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1 Department of Thoracic and Cardiovascular Surgery2 Department of Internal Medicine3 Department of Physiology4 Department of Pathology, Osaka Medical College, Takatsuki 569-8686, Japan
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Abstract |
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(Received 14 July 2004;
accepted after revision 6 September 2004; first published online 13 September 2004)
Corresponding author T. Nakahari: Department of Physiology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, Japan. Email: takan{at}art.osaka-med.ac.jp
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Introduction |
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Ciliary beat frequency (CBF) was previously reported to be increased by many substances, such as ß-adrenergic agonists, muscarinic agonists and ATP (Wanner et al. 1996; Shiima-Kinoshita et al. 2004). Muscarinic agonists such as acetylcholine (ACh) were reported to increase CBF mediated by intracellular Ca2+ concentration ([Ca2+]i) (Salathe & Bookman, 1995; Salathe et al. 1997). However, most of the studies were conducted using primary-cultured cells from the trachea and nasal epithelia (Wanner et al. 1996). In the present study, we attempted to directly measure the CBF of tracheal ciliary cells using a slice preparation. We can observe the ciliary beatings of the tracheal apical surface and directly measure CBF from video frame images (30 Hz) using video-enhanced contrast (VEC) optical microscopy.
Hypo-osmotic stress modulates some cellular functions, such as cell volume regulation (Wang et al. 1996; Dezaki et al. 2000), ion channel and transporters (Matthews et al. 1998; Hazama et al. 2000; Sabirov et al. 2001), exocytosis (Fujiwara et al. 1999) and CBF (Shiima-Kinoshita et al. 2004). In some cells, these modulations were reported to be induced by ATP, which is released from cells by a hypo-osmotic cell swelling (Wang et al. 1996; Dezaki et al. 2000; Hazama et al. 2000; Sabirov et al. 2001). On the other hand, ATP increases CBF in tracheal ciliary cells in primary culture (Weiss et al. 1992; Tarasiuk et al. 1995; Wanner et al. 1996; Evans & Sanderson, 1999; Lieb et al. 2002). We examined the effects of hypo-osmotic stress on tracheal CBF, but no increase in CBF was detected during the hypo-osmotic stress. However, in the course of experiments we found that a hypo-osmotic stress enhances the ACh-stimulated CBF increase.
The inhalation of aerosolized drugs is widely used as a therapy to improve respiratory problems. The osmolarity of a solution containing a drug (e.g. bromhexine) that is widely used for the inhalation is approximately 220 mosM, although the final osmolarity of the solution deposited within the airway is unknown, given complex evaporation and condensation from inhaled aerosol droplets prior to final deposition. However to the best of our knowledge, it is not known whether a hypo-osmotic solution is preferable to a hyper- or an isosmotic solution. These questions drove us to study the effects of hypo-osmotic stress on the CBF on the tracheal surface during ACh stimulation.
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Methods |
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Solution I contained (mM): NaCl 121, KCl 4.5, MgCl2 1, CaCl2 1.5, NaHCO3 25, NaHepes 5, HHepes 5 and glucose 5; pH 7.4. Solution II contained (mM): KCl 4.5, MgCl2 1, CaCl2 1.5, NaHCO3 25, NaHepes 5, HHepes 5 and glucose 5; pH 7.4. To reduce the osmolarities of test solutions, an appropriate amount of solution II was added to solution I. Hypo-osmotic stresses applied were expressed as the reduced osmolarities, such as –40 mosM. Solution III contained (mM): NaCl 121, KCl 4.5, MgCl2 1, NaHCO3 25, NaHepes 5, HHepes 5 and glucose 5; pH 7.4. For a Ca2+-free solution, EGTA (1 mM) was added to solution III and pH was adjusted to 7.4 by adding 1 M NaOH. All the solutions were aerated with a gas mixture of 95% O2–5% CO2. Acetylcholine chloride (ACh), ATP, ionomycin, thapsigargin, heparin, suramin and bovine serum albumin (BSA) were purchased from Wako Pure Chemicals (Osaka, Japan), and apyrase (Grade VII, ATPase/ADPase ratio 
1), 5-nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB) from Sigma Chemical Co (St Louis, MO, USA). Some reagents were dissolved with dimethylsulfoxide (DMSO). Before the experiments, the reagents were diluted to their final concentrations and the final DMSO concentration never exceeded 0.1%. All the experiments were performed at 37°C.
