with culture medium. The motility of ink-labeled cilia tips was recorded by high-speed cameras.ResultsShort-term IAV infection significantly increased cilia-driven flow and ciliary beat frequency (CBF) compared with the control level in WT culture. Whereas IAV infection did not elicit any increases of cilia-driven flow and CBF in TLR3-KO culture, indicating that TLR3 was essential to elicit an increase of cilia-driven flow and CBF in response to IAV infection. TLR3 activation by polyI:C readily induced adenosine triphosphate (ATP) release from the trachea and increases of cilia-driven flow and CBF in WT culture, but not in TLR3-KO culture. Moreover, blockade of purinergic P2 receptors (P2Rs) signaling using P2R antagonist, suramin, suppressed polyI:C-mediated increases of cilia-driven flow and CBF, indicating that TLR3-mediated ciliary activation depended on released extracellular ATP and the autocrine ATP-P2R loop.ConclusionsIAV infection readily increases ciliary activity and cilia-driven flow via TLR3 activation in the airway epithelium, thereby hastening mucociliary clearance and “sweeping” viruses from the airway as an initial host defense response. Mechanically, extracellular ATP release in response to TLR3 activation promotes ciliary activity through autocrine ATP-P2R loop.

Loss of cAMP-dependent stimulation of isolated cilia motility by alcohol exposure is oxidant-dependent.

Alcohol exposure is associated with decreased mucociliary clearance, a key innate defense essential to lung immunity. Previously, we identified that prolonged alcohol exposure results in dysfunction of airway cilia that persists at the organelle level. This dysfunction is characterized by a loss of 3',5'-cyclic adenosine monophosphate (cAMP)-mediated cilia stimulation. However, whether or not ciliary dysfunction develops intrinsically at the organelle level has not been explored. We hypothesized that prolonged alcohol exposure directly to isolated demembranated cilia (axonemes) causes ciliary dysfunction. To test this hypothesis, we exposed isolated axonemes to alcohol (100mM) for 1-24h and assessed ciliary beat frequency (CBF) in response to cAMP at 1, 3, 4, 6, and 24h post-exposure. We found that after 1h of alcohol exposure, cilia axonemes do not increase CBF in response to cAMP. Importantly, by 6h after the initial exposure to alcohol, cAMP-mediated CBF was restored to control levels. Additionally, we found that thioredoxin reverses ciliary dysfunction in axonemes exposed to alcohol. Finally, we identified, using a combination of a xanthine oxidase oxidant-generating system, direct application of hydrogen peroxide, and electron paramagnetic resonance, that hydrogen peroxide versus superoxide, is likely the key oxidant species driving alcohol-induced ciliary dysfunction in isolated axonemes. These data highlight the role of alcohol to stimulate local production of oxidants in the axoneme to cause ciliary dysfunction. Additionally, these data specifically add hydrogen peroxide as a potential therapeutic target in the treatment or prevention of alcohol-associated ciliary dysfunction and subsequent pneumonia.

The Role of Quinine-Responsive Taste Receptor Family 2 in Airway Immune Defense and Chronic Rhinosinusitis.

Bitter (T2R) and sweet (T1R) taste receptors in the airway are important in innate immune defense, and variations in taste receptor functionality in one T2R (T2R38) correlate with disease status and disease severity in chronic rhinosinusitis (CRS). Quinine is a bitter compound that is an agonist for several T2Rs also expressed on sinonasal cells, but not for T2R38. Because of this property, quinine may stimulate innate immune defense mechanisms in the airway, and functional differences in quinine perception may be reflective of disease status in CRS. Demographic and taste intensity data were collected prospectively from CRS patients and non-CRS control subjects. Sinonasal tissue from patients undergoing rhinologic surgery was also collected and grown at an air-liquid interface (ALI). Nitric oxide (NO) production and dynamic regulation of ciliary beat frequency in response to quinine stimulation were assessed in vitro. Quinine reliably increased ciliary beat frequency and NO production in ALI cultures in a manner consistent with T2R activation (p < 0.01). Quinine taste intensity rating was performed in 328 CRS patients and 287 control subjects demonstrating that CRS with nasal polyps (CRSwNP) patients rated quinine as significantly less intense than did control subjects. Quinine stimulates airway innate immune defenses by increasing ciliary beat frequency and stimulating NO production in a manner fitting with T2R activation. Patient variability in quinine sensitivity is observed in taste intensity ratings, and gustatory quinine "insensitivity" is associated with CRSwNP status. Thus, taste tests for quinine may be a biomarker for CRSwNP, and topical quinine has therapeutic potential as a stimulant of innate defenses.

