The regulation of CBF has been studied extensively by many investigators using ciliated ependymal cells (Nguyen et al. 2001), ciliated nasal or tracheal epithelial cells (Wanner et al. 1996; Wong et al. 1998; Lieb et al. 2002; Mwimbi et al. 2002) and frog ciliated oesophageal cells (Zagoory et al. 2002); however, most of the studies were conducted using ciliated cells in primary culture (Wanner et al. 1996). In the present study, single ciliary cells were obtained from rat lungs, and the ciliary beatings of bronchiolar ciliary cells were observed by video-enhanced contrast (VEC) optical microscopy, which allowed the measurement of CBF from video frame images (30 Hz) and changes in cell morphology, such as cellular shape or size (Nakahari et al. 1990, 2002; Fujiwara et al. 1999; Hosoi et al. 2002). In the present study, we examined the effects of terbutaline (a specifically selective β₂-adrenergic agonist) on the CBF of bronchiolar ciliary cells. In the course of the experiments, we found that terbutaline not only increased CBF but also decreased the volume of bronchiolar ciliary cells.

The isosmotic cell shrinkage coincides with the activation of cellular functions, such as fluid secretion in salivary acinar cells and sweat gland cells (Nakahari et al. 1990; Suzuki et al. 1991; Takemura et al. 1991; Foskett & Melvin, 1989), and fluid absorption in lung cells (Nakahari & Marunaka, 1996, 1997; Hosoi et al. 2002). Moreover, isosmotic cell shrinkage modulates some cellular functions such as exocytosis in antral mucous cells (Fujiwara et al. 1999), non-selective cation channels in fetal lung cells (Tohda et al. 1994; Marunaka et al. 1999), apoptosis (McCarthy & Cotter, 1997; Maeno et al. 2000), and Na⁺–H⁺ exchange (Robertson & Foskett, 1994; Shrode et al. 1997). The cell shrinkage of bronchiolar ciliary cells induced by terbutaline may also modulate CBF.

Our hypothesis is that cell volume modulates CBF in bronchiolar ciliary cells under isosmotic conditions; that is, cell shrinkage enhances terbutaline-stimulated CBF, and in contrast, cell swelling suppresses it. In the present study, we measured the CBF and volume of single bronchiolar ciliary cells using VEC microscopy, and the effects of cell volume on terbutaline-stimulated CBF were examined by altering the volume of single bronchiolar ciliary cells under isosmotic conditions. The goal of this study was to confirm that isosmotic cell shrinkage enhances the CBF in terbutaline-stimulated rat bronchiolar ciliary cells.

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.

The video frame images recorded using a video recorder were stored in a computer via a video A/D converter. CBF was measured from 60–90 video frame images (30 frames s⁻¹) as shown in Fig. 2B, and expressed in Hz. Before the experiments, CBF was measured every 30 s for 2 min and the averaged value for CBF for 2 min was used for unstimulated CBF (CBF₀). CBF ratio was calculated (CBF_t/CBF₀). The subscript t indicates the time after the start of the experiment.

Figure 2 Video DIC images of a single ciliary cell

To estimate cell volume, cell area was measured by tracing the outline of the cell on the video image at 20 s to 1 min intervals. The outline of a cell was traced three times and the mean value was used for calculation. Cells were first perfused with the control solution for 2 min, and the mean value from five images measured every 30 s in the first 2 min was used as the control value (A₀). The variation in the traced areas of the five images in the first 2 min was less than 1.5%. The relative volume of a ciliary cell was expressed as V/V₀= (A/A₀)^1.5, where V is the volume, A is the area, and subscript 0 indicates the control value. The volume changes of a ciliary cell were estimated based on the assumption that the volume changed to the same extent in all three dimensions. This method has been described in detail previously (Foskett & Melvin, 1989; Suzuki et al. 1991). The number of cells used for one experiment is shown as n and the V/V₀ values calculated are presented as means ± s.e.m.

The statistical significance of differences between the mean values was assessed using ANOVA. Differences were considered significant at P < 0.05.