Cell preparations
Rats (Slc:Wistar/ST) weighing approximately 150–200 g were purchased from SLC (Shizuoka, Japan) and fed a standard diet and water. The rats were anaesthetized by an intraperitoneal injection of pentobarbital sodium (Nembutal, 60–70 mg (kg body weight)–1). The animals were then heparinized (1000 units (kg body weight)–1, I.V.) (Hosoi et al. 2002, 2004) and the lungs and trachea were cleared of blood by transcardial perfusion. The lungs, trachea and heart were removed from the rats, and then the removed trachea was opened and cut into small pieces (5–6 mm square blocks) and stored in solution I (4°C). The tracheal blocks were used in experiments within 3 h.
The experiments were approved by the Animal Research Committee of Osaka Medical College, and the animals were cared for according to the guidelines of this committee.
CBF measurement
Before the experiments, a tracheal block was cut into thin slices using two razor blades, and the thin slices were placed on a coverslip precoated with neutralized Cell-Tak (Becton Dickinson Labware, Bedford, MA, USA) to allow the slices to adhere firmly to the coverslip. The coverslip with the slices was set in a perfusion chamber as described elsewhere (Fujiwara et al. 1999; Nakahari et al. 2002; Hosoi et al. 2002, 2004; Shiima-Kinoshita et al. 2004). The perfusion chamber was placed on the stage of a differential interference contrast (DIC) microscope (BX50Wi, Olympus, Osaka) connected to a VEC system (ARGUS-10, Hamamatsu Photonics, Hamamatsu, Japan). Images were recorded continuously using a video recorder. The volume of the perfusion chamber was approximately 20 µl, and the rate of perfusion was 200 µl min–1. The images recorded by a video recorder were stored in a computer via a video A/D converter. The CBF was measured from 60–90 video frame images (30 frames s–1) as shown in Fig. 1B, and were expressed in Hz (Shiima-Kinoshita et al. 2004). Before the experiments, CBF was measured every 30 s for 3 min and the averaged CBF for 3 min was used as unstimulated CBF (CBF0). The CBF changes were expressed as CBF ratio (CBFt/CBF0), where the subscript t indicates the time after the start of experiments.
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Tracheal blocks were incubated in solution I containing 2% BSA and 10 µM acetoxymethyl ester form of fura 2 (fura 2-AM; Dojindo, Kumamato, Japan) for 60 min at room temperature (22–24°C), and then washed three times with solution I containing 2% BSA. The tracheal blocks were resuspended and stored in solution I containing 2% BSA at 4°C. A fura 2-loaded tracheal block was cut into thin slices using two razor blades, and the thin slices were placed on a coverslip precoated with neutralized Cell-Tak to allow the cells to adhere firmly to the coverslip. The coverslip with slices was set in a perfusion chamber, which was then mounted on the stage of an inverted microscope (IX70, Olympus, Tokyo, Japan) connected to an image analysis system (ARGUS/HiSCA, Hamamatsu Photonics, Hamamatsu, Japan) (Fujiwara et al. 1999; Nakahari et al. 1999, 2002; Yoshida et al. 2003). All experiments were performed at 37°C. The volume of the perfusion chamber was approximately 80 µl and the rate of perfusion was 500 µl min–1. Fura 2 was excited at 340 nm and 380 nm, and emission was measured at 510 nm. Fluorescence ratio (F340/F380) was calculated and stored in an image analysis system. One experiment was performed using five to six coverslips from two to three rats, and the F340/F380 values of three cells from two to three coverslips were expressed as means ± S.E.M.