TNFα Affects Ciliary Beat Response to Increased Viscosity in Human Pediatric Airway Epithelium.

In airway epithelium, mucociliary clearance (MCC) velocity depends on the ciliary beat frequency (CBF), and it is affected by mucus viscoelastic properties. Local inflammation induces secretion of cytokines (TNFα) that can alter mucus viscosity; however airway ciliated cells have an autoregulatory mechanism to prevent the collapse of CBF in response to increase in mucus viscosity, mechanism that is associated with an increment in intracellular Ca+2 level ([Ca2+]i). We studied the effect of TNFα on the autoregulatory mechanism that regulates CBF in response to increased viscosity using dextran solutions, in ciliated cells cultured from human pediatric epithelial adenoid tissue. Cultures were treated with TNFα, before and after the viscous load was changed. TNFα treatment produced a significantly larger decrease in CBF in cultures exposed to dextran. Furthermore, an increment in [Ca2+]i was observed, which was significantly larger after TNFα treatment. In conclusion, although TNFα has deleterious effects on ciliated cells in response to maintaining CBF after increasing viscous loading, it has a positive effect, since increasing [Ca2+]i may prevent the MCC collapse. These findings suggest that augmented levels of TNFα associated with an inflammatory response of the nasopharyngeal epithelium may have dual effects that contribute to maintaining the effectiveness of MCC in the upper airways.

The effects of vitamins C and B12 on human nasal ciliary beat frequency

BackgroundThis study was designed to investigate the effects of the vitamins C and B12 on the regulation of human nasal ciliary beat frequency (CBF).MethodsHuman nasal mucosa was removed endoscopically and nasal ciliated cell culture was established. Changes of CBF in response to different concentrations of vitamin C or vitamin B12 were quantified by using high-speed (240 frames per second) digital microscopy combined with a beat-by-beat CBF analysis.ResultsAt the concentrations of 0.01% and 0.10%, vitamin C induced an initial increase, followed by a gradual decrease of CBF to the baseline level, while 1.00% vitamin C induced a reversible decrease of CBF. Vitamin B12, at the concentrations of 0.01% and 0.10%, did not influence CBF during the 20-min observation period, while a 1.00% vitamin B12 treatment caused a time-dependent but reversible decrease of CBF.ConclusionsTreatment with vitamin C or vitamin B12 caused a concentration-dependent but reversible decrease of CBF in cultured human nasal epithelial cells. Therefore, it is necessary to choose a concentration that is safe, effective, and non-ciliotoxic when applying these drugs topically in the nasal cavity.

Different Cilia Response to Adenosine Triphosphate or Benzalkonium Chloride Treatment in Mouse Nasal and Tracheal Culture

Objectives: Our purpose was to compare the ciliary function between the upper and lower airway. Methods: Basal ciliary beat frequency (CBF) and changes in CBF in response to exogenous stimuli [adenosine triphosphate (ATP) or benzalkonium chloride (BAC)] of primary cultured mouse nasal and tracheal epithelial cells were quantified using digital microscopy combined with a beat-by-beat CBF analysis. Results: The basal CBF of the mouse tracheal culture was statistically higher than the nasal culture. Adding 10 or 100 µ<span style="font-variant:small-caps; text-transform:lowercase">m</span> ATP caused an increase of CBF in both mouse nasal and tracheal cultures; however, nasal culture induced a more robust increase of CBF than tracheal culture. Treatment with 0.001% BAC increased CBF within 2 min after treatment and then CBF gradually decreased; nasal and tracheal culture manifested a similar change in CBF. With a concentration of 0.01%, BAC induced a rapid and time-dependent decrease of CBF in both mouse nasal and tracheal cultures; however, tracheal culture showed a blunter response to this ciliotoxic agent, with the ciliary beating duration time distinguishably longer than in the nasal culture. Conclusion: The inherent ciliary and physiological function is different in the upper and lower airway in mice.

