Introduction

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Normal airway mucociliary clearance plays an important role in the host defense mechanism that clears the upper airways of inhaled particles, such as dusts, aerosols, bacteria, and air pollutants (1). This defense mechanism is dependent upon the production of mucus and the coordinated beating of ciliated cells that line the surface of the airway lumen. Cilia in the airway continually beat at a baseline frequency to sweep debris toward the glottis under normal physiologic conditions (2). In disease states, such as airway inflammation or infection, the mucociliary clearance function is altered either by increased mucus production or by damaged ciliary beat frequency (CBF) (1, 3).

Nitric oxide (NO) is an important cell-signaling molecule that affects many critical cell functions (4). NO is present in the exhaled air of humans, and low levels of nasal NO correlate with impaired mucociliary function in the upper airways (5). Moreover, there is no NO production in patients with Kartagener's syndrome (6, 7). There is increasing evidence that the NO-cyclic guanosine monophosphate (cGMP) signaling pathway plays an important role in CBF regulation. NO is released by an enzyme family of NO synthases (NOSs) from the substrate L-arginine (L-Arg). There are at least three known isoforms of NOS and all of themthe neuronal, inducible, and endothelial NOS (eNOS)have been found in airway epithelial cells (8). Further, release of NO in rabbit tracheal mucosa (11) and synthesis of cGMP in human airway epithelium (12) have been reported previously. In addition, inhibition of NOS has been shown to attenuate CBF stimulation induced by -adrenergic stimulants (11), cytokines (13, 14), and methacholine (15, 16). Direct application of 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP) enhances CBF in cultured human nasal airway epithelial cells (12).

Most of the biologic functions of NO are mediated through its receptor protein, soluble guanylate cyclase (sGC), which catalyzes guanosine triphosphate into cGMP. cGMP activates protein kinase (PK) G, which may participate in the regulation of CBF. Moreover, a recent study from our laboratory demonstrated that eNOS, sGC, and PKG I are colocalized in rat airway ciliated epithelial cells, implicating the involvement of the NO-cGMP signaling pathway in the regulation of CBF (17). Previous studies have suggested that CBF is regulated by the parasympathetic nervous system (15), NO (5, 13, 14), and PKA and PKG (16, 18). Recently, a new fluorescent indicator for NO, diaminofluorescein (DAF), has been developed by Kojima and colleagues (19, 20). DAF has been shown to visualize intracellular NO with high sensitivity (detection limit, 5 nM) and specificity in cultured vascular smooth-muscle cells. For verifying the NO production by ciliated cells, we stained the ciliated cells with DAF-diacetate (2DA), a cell-permeable NO indicator of DAF. We demonstrated for the first time that ciliated cells produce NO, and we also explored the regulatory effects of different components of the NO-cGMP signaling pathway on CBF in cultured rat airway ciliated cells. The results indicate that ciliated airway epithelial cells actively produce NO and that NO regulates CBF in the rat airway ciliated epithelial cells.

	Materials and Methods

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Reagents

Dulbecco's modified Eagle's medium (DMEM) and antibiotics were from GIBCO BRL (Grand Island, NY). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). 4,5-DAF-2DA was from Calbiochem (La Jolla, CA). S-nitroso-L-glutathione (GSNO), 1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene (NOC 12), N^G-monomethyl-L-arginine (L-NMMA), 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ), KT5823, 8-Br-cGMP, and Zaprinast were from Alexis Corp. (San Diego, CA). L-Arg, D-arginine (D-Arg), dimethyl sulfoxide (DMSO), and all other chemicals were obtained from Sigma (St. Louis, MO).

DAF-2DA, ODQ, and KT5823 were dissolved in DMSO/H₂O (1:1) first and diluted further in phosphate-buffered saline (PBS), pH 7.5. All the other mediators used were dissolved in PBS and were freshly prepared before each experiment. The final concentration of DMSO was not more than 0.1%.

Preparation and Culture of Rat Tracheal Epithelial Cells

The rat tracheal epithelial explant culture followed the methods described by Dirksen and associates (21) with minor modifications. Briefly, adult Sprague-Dawley rats (300 to 350 g body weight; Hilltop, Scottdale, PA) were anesthetized with isoflurane (Ohmeda PPD, Liberty Corner, NJ) and the chests were opened, then the heart, lung, and trachea were quickly removed. The tracheas were cut into 0.5- to 1-mm³ pieces of tissue explants, which were placed on coverslips precoated with rat-tail collagen. The explants were incubated in DMEM containing 10% FBS, 25 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, and 0.37% NaHCO₃ at 37°C, 5% CO₂. After 3 to 6 d of culture, the single-layered ciliated cells appeared at the edge of the explants. Clusters of three to six ciliated cells within a ×40 objective field were selected for CBF measurement. Only explants free of any bacterial or fungal contamination were used for the experiments.