The statistical significance of differences was assessed by paired or unpaired Student's t test, as appropriate. Differences were considered significant at 0.05.
Immunohistological examinations
The trachea was fixed in 10% formalin buffered with 150 mM phosphate for 24 h, dehydrated in graded series of ethanol concentrations and embedded in paraffin, according to a standard protocol. Some sections were then stained with haematoxylin–eosin (HE). Other sections were immunostained for the P2X4 receptor. Briefly, 5-µm thick deparaffinized sections were tested preliminarily for antigen retrieval either by protease digestion (0.01% protease XXIV (Sigma) solution) at 37°C for 30 min, trypsin digestion (0.1% trypsin (Nacalai Tesque, Kyoto, Japan)/0.01 M PBS) at 37°C for 15 min or heat-mediated epitope retrieval (10 mM pH 6.0 citrate buffer) at full pressure at 121°C for 15 min. However, the best staining was achieved without any pretreatment. The sections were incubated with 0.3% H202 in methanol for 10 min to block endogenous peroxidase, and then incubated with a polyclonal antibody against the rat P2X4 receptor raised in rabbits (Alomone Laboratories, Israel; dilution 1: 200) for 1 h at room temperature in a moist chamber. Secondary immunoreaction was performed by the avidin–biotin–peroxidase complex method using a Vectastain universal Elite ABC kit (Vector Laboratory, Burlingame, CA, USA) for 30 min. Colour development was performed for 5–8 min using 0.02% 3,3'-diaminobenzidine tetrahydrochloride (Dako)/0.05 M Tris-HCl (pH 7.6) with 0.005% H202 (Yoshii et al. 2003). Negative control was obtained by substituting the primary antibody with normal serum or PBS. The sections were finally counterstained with haematoxylin.
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Results |
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Figure 1A shows a video frame image of a ciliary cell lining on the luminal side of a tracheal slice. Beating cilia were observed on the apical surface of the tracheal ciliary cells. Figure 1B shows nine consecutive frame images taken every 30–40 ms. A cilium in frame images (panels B1–B9) is traced over the features. Panels B1–B5 and B5–B9 show two cycles of ciliary beating and the CBF of this cell is approximately 7.7 Hz. Thus, we could measure CBF in a tracheal ciliary cell in a slice preparation. In unstimulated tracheal ciliary cells, the CBF was 4–12 Hz. In the present study, we used ciliary cells whose CBF was 6–12 Hz.
Effects of ACh
Figure 2 shows a typical CBF response in a tracheal ciliary cell stimulated with ACh or ionomycin. ACh (10 µM) rapidly increased CBF from 10 Hz to 12 Hz. Upon ACh removal, the CBF decreased to a prestimulation level within 1.5 min. The CBF ratio 3 min after stimulation with ACh was 1.17 ± 0.02 (n = 8), which was significantly different from that of unstimulated CBF (P < 0.02). Ciliary cells in the tracheal slice were stimulated with the Ca2+ ionophore ionomycin (5 µM). Ionomycin increased CBF from 11 Hz to 14 Hz within 2 min (Fig. 2B). Thapsigargin (2 µM) also increased CBF (data not shown). The CBF ratio (CBF/CBF0) 4 min after the stimulation with ionomycin or thapsigargin was 1.26 ± 0.06 (n = 5) or 1.15 ± 0.02 (n = 4), respectively; these values were significantly different from that of unstimulated CBF, P < 0.02.
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The effects of Ca2+ on the sustained CBF ratios (3–4 min after the switch to agonists or inhibitors) of tracheal ciliary cells are summarized in Fig. 3, in which the CBF ratios were obtained from four to eight experiments. ACh (10 µM), thapsigargin (2 µM) and ionomycin (5 µM) increased CBF significantly (P < 0.02), and the CBF increase induced by 10 µM ACh was suppressed by Ca2+-free solution or Ni2+ (1 mM).
