Cell to cell communication in response to mechanical stress via bilateral release of ATP and UTP in polarized epithelia

Airway epithelia are positioned at the interface between the body and the environment, and generate complex signaling responses to inhaled toxins and other stresses. Luminal mechanical stimulation of airway epithelial cells produces a propagating wave of elevated intracellular Ca(2+) that coordinates components of the integrated epithelial stress response. In polarized airway epithelia, this response has been attributed to IP(3) permeation through gap junctions. Using a combination of approaches, including enzymes that destroy extracellular nucleotides, purinergic receptor desensitization, and airway cells deficient in purinoceptors, we demonstrated that Ca(2+) waves induced by luminal mechanical stimulation in polarized airway epithelia were initiated by the release of the 5' nucleotides, ATP and UTP, across both apical and basolateral membranes. The nucleotides released into the extracellular compartment interacted with purinoceptors at both membranes to trigger Ca(2+) mobilization. Physiologically, apical membrane nucleotide-release coordinates airway mucociliary clearance responses (mucin and salt, water secretion, increased ciliary beat frequency), whereas basolateral release constitutes a paracrine mechanism by which mechanical stresses signal adjacent cells not only within the epithelium, but other cell types (nerves, inflammatory cells) in the submucosa. Nucleotide-release ipsilateral and contralateral to the surface stimulated constitutes a unique mechanism by which epithelia coordinate local and distant airway defense responses to mechanical stimuli.     

Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO(2)/HCO(3)(-) during exacerbations of airway diseases, but the role of CO(2)/HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO(2)/HCO(3)(-)-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO(2)/HCO(3)(-)-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO(2)/HCO(3)(-) increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells.     

Stimulus-response coupling in mammalian ciliated cells. Demonstration of two mechanisms of control for cytosolic [Ca2+]

Changes of cytosolic [Ca2+] have been proposed to couple stimulation of ciliary movement, however, quantitative measurements of fluctuations of intracellular free [Ca2+] associated with stimulation of ciliated cells have not been investigated. In primary cultures of rabbit oviductal ciliated cells, the stimulation of ciliary activity produced by micromolar concentrations of adenosine triphosphate (ATP) and prostaglandin F2 alpha (PGF2 alpha) was associated with a transient increase of intracellular [Ca2+]. Whereas the increase of cytosolic [Ca2+] and beat frequency produced by ATP were inhibited by the Ca-channel blocker LaCl3, the rise of cytosolic [Ca2+] and frequency of ciliary beat produced by PGF2 alpha was not affected by LaCl3. These results are the first direct demonstration that fluctuations of cytosolic [Ca2+] are associated with increased ciliary beat frequency in mammalian epithelial cells. The present findings suggest two different calcium-dependent mechanisms for stimulus-coupling in ciliary epithelium: ATP acting via purinergic receptor coupled to transmembrane influx of Ca2+, and PGF2 alpha acting via receptor-mediated release of intracellular sequestered Ca.     

Ciliary beat co-ordination by calcium

Motile cilia in the airway epithelium are the engine for mucociliary clearance, the mechanism responsible for cleaning the airways from inhaled particles. Human airway epithelial cilia appear to have a slow constitutive rate of beating, driven by inherent and spontaneous dynein ATPase activity. Additionally, cilia can increase their beating frequency by activation of several different control mechanisms. One of these controllers is calcium. Its intracellular concentration is regulated by purinergic and acetylcholine receptors. Besides the rate regulatory effect of calcium on ciliary beat, calcium is also involved in synchronizing the beat among cilia of one single cell as well as between cilia on different cells. This article gives an overview of the complex effects of calcium on the beating of motile cilia in the airways.     