Measurement of NO Production and CBF

For measuring NO production, coverslips with cultured rat airway ciliated epithelial cells were washed with Krebs-Ringer phosphate (KRB) buffer (120 mM NaCl, 4.8 mM KCl, 0.54 mM CaCl₂, 1.2 mM MgSO₄, 11 mM glucose, and 15.9 mM sodium phosphate, pH 7.2). Then the cells were incubated at 37°C in 10 µM DAF-2DA in KRP buffer for 1 h and mounted under an inverted phase contrast Olympus IMT-2 fluorescence microscope (Olympus, Tokyo, Japan) fitted with a cube containing a 495-nm excitation filter, 515-nm emission filter, and 505-nm dichroic mirror. The microscope was linked to a Toshiba 3CCD camera (Toshiba, Tokyo, Japan) and I-Cube computer image analysis system (I-Cube, Cambridge, MA). A ×40 objective was used to obtain the image of the cells. For observation of the fluorescence of NO produced by the ciliated cells, 10 mM L-Arg was added to the KRP buffer, the fluorescence was observed with ultraviolet (UV) light, and the image was captured with the video camera at 0 and at 25 min after adding L-Arg. To avoid damage to the ciliated cells, the exposure to UV light was only for 2 to 4 s. Only the intensity of the fluorescence of the ciliated cells in the selected fields was measured with Image-Pro software (Media Cybernetics, Silver Spring, MD), then compared with the 0 time-point results.

For the analysis of CBF in the ciliated cells, the baseline CBF was recorded with a JVC VCR HR-S5400U tape recorder (JVC, Tokyo, Japan) for 5 min, then L-Arg was added and recording continued for an additional 25 min with phase contrast illumination only. The videorecorded CBF was analyzed frame by frame using Adobe Premiere software (Adobe Systems, Inc., San Jose, CA). The analyzers were blinded during the CBF measurements. NO production (fluorescence intensity) and the CBF were correlated later.

Measurement of CBF Only

For the analysis of CBF in rat airway ciliated epithelial cells, tracheal explants were cultured on coverslips for 3 to 6 d in DMEM plus 10% FBS. Before experimentation, the coverslips were marked on the edges with a Dako pen (Dako, Carpinteria, CA) to avoid leaking of medium and then 500 µl of DMEM with 0.037% NaHCO₃ was added on the top. The coverslips were mounted under an inverted phase contrast Olympus microscope and the image was observed and videotaped as described earlier. Actively beating ciliated cells were selected from the field with at least three ciliated cells close to each other. All the experiments were carried out at a constant temperature of 24 ± 1°C, and at pH 7.4 to 7.6. The experiments were repeated three to 12 times (expressed as n number) according to different mediators tested. NO elevating agents and stimulants or inhibitors of the NO-cGMP signal transduction pathway were applied to examine their effects on ciliary frequency. NO donors such as L-Arg or NOC 12 (0.1 mM), and GSNO (0.1 mM), were applied. A dose-related range of different concentrations of L-Arg (at final concentrations of 0.01, 0.1, 1, and 10 mM) was added into the 500 µl of medium on top of the coverslips. Both 8-Br-cGMP (1 mM) and Zaprinast, a cGMP-specific phosphodiesterase (PDE) V inhibitor (0.1 mM), were tested for their CBF stimulating effects.

The effects of different inhibitors of the NO-cGMP signal transduction pathway, including the NOS inhibitor L-NMMA (1 mM), sGC inhibitor ODQ (from 0.1 to 100 µM), and PKG inhibitor KT5823 (0.03 to 30 µM), on L-Arg-stimulated CBF enhancement were examined. D-Arg (0.1 mM) and N^G-monomethyl-D-arginine (D-NMMA) (10 mM), the dextrorotatory enantiomers of L-Arg and L-NMMA, were also used as controls.

