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Am J Physiol Lung Cell Mol Physiol 288: L546-L551, 2005. First published November 12, 2004; doi:10.1152/ajplung.00107.2004
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Both cAMP and cGMP are required for maximal ciliary beat stimulation in a cell-free model of bovine ciliary axonemes

Todd A. Wyatt,1,2 Mary A. Forgèt,2 Jennifer M. Adams,2 and Joseph H. Sisson2

1Research Service, Department of Veterans Affairs Medical Center; and 2Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska

Submitted 24 March 2004 ; accepted in final form 8 November 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previously, we have shown that the ATPase-dependent motion of cilia in bovine bronchial epithelial cells (BBEC) can be regulated through the cyclic nucleotides, cAMP via the cAMP-dependent protein kinase (PKA) and cGMP via the cGMP-dependent protein kinase (PKG). Both cyclic nucleotides cause an increase in cilia beat frequency (CBF). We hypothesized that cAMP and cGMP may act directly at the level of the ciliary axoneme in BBEC. To examine this, we employed a novel cell-free system utilizing detergent-extracted axonemes. Axoneme movement was whole-field analyzed digitally with the Sisson-Ammons video analysis system. A suspension of extracted axonemes remains motionless until the addition of 1 mM ATP that establishes a baseline CBF similar to that seen when analyzing intact ciliated BBEC. Adding 10 µM cAMP or 10 µM cGMP increases CBF beyond the established ATP baseline. However, the cyclic nucleotides did not stimulate CBF in the absence of ATP. Therefore, the combination of cAMP and cGMP augments ATP-driven CBF increases at the level of isolated axoneme.

cilia; adenosine 3',5'-cyclic monophosphate; guanosine 3',5'-cyclic monophosphate

MUCOCILIARY CLEARANCE SERVES as a primary host defense mechanism. It is through the production of mucus and the synchronized beating of ciliated airway epithelium that the body removes dust, debris, and microorganisms from the lungs. Like many host defense mechanisms, mucociliary clearance can be regulated in response to external stimuli (28). In the intact airway epithelial cell it has been shown that during periods of normal function, baseline ciliary beat frequency (CBF) maintains resting airway clearance. During periods of stress, ciliary motility increases and enhances clearance (19).

One of the mechanisms responsible for this stimulation of ciliary motility, as studied in the intact ciliated epithelial cell, is an increase in intracellular cyclic nucleotides (12, 25). Increases in cAMP have been associated with increases in mammalian cilia motility in the rat (1), rabbit (18, 23), dog (26), ovine (17), bovine (25), and human (4). Similarly, cGMP also elevates mammalian CBF (6, 25). We initially reported that cGMP-mediated cilia stimulation pathway was linked to elevations in nitric oxide (8, 9). Such increases in cyclic nucleotides result in the activation of the cAMP- and cGMP-dependent protein kinases (PKA and PKG, respectively) (3, 11, 13, 14, 25). The exact mechanisms linking PKA and PKG activation to CBF increases in the intact mammalian ciliated cell are not fully understood, although dual cyclic nucleotide kinase interplay has been reported (3, 24, 27).

In an effort to define the relationship between PKA, PKG, and increased motility, a variety of nonmammalian cilia systems have been examined. In prokaryotic organisms, increases in cAMP levels have been associated with changes in axonemal motility. In the paramecium, increases in cAMP result in increases in motility and are thought to influence movement of the outer dynein arm (20). Although both cAMP and cGMP similarly stimulate forward beating in paramecium, the nucleotides have different roles in the calcium antagonism of backward beating (2). In Chlamydomonas, increases in cAMP decrease flagellar motion (16). In this organism PKA activity is associated with the motion of inner dynein arm, via an A-kinase-anchoring protein (AKAP) bound to the complex of proteins that make up the radial spoke of the axoneme (5). PKA has also been localized on human ciliary axonemes via a novel AKAP28 (11). No localization of PKG activity has been previously reported on ciliary axonemes.