Functional apical large conductance, Ca2+-activated, and voltage-dependent K+ channels are required for maintenance of airway surface liquid volume

Large conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels control a variety of physiological processes in nervous, muscular, and renal epithelial tissues. In bronchial airway epithelia, extracellular ATP-mediated, apical increases in intracellular Ca(2+) are important signals for ion movement through the apical membrane and regulation of water secretion. Although other, mainly basolaterally expressed K(+) channels are recognized as modulators of ion transport in airway epithelial cells, the role of BK in this process, especially as a regulator of airway surface liquid volume, has not been examined. Using patch clamp and Ussing chamber approaches, this study reveals that BK channels are present and functional at the apical membrane of airway epithelial cells. BK channels open in response to ATP stimulation at the apical membrane and allow K(+) flux to the airway surface liquid, whereas no functional BK channels were found basolaterally. Ion transport modeling supports the notion that apically expressed BK channels are part of an apical loop current, favoring apical Cl(-) efflux. Importantly, apical BK channels were found to be critical for the maintenance of adequate airway surface liquid volume because continuous inhibition of BK channels or knockdown of KCNMA1, the gene coding for the BK α subunit (KCNMA1), lead to airway surface dehydration and thus periciliary fluid height collapse revealed by low ciliary beat frequency that could be fully rescued by addition of apical fluid. Thus, apical BK channels play an important, previously unrecognized role in maintaining adequate airway surface hydration.     

Effect of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) on airway epithelial cells

Acetylcholine (ACh) exerts various anti-inflammatory effects through α7 nicotinic ACh receptors (nAChRs). We have previously shown that secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of α7 nAChR signaling, is down-regulated both in an animal model of asthma and in human epithelial cells treated with an inflammatory cytokine related to asthma. Our aim of this study was to explore the effect of SLURP-1, signal through α7 nAChR, in the pathophysiology of airway inflammation. Cytokine production was examined using human epithelial cells. Ciliary beat frequency of murine trachea was measured using a high speed camera. The IL-6 and TNF-α production by human epithelial cells was augmented by siRNA of SLURP-1 and α7 nicotinic ACh receptor. The cytokine production was also dose-dependently suppressed by human recombinant SLURP-1 (rSLURP-1). The ciliary beat frequency and amplitude of murine epithelial cells were augmented by PNU282987, a selective α7 nAChR agonist. Those findings suggested that SLURP-1 and stimulus through α7 nicotinic ACh receptors actively controlled asthmatic condition by stimulating ciliary beating and also by suppressing airway inflammation.     

Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO₃⁻/CO₂ stimulation to increase ciliary beat frequency (CBF). Because apical HCO₃⁻ exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO₃⁻/CO₂ exposure in part because of greater intracellular acidification from unbalanced CO₂ influx (estimated by 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO₃⁻/CO₂ perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTR_inh172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO₃⁻/CO₂ rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.     

Relaxin stimulates bronchial epithelial cell PKA activation,migration, and ciliary beating

Relaxin is an insulin-like serum protein secreted during pregnancy and found in many tissues, including the lung. Relaxin is reported to stimulate epithelial cell proliferation, but the effects of relaxin on airway epithelium are unknown. We tested the hypothesis that relaxin would stimulate the increased migration of bronchial epithelial cells (BEC) in response to wounding. Using monolayers of BEC in a wound-healing model, relaxin augmented wound closure with maximal closure occurring at 12 hr (1 micro M). Unlike cytokines, relaxin did not stimulate increased BEC interleukin-8 (IL-8) release. Relaxin caused a significant stimulation of ciliary beat frequency (CBF) in BEC. Because protein kinase (PKA) activation increases CBF and relaxin can elevate intracellular cAMP levels, we measured PKA activity in BEC treated with relaxin. Relaxin increased PKA activity 3-4 fold by approximately 4 hr, with a return to baseline levels by 8-10 hr. Relaxin-stimulated PKA activity differs temporally from the rapid (1 hr) beta-adrenergic activation of PKA in BEC. These data suggest that relaxin augments epithelial repair by increasing airway cell migration and CBF via PKA-dependent mechanisms.     

Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection

Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where - in pneumococcal infection - persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.     

SERCA2 regulates non-CF and CF airway epithelial cell response to ozone

Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target.     

Regulation of airway ciliary activity by Ca2+: simultaneous measurement of beat frequency and intracellular Ca2+.