Statistical Analysis

All data are expressed as the mean ± standard error of mean. For the correlation of NO production and CBF stimulation, simple regression analysis was used. For the time-course response, the CBF was expressed as Hz (beats/s) and the comparisons were carried out between baseline and different time points using two-factor repeated-measures analysis of variance (ANOVA). For the dose-dependent response, the percentage change (baseline = 100%) in CBF was used as the measurement of the response of CBF to stimulus by the various mediators. This was done to permit the comparison of preparations with differing initial CBF. The comparisons were taken between 5 and 0 min (baseline) and 25 min after adding the mediators for the dose-dependent response of various mediators, and the time-matched data were compared using one-way ANOVA after the Bonferroni test. A value of P < 0.05 was considered statistically significant.

	Results

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NO Production by Ciliated Epithelial Cells and Its Correlation with CBF Enhancement

For the demonstration of NO production by the cultured ciliated epithelial cells, DAF-2DA (10 µM) was added to the cells, then 10 mM L-Arg was applied. The ciliated cells of the rat airway produced NO and became fluorescent (Figure 1, arrows). The other cells without cilia in the same culture only showed very low levels of NO production. To further elicit the effects of NO produced by ciliated cells, CBF was measured and only the changes in intensity of fluorescence in the ciliated cells were quantified and compared before and after L-Arg stimulation. These experiments demonstrate that 10 mM L-Arg (n = 6) increased CBF 28% compared with basal (n = 7, vehicle only) CBF at the 25-min time point (P < 0.0001, one-way ANOVA, Bonferroni test). At the same time, NO production, as measured by its fluorescent intensity in the ciliated cells, increased 30% (P = 0.0041, one-way ANOVA, Bonferroni test) (Figure 2). Comparison by correlation of the increase of CBF with NO production resulted in the highly significant r value of 0.76 (P = 0.0026), as shown in Figure 3. The NOS inhibitor L-NMMA (10 mM) inhibited L-Arg- induced NO production (DAF fluorescent intensity) and attenuated CBF significantly compared with D-NMMA (P < 0.0001; Figure 4). In contrast, D-Arg and D-NMMA had no effects on CBF and DAF fluorescent intensity.

View larger version (43K): [in this window] [in a new window]   Figure 1.   DAF staining in cultured rat tracheal epithelial cells. (Arrows indicate the ciliated cells; A: phase contrast brightfield; B: darkfield for DAF under UV light.) Bar = 25 µm.

View larger version (14K): [in this window] [in a new window]   Figure 2.   Effect of L-Arg on NO production (measured by DAF staining) and changes of CBF in rat tracheal ciliated epithelial cells. (P = 0.0041, and P < 0.0001 for DAF staining and CBF changes, respectively; one-way ANOVA, Bonferroni test.)

View larger version (23K): [in this window] [in a new window]   Figure 3.   Relationship between NO production and CBF changes. (r = 0.760, P = 0.0026; simple regression analysis.)

View larger version (35K): [in this window] [in a new window]   Figure 4.   Relationship between DAF fluorescent intensity and CBF in L-Arg stimulation and NOS inhibition with L-NMMA. L-NMMA at 10 mM completely inhibited CBF increase induced by L-Arg (P < 0.0001 compared with D-NMMA.)

Effects of NO Donors Such as L-Arg, NOC 12, and GSNO on CBF

To determine the effects of NO on CBF, different NO donors and stimulators were used. L-Arg at 1 mM induced a significant (20%) increase of CBF compared with baseline CBF. Moreover, the L-Arg increase in CBF was time-dependent; an increase of CBF over baseline was detectable by 5 min and reached a peak at 25 min after L-Arg stimulation (P < 0.05). Further, to determine whether the increase of CBF in response to L-Arg was dose-dependent, 0.01 (n = 4), 0.1 (n = 6), 1 (n = 7), and 10 mM (n = 3) of L-Arg were used. At the concentrations of 0.01 and 0.1 mM, L-Arg caused a slight (from 5 to 10%) increase but no significant effect on CBF stimulation. With the increase of the concentration to 1 and 10 mM of L-Arg, the CBF increased considerably from 15 to 20% (P < 0.05 compared with baseline CBF; Figure 5). To determine whether the increase in CBF seen with L-Arg is NOS-specific, the ciliated cells of the explants were exposed to equimolar (1 mM) concentrations of D-Arg. The results, as expected, demonstrated that D-Arg (n = 6) had no effect on CBF stimulation (Figure 5). Similar stimulating effects of NO were also seen with different NO donors, such as NOC 12 (0.1 mM, n = 7) and GSNO (0.1 mM, n = 5). All the NO donors increased CBF significantly and in a time-dependent manner (Figure 6).