Rapid effects of progesterone on ciliary beat frequency in the mouse fallopian tube

BackgroundThe physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations.MethodsA method to assess tubal CBF of mice was developed. Fallopian tubes were dissected and the tissue was cut in small pieces. Tissue samples with moving cilia were located under an inverted bright field microscope and held still against the bottom of a petri dish by a motorized needle system. Images were acquired over 90 minutes at 35 degrees C with a high-speed camera and used for assessing changes in the CBF in response to the addition of hormone.ResultsThe baseline CBF of the mouse fallopian tube was 23.3 +/- 3.8 Hz. The CBF was stable over at least 90 minutes allowing establishment of a baseline frequency, addition of hormone and subsequent recordings. Progesterone at concentrations of 20 micromolar and 100 nM significantly reduced the CBF by 10% and 15% respectively after 30 minutes compared with controls.ConclusionsThe present study demonstrates that the mouse, despite its small size, is a useful model for studying the fallopian tube CBF ex vivo. The rapid reduction in CBF by 100 nM progesterone suggests that gamete transport in the fallopian tube could be mediated by progesterone via a non-genomic receptor mechanism.

The Effect of Myrtol Standardized on Human Nasal Ciliary Beat Frequency and Mucociliary Transport Time

This study was designed to observe the effects of myrtol standardized (Gelomyrtol forte), a secretomucolytic phytomedicine, on both ciliary beat frequency (CBF) in vitro and mucociliary transport time (MTT) in vivo. Changes in cultured human nasal CBF in response to immediate treatment with 75, 150, or 300 ng/mL of myrtol standardized and prolonged treatment (12 or 24 hours) with 300 ng/mL of myrtol standardized were quantified by using high-speed digital microscopy. In addition, MTT before and after oral application of myrtol standardized (three times a day, 900 mg/day, 10 days) was determined using the saccharine test, and the effects of this treatment regime on nasal patency was measured by acoustic rhinometry and active anterior rhinomanometry in 22 patients with nonallergic chronic rhinitis. Another 10 patients without medication, who had the same examinations twice with a 10-day interval, were involved as controls. Neither immediate nor prolonged treatment with myrtol standardized produced a distinguishable change in CBF. Meanwhile, only in patients with treatment, MTT, as well as a unilateral minimum cross-sectional area, the volume of 0-5 cm inside the nasal cavity, the unilateral nasal resistance at 75 Pa and total symptom visual analog score were significantly improved after treatment. Based on these results we propose that a 10-day treatment with an herbal medicine, myrtol standardized, improves nasal mucociliary clearance as well as nasal patency in patients with chronic rhinitis. However, it has no impact on ex vivo CBF.

Increased nasal epithelial ciliary beat frequency associated with lifestyle tobacco smoke exposure

The ciliated epithelium of the respiratory airways is one of the first vital systemic surfaces in contact with the ambient air. Ex vivo nasal epithelial ciliary beat frequency (CBF) at room temperature is on the order of 7–8 Hz but may be stimulated by irritant exposure. The upregulation of CBF in response to acute irritant exposure is generally considered to be a transient event with eventual return to baseline. However, studies of CBF dynamics in response to typical lifestyle exposures are limited. This study assessed nasal epithelial CBF among human subjects as a function of quantifiable lifestyle tobacco smoke exposure. Nasal epithelial biopsies were obtained from human subjects with well documented histories of tobacco smoke exposure. CBF was determined using a digital photometric technique and concurrent assays of nasal nitric oxide and urine cotinine and creatinine were performed. Mean CBF among active smokers and non-smokers exposed to environmental tobacco smoke (ETS) was elevated over non-smokers. Although there were dramatic differences in relative levels of tobacco smoke exposure, CBF values among tobacco smoke-exposed groups were comparable. Parallel in vitro studies of cultured nasal epithelium exposed to cigarette smoke condensate further supported these observations. These studies suggest that persistent elevation in nasal epithelial CBF is an early, subtle, physiologic effect associated with lifestyle tobacco smoke exposure. The molecular mechanisms that upregulate CBF may also create a cell molecular milieu capable of provoking the eventual emergence of more overt adverse health effects and the pathogenesis of chronic airway disease.