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ACh (1 µM) increased CBF from 10.25 Hz to 10.5–10.75 Hz. The subsequent application of a hypo-osmotic stress (–60 mosM) induced a further increase in ACh-stimulated CBF to 11.5 Hz (Fig. 4A). The CBF ratios before and 3 min after the application of a hypo-osmotic stress (–60 mosM) were 1.05 ± 0.01 and 1.16 ± 0.02 (n = 6), respectively. Thus, the –60 mosM stress significantly enhanced the ACh-stimulated CBF ratio (P < 0.05). The application of a hypo-osmotic stress (–60 mosM) prior to stimulation with ACh (1 µM) increased CBF slightly and then decreased CBF from 10 Hz to 9.5 Hz within 2 min (Fig. 4B). The subsequent stimulation with 1 µM ACh rapidly increased CBF from 9.5 Hz to 11.5 Hz. The CBF ratios before and 3 min after the ACh stimulation during a hypo-osmotic stress (–60 mosM) were 0.93 ± 0.02 and 1.12 ± 0.03 (n = 11), respectively. The ACh-stimulated CBF ratios during the –60 mosM stress were similar to those shown in Fig. 4A and B, and were significantly higher than without any hypo-osmotic stress (P < 0.05).
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A hypo-osmotic stress induces ATP release in many cell types. The hypo-osmotic potentiation of ACh-stimulated CBF may be caused by ATP released from the tracheal slice. We examined the effects of suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) to inhibit ATP actions on ACh-stimulated CBF. Ciliary cells were pretreated with suramin (100 µM), which decreased CBF by approximately 0.5 Hz. The CBF ratio 5 min after the addition of suramin was 0.93 ± 0.02 (n = 5). Cells were stimulated with 5 µM ACh prior to the application of a hypo-osmotic stress (–40 mosM). ACh (1 µM) induced a rapid increase in CBF followed by a gradual decrease. The CBF ratios 1 min and 4 min after the stimulation with ACh were 1.0 ± 0.03 (significantly different from the value before the addition of ACh, P < 0.05) and 0.88 ± 0.03 (n = 5), respectively. The subsequent application of a hypo-osmotic stress did not induce any increase in CBF, but induced a continuous decrease. The CBF ratios 1.5 min and 5 min after the –40 mosM stress were 0.87 ± 0.03 and 0.79 ± 0.05 (significantly different from the value before the –40 mosM stress, P < 0.05), respectively. Thus, suramin eliminated further increases in ACh-stimulated CBF induced by the hypo-osmotic stress (Fig. 8A). Similar experiments were also performed in the presence of apyrase (1 U ml–1), which had a low ATPase/ADPase ratio (approximately 1) (Fig. 8B). Apyrase decreased CBF by approximately 1 Hz. The CBF ratio 4 min after the addition of apyrase was 0.89 ± 0.03 (n = 4). ACh (5 µM) increased CBF transiently, which subsequently decreased gradually. The CBF ratios 1 min and 5 min after the ACh stimulation were 0.98 ± 0.03 (significantly different from the value before the addition of ACh, P < 0.05) and 0.86 ± 0.04, respectively. The subsequent application of a hypo-osmotic stress did not induce any increase in CBF but induced a continuous decrease. The CBF ratios 1.5 min and 5 min after the application of hypo-osmotic stress (–40 mosM) were 0.82 ± 0.03 and 0.70 ± 0.05 (significantly different from the value before the application of the –40 mosM stress, P < 0.05), respectively. Suramin and apyrase eliminated the hypo-osmotic potentiation of ACh-stimulated CBF increase, although they decreased CBF throughout the application of suramin or apyrase.
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Measurement of [Ca2+]i
The change in [Ca2+]i was monitored by the fluorescence ratio (F340/F380) in fura 2-loaded tracheal slices. A hypo-osmotic stress (–40 mosM) transiently increased F340/F380 and the subsequent addition of ACh induced a sustained increase in the ratio (Fig. 10A). In Fig. 10B, ciliary cells were stimulated with 1 µM ACh prior to the application of the hypo-osmotic stress (–40 mosM). ACh caused a sustained increase in F340/F380 and the subsequent application of a hypo-osmotic stress (–40 mosM) produced a rapid increase in F340/F380 followed by a gradual decrease to a plateau (Fig. 10B). ATP (1 µM) increased F340/F380 immediately, which thereafter decreased gradually (Fig. 10C). Thus, ATP transiently increased F340/F380.