Figure 1 Histological examination of rat lung (HE staining)

Figure 2 shows video images of a single bronchiolar ciliary cell obtained by elastase treatment. The beating cilia were observed on one side of the single bronchiolar ciliary cell, that is, the apical surface (Fig. 2A). From a morphological viewpoint, single bronchiolar cells after elastase treatment are likely to maintain cellular polarity (Figs 1 and 2). Figure 2B shows 12 consecutive images taken at 30–40 ms intervals. Panels B1–6 and B7–11 show two beating cycles and the CBF was 5.4 Hz in this case. Thus, we could measure CBF in single ciliary cells. In unstimulated bronchiolar ciliary cells, the CBFs were 3–10 Hz. We used ciliary cells with CBFs of 5–8 Hz.

Figure 3 Changes in CBF and cell volume during 10 μm terbutaline stimulation

A video frame image of a single brochiolar ciliary cell before terbutaline stimulation is shown in Fig. 4A, in which the outline of the cell is traced. Figure 4B shows the video frame image 2 min after terbutaline (10 μm) stimulation, and the line shows the outline of the cell before terbutaline stimulation as obtained in Fig. 4A. Terbutaline-induced cell shrinkage is shown in Fig. 4B. V/V₀ in Fig. 4B was 0.84.

Figure 4 Video DIC images of a single ciliary cell

Figure 5A shows the effects of 10 μm H-89 (an inhibitor of PKA). The addition of H-89 did not change the CBF ratio (0.98 ± 0.03, n = 4, 5 min after addition), although it decreased V/V₀ slightly (0.98 ± 0.00, 5 min after addition). The subsequent stimulation with 10 μm terbutaline did not induce any increase in CBF ratio (0.95 ± 0.03, 5 min after stimulation) and V/V₀ (0.97 ± 0.02, 5 min after stimulation). Thus, the effects of terbutaline were inhibited by H-89 (10 μm). Moreover, forskolin (10 μm) increased CBF and decreased V/V₀ (Fig. 5B). The CBF ratio and V/V₀ 5 min after the addition of forskolin were 1.54 ± 0.06 and 0.81 ± 0.01 (n = 3), respectively.

Figure 5 Effects of H-89 (a PKA inhibitor) and forskolin

The effects of terbutaline and cAMP on CBF ratio (5 min after addition of the agonists) are summarized in Fig. 6. The increase in CBF during terbutaline stimulation was not affected by the Ca²⁺-free solution, while it was inhibited by H-89 (10 μm). Forskolin (10 μm), DBcAMP (0.5 mm) and IBMX (1 mm) increased CBF to the same extent as terbutaline (10 μm). These observations indicate that terbutaline increases the CBF mediated by cAMP accumulation in rat bronchiolar ciliary cells.

Figure 6 Effects of cAMP accumulation on CBF

Figure 7 Changes in CBF and cell volume induced by inhibition of K+ channels (quinidine) or Na+ entry pathways (amiloride and bumetanide)

The effects of amiloride (1 μm), a Na⁺ channel inhibitor, and bumetanide (20 μm), an inhibitor of Na⁺–K⁺–2Cl⁻ cotransport, on CBF ratio and V/V₀ were also examined (Fig. 7C and D). In unstimulated cells, the addition of amiloride and bumetanide did not induce any change in CBF ratio (1.00 ± 0.00, n = 3, 5 min after addition), although they gradually decreased V/V₀ (0.92 ± 0.01, 5 min after addition) (Fig. 7C). Stimulation with terbutaline (0.5 μm) increased the CBF ratio (1.35 ± 0.02, n = 5, 5 min after stimulation) and decreased V/V₀ (0.85 ± 0.01, 5 min after stimulation), and the subsequent addition of both amiloride (1 μm) and bumetanide (20 μm) induced a further increase in CBF ratio (1.50 ± 0.04, 5 min after addition) and a further decrease in V/V₀ (0.78 ± 0.02, 5 min after addition) (Fig. 7D). These observations suggest that cell shrinkage enhances terbutaline-stimulated CBF, and in contrast, cell swelling decreases it.