Airway ciliary activity is influenced by [Ca2+]i, but this mechanism is not fully understood. To investigate this relationship, ciliary activity and [Ca2+]i were measured simultaneously from airway epithelial ciliated cells. Ciliary beat frequency was determined, for each beat cycle, with phase-contrast optics and high-speed video imaging (at 240 images s-1) and correlated with [Ca2+]i determined, at the ciliary base, by fast imaging (30 images s-1) of fura-2 fluorescence. As a mechanically induced intercellular Ca2+ wave propagated through adjacent cells, [Ca2+]i was elevated from a baseline concentration of 45 to 100 nM, to a peak level of up to 650 nM. When the Ca2+ wave reached the ciliary base, the beat frequency rapidly increased, within a few beat cycles, from a basal rate of 6.4 to 11.6 Hz at 20-23 degrees C, and from 17.2 to 26.7 Hz at 37 degrees C. Changes in [Ca2+]i, above 350 nM, had no effect on the maximum beat frequency. We suggest that airway ciliary beat frequency is 1) controlled by a low range of [Ca2+]i acting directly at an axonemal site at the ciliary base and 2) that a maximum frequency is induced by a change in [Ca2+]i of approximately 250-300 nM.     

Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology

Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.     

Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, contain primary cilia, which are mechano- and osmosensory organelles detecting changes in bile flow and osmolality and transducing them into intracellular signals. Here, we asked whether cholangiocyte cilia are chemosensory organelles by testing the expression of P2Y purinergic receptors and components of the cAMP signaling cascade in cilia and their involvement in nucleotide-induced cAMP signaling in the cells. We found that P2Y(12) purinergic receptor, adenylyl cyclases (i.e., AC4, AC6, and AC8), and protein kinase A (i.e., PKA RI-beta and PKA RII-alpha regulatory subunits), exchange protein directly activated by cAMP (EPAC) isoform 2, and A-kinase anchoring proteins (i.e., AKAP150) are expressed in cholangiocyte cilia. ADP, an endogenous agonist of P2Y(12) receptors, perfused through the lumen of isolated rat intrahepatic bile ducts or applied to the ciliated apical surface of normal rat cholangiocytes (NRCs) in culture induced a 1.9- and 1.5-fold decrease of forskolin-induced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-gammaS, a nonhydrolyzed analog of ATP but was not affected by UTP. The ADP-induced changes in cAMP levels in cholangiocytes were abolished by chloral hydrate (a reagent that removes cilia) and by P2Y(12) siRNAs, suggesting that cilia and ciliary P2Y(12) are involved in nucleotide-induced cAMP signaling. In conclusion, cholangiocyte cilia are chemosensory organelles that detect biliary nucleotides through ciliary P2Y(12) receptors and transduce corresponding signals into a cAMP response.     

LRRC6 Mutation Causes Primary Ciliary Dyskinesia with Dynein Arm Defects

Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects.     

Adenosine A(3) receptor-mediated potentiation of mucociliary transport and epithelial ciliary motility

To examine the effect of adenosine A(3) receptor stimulation on airway mucociliary clearance, we measured transport of Evans blue dye in rabbit trachea in vivo and ciliary motility of epithelium by the photoelectric method in vitro. Mucociliary transport was enhanced dose dependently by the selective A(3) agonist N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) and to a lesser extent by the less-selective N(6)-2-(4-amino-3-iodophenyl)ethyladenosine, whereas the A(1) agonist N-cyclopentyladenosine (CPA) and the A(2) agonist CGS-21680 had no effect. The effect of IB-MECA was abolished by pretreatment with the selective A(3) antagonist MRS-1220 but not by the A(1) antagonist 1,3-dipropyly-8-cyclopentylxanthine or the A(2) antagonist 3,7-dimethyl-L-propargylxanthine. Epithelial ciliary beat frequency was increased by IB-MECA in a concentration-dependent manner, the maximal increase being 33%, and this effect was inhibited by MRS-1220. The IB-MECA-induced ciliary stimulation was not altered by the Rp diastereomer of cAMP but was greatly inhibited by Ca(2+)-free medium containing BAPTA-AM. Incubation with IB-MECA increased intracellular Ca(2+) contents. Therefore, A(3) agonist enhances airway mucociliary clearance probably through Ca(2+)-mediated stimulation of ciliary motility of airway epithelium.     

Characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling

Background

In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However, undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study, we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients.

Methods

Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated, mucus-secreting and basal cells, and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally, epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay.

Results

Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also, immunofluorescence analysis revealed the presence of ciliated, mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight.

Conclusion

In summary, primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated, mucus-producing and basal cells, which adequately reflect the in vivo properties of the human respiratory epithelium.