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The thin sections of the trachea were stained with HE (Fig. 11A). Cilia were detected on the apical surface of ciliary cells, which lined the luminal surface of the trachea. A previous report showed that rat tracheal epithelium expresses the P2X4 and P2Y2 receptors (Marino et al. 1999). Immunohistochemical examinations demonstrated that the apical part of a ciliary cell was immunopositively stained for only the P2X4 receptor (Fig. 11B). However, rat tracheal ciliary cells were not immunopositively stained for the P2Y2 receptor.
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Discussion |
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The CBF of unstimulated tracheal ciliary cells was mainly maintained by Ca2+-independent mechanisms, as the Ca2+-free solution decreased CBF only by 10%. However, the augmentation of [Ca2+]i induced by ionomycin, thapsigargin or ACh increased CBF in tracheal ciliary cells as previously reported (Salathe & Bookman, 1995; Salathe et al. 1997, 2001). An adequate hypo-osmotic stress enhanced ACh-stimulated CBF and [Ca2+]i increases and shifted the ACh dose–response curve of CBF to the left (lower concentrations) in tracheal ciliary cells. However the hypo-osmotic stress did not induce any increase in ACh-stimulated CBF in the absence of extracellular Ca2+. This indicates that the shift of the ACh dose–response curve during a hypo-osmotic stress is induced by further increases in [Ca2+]i in tracheal ciliary cells.
The extent of the hypo-osmotic potentiation of ACh-stimulated CBF did not depend on the osmolarities of solutions, as the CBF ratios were similar (approximately 1.2) within the hypo-osmotic stresses tested (–20 mosM to –90 mosM). The potentiation of ACh-stimulated CBF is likely to be induced by other factors that are stimulated by a hypo-osmotic stress, such as the release of biologically active substances.
Hypo-osmotic stress is a well-known stimulus of ATP release in many cell types (Wang et al. 1996; Roman et al. 1999; Dezaki et al. 2000; Guyot & Hanrahan, 2002). ATP release is considered to be stimulated by osmotic cell swelling (Wang et al. 1996; Mitchell et al. 1998; Hazama et al. 2000; Sabirov et al. 2001). In tracheal ciliary cells, the effects of hypo-osmotic stress on ACh-stimulated CBF were eliminated by suramin (an inhibitor of purinergic receptors) or apyrase (an enzyme that breaks down extracellular ATP), and were mimicked by ATP addition. Moreover, immunohistochemical examinations demonstrated that the P2X4 purinergic receptor exists on the apical surface of tracheal ciliary cells (Marino et al. 1999). These observations suggest that the potentiation of ACh-stimulated CBF may be induced by ATP released by hypo-osmotic stress.
On the other hand, both suramin and apyrase decreased unstimulated CBF. ATP may be released from tracheal cells continuously, although the non-specific effects of both inhibitors may also decrease CBF, as inhibitor agents may not be as specific as assumed.
A pathway for ATP release was reported to be volume-sensitive anion channels, which are inhibited by NPPB (a Cl– channel blocker) (Sabirov et al. 2001). However, NPPB did not inhibit the hypo-osmotic potentiation of ACh-stimulated CBF in tracheal ciliary cells. Several pathways for ATP release may exist in tracheal ciliary cells, such as NPPB-insensitive channels, exocytosis or other mechanisms.
Hypo-osmotic stress did not potentiate ACh-stimulated CBF in cells stimulated with 100 µM ACh. A high concentration of ACh, such as 100 µM, maintains a high level of inositol 1,4,5-trisphosphate and empties intracellular Ca2+ stores, which completely activate the store-operated Ca2+ entry (Putney, 1986). Under this condition, ATP stimulation may not induce further increases in [Ca2+]i.