The effects of Cl⁻ channel inhibitors were examined using NPPB (10 μm) and glybenclamide (200 μm). Cells were treated with NPPB (10 μm, an inhibitor of Cl⁻ channels) for 5 min prior to terbutaline stimulation (Fig. 8A). NPPB (10 μm) did not induce any change in CBF ratio or V/V₀ in unstimulated cells. Terbutaline (0.5 μm) increased the CBF ratio (1.29 ± 0.01, n = 3, 5 min after stimulation) and decreased V/V₀ (0.90 ± 0.01, 5 min after stimulation). Cells were also treated with glybenclamide (200 μm) (Fig. 8B). Glybenclamide (200 μm) did not induce any change in V/V₀; however, it decreased the CBF ratio gradually (0.85 ± 0.03, n = 3, 5 min after addition). Terbutaline (0.5 μm) increased the CBF ratio (1.12 ± 0.03, 5 min after addition) and decreased V/V₀ (0.91 ± 0.02, 5 min after addition). Thus, NPPB and glybenclamide did not inhibit terbutaline actions, although the cell shrinkage and CBF increase induced by 0.5 μm terbutaline were slightly smaller than those in the absence of Cl⁻ channels blockers (Figs 7D and 8).

Figure 8 Effects of Cl− channel blockers

Figure 9 Dose effects of terbutaline on CBF and V/V0

The V/V₀ values and CBF ratios before and after the addition of amiloride (1 μm) and bumetanide (20 μm) were plotted against terbutaline concentration (Fig. 9E and F). The cell shrinkage and CBF increase depended on terbutaline concentration; V/V₀ decreased and CBF ratio increased with increasing terbutaline concentration. The subsequent addition of amiloride and bumetanide shifted the terbutaline dose–response curve of V/V₀ downwards (Fig. 9E). Moreover, it shifted the terbutaline dose–response curve of the CBF ratio to the lower-concentration side (Fig. 9F), and the half-maximum concentrations (EC₅₀ values) of terbutaline before and after the addition of amiloride and bumetanide were approximately 0.15 μm and 0.04 μm, respectively.

Figure 10 Effects of cell swelling on CBF

In Fig. 10B, the perfusion solution was switched to the KCl solution, which contained 1 μm amiloride, 100 μm strophanthidin and 0.5 μm terbutaline, to block Na⁺ channels and inhibit the Na⁺ pump. Stimulation with 0.5 μm terbutaline increased V/V₀ to a plateau within 3 min (1.13 ± 0.02, n = 4), and it also increased the CBF ratio to a plateau within 3 min (1.17 ± 0.02); the subsequent removal of amiloride decreased V/V₀ (1.02 ± 0.00, 3 min after the removal) and increased CBF gradually (1.25 ± 0.03, 3 min after removal) (Fig. 10B). In Fig. 10C, strophanthidin was removed instead of amiloride. The subsequent removal of strophanthidin also decreased V/V₀ and increased the CBF ratio gradually (Fig. 10C). The CBF ratio and V/V₀ 3 min after the terbutaline stimulation were 1.19 ± 0.01 and 1.17 ± 0.01 (n = 3), and those 3 min after the removal of strophanthidin were 1.36 ± 0.02 and 0.99 ± 0.01, respectively.

The relationship between the CBF ratio and V/V₀ under isosmotic conditions is summarized in Fig. 11, in which the CBF ratio during 0.5 μm terbutaline stimulation was plotted against V/V₀. As V/V₀ decreased, the CBF ratio increased as shown in Fig. 10; cell shrinkage increased the terbutaline-stimulated CBF, and in contrast, cell swelling suppressed it.

Figure 11 Relationship between CBF ratio and V/V0 during stimulation with 0.5 μm terbutaline

Figure 12 Osmotic stress

The application of osmotic stresses (±50 mosmol l⁻¹) in terbutaline-stimulated bronchiolar ciliary cells demonstrated that hyperosmotic stress (cell shrinkage) suppresses the CBF increase and hyposmotic stress (cell swelling) enhances it. Thus, the results of direct manipulation of cell volume by changing the osmolarity were the opposite of the findings of the isosmotic experiments: hyposmotic cell swelling enhanced the terbutaline-stimulated CBF increase, while isosmotic cell swelling suppressed it (hyperosmotic cell shrinkage suppressed the terbutaline-stimulated CBF increase, while isosmotic shrinkage enhanced it).