Although mechanistic studies have been performed on cell-extracted axonemal models of single-celled organisms (15, 20), few mammalian axonemal models exist. Given that PKA activity has been associated with an increase in CBF in the whole cell and that the regulatory subunit of type II PKA has been localized on the axonemes of human airway cilia, we hypothesize that the activation of PKA directly on isolated ciliary axonemes in suspension will result directly in an increase in CBF. To test this hypothesis, we have applied our recently developed method of whole field analysis (WFA) of populations of beating cilia (22) to quantitate isolated axonemal beating in a cell-free system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Axonemal extraction and preparation. Axonemes were isolated from bovine ciliated epithelium by a modification of a previously described method (7). To obtain isolated axonemes from bovine ciliated cells, we obtained lungs at a local abattoir and extracted tracheae. After the removal of excess adipose and connective tissue, each trachea was washed twice in PBS, and one tracheal orifice was closed with a clamp. An extraction buffer containing 20 mM Tris·HCl, 50 mM NaCl, 10 mM calcium chloride, 1 mM EDTA, 7 mM 2-mercaptoethanol, 100 mM Triton X-100, and 1 mM dithiothreitol was utilized to extract the axonemes from the ciliated cells. To accomplish this, we added 25 ml of extraction buffer to the open end of a bovine trachea. After all orifices were closed, the trachea was vigorously shaken for 90 s, buffer containing axonemes was filtered through a 100-µm mesh and centrifuged at 17,250 g for 7 min, and the supernatant was removed. The pelleted axonemes were resuspended to a concentration of 1 mg/ml in resuspension buffer consisting of 20 mM Tris·HCl, 50 mM KCl, 4 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, 10 mM soybean trypsin inhibitor, and 25% sucrose by volume. Resuspended axonemes were aliquoted and stored at –80°C until needed for up to 3 wk without alteration in beating responsiveness. Axoneme preparations from five different tracheae were used in this study with consistent results. Because this preparation removes the membranes and leaves a relatively pure preparation of isolated cilia axonemes, there is no source of ATP, nor are receptors on the membrane involved in controlling the signaling. The loss of membranes has been demonstrated with transmission electron micrographs of the cilia preparation (data not shown). For this reason, external ATP is required as a substrate for the activation of the axonemal ATPase.

Experimental treatment of axonemes. Frozen aliquots of axonemes stored at –80°C were thawed on ice, maintained at 4°C, and pipetted up and down to minimize axoneme clumping. For each experimental condition, we diluted axoneme samples to a final concentration of 0.25–0.5 mg/ml in microcentrifuge tubes by adding various reagents in resuspension buffer and incubating them at room temperature. At each time point measured (2–30 min range), 15 µl were removed from the sample microcentrifuge tube and pipetted onto one well of a 48-well polystyrene tissue culture plate. The meniscus of the drop was broken to allow even distribution of the sample. Axonemes were adhered to the bottom of the plate by centrifugation (2 min at 400 g), ensuring that observed motion was attached axonemal beating and not axoneme drift.

Assay for the quantification of axonemal motion. Axoneme samples were maintained at a constant temperature (25°C ± 0.5°C) during all experimental procedures with the use of a thermostatically controlled heated stage. The motion of axonemes was processed using the Sisson-Ammons video analysis (SAVA) system (22). Axonemes were anchored by centrifugation to the plastic well with one end adhering to the dish and the free end beating. Each axoneme appears to be anchored to the dish only on one end, with the other free end motile and responsible for the observed frequency measurements. This appearance very closely resembles the normal cilia attached to intact cells. Images of axonemes were visualized on an Olympus IMT-2 inverted phase-contrast microscope with a x20 objective lens with a x1.5 tube multiplier, and images were captured with a Kodak 310 analog/digital video camera (Eastman Kodak Motion Analysis System Division, San Diego, CA). The sampling rate was set at 85 frames per second for all experimental conditions. Captured digital video was transmitted from the camera directly into an IMACQ OCI/PXI-1422 digital acquisition board (National Instruments, Austin, TX) within a Dell Precision 420 Workstation. The entire captured image of 640 x 480 pixels was automatically analyzed for motion by SAVA using a process known as WFA. Axonemes with a frequency of ≤2 Hz were not analyzed and considered nonmotile. The WFA technique has been validated against specific region-of-interest analysis as described (25) and found to correlate for axonemal beating as well (R2 = 0.98, Fig. 1). The SAVA software analyzed each image containing thousands of motion points to determine the average frequency, and the standard error of the mean for each field was captured. For each experimental condition, a minimum of six separate fields were captured, analyzed, and expressed as a data point. ANOVA was run on each data point and was considered significant with a P value ≤ 0.05.