Addition of exogenous ATP induced a sustained increase in CBF, although it increased [Ca2+]i transiently. Similar observations were reported in ciliary cells of the human trachea and frog oesophagus (Lieb et al. 2002; Zagoory et al. 2002). In these reports, an ATP-induced [Ca2+]i transient activated signalling cascades, including protein kinase A (PKA) and protein kinase G (PKG), which sustained increases in CBF. These observations suggest that PKA and PKG may be involved in the hypo-osmotic potentiation of ACh-stimulated CBF in tracheal ciliary cells.
Mechanical stimulation was reported to increase CBF significantly in rabbit tracheal ciliary cells in primary culture (Felix et al. 1998; Lansley & Sanderson, 1999) and ATP (10 µM) increased CBF significantly. In these experiments, ATP is released from tracheal ciliary cells. In the present study, we used the tracheal slice, which contains other cells, such as submucosal cells, goblet cells and connective tissue cells. ATP may also be released from these cells. Although we did not identify the ATP-releasing cells during hypo-osmotic stress, the tracheal ciliary cells at least appear to release ATP during the hypo-osmotic stress.
On the other hand, a hypo-osmotic stress alone does not induce any increase in CBF, although it increases [Ca2+]i transiently. In the present study, a low concentration of ATP (0.1 µM) did not increase CBF, although it potentiated ACh-stimulated CBF increase significantly. ATP release induced by a hypo-osmotic stress may be insufficient to increase CBF in rat tracheal ciliary cells.
The present study demonstrated that the P2X4 purinergic receptor exists in tracheal ciliary cells. A previous report showed the P2Y2 purinergic receptors also exist in rat tracheal epithelium (Marino et al. 1999). We examined the P2Y2 purinergic receptor by the immunohistochemistry of tracheal ciliary cells. However, the tracheal ciliary cells were not immunopositively stained for the P2Y2 receptor. As the anti-P2Y2 receptor antibody used was for human purinergic receptors, the specificity for rat receptors may be insufficient.
The inhalation of hypo-osmotic aerosolized drugs such as bromhexine (220 mosM), is widely used to improve respiratory problems. The present study demonstrated that an adequate hypo-osmotic stress (–20 to –90 mosM) enhances ACh-stimulated CBF increase. As the tracheal surface is covered with surface liquid, the final osmolarity of surface liquid remains unclear even during the inhalation of bromohexine. The hypo-osmotic potentiation of CBF may be an important finding with respect to the enhancement of the effects of inhalation therapy, although we require further knowledge to apply it as a therapy.
The CBF of ciliary cells is activated by increases in [Ca2+]i in tracheal epithelium during ACh stimulation. A hypo-osmotic stress appears to induce ATP release from tracheal ciliary cells, and the released ATP induces further augmentation of [Ca2+]i in ACh-stimulated ciliary cells, which may be mediated by purinergic receptors, such as P2X4. This augmentation potentiates ACh-stimulated CBF in tracheal ciliary cells.
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 A. H. Saad, C. Shimamoto, T. Nakahari, S. Fujiwara, K.-i. Katsu, and Y. Marunaka cGMP modulation of ACh-stimulated exocytosis in guinea pig antral mucous cells Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1138 - G1148. [Abstract] [Full Text] [PDF]
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 S. Fujiwara, C. Shimamoto, Y. Nakanishi, K.-i. Katsu, M. Kato, and T. Nakahari Enhancement of Ca2+-regulated exocytosis by indomethacin in guinea-pig antral mucous cells: arachidonic acid accumulation Exp Physiol, January 1, 2006; 91(1): 249 - 259. [Abstract] [Full Text] [PDF]
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T. Hayashi, M. Kawakami, S. Sasaki, T. Katsumata, H. Mori, H. Yoshida, and T. Nakahari ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium Exp Physiol, July 1, 2005; 90(4): 535 - 544. [Abstract] [Full Text] [PDF]
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