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1 Central Clinical Laboratory2 Department of Internal Medicine3 Department of Physiology, and Osaka Medical College, Takatsuki 569-8686, Japan
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(Received 17 August 2004;
accepted after revision 10 November 2004; first published online 7 January 2005)
Corresponding author T. Nakahari: Department of Physiology, Osaka Medical College, Takatsuki, 569-8686, Japan. Email: takan{at}art.osaka-med.ac.jp
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Introduction |
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In exocrine glands, NaCl enters cells via the coupled exchanges located in the basolateral membrane. This NaCl entry accumulates Cl– in the cells, which maintains the driving force for Cl– secretion, since the Na+ that enters is pumped out by Na+–K+-ATPase. Previous reports showed that AT-II cells secrete Cl– in the presence of amiloride (Nielsen et al. 1998), and that a ß-adrenergic agonist activates Cl– channels (O'Grady & Lee, 2003). The Na+–H+ exchange may play an important role in Cl– secretion in AT-II cells when Na+ channels are inactivated or inhibited under pathogenic conditions, such as infections, shock or acute lung injury.
The Na+–H+ exchange is activated by an increase in [Ca2+]i in many cell types (Putney et al. 2002). In our previous report, a Ca2+-free solution was found to induce shrinkage of the AT-II cells, which was blockable by MIA (Hosoi et al. 2002). This suggests that activity of the Na+–H+ exchange in AT-II cells appears to be regulated by [Ca2+]i. At present, however, the effects of [Ca2+]i on the Na+–H+ exchange in AT-II cells remain uncertain. The present study was designed to determine whether [Ca2+]i regulates the Na+–H+ exchange in AT-II cells by measuring cell volume and pHi.
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Methods |
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The control solution (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). To prepare a Ca2+-free solution (solution II), CaCl2 was removed from the control solution and 1 mM EGTA was added. The HCO3–-free solution (solution III) contained (mM): NaCl, 146; KCl, 4.5; MgCl2, 1; CaCl2, 1.5; NaHCO3, 25; NaHepes, 5; HHepes, 5; and glucose, 5 (pH 7.4). Solutions containing 25 mM HCO3– were aerated with 95% O2 and 5% CO2, and the HCO3–-free solution was aerated with 100% O2 at 37°C. For the NH4+ pulse experiment (acid loading), 25 mM NaCl of solution III was replaced with 25 mM NH4Cl. Ionomycin was from Calbiochem-Novabiochem (La Jolla, CA, USA), 5-(N-methyl-N-isobutyl)-amiloride (MIA) and DNase I were from Sigma (St Louis, MO, USA), and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-2Na (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid-2Na (SITS), elastase, heparin, and bovine serum albumin (BSA) were from Wako (Osaka, Japan). MIA, DIDS, SITS, amiloride and ionomycin were dissolved in dimethyl sulphoxide (DMSO). All the reagents were prepared at their final concentrations immediately before the experiments. The final concentration of DMSO never exceeded 0.1%, which was previously confirmed to have no effect on cell volume.
Cell preparations
Alveolar type II cells (AT-II cells) were isolated from the lungs of male rats (Slc:Wistar/ST, 150–200 g from SLC Inc., Hamamatsu, Japan) according to previous reports (Kikkawa & Yoneda, 1974; Dobbs et al. 1980; Hosoi et al. 2002, 2004). Briefly, the rats were anaesthetized by intraperitoneal injection of pentobarbitone sodium (60–70 mg kg–1) and then heparinized (1000 i.u. kg–1). Lungs were cleared of blood by perfusion, lavaged and incubated with endotracheally infused elastase (0.15 mg ml–1) and DNase I (0.03 mg ml–1) for 30 min at 37°C. Following this incubation, both lungs were placed in the control solution containing DNase I (0.08 mg ml–1) and 3% BSA and were minced using fine forceps. The minced tissues were filtered through a nylon mesh with a pore size of 150 µm2. Isolated cells were washed three times with centrifugation (160 g for 5 min). The cells were resuspended in the control solution (4°C) and used for experiments within 3 h after preparation.