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  		Fig. 1. Comparison of cilia beat frequency (CBF) determined by the region of interest (ROI) and whole field analysis (WFA) methods. CBF was determined on ATP-activated axonemes (2.5 mM ATP) and analyzed over a range of temperatures (12–50°C) and microscope magnifications to obtain a broad range of frequencies and image capture conditions. CBF was determined from identical digital images by 2 methods: 1) Six 3-x-3 pixel ROI were chosen from each screen. The means and SE of these values are displayed on the x-axis. 2) WFA was performed on the same images described above. The means and SE of these values are displayed on the y-axis. The dashed line represents the linear regression "best fit" to the data. Images were obtained at x400 ({circ}), x300 ({blacksquare}), and x150 ({blacktriangleup}) image magnification, respectively. A high correlation (R2 = 0.98) was observed between these 2 methods of CBF determination.

 
Cyclic nucleotide-dependent kinase activity assay. PKA activity was determined in extracted axonemes from bovine trachea prepared as described above. The axoneme protein (1 mg/ml) was assayed in the presence or absence of 10 µM cAMP by a modification of the methods previously described (10) using a reaction mix consisting of 130 µM PKA heptapeptide substrate (LRRASLG; Peninsula, Belmont, CA) in a buffer containing 20 mM Tris·HCl (pH 7.5), 100 µM IBMX, 20 mM magnesium-acetate, and 200 µM [{gamma}-32P]ATP. PKG activity was assayed in a similar manner to PKA, with the substitution of the peptide RKRSRAE for the heptapeptide substrate, the addition of 10 µM cGMP, and the presence of protein kinase inhibitor peptide (PKI) (24). Axonemes (20 µl) were added to 50 µl of the reaction mix described above and incubated for 15 min at 30°C. Spotting the assay mix (60 µl) onto P-81 phosphocellulose papers (Whatman, Hillsboro, OR) halted the reaction. Papers were then washed five times for 5 min in 75 mM phosphoric acid and washed once in ethanol. Dried papers were counted in nonaqueous scintillation fluid, and enzyme activity was expressed as pmol·min–1·mg–1. Significance was determined using a one-way ANOVA.

ATPase activity assay. Dynein Mg2+-ATPase activity was determined in detergent-extracted bovine bronchial epithelial cell axonemes. Axoneme samples were added to a reaction mixture consisting of Mg2+, [{gamma}-32P]ATP, and Tris·HCl and incubated for 15 min at 30°C. The reaction was halted by the addition of silicotungstic acid, KH2PO4, and molybdate-sulfuric acid. The aqueous phase was separated by the addition of an isobutanol-benzene solution, and the aqueous phase was counted in aqueous scintillant (Ecolume; ICN, Irvine, CA). Results were expressed as nmol·min–1·mg–1 of ATPase activity in relationship to total cellular protein. Data were analyzed for significance by one-way ANOVA with a confidence interval of 95%.

Materials. The [{gamma}-32P]ATP was purchased from ICN, the phosphocellulose P-81 paper from Whatman (Clifton, NJ), and the peptides for kinase assays from Peninsula Laboratories (Belmont, CA). All other reagents were purchased from Sigma Chemical (St. Louis, MO).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of ATP on axoneme beating. Because it has been shown that ATP alone can induce baseline beating of bovine ciliary axonemes via the activity of an ATPase, we determined the optimal concentration of ATP required for a steady axonemal beating. Final concentrations of ATP from 0.1–1.25 mM were added to a suspension of axonemes, and CBF measurements were taken at intervals from 3 to 15 min or until axoneme activity ceased. Without the addition of ATP, no motion is observed in extracted axonemes. Increasing the concentration of ATP resulted in higher frequency measurements (Fig. 2). A concentration of 0.15 mM ATP resulted in 2.8 ± 0.2 Hz of axoneme beating at 4 min and slowed to a halt by 10 min. Increasing the concentration of ATP to 0.3 mM resulted in an initial increased activity of 3.9 ± 0.3 Hz at 4 min decreasing to 2.7 ± 0.2 Hz by 10 min before beating stopped at 13 min. A concentration of 0.6 mM stimulated axoneme motility of 5.6 ± 0.6 Hz at 4 min followed by decreasing activity until no movement occurred at 30 min. A baseline of ~5 Hz was maintained with the addition 1.25 mM ATP from 3 to 15 min with little decline until 40 min. Axoneme activity decreased at 45 min, and by 50 min the motion of the axonemes had stopped. These results demonstrate that the consumption of exogenous ATP is required for the activation of isolated axonemal beating.