The regulations of CBF in airway epithelia have been studied extensively by many investigators (Wanner et al. 1996; Wong et al. 1998; Lieb et al. 2002; Mwimbi et al. 2002). However, most of the studies have been carried out using tracheal or nasal mucosal ciliary cells in primary culture (Wanner et al. 1996). In the present study, single-bronchiolar ciliary cells were obtained from rat lungs by elastase treatment, and CBF and cell volume were measured from video images using VEC microscopy. This is the first report measuring CBF and cell volume using freshly isolated single bronchiolar ciliary cells.

Isolated epithelial cells may loose their polarity, which may change their function. The light micrographs of the lung sections show that cilia only exist on the apical membrane (Fig. 1). The video images of single ciliary cells (Figs 2 and 4) also show that cilia are only present on one side of the isolated bronchiolar ciliary cells, probably the apical side. Reuss (2001) reported that apical ion channel proteins and an apical glycoprotein remain in the apical membrane of isolated polarized gallbladder epithelial cells, while they are distributed across the entire surface of non-polarized cells, and the freeze-fracture studies demonstrated that apical structures, that is, the microvilli and folds, still existed only in the plasma membrane of the small apical domain in the isolated polarized gallbladder cells (Reuss, 2001). Thus, from a morphological viewpoint, the apical structures, that is, the beating cilia, remain in the apical membrane of bronchiolar ciliary cells after elastase treatment, suggesting that the single bronchiolar ciliary cells maintain their polarity. Moreover, when we measured the bronchiolar CBF of lung slices (without elastase treatment) using the same experimental setup (data not shown), terbutaline increased the CBF of bronchiolar ciliary cells in the lung slices to a rate similar to that of the single bronchiolar ciliary cells. The freshly isolated bronchiolar ciliary cells remained dependence of CBF on terbutaline. These observations suggest that cellular functions, particularly with regard to the polarity of the cells and the CBF response to terbutaline, are maintained in the freshly isolated single ciliary cells used in the present experiments.

The CBF of unstimulated bronchiolar ciliary cells was in the 3–10 Hz range, and the maximum CBF during terbutaline stimulation was approximately 12 Hz. The CBF of the single bronchiolar ciliary cells was similar to that reported in tracheal ciliary cells in primary culture. Since the National TV Standards Committee (NTSC) video frame rate is 30 Hz, we are able to measure CBF using standard video equipment, and also to observe the shape of single bronchiolar ciliary cells on the video monitor.

In the present study, terbutaline increased the CBF of single bronchiolar ciliary cells, which was mimicked by forskolin, IBMX and DBcAMP, and inhibited by a PKA inhibitor (H-89). However, the terbutaline-stimulated CBF increase was not affected by the Ca²⁺-free solution or KCl solution, which decreases Ca²⁺ entry. These suggest that terbutaline increases the CBF mediated via cAMP accumulation in bronchiolar ciliary cells.

On the other hand, terbutaline decreased cell volume, as did forskolin. However, terbutaline increased cell volume in the presence of quinidine (a K⁺ channel blocker). This suggests that terbutaline activates K⁺ channels mediated via cAMP accumulation in bronchiolar ciliary cells. A similar isosmotic cell shrinkage induced by K⁺ channel activation during terbutaline stimulation was reported in fetal lung cells (Nakahari & Marunaka, 1996, 1997) and rat alveolar type II cells (Hosoi et al. 2002).

We also examined the effects of Cl⁻ channel inhibitors (NPPB and glybenclamide) on CBF and cell volume. In unstimulated ciliary cells, NPPB had no effects on CBF or cell volume. Glybenclamide also had no effects on cell volume but it decreased CBF slightly. Glybenclamide may have some adverse effects on CBF. However, NPPB or glybenclamide slightly suppressed the cell shrinkage induced by terbutaline, although it did not inhibit the CBF increase induced by terbutaline. Thus, terbutaline activated Cl⁻ channels in bronchiolar ciliary cells, which were partially inhibited by NPPB or glybenclamide partially. Since the complete inhibition of Cl⁻ channels is unlikely to induce cell shrinkage, most of the Cl⁻ channels of the bronchiolar ciliary cells seem to be insensitive to NPPB or glybenclamide. Based on these observations, terbutaline stimulates KCl release via the activation of K⁺ channels and Cl⁻ channels, which decreases the volume of bronchiolar ciliary cells.