Muscarinic receptor subtypes in cilia-driven transport and airway epithelial development

Ciliary beating of airway epithelial cells drives the removal of mucus and particles from the airways. Mucociliary transport and possibly airway epithelial development are governed by muscarinic acetylcholine receptors but the precise roles of the subtypes involved are unknown. This issue was addressed by determining cilia-driven particle transport, ciliary beat frequency, and the composition and ultrastructural morphology of the tracheal epithelium in M1-M5 muscarinic receptor gene-deficient mice. Knockout of M3 muscarinic receptors prevented an increase in particle transport speed and ciliary beat frequency in response to muscarine. Furthermore, the ATP response after application of muscarine was blunted. Pretreatment with atropine before application of muscarine restored the response to ATP. Additional knockout of the M2 receptor in these mice partially restored the muscarine effect, most likely through the M1 receptor, and normalised the ATP response. M1, M4 and M5 receptor-deficient mice exhibited normal responses to muscarine. None of the investigated mutant mouse strains had any impairment of epithelial cellular structure or composition. In conclusion, M3 receptors stimulate whereas M2 receptors inhibit cilia-driven particle transport. The M1 receptor increases cilia-driven particle transport if the M3 and M2 receptors are missing. None of the receptors is necessary for epithelial development.

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.

Effect of Oxymetazoline on Healthy Human Nasal Ciliary Beat Frequency Measured with High-Speed Digital Microscopy and Mucociliary Transport Time

We investigated the effects of oxymetazoline hydrochloride on the regulation of healthy human nasal ciliary beat frequency (CBF) and its influence on nasal mucociliary transport time (MTT). Changes in (cultured) human nasal CBF in response to increasing concentrations of oxymetazoline within 20 minutes were quantified by use of high-speed digital microscopy. Moreover, the MTT before and after application of 0.05% oxymetazoline was determined by use of the saccharin test. Whereas no statistically significant difference was identified when compared to basal CBF at the concentration of 0.025% or 0.05%, both 0.10% and 0.20% oxymetazoline induced a significantly lower CBF at the end of the observation period. The decrement induced by 0.20% oxymetazoline appeared earlier. At concentrations ranging from 0.025% to 0.20%, the inhibitory effect was dependent on the concentration of oxymetazoline. In addition, the use of 0.05% oxymetazoline increased the mean (+/- SD) human nasal MTT from 474 +/- 21 seconds to 572 +/- 41 seconds (n = 29). The clinical concentration of oxymetazoline, 0.05%, has no obvious inhibitory effect on human nasal CBF in vitro. The increased MTT caused by 0.05% oxymetazoline in vivo is within the normal range.

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)

Ciliary beat frequency desensitization in an ovine alcohol exposure model

Mucociliary clearance is regulated in part by changes in the ciliary beat frequency (CBF) of epithelial cells lining the airways. We have demonstrated that exposure to human intoxicating concentrations of ethanol (100 mM) can result in chronic desensitization of CBF in response to beta agonists in both rat and mouse models of alcohol exposure. We hypothesized that maternal exposure to alcohol in an ovine model would result in desensitized CBF in newborn lambs. Ewes were exposed to 1mg ethanol/kg body weight of 40% ethanol solution during the last 5 weeks of pregnancy by cannulation in the last ruminant stomach. Ewes from a second set were transdermally exposed to 21 mg nicotine for the same time. At full-term parturition, lambs were sacrificed and trachea necropsied. CBF was assayed ex vivo using digital whole-field analysis of tracheal ring cilia in the presence or absence of 100 μM isoproterenol (ISO) for 30 m. The remainder of the tracheal epithelium was extracted for direct assay of cAMP-dependent protein kinase (PKA). Baseline CBF was approximately 6–7 Hz in control lamb trachea. ISO stimulated a 4–5 Hz increase in CBF from control or lambs maternally exposed to nicotine. Conversely, maternal alcohol-exposed lambs showed no response to ISO. Likewise, epithelial cell PKA activity was elevated twofold in response to ISO, but not in maternal alcohol-exposed lamb trachea. These data suggest that maternal alcohol exposure can cause cilia dysfunction in the newborn, potentially impacting early host lung defense. Supported by VA Merit, NIH-AA08769, and ALA grants