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  		Fig. 2. Increasing ATP concentration increases frequency measurements. The x-axis depicts time from 0 to 18 min; the y-axis depicts the CBF as measured in hertz (Hz). Experiments (n = 9) were performed in triplicate.

 
Effect of cAMP on axoneme beating. To determine the effect of cAMP on the activity of bovine ciliary axonemes, a time course was performed, and readings were taken from 2 to 20 min in the presence and absence of various concentrations of cAMP. The addition of 1 mM ATP established a baseline (4.5 ± 0.3 Hz) at 2 min. The addition of 10 µM cAMP significantly increased the motion of the bovine ciliary axonemes at 10 min to 8 ± 0.5 Hz (Fig. 3). The addition of cAMP (10 µM) significantly increased cilia motility, with the observed motion ranging from 7.9 ± 0.7 Hz at 2 min to a maximal 8.3 ± 0.6 Hz at 5 min and continued at 20 min (Fig. 3, inset). Concentrations of cAMP ranging from 1 to 10 were determined to be optimal for increasing axoneme activity above baseline. Higher cAMP concentrations (100 µM) did not further enhance activity at 2, 5, or 10 min beyond that of 10 µM.



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  		Fig. 3. Effect of cAMP on axoneme beating. The x-axis represents the amount of cAMP measured in µM; the y-axis is CBF measured in Hz. The filled bars represent the addition of 1, 10, and 100 µM cAMP for 10 min. The addition of 10 and 100 µM is significant compared with baseline (*P ≤ 0.05). The open bar depicts the baseline CBF observed from the addition of 1 mM ATP only. In the inset, the x-axis demonstrates time from 0 to 20 min; the y-axis depicts CBF as measured in Hz. ATP (1 mM, {square}) establishes a baseline of 4.5–5.5 Hz from 2–20 min, whereas 10 µM cAMP further stimulates CBF over the same time course. All experiments (n = 9) were performed in triplicate.

 
Effect of cGMP on axoneme beating. To determine the effect of cGMP on axonemal motility, detergent-extracted bovine ciliary axonemes were examined, and their motion was assayed in the presence and absence of various concentrations of cGMP for various times. Baseline activity was established with 1 mM ATP, and motility readings were taken from 2 to 15 min. At 5 min, CBF was increased ~1 ± 0.4 Hz over baseline with the addition of 1 µM cGMP (Fig. 4). Increasing the concentration of cGMP to 10 µM resulted in an increase in CBF to 8+0.5 Hz compared with a baseline ATP-only CBF of 5.7 ± 0.3 Hz. The addition of 100 µM cGMP further enhanced the activity of ciliary axonemes, resulting in an increase to 8.6 ± 0.6 Hz. Stimulation of axoneme beating occurred rapidly (within 2 min) and continued to 15 min (Fig. 4, inset). Concentrations greater than 100 µM did not have a significant effect on baseline beating of ciliary axonemes. Aberrantly large concentrations of either cyclic nucleotide interfere via competition with the accessibility of ATP to its binding site in vitro. For this fundamental reason, increasing concentrations to the millimolar range actually decreases axonemal CBF by hindering the axoneme's ability to process the exogenous ATP (data not shown).



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  		Fig. 4. Effect of cGMP on axoneme beating. Detergent-extracted bovine ciliary axonemes were examined, and their motion was assayed in the presence and absence of various concentrations of cGMP for various times. All experiments (n = 9) were performed in triplicate. The open bar depicts the baseline CBF observed from the addition of 1 mM ATP only. The filled bars represent CBF at 5 min in the presence of 1, 10, and 100 µM cGMP. The inset demonstrates baseline CBF in the presence of 1 mM ATP and 10 µM cGMP-stimulated CBF from 2–15 min. *P ≤ 0.05.