Moreover, amiloride (Na⁺ channel inhibitor) as well as bumetanide (Na⁺–K⁺–2Cl⁻ cotransport inhibitor) decreased cell volume, suggesting that bronchiolar ciliary cells have Na⁺ channels and Na⁺–K⁺–2Cl⁻ cotransport as Na⁺ entry pathways. Na⁺–H⁺ exchange is also a well-known Na⁺ entry pathway. However, the present experiments were performed using a -free solution, in which the activity of the Na⁺–H⁺ exchanger is negligible. Moreover, methylisobutyl amiloride (10 μm, an inhibitor of the Na⁺–H⁺ exchanger) does not induce any change in cell volume. A low amiloride concentration, such as 1 μm, is known to inhibit only Na⁺ channels selectively, although a high concentration of amiloride, such as 100 μm, inhibits Na⁺–H⁺ exchange. These observations suggest that Na⁺ enters cells via the amiloride-blockable Na⁺-permeable channels and bumetanide-sensitive Na⁺–K⁺–2Cl⁻ cotransport under the present experimental conditions.

When Na⁺ entry is suppressed by inhibitors of Na⁺ entry pathways and the K⁺ released maintained, the amount of K⁺ released becomes larger than that of Na⁺ entering, which decreases cell volume. Amiloride (1 μm) has been reported to decrease the volume of lung cells (Nakahari & Marunaka, 1996, 1997; Hosoi et al. 2002). A similar cell shrinkage occurred in bronchiolar ciliary cells upon the addition of both amiloride and bumetanide.

In terbutaline-stimulated bronchiolar ciliary cells, the KCl solution alone decreased cell volume, and the KCl solution containing either amiloride or strophanthidin still decreased cell volume, although the KCl solution containing both amiloride and strophanthidin increased cell volume. These results suggest that bronchiolar ciliary cells may maintain a high intracellular Na⁺ concentration during perfusion with the control solution and high activities of Na⁺ extrusion mechanisms during perfusion with the KCl solution, that is, high activities of Na⁺ channels and the Na⁺ pump. These Na⁺ extrusion mechanisms may decrease the volume of the bronchiolar ciliary cells perfused with the KCl solution, and their inhibition (amiloride and strophanthidin) appears to increase cell volume because K⁺ enters the cell via K⁺ channels activated by terbutaline and no Na⁺ is extruded. At present, however, we have a limited knowledge of intracellular Na⁺ concentration, amiloride-blockable Na⁺ channels and the Na⁺ pump in bronchiolar ciliary cells. Further studies are required.

On the other hand, bumetanide and amiloride, which enhance terbutaline-induced cell shrinkage, when added during 0.5 μm terbutaline stimulation potentiated the CBF increase in bronchiolar ciliary cells. In contrast, quinidine added during terbutaline stimulation, which induced cell swelling, decreased CBF. During perfusion with the KCl solution containing amiloride and strophanthidin, 0.5 μm terbutaline increased the volume and CBF of the bronchiolar ciliary cells. However, the CBF increase induced by 0.5 μm terbutaline was small compared with that induced during perfusion with the KCl solution alone, in which terbutaline decreased the volume of bronchiolar ciliary cells. Moreover, the subsequent removal of either strophanthidin or amiloride, which decreased cell volume, induced a further increase in CBF. Thus, cell swelling inhibits the CBF increase in terbutaline-stimulated bronchiolar ciliary cells, and in contrast, cell shrinkage enhances it.

Moreover, the terbutaline dose–response study demonstrated that amiloride and bumetanide shift the dose–response curve of CBF to the lower-concentration side. However, amiloride and bumetanide did not induce any further increase in CBF during stimulation with 10 μm or 100 μm terbutaline. A high concentration of terbutaline leads to a maximum accumulation of cAMP, which induces the maximum activation of CBF, and cell shrinkage induces no potentiation of the CBF increase. Thus, cell shrinkage increases the cAMP sensitivity of the CBF increase, although it does not increase CBF directly. Moreover, CBF and cell volume showed a linear relationship in bronchiolar ciliary cells stimulated with 0.5 μm terbutaline, although the experimental conditions were different. Thus, the present experiments demonstrated that cell volume modulates the CBF increase during terbutaline stimulation.