View larger version (19K): [in this window] [in a new window]   Figure 5.   Effects of different concentrations of L-Arg on CBF. One-way ANOVA, *P < 0.05 versus D-Arg and vehicle.

View larger version (15K): [in this window] [in a new window]   Figure 6.   Effects of NOC 12 (0.1 mM) and GSNO (0.1 mM), NO donors, on CBF. *P < 0.05 versus vehicle (one-way ANOVA).

Effects of cGMP Elevating Agents on CBF

To determine whether the CBF-stimulating actions of the NO-cGMP signal transduction pathway were mediated through cGMP, different concentrations of 8-Br-cGMP (10 µM, n = 4; 100 µM, n = 5; and 1,000 µM, n = 12) were applied to the cultured epithelial cells. 8-Br-cGMP increased CBF in a time- and concentration-dependent manner with a significant (10%) increase over control occurring at 100 µM (Figure 7). The increase of CBF was detectable at 5 min and lasted until 25 min after 8-Br-cGMP administration. In addition, Zaprinast, a cGMP-specific PDE V inhibitor, showed the same effect. At 0.1 mM concentration, Zaprinast increased CBF 30% from 7.8 ± 0.5 to 9.3 ± 0.3 Hz. The increase became detectable at 5 min and peaked at 20 min after Zaprinast addition (Figure 8).

View larger version (14K): [in this window] [in a new window]   Figure 7.   Time- and dose-dependent effects of 8-Br-cGMP on CBF (one-way ANOVA).

View larger version (20K): [in this window] [in a new window]   Figure 8.   Effects of Zaprinast, a cGMP-specific PDE (type V) inhibitor on CBF. *P < 0.05 versus vehicle.

Effects of Different Inhibitors of the NO-cGMP Signaling Pathway on CBF

To determine whether the L-Arg-induced increase of CBF was dependent upon the NO produced by NOS from ciliated cells, the cells were exposed to media containing 1 mM L-Arg followed by 0.1 to 10 mM L-NMMA, a stereospecific NOS inhibitor, or 10 mM D-NMMA, the inactive enantiomer of L-NMMA. L-NMMA had no effect on baseline CBF, but dose-dependently inhibited the CBF stimulated by 1 mM L-Arg (Figure 9). Pretreatment of ciliated cells with L-NMMA prevented L-Arg-induced increase of CBF (data not shown). D-Arg did not enhance CBF and D-NMMA had no effect on L-Arg-stimulated CBF.

View larger version (31K): [in this window] [in a new window]   Figure 9.   Effects of D-NMMA and L-NMMA on L-Arg-induced enhancement of CBF.

To determine whether the L-Arg-induced increase in CBF was mediated by sGC and PKG, ODQ (a specific inhibitor of sGC) and KT5823 (a specific PKG inhibitor) were added. In addition, the inhibitory effects of ODQ (inhibition constant [K_i] = 3 µM) on the L-Arg-induced increase in CBF occurred in a dose-dependent manner. At 0.1 µM (n = 4), ODQ had no inhibitory effects at all; at 1 µM (n = 4) it inhibited L-Arg-induced CBF by 8 ± 2.2%; at 10 µM (n = 4), by 15 ± 1.8%; and at 100 µM (n = 5), complete inhibition was achieved (Figure 10).

View larger version (33K): [in this window] [in a new window]   Figure 10.   Inhibitory effect of ODQ, an sGC inhibitor, on L-Arg- induced CBF enhancement.

KT5823 (K_i = 0.2 µM) showed similar dose-dependent inhibitory effects on L-Arg-induced increase of CBF: at 0.03 µM (n = 4) KT5823 exhibited no inhibition, but at 0.3 µM (n = 5) it reached 15 ± 1.9% inhibition. At 3 (n = 4) and 30 µM (n = 6), KT5823 completely inhibited L-Arg- induced increase of CBF (Figure 11).

View larger version (31K): [in this window] [in a new window]   Figure 11.   Inhibitory effect of KT5823 on L-Arg-induced enhancement of CBF.