 
Dual cyclic nucleotide regulation of axoneme beating. To determine the effect of PKA and PKG inhibition on cyclic nucleotide enhanced axoneme activity, the motion of detergent-extracted axonemes was measured in the presence and absence of kinase inhibitors and cyclic nucleotides. Axonemes were preincubated for 5 min at room temperature with inhibitors of PKA (1 µM KT-5720) and PKG (1 µM Rp-cGMPS) or buffer. After preincubation with inhibitors, samples were treated in the presence or absence of 10 µM cAMP, 10 µM cGMP, or both. Finally, axonemes were activated with 1 mM ATP, and their motility was measured 10 min after addition of ATP. As shown in Fig. 5, the addition of either cyclic nucleotide alone enhances axonemal CBF over axonemes stimulated with ATP alone (9.6 ± 0.8 vs. 5.8 ± 0.7 Hz, P ≤ 0.05). The combination of cAMP and cGMP stimulates the maximum elevation in axoneme beating (11.4 ± 0.5 Hz, P ≤ 0.005). This augmented CBF is not due to an additive effect as 20 µM of either cAMP or cGMP alone do not produce the same response increase of the combination of 10 µM cAMP plus 10 µM cGMP (9.7 ± 0.5 Hz, P ≤ 0.05 vs. ATP control). The addition of KT-5720, a specific inhibitor of PKA activity, blocked the ability of 10 µM cAMP to enhance CBF. Likewise, PKI also blocked cAMP-stimulated increases in axoneme beating (data not shown). As expected, KT-5720 had no effect on cGMP-enhanced CBF; however, KT-5720 was able to decrease the enhanced motility observed when a combination of cAMP and cGMP was used. Preincubating suspended axonemes with Rp-cGMPS inhibited the ability of cGMP to enhance axoneme CBF. Rp-cGMPS significantly blocked the augmented beat frequency produced by the combination of both cyclic nucleotides. Neither KT-5720 nor Rp-cGMPS altered ATP-stimulated baseline beating.



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  		Fig. 5. Dual cyclic nucleotide regulation of axoneme beating. The motion of detergent-extracted axonemes was measured in the presence and absence of kinase inhibitors and cyclic nucleotides. All experiments (n = 9) were performed in triplicate. Axonemes were preincubated for 5 min at room temperature with inhibitors of PKA (1 µM KT-5720, gray bars) and PKG (1 µM Rp-cGMPS, black bars) or no inhibitor (open bars). After preincubation with inhibitors, samples were treated in the presence or absence of 10 µM cAMP, 10 µM cGMP, or both. Finally, axonemes were activated with 1 mM ATP, and their motility was measured 10 min after adding ATP. *Significant difference from cyclic nucleotide in the absence of inhibitor; P ≤ 0.05.

 
Isolated axonemes contain endogenous PKA and PKG activity. Because the direct application of cyclic nucleotides to axonemes results in elevated beating, we hypothesized that the target kinases for these axonemes exist in an activatable state on the axoneme. To test this hypothesis, direct kinase activity assays were performed to identify PKA and PKG catalytic activity from extracted axonemes. In the presence of 10 µM cAMP, total axonemal PKA activity was approximately tenfold greater than baseline PKA in the absence of exogenous cAMP (Fig. 6). To evaluate total stimulatable PKG activity, we added 10 µM cGMP to the reaction mix, resulting in a nearly threefold greater activation of PKG than in the absence of cGMP. Axonemes subjected to multiple cycles of freeze-thaw lost this elevated kinase response to cyclic nucleotide (data not shown). Likewise, the response of axonemal beating to cyclic nucleotide was abolished by freeze-thawing. These data suggest that target kinases for the cyclic nucleotides exist in a localized state on the ciliary axoneme.



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  		Fig. 6. Isolated axonemes contain endogenous PKA and PKG activity. Direct kinase activity assays were performed to identify PKA and PKG catalytic activity from extracted axonemes. Extracted axonemal protein was incubated in the presence or absence of 10 µM cAMP and assayed for PKA as described in MATERIALS AND METHODS. Similarly, axonemal protein was assayed for PKG activity in the presence or absence of 10 µM cGMP. The data represent the fold ratio increase in activity of the presence over the absence of cyclic nucleotide. All experiments (n = 9) were performed in triplicate. The y-axis depicts fold activity increases over baseline activity; the x-axis depicts the 2 measured signals. *P ≤ 0.005; **P ≤ 0.05.