The surfactant in isolated AT-II cells was stained by the modified Papanicolaou method and an immunocytochemical method using surfactant protein D (SP-D) (Kikkawa & Yoneda, 1974; Kasper et al. 1995; Hosoi et al. 2002). The results showed that 60–70% of the cells contained surfactant granules as previously reported (Dobbs et al. 1980; Hosoi et al. 2002). Under video-enhanced contrast (VEC) microscopy, the AT-II cells, which have intracelluar granules and microvilli, were distinguished from other cells as previously reported (Hosoi et al. 2002, 2004).
Cell volume measurement
Isolated AT-II cells were placed on a coverslip precoated with Cell-Tak (Becton Dickinson Labware, Bedford, MA, USA). The coverslip with cells was set in a perfusion chamber (Fujiwara et al. 1999; Hosoi et al. 2002, 2004; Shiima-Kinoshita et al. 2004) that was mounted on the stage of a differential interference contrast microscope connected to a VEC system (ARGUS-10, Hamamatsu Photonics, Hamamatsu, Japan), and images were recorded continuously by a video recorder. The volume of the perfusion chamber was approximately 20 µl, and the rate of perfusion was 200 µl min–1. Experiments were carried out at 37°C. The focus of the microscope was frequently adjusted to observe the cells at the same focal plane.
To estimate cell volume, the area of the cell was measured by tracing its outline on the video image every 10–30 s. The average value obtained from five images measured in the first 2 min was used as the control value (A0). The relative volume of the AT-II cell was expressed as V/V0 = (A/A0)1.5, where V is the volume, A the area, and the subscript 0 indicates the control value. Thus, the values of relative cell volume (V/V0) were normalized to the control value. Volume changes in the AT-II cell were estimated, assuming that the volume changed to the same extent in all three dimensions. This method has already been described in detail previously (Foskett & Melvin, 1989; Nakahari et al. 1990; Suzuki et al. 1991; Nakahari & Marunaka, 1996, 1997; Hosoi et al. 2002, 2004). The values of V/V0 from four experiments were expressed as means ± S.E.M.
pHi and [Ca2+]i measurement
Cells were incubated with 5 µM 3'-O-acetyl-2',7'-bis(carboxyethyl)-5-carboxyfluorescein diacetoxymethyl ester (BCECF-AM) or 5 µM fura 2-AM (Dojindo, Kumamato, Japan) for 30 min at room temperature (22–24°C) in solution I containing 2% BSA and then washed three times with solution I containing 2% BSA. Cells were resuspended and stored in solution I containing 2% BSA at 4°C, and 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; Yoshida et al. 2003). All the 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. BCECF was excited at 450 and 490 nm, and emission was measured at 530 nm. Fura 2 was excited at 340 and 380 nm, and emission was measured at 510 nm. The fluorescence ratio (F490:F450 for BCECF or F340:F380 for fura 2) was calculated and stored in an image analysis system. The calibration curve for pHi was obtained from the F490:F450 values of the BCECF-AM-loaded cells, which were perfused with solution IV containing nigericin (10 µg ml–1). The pH of solution IV was set at 6.6, 7.0, 7.2, 7.4 or 7.8 by adding 1M KOH. Solution IV contained (mM): KCl, 130; NaCl, 20; MgSO4, 1; and Hepes, 10. In the [Ca2+]i measurement, changes in [Ca2+]i were expressed as fura 2 fluorescence ratio (F340:F380). One experiment was performed using five or six coverslips, and the pHi or F340:F380 values of three cells from two or three coverslips were expressed as means ± S.E.M.
The statistical significance of the differences between the mean values was assessed using paired and unpaired Student's t test, as appropriate. Differences were considered significant at P < 0.05.