     

Control of the ciliary beat by cyclic nucleotides in intact cortical sheets from Paramecium

The locomotor behavior of Paramecium depends on the ciliary beat direction and beat frequency. Changes in the ciliary beat are controlled by a signal transduction mechanism that follows changes in the membrane potential. These events take place in cilia covered with a ciliary membrane. To determine the effects of second messengers in the cilia, cortical sheets were used with intact ciliary membrane as a half-closed system in which each cilium is covered with a ciliary membrane with an opening to the cell body. Cyclic nucleotides and their derivatives applied from an opening to the cell body affected the ciliary beat. cAMP and 8-Br-cAMP increased the beat frequency and the efficiency of propulsion and acted antagonistically to the action of Ca(2+). cGMP and 8-Br-cGMP increased the efficiency of propulsion accompanying clear metachronal waves but decreased the beat frequency. These results indicate that the cyclic nucleotides affect target proteins in the ciliary axonemes surrounded by the ciliary membrane without a membrane potential and increase the efficiency of propulsion of the ciliary beat. In vitro phosphorylation of isolated ciliary axonemes in the presence of cyclic nucleotides and their derivatives revealed that the action of cAMP was correlated with the phosphorylation of 29-kDa and 65-kDa proteins and that the action of cGMP was correlated with the phosphorylation of a 42-kDa protein.     

Regulation of ciliary motility by membrane potential in Paramecium: a role for cyclic AMP

The membrane potential of Paramecium controls the frequency and direction of the ciliary beat, thus determining the cell's swimming behavior. Stimuli that hyperpolarize the membrane potential increase the ciliary beat frequency and therefore increase forward swimming speed. We have observed that 1) drugs that elevate intracellular cyclic AMP increased swimming speed 2-3-fold, 2) hyperpolarizing the membrane potential by manipulation of extracellular cations (e.g., K+) induced both a transient increase in, and a higher sustained level of cyclic AMP compared to the control, and 3) the swimming speed of detergent-permeabilized cells in MgATP was stimulated 2-fold by the addition of cyclic AMP. Our results suggest that the membrane potential can regulate intracellular cAMP in Paramecium and that control of swimming speed by membrane potential may in part be mediated by cAMP.     

Characterization of an A-kinase anchoring protein in human ciliary axonemes

Although protein kinase A (PKA) activation is known to increase ciliary beat frequency in humans the molecular mechanisms involved are unknown. We demonstrate that PKA is associated with ciliary axonemes where it specifically phosphorylates a 23-kDa protein. Because PKA is often localized to subcellular compartments in proximity to its substrate(s) via interactions with A-kinase-anchoring proteins (AKAPs), we investigated whether an AKAP was also associated with ciliary axonemes. This study has identified a novel 28 kDa AKAP (AKAP28)that is highly enriched in airway axonemes. The mRNA for AKAP28 is up-regulated as primary airway cells differentiate and is specifically expressed in tissues containing cilia and/or flagella. Additionally, both Western blot and immunostaining data show that AKAP28 is enriched in airway cilia. These data demonstrate that we have identified the first human axonemal AKAP, a protein that likely plays a role in the signaling necessary for efficient modulation of ciliary beat frequency.     

Effect of cAMP on ciliary function in rabbit tracheal epithelial cells

To study the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on respiratory ciliary activity, we measured ciliary beat frequency (CBF) of rabbit tracheal epithelium by a photoelectric method in response to cAMP analogues and agents that can increase endogenous cAMP production. Addition of 8-bromo-cAMP dose dependently enhanced CBF, with the maximal increase and the concentration necessary to produce a half-maximal response (KD) being 31.0 +/- 3.4% (SE) (P less than 0.001) and 3.2 +/- 1.5 x 10(-7) M, respectively. Other structurally dissimilar cAMP analogues dibutyryl cAMP and chlorophenylthio-cAMP likewise caused increases in CBF. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the adenylate cyclase stimulator forskolin also augmented CBF in a dose-dependent fashion and were accompanied by the increases in intracellular concentrations of cAMP. Ciliary discoordination was not observed in any of the experiments. These results suggest that cAMP may accelerate mucociliary clearance through the activation of ciliary motility and that intracellular cAMP levels appear to be an important determinant for the lung mucociliary transport functions.