 
Effects of activated PKA on axoneme beating. Because cAMP enhances axoneme CBF and PKA catalytic activity is found on the bovine ciliary axoneme, the direct addition of the active catalytic subunit (C-subunit) of PKA to the axonemes should result in increased axoneme CBF in the absence of cAMP. Baseline CBF was established using 1 mM ATP, and increasing concentrations of C-subunit (0.12–1 µg/ml) were added to the axoneme suspensions. Adding the C-subunit of PKA to suspended axonemes dose dependently increased CBF on the axoneme (Fig. 7). The addition of 0.12 and 0.25 µg/ml of C-subunit slightly increased axoneme CBF (0.5 and 1 Hz, respectively). Significant increases in CBF were observed with the addition of C-subunit at concentrations of 0.5 and 1.0 µg/ml (1.5 and 3 Hz).



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  		Fig. 7. Effects of activated PKA on axoneme beating. The addition of the active catalytic subunit (C-Sub) of PKA to the axonemes in the absence of cAMP was measured. All experiments (n = 9) were performed in triplicate. The y-axis represents CBF measured in Hz; the x-axis represents the addition of C-Sub to 1 mM ATP. Baseline CBF was established using 1 mM ATP, and increasing concentrations of C-Sub (0.12–1.0 µg/ml) were added to the axoneme suspensions. *P ≥ 0.05.

 
Effects of activated PKA on axoneme ATPase activity. Ciliary beating requires dynein ATPase activity. If the addition of the C-subunit of PKA enhances beating in a suspension of axonemes, we hypothesized that PKA might be acting to increase ATPase activity on the axoneme. To test this hypothesis, C-subunit was added in increasing concentrations from 0.1 to 2 µg/ml to a suspension of axonemes and ATPase activity was measured. ATPase activity increased in a concentration-dependent manner with the addition of 0.1–0.4 µg/ml C-subunit and reached a plateau from 0.4 to 2 µg/ml C-subunit (Fig. 8). As a control for endogenous ATPase activity of the kinase, C-subunit alone at the concentrations used did not significantly elevate ATPase activity in our assay.



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  		Fig. 8. Effects of activated PKA on axoneme ATPase activity. The effect of activated PKA on axoneme ATPase activity was measured. All experiments (n = 9) were performed in triplicate. The y-axis represents ATPase activity measured in nmol·min–1·mg–1; the x-axis represents increasing concentrations of C-subunit (0–2 µg/ml) added to a suspension of axonemes. C-subunit alone was used as control. *P ≤ 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our study focuses on the signaling elements present in the axoneme that regulate the stimulation of ciliary motility that occurs with stress. In ciliated airways and in whole ciliated cells CBF stimulation by {beta}-agonists is the prototype of this ciliary "fight or flight" response and is dependent on the activation of PKA and/or PKG (25). Indeed, desensitization of this stress response results in impaired mucociliary function in vivo (21). Our findings demonstrate that these key ciliary regulatory kinases are present on isolated demembranated axonemes and, importantly, remain functional regulators of ciliary motility even in this cell-free organelle system. This cell-free ex situ organelle motility assay provides an important tool to study cilia regulation and the location of the regulatory elements.

As with any ex situ assay, we developed specific handling procedures for the axonemes to ensure the reproducibility of the results. The axonemes must be detergent-extracted from the intact trachea for a period not longer than 1.5 min. Longer extraction times "contaminate" the axoneme preparation with other organelles and result in excessive debris, which could contribute to particle drift artifact during the video analysis as previously reported (22). Similarly, drift of the axonemes is of concern, since this model does not anchor the axonemes to the culture dish by any means other than gravity. Visualization of the axonemes is also a challenge in large volumes impairing clear image focus. Although a small volume promotes rapid axoneme settling to the bottom of the dish, the sample dries out too rapidly for extended time courses. The optimal working conditions existed when 15 µl of axonemes (0.2 mg/ml) were visualized in a 48-well tissue culture plate.