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Results |
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Before experiments, cells were perfused with the control solution for 5 min. During the control perfusion, changes in the AT-II cell volume were not detected. The switch to the Ca2+-free solution decreased the volume of AT-II cells (V/V0 3 min after the switching was 0.92 ± 0.01, n = 4), and the subsequent addition of amiloride (1 µM) further decreased it (V/V0 3 min after the addition of amiloride was 0.86 ± 0.00; Fig. 1A). In Fig. 1B, cells were first perfused with the control solution containing 1 µM amiloride, and then with the Ca2+-free solution containing 1 µM amiloride. The addition of amiloride (1 µM) decreased the volume of AT-II cells (V/V0 3 min after the addition of amiloride was 0.93 ± 0.01, n = 4), and the subsequent removal of extracellular Ca2+ further decreased it (V/V0 3 min after the removal of extracellular Ca2+ was 0.88 ± 0.01). In Fig. 1C, cells were perfused with the Ca2+-free solution and then MIA (10 µM), not amiloride (1 µM), was added. The switch to the Ca2+-free solution decreased the AT-II cell volume (V/V0 3 min after the removal of extracellular Ca2+ was 0.94 ± 0.01, n = 4) and the subsequent addition of amiloride (1 µM) did not induce any change in the AT-II cell volume (V/V0 3 min after the addition of MIA was 0.93 ± 0.01). In Fig. 1D, cells were first perfused with the control solution containing MIA and then with the Ca2+-free solution. MIA (10 µM) decreased the volume of AT-II cells (V/V0 3 min after the addition of MIA was 0.93 ± 0.01, n = 4), and the subsequent switch to the Ca2+-free solution did not induce any change in the AT-II cell volume (V/V0 3 min after the removal of extracellular Ca2+ was 0.93 ± 0.01). Thus, the cell shrinkage induced by the Ca2+-free solution was MIA sensitive, but amiloride insensitive, suggesting that Na+ entry via the Na+–H+ exchange is Ca2+ sensitive.
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The Na+–H+ exchange is coupled with Cl––HCO3– exchange, which results in movement of NaCl into the AT-II cells. Inhibition of Cl––HCO3– exchange is expected to induce shrinkage of the AT-II cell. The effects of inhibitors of Cl––HCO3– exchange (200 µM DIDS and 1 mM SITS) on AT-II cell volume were examined (Fig. 3). DIDS (200 µM) decreased AT-II cell volume (V/V0 = 0.94 ± 0.01 at 3 min, n = 4), and the subsequent switch to Ca2+-free solution containing 200 µM DIDS did not induce any change in AT-II cell volume (V/V0 = 0.94 ± 0.01 at 3 min after switching; Fig. 3A). SITS (1 mM) also induced similar shrinkage of the AT-II cell (V/V0 = 0.95 ± 0.00 at 3 min, n = 4) and the subsequent switch to a Ca2+-free solution containing 1 mM SITS also did not induce any change in AT-II cell volume (V/V0 = 0.94 ± 0.01 at 3 min after the switching; Fig. 3B). Thus, DIDS and SITS induced shrinkage of the AT-II cell like MIA.
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AT-II cells were pretreated with 1 mM quinine (a K+ channel blocker) and 1 µM amiloride. In these conditions, Na+ entry via Na+ channels and K+ release via K+ channels are minimized (Hosoi et al. 2002) and Na+ enters cells via MIA-blockable Na+–H+ exchange, since inhibition of Na+–K+–Cl– cotransport (using bumetanide) has no effect on AT-II cell volume during perfusion with HCO3–-containing solution (Hosoi et al. 2002). Addition of both quinine and amiloride induced swelling of AT-II cells (V/V0 = 1.08 ± 0.02 at 2 min, n = 4). Then, addition of ionomycin (1 µM) induced further cell swelling (V/V0 = 1.13 ± 0.00 at 3 min after ionomycin addition; Fig. 5A).