Changes in cell volume have already been reported to modulate some cellular functions: amiloride-blockable non-selective cation channels in fetal lung cells (Tohda et al. 1994; Marunaka et al. 1999), exocytosis in antral mucous cells (Fujiwara et al. 1999), and apoptosis; (McCarthy & Cotter, 1997; Maeno et al. 2000), Na⁺–H⁺ exchange in rat astrocytes (Shrode et al. 1997) and salivary acinar cells (Robertson & Foskett, 1994).

We also applied hyper- (+50 mosmol l⁻¹) and hyposmotic (–50 mosmol l⁻¹) stresses by adding or removing 50 mm saccharose during 0.5 μm terbutaline stimulation. The application of a hyperosmotic stress induced cell shrinkage, but it decreased terbutaline-stimulated CBF. The application of a hyposmotic stress induced cell swelling, and it potentiated the terbutaline-stimulated CBF increase. Thus, the effects of osmotic cell volume changes on terbutaline-stimulated CBF were the opposite of those of isosmotic cell volume changes; that is, hyposmotic cell swelling enhanced the CBF increase, while isosmotic swelling suppressed it. The signalling pathways triggered by isosmotic volume changes may be different from those triggered by non-isosmotic volume changes in terbutaline-stimulated bronchilar ciliary cells. A similar observation has been reported in antral mucous cells; Ca²⁺-regulated exocytosis was enhanced by isosmotic cell shrinkage, but it was also enhanced by hyposmotic cell swelling (Fujiwara et al. 1999). The cellular events induced by cell volume changes under isosmotic conditions may not be the same as those induced during osmotic stress, such as a change in intracellular ionic concentration.

Isosmotic cell shrinkage has already been reported to decrease intracellular Cl⁻ concentration, [Cl⁻]_i, in salivary acinar cells (Foskett, 1990) and fetal lung cells (Tohda et al. 1994; Marunaka, 1997). The decrease in intracellular Cl⁻ concentration was reported to modulate non-selective cation channels in fetal lung cells (Tohda et al. 1994; Marunaka et al. 1999), Na⁺ channels in salivary duct cells (Dinudom et al. 1993), G-proteins (Higashijima et al. 1987; Treharne et al. 1994), and Na⁺–K⁺–2Cl⁻ cotransport in airway cells (Haas & McBrayer, 1994). With respect to [Cl⁻]_i during osmotic stress, hyposmotic stress decreases [Cl⁻]_i, and in contrast, hyperosmotic stress increases [Cl⁻]_i. The present study demonstrated that the CBF increase in terbutaline-stimulated bronchiolar ciliary cells was potentiated by hyposmotic stress and suppressed by hyperosmotic stress. These observations suggest that the [Cl⁻]_i decrease induced by isosmotic cell shrinkage may enhance the CBF increase in terbutaline-stimulated bronchiolar ciliary cells. Although, at present, we do not know the intracellular ionic concentrations in bronchiolar ciliary cells, [Cl⁻]_i may be a modulator of CBF during terbutaline stimulation. However, we also have to consider other factors activated by isosmotic cell shrinkage which may modulate CBF. Further study is required to elucidate the underlying mechanisms of cell shrinkage-induced CBF potentiation.

In unstimulated ciliary cells, cell swelling induced by quinidine decreased CBF, although cell shrinkage induced by bumetanide and amiloride did not induce any increase in CBF. Cell swelling may decrease CBFs in unstimulated cells, although we cannot neglect the direct inhibitory effects of quinidine on CBF. Thus, it remains uncertain whether or not cell volume modulates CBF in unstimulated bronchiolar ciliary cells.

Upon stimulation with terbutaline, cell shrinkage preceded the CBF increase. This observation suggests that cell shrinkage during terbutaline stimulation plays an important role in the CBF increase; that is, cell shrinkage may cause a rapid increase in CBF when ciliary cells are stimulated with a small amount of cAMP, such as in the first 30 s of the terbutaline stimulation.

In bronchiolar ciliary cells under isosmotic conditions, terbutaline stimulates cAMP accumulation, which increases CBF and decreases cell volume, and cell shrinkage enhances the CBF increase by increasing cAMP sensitivity.

We thank Dr S. Ito for his technical support in connecting the video A/D converter.