Baseline beating in response to exogenous ATP was carefully established. We determined a time frame in which the frequency and the number of motile points were consistently present by examining the effects of ATP concentration on the time period of consistent baseline beat frequency and total number of motion points detected by WFA. A consistent baseline of stimulated motility is obtained by 1 mM ATP for a period of ~15 min. Decreasing the ATP concentration results in an increasingly rapid return of the axonemes to a static nonmotile state. This is most likely due to the rapid dynein ATPase-mediated consumption of all available exogenous ATP. Baseline beating is also sensitive to the storage conditions of the axonemes and the temperature of the assay. Increasing the sucrose concentration of the frozen axoneme storage buffer from 8 to 25% elevated baseline ATP-stimulated beating by ~3 Hz. Such elevated sucrose (or glycerol) would protect enzyme "survival" from freeze-thaw degradation. Likewise, increasing the assay temperature from 25 to 30°C (without increasing the ATP concentration) increased the beat frequency of the axonemes and decreased the duration of beating. Incubating the axonemes with cyclic nucleotides at 4°C did not result in an elevation in ATP-stimulated beating. Both the storage and assay temperature conditions indicate the presence of temperature-sensitive enzymes, further supporting our observation that cyclic nucleotide kinases are directly located on the axonemes.

The combination of cAMP and cGMP generated the maximal stimulation of axoneme beating. This does not appear to be an additive effect of increasing cyclic nucleotide concentrations. Our dose-response studies suggest that no significant increase in CBF is obtained with 100 µM than with 10 µM of any single cyclic nucleotide. Therefore, it is unlikely that even the 20 µM combined concentration of cAMP and cGMP is responsible for the augmented effect of both cyclic nucleotides on axoneme CBF. This is consistent with our published findings that substimulatory concentrations of cAMP and cGMP elevate CBF in combination, but not separately (24). The order of addition of cyclic nucleotides has an effect on total beating. Maximal augmented beating occurs when cAMP and cGMP are added at the same time. The sequential addition of one cyclic nucleotide followed by the other augmented axoneme beating to a lesser extent (data not shown). Because the inhibition of PKA or PKG does not inhibit axoneme CBF to baseline ATP-only levels when both cyclic nucleotides are present (Fig. 5), it suggests that precedent activation of the cilia by one cyclic nucleotide is not required for activation by the other.

The PKA-dependent cilia axoneme stimulation pathway can be activated without cyclic nucleotide stimulation since the C-subunit of PKA (which does not require activation) stimulated CBF in addition to exogenous cAMP (Fig. 7). This indicates that, despite extraction of the axonemes from the cell, exogenous PKA C-subunit still localizes to the cilia activation sites that are likely closely associated with the dynein ATPase motor complexes. This hypothesis is supported by the observation that exogenous PKA C-subunit dramatically stimulated axoneme dynein ATPase activity (Fig. 8). The concentrations of exogenous C-subunit activity were greater than the endogenous activity stimulated by cAMP. This might suggest that the highly ordered or targeted compartmentalization of PKA on the axoneme represents a more efficient apparatus for substrate phosphorylation than media diffusion of active kinase. Both stimulated ATPase activity and CBF increases in our studies demonstrate the preservation of functional roles in these detergent-extracted cilia, suggesting that the isolation protocol does not induce artifacts in the signal response mechanisms. However, as with any ex situ system, some complexities of intact cell cilia function, such as phosphatases or phosphodiesterases, may be lost due to the extraction method.

In summary, our findings indicate that ciliary beating of isolated bovine tracheal cilia axonemes can be upregulated through either PKA- or PKG-dependent pathways in a cell-free organelle system. This dual cyclic nucleotide-dependent increase in axoneme beating is tightly coupled with the activation of axoneme PKA and/or PKG enzyme activity and, in the case of PKA, is associated with stimulation of axoneme dynein ATPase enzyme activity. Taken together, basic regulatory signaling elements for stimulation of ciliary beating are present in the isolated cilia organelle and make this an ideal model system for studying the regulation of ciliary beating at the subcellular level.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Veterans Affairs Merit Review Grant (T. A. Wyatt) and National Institute on Alcohol Abuse and Alcoholism Grant 5 RO1 AA-08769-12 (J. H. Sisson).


   ACKNOWLEDGMENTS
 
We acknowledge Jacqueline A. Pavlik for technical expertise and contribution to this manuscript. T. A. Wyatt is an American Lung Association Career Investigator.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. A. Wyatt, Dept. of Internal Medicine, Pulmonary and Critical Care Medicine Sect., Univ. of Nebraska Medical Center, 985300 Nebraska Medical Center, Omaha, NE 68198-5300 (E-mail: twyatt{at}unmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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