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Intracellular pH and [Ca2+]i
The pHi of AT-II cells was measured using the BCECF fluorescence ratio. During perfusion with the HCO3–-containing control solution, the pHi of AT-II cells was approximately 7.2–7.4. When the perfusion solution was switched from the control solution to Ca2+-free solution, pHi decreased (Fig. 6A). The pHi values during perfusion with control solution (4 min) and Ca2+-free solution (13 min) were 7.24 ± 0.01 and 7.19 ± 0.01 (n = 3), respectively. The addition of MIA (10 µM) also decreased pHi (Fig. 6B). The pHi values before and after addition of 10 µM MIA were 7.30 ± 0.02 and 7.18 ± 0.02 (n = 4), respectively. The addition of ionomycin (1 µM) increased pHi. The pHi values before and after addition of 1 µM ionomycin were 7.31 ± 0.03 and 7.48 ± 0.02 (n = 4), respectively. The further addition of MIA (10 µM) decreased pHi to 7.44 ± 0.02 (Fig. 6C). The addition of DIDS (200 µM) increased pHi slightly, and then decreased it gradually (Fig. 6D). The pHi values before and after the addition of DIDS were 7.31 ± 0.03 (n = 3) and 7.21 ± 0.01, respectively.
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Discussion |
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The present study showed that inhibition of Na+–H+ exchange decreases cell volume and, in contrast, its activation increases cell volume, and that DIDS-induced cell shrinkage is MIA sensitive. This indicates that coupling of Na+–H+ exchange and Cl––HCO3– exchange takes Na+ and Cl– into the cells, and that the effects of Na+–HCO3– cotransport on AT-II cell volume are negligibly small. Thus, not only Na+–H+ exchange but also Cl––HCO3– exchange may be regulated by [Ca2+]i, since activation of Cl––HCO3– exchange appears to increase cell volume. The present study demonstrated that an increase in [Ca2+]i enhances the recovery of pHi after acid loading in the absence of CO2–HCO3–. Since the activity of the Cl––HCO3– exchanger depends on CO2 and intracellular HCO3, its activity is low under CO2–HCO3–-free conditions. Also, CO2–HCO3–-free conditions, the Na+–HCO3– cotransport is less active. These results indicate that the Na+–H+ exchange is Ca2+ sensitive, at least in AT-II cells.
The NaCl entry via the coupled exchanges (Na+–H+ exchange and Cl––HCO3– exchange), which induces intracellular Cl– accumulation, plays an important role in maintaining the driving force for Cl– secretion in exocrine acinar cells, and is Ca2+ sensitive (Cook et al. 1994; Robertson et al. 1997; Putney et al. 2002). A subtype of Na+–H+ exchanger (NHE1) found in exocrine glands has a calmodulin binding site and is activated by the Ca2+–calmodulin complex (Wakabayashi et al. 1994; Putney et al. 2002). The AT-II cells were reported to express NHE1 in the basolateral membrane (Lubman & Crandall, 1994). These observations are consistent with our findings, that is, an increase in [Ca2+]i stimulates the Na+–H+ exchange in AT-II cells.
Protein kinase C has been reported to activate Na+–H+ exchange in AT-II cells (Wadsworth et al. 1996). The pH recovery after the acid loading (NH4+ pulse method) demonstrated that the Na+–H+ exchange activation occurred 15 min after the start of PKC stimulation. However, activation of Na+–H+ exchange induced by an increase in [Ca2+]i occurred within 1 min, which is faster than that induced by the PKC.
Surfactant exocytosis from AT-II cells was reported to be activated by an increase in [Ca2+]i (Dietl et al. 2001; Haller et al. 2001). Some agonists, such as a combination of PKC activation and terbutaline, ATP and prostaglandin E2, increase [Ca2+]i in AT-II cells (Isohama et al. 2001; Morsy et al. 2001). Expansion of the lung, which occurrs during respiration, induces [Ca2+]i oscillations in the AT-II cells (Ashino et al. 2000). Changes in the partial pressure of CO2 during respiration should induce changes in pHi of the AT-II cells. This suggests that changes in the Na+–H+ exchange activity controls H+ ion extrusion from AT-II cells to minimize pHi changes and that [Ca2+]i may play an important role in the control of pHi in response to the rapid changes in CO2 partial pressure during respiration.