Ciliary Beat Frequency Is Maintained at a Maximal Rate in the Small Airways of Mouse Lung Slices

Ciliary beat frequency (CBF) is a key factor in the defense of the airways, and ATP can stimulate CBF by increasing intracellular calcium concentration ([Ca2+]i). However, the regulatory effects of ATP have been mainly studied in cultured or isolated epithelial cells from the large cartilaginous airways. The aim of this study was to evaluate the regulation of CBF in small airways of lung slices that are representative of in vivo tissue. Mice lungs were inflated with agarose and cut into thin slices with a vibratome. CBF in the small bronchioles was observed with differential interference contrast microscopy and quantified using high-speed digital imaging (at 240 images s(-1)). We found that the in situ organization of the ciliated cells was well preserved and that their CBF was high. We verified the fidelity of our recording system by analyzing rapid changes in CBF in response to temperature. However, we found that ATP had no effect on CBF, despite the fact that the [Ca2+]i, measured with confocal fluorescence imaging, was increased. Ionomycin and purinergic or beta-adrenergic agonists also failed to increase CBF. Similar results were obtained in outgrowths of cells cultured from lung slices. By contrast, ATP increased the slower CBF of outgrowths of ciliated cells cultured from tracheal rings. Therefore, we conclude that CBF in intrapulmonary airways of mice is maintained at a maximum rate and cannot be further increased by agonist stimulation. These conditions would ensure that mucociliary clearance is constantly active to provide continuous airway protection.

Making human nasal cilia beat in the cold: a real time assay for cell signalling

Human nasal epithelium must adapt to cold climates, and yet, in vitro, human ciliary beat frequency (CBF) is zero at 4 °C. Similarly, hibernating mammals do not die of pneumonia despite a core body temperature as low as 6 °C, implying that cilia continue to beat. Here, we show that protein kinase C (PKC) and Ca 2+/calmodulin-dependent kinase II (CaMKII) regulate the profile of human nasal CBF in response to rising temperature from 4 °C. Onset of ciliary beat was at 10 °C in Medium 199, 7 °C in the presence of the PKC activator phorbol 12-myristate 13-acetate (PMA), the calcium ionophore ionomycin, or the CAMKII blocker myristoylated autocamtide-2 related inhibitory peptide (MACI), and 6 °C for the myristoylated peptide PKC inhibitor EGF-R Fragment 651–658 (MyrPKCI). During cell warming to 32 °C, the thermal profile was sigmoid in all solutions except those containing MACI+PMA. Surprisingly, cilia continued to beat despite 4 °C and were significantly more responsive to rising temperature with either MACI+PMA, or MACI+MyrPKCI. Our data suggest that CaMKII and PKC regulate the thermal slope and profile of CBF in vitro, and that when these protein kinases are manipulated, cilia can continue to beat despite hypothermia. These findings may relate to adaptive responses to cold climates.

Hyaluronic acid in cultured ovine tracheal cells and its effect on ciliary beat frequency in vitro.

Hyaluronic acid (hyaluronan, or HA) is secreted by submucosal glands, but its function in airway secretions other than influencing the rheology of mucus is not fully understood. HA is known to modulate cell behavior and to enhance sperm motility. Because sperm tails and cilia have the same microtubular structure, we studied the effect of HA on ciliary beat frequency (CBF) in vitro. CBF of cultured ovine airway epithelial cells was measured continuously by digital video microscopy. After removal of endogenous HA by hyaluronidase, cells were exposed to 50 to 100 microg/mL of HA at different times in culture. No change in CBF in response to HA was seen in cells cultured less than 7 days. After 7 days, however, 6 of 10 measured cells (from three different sheep) showed a transient CBF increase from a baseline of 6.4 +/- 0.3 Hz (mean +/- SE) to 7.4 +/- 0.4 Hz or 16% above baseline (p < 0.05). At these time points (but not before), cytochemical staining was positive for endogenous HA using a biotinylated HA-binding protein. These data suggest that HA can increase CBF of tracheal epithelial cells only late in culture when HA is able to bind to an unspecified cell surface structure. Because this binding has a physiological effect, we hypothesize that it is an HA-binding receptor, that is either transiently expressed late in culture or initially destroyed by the protease treatment for cell dispersion.