	Discussion

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The current studies provide a physiologic correlation to an initial observation (using immunoelectron microscopy for eNOS) that the eNOS protein is specifically localized to the basal microtubule membrane in ciliated epithelium of rat lung (10). Other studies have indirectly suggested a role for NO in regulating ciliary motility (5, 8, 10). Recently, a novel NO indicator, DAF, was described which has high specificity and sensitivity in detecting NO produced by living cells (19, 20). DAF was first used for measurement of NO production in cultured rat aortic smooth-muscle cells after activation with interleukin-1, tumor necrosis factor-, and lipopolysaccharide. Then the cells were treated with DAF-2DA, a cell-permeable and nonfluorescent derivative of DAF. In the cells DAF-2DA is converted by intracellular esterase to DAF-2, which is nonfluorescent and remains in the cells. NO produced by the cells rapidly converts DAF-2 to highly fluorescent triazolofluorescein (DAF-2 T). The NO production and DAF fluorescence were observed and the fluorescence intensity in the activated smooth-muscle cells increased with time owing to DAF-2 T production from DAF-2 by reaction with NO. The intensity was suppressed by the NOS inhibitor L-NMMA. The DAF fluorescent staining is specific and sensitive (5 nM). Stable oxidized forms of NO, such as NO₂ and NO₃, and other reactive oxygen species, such as O₂, H₂O₂, and ONOO, do not react with DAF-2 to yield any fluorescent product. Under physiologic conditions, DAF-2 T is not formed in the absence of NO (19, 20).

In this study, using this new NO indicator, we indirectly demonstrated NO production in cultured rat airway ciliated epithelial cells. Further, we also showed that the increased NO levels are correlated with the enhancement of CBF induced by L-Arg. Activation of NOS and PKG enhanced this response, and inhibition of NOS, sGC, and PKG inhibited it. We conclude that NO produced by rat airway ciliated epithelial cell may act as a modulator to stimulate CBF in an autocrine/paracrine manner.

A variety of stimulants can increase CBF. Previous reports have shown that ethanol (22), bradykinin (11, 23), Zaprinast (24), cytokines (14), methacholine (15), prostacyclin (25), and isoprenaline (26) can stimulate CBF. NOS inhibition has been shown to inhibit ethanol-elicited increase of CBF (27). A recent study reported by Venkov and coworkers (27) demonstrated that ethanol can stimulate eNOS protein and messenger RNA expression in cultured bovine endothelial cells. Whether this accounted for the NO-dependent ethanol response in airway epithelial cells is unknown. Also, L-Arg and other NO donors, such as sodium natroprusside, have been shown to reverse the effects of NOS inhibition on ethanol-induced enhancement of ciliary motility (22). In the present study we demonstrated that L-Arg-induced CBF activity in a dose- and time-dependent manner (see Figure 5), and that inhibition of NOS reduced NO production (DAF staining intensity) and attenuated CBF. In addition, an increase of intracellular cGMP levels, either by adding 8-Br-cGMP or by inhibiting cGMP-specific PDE using Zaprinast, resulted in effects identical to those from addition of NO to the ciliated epithelial cells (see Figures 7 and 8). In contrast, D-Arg did not show any cilia-stimulating effects, and inhibition of NOS activity with L-NMMA but not D-NMMA, inhibition of sGC activity with ODQ, or inhibition of PKG activity with KT5823 attenuated the cilio-stimulating effects of L-Arg (see Figures 9-11). These results are in agreement with previous suggestions (11, 12, 15, 16, 18) that the NO-cGMP signaling pathway regulates CBF.

The controversial effects of cGMP and cGMP-elevating agents, such as C-type natriuretic peptide (CNP) and atrial natriuretic factor (ANF), on CBF are very difficult to explain, as CNP stimulates and ANF inhibits CBF (28, 29). In the present study we demonstrated that 8-Br-cGMP stimulates CBF in a time- and dose-dependent manner (see Figure 7). This result is in agreement with a previous report by Geary and colleagues (12) which demonstrated that direct application of 8-Br-cGMP enhanced CBF in cultured nasal airway epithelial cells. A recent study by Uzlaner and Priel (30), in contrast, showed that use of 100 µM dibutyryl-cGMP (db-cGMP), another analogue of cGMP, failed to increase CBF in cultured rabbit tracheal epithelial cells. At the same time, db-cGMP did not change the plateau of intracellular calcium. Uzlaner and Priel suggested that the failure of db-cGMP to induce Ca²⁺ influx and to stimulate CBF might indicate that an additional factor other than activated PKG is at work (30).