ß-Adrenergic agonists activate amiloride-sensitive Na+ channels and Cl– channels in the apical membrane of AT-II cells (Nielsen et al. 1998; Lazrak et al. 2000; O'Grady & Lee, 2003). However, it remains uncertain whether the AT-II cell secretes Cl– into the alveolar cavity under physiological conditions. A short-circuit current measurement demonstrated that Cl– secretion occurs in rabbit alveolar epithelium treated with amiloride (Nielsen et al. 1998; Lazrak et al. 2000). Fetal lung cells, in which Na+ channels are inactivated, secrete Cl– into the alveolar cavity (O'Grady & Lee, 2003). From these observations, Cl– secretion may be activated when Na+ absorptive pathways are damaged, such as in the case of acute lung injury (Lazrak et al. 2000). Under such pathological conditions, Na+–H+ exchange may play an important role in Cl– secretion.
Hosoi et al. (2002, 2004) reported that terbutaline induces triphasic volume changes in AT-II cells caused by activation of ion channels and the Na+–K+ pump, mediated by cAMP accumulation. The present results show that changes in Na+–H+ exchange activity induce changes in AT-II cell volume. This indicates that the Na+–H+ exchange activity also regulates or maintains the volume of AT-II cells. Moreover, H+ production induced by a, increase partial pressure of CO2 during respiration may also stimulate Na+ entry via Na+–H+ exchange in AT-II cells, which increases cell volume.
Changes in cell volume modulate various cellular functions, such as Na+-permeable channels in fetal lung cells, exocytosis in antral mucous cells and ciliary beat frequency in bronchiolar ciliary cells (Tohda et al. 1994; Fujiwara et al. 1999; Shiima-Kinoshita et al. 2004). The cell volume changes induced by Na+–H+ exchange may also modulate some cellular functions of AT-II cells, such as surfactant secretion and Na+ absorption. Moreover, cell swelling inhibited the Na+–H+ exchange activities in erythrocytes (Dunham et al. 2004) and cell shrinkage activated it in salivary acinar cells (Robertson & Foskett, 1994). Our previous report (Hosoi et al. 2002) demonstrated that an increase in [Ca2+]i induces cell shrinkage in AT-II cells. These observations suggest that activity of the Na+–H+ exchanger stimulated by an [Ca2+]i increase are enhanced by shrinkage of the AT-II cell, such as during surfactant secretion or stimulation with PGE2, ATP, PKC or ß2-adrenergic agonists. Further studies are needed to clarify the physiological role of volume changes in AT-II cells.
The function of the AT-II cell is summarized in Fig. 9. In AT-II cells, NaCl enters via Na+–H+ exchange and via Cl––HCO3– exchange, and also via Na+ channels and Cl– channels. Na+ ions are extruded via the Na+–K+ pump (Hosoi et al. 2002, 2004). K+ and Cl– ions are released via K+ channels and Cl– channels, respectively, and K+ ions released enter via the Na+–K+ pump. Thus, the Na+, K+, Cl– and HCO3– ion effluxes balance the influxes of these ions, which maintains the steady-state cell volume. The present study demonstrates that Ca2+-free solution or MIA decreases AT-II cell volume by inhibiting Na+ entry via the Na+–H+ exchanger.
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Footnotes |
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are significantly different (P < 0.05).
and marked as ‘Control’), the application of an NH4Cl pulse increased pHi and its removal induced rapid decrease in pHi, which recovered gradually (n
= 4). A, the pHi recovery after the NH4Cl pulse was inhibited by 10 µM MIA. In the presence of MIA, ionomycin had no effects on pHi (•, n
= 5). B, the pHi recovery after the NH4Cl pulse was accerelated by the addition of ionomycin alone (n
= 5). Values marked by * were significantly different from those of the respective control experiments (P < 0.05).