The regulation and control of CBF in the airways is a very complicated process and many pathways have been implicated to play a role in the modulation of CBF (2). Several PKs have been implicated. Activation of PKG or PKA will result in an increase of CBF (16, 18, 30). PKC actions have been controversial. Levin and colleagues (31) demonstrated that activation of PKC induced calcium influx and sustained enhancement of CBF by extracellular adenosine triphosphate (ATP) in frog esophagus epithelium; in contrast, Uzlaner and Priel (30) reported inhibitory effects of PKC on CBF enhancement in rabbit tracheal ciliated epithelial cells. An explanation for this discrepancy may be based upon the species differences, but other unknown factors cannot be excluded. Several reports have demonstrated a dual regulation of CBF by PKA and PKG (16, 18). This may not be simply explained by phosphorylation or dephosphorylation of the components involved in the ciliary motility. Although our results clearly show that CBF enhancement by L-Arg can be attenuated by sGC or PKG inhibition with ODQ or KT5823, respectively, the exact mechanism(s) of how the NO-cGMP signal transduction pathway regulates CBF is still unknown. Particularly, the inhibitors used in our experiments, such as PKG inhibitor KT5823 (K_i = 0.2 µM), attenuated L-Arg- induced CBF at 0.3 µM and completely inhibited L-Arg- induced increase of CBF at 3- and 30-µM concentrations (see Figure 11), but also inhibited bromo-cyclic adenosine monophosphate (Br-cAMP)-induced increase of CBF (data not shown) in our system. The lack of relative specificity of this inhibitor and the cross-talks between PKA and PKG signaling pathways in the regulation of CBF need to be further elucidated.

Previous reports found that CBF may be regulated by modulating the intracellular Ca²⁺ concentration ([Ca²⁺]i). Korngreen and Priel (32, 33) found that extracellular ATP induced a rapid and strong (4- to 5-fold) increase in [Ca²⁺]i in the rabbit airway epithelia. This effect was transient, decaying after 3 to 4 min, whereas CBF enhancement induced by extracellular ATP was rapid and prolonged, continuing for 20 to 30 min. Using a technique that allows simultaneous measurement of [Ca²⁺]i and CBF, these investigators revealed that [Ca²⁺]i decayed to a sustained, higher-than-basal plateau. Manipulations that lowered this sustained plateau of [Ca²⁺]i caused the CBF to decay rapidly to its basal value. Most recently, using the same system, Uzlaner and Priel reported that there is an interplay between the NO-cGMP signal transduction pathway and elevated [Ca²⁺]i that enhances ciliary activity in rabbit trachea (30). They found that PKC is not involved in the regulation of CBF and [Ca²⁺]i. KT5823 and LY-83583, an sGC inhibitor, and N-nitro-L-arginine methylester, a NOS inhibitor, did not change the peak of [Ca²⁺]i but altered the plateau of [Ca²⁺]i; meanwhile, ATP-induced CBF enhancement was significantly attenuated. They concluded that the elevated [Ca²⁺]i is a strong stimulator of NOS, which activated sGC and PKG via the NO-cGMP signal transduction pathway. The activation of PKG plays a crucial role by inducing the elevated [Ca²⁺]i levels required for sustained activation of NOS and CBF enhancement (positive feedback). Unfortunately, these data still do not provide a detailed mechanism of how the NO-cGMP signal transduction pathway regulates CBF.

Both cAMP and cGMP increase the swimming velocity of permeable cells of paramecium reactivated with ATP and also increase dynein-like adenosine triphosphatase activity in permeable cells of paramecium and in motile cilia preparations (34). Studies reported that phosphorylation of ciliary dyneins by PKs from paramecium (34), from Chlamydomonas flagella (35), and from Ciona sperm plays an important role in regulating their motility. Whether the NO-cGMP signal transduction pathway stimulates CBF by phosphorylating these ciliary motor components, and whether this is related to the elevation of [Ca²⁺]i in rat airway ciliated cells, need further study.

In summary, in this study we demonstrated that NO production by cultured airway ciliated epithelial cells and the increased NO correlated with CBF stimulation. Increases in NO or cGMP stimulate CBF, and inhibition of the activity of the NO-cGMP signaling pathway attenuates L-Arg-induced CBF stimulation. The limitation of this study is that there is a mixed cell population in the culture system; other cells such as fibroblasts, endothelial cells and smooth-muscle cells may produce NO, which may affect CBF. Using single ciliated cells or cultured pure ciliated cells may eliminate the interference of CBF by the other cell types. In addition, measuring CBF in vivo under different conditions of the NO-cGMP stimulation/inhibition would provide valuable information to answer how and why NO produced by ciliated epithelial cells regulates CBF.