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MUCOSAL BIOLOGY

[Cl^–]_i modulation of Ca²⁺-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

¹Central Research Laboratory (Nakahari Project), ²Department of Internal Medicine (Division II), and ³Department of Physiology, Osaka Medical College, Takatsuki, Japan

Submitted 13 March 2007 ; accepted in final form 1 August 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The effects of intracellular Cl^– concentration ([Cl^–]_i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca²⁺-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 µM) or a Cl^– -free (NO₃^–) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl^– channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO₃^– solution. ACh and Ca²⁺ dose-response studies demonstrated that NO₃^– solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO₃^– solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO₃^– solution after ATP depletion induced only a sustained phase. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) measurements showed that bumetanide and NO₃^– solution enhanced the ACh-stimulated [Ca²⁺]_i increase. Measurements of [Cl^–]_i revealed that ACh decreases [Cl^–]_i and that bumetanide and NO₃^– solution decreased [Cl^–]_i and enhanced the ACh-evoked [Cl^–]_i decrease; in contrast, NPPB increased [Cl^–]_i and inhibited the [Cl^–]_i decrease induced by ACh, bumetanide, or NO₃^– solution. These suggest that [Cl^–]_i modulates [Ca²⁺]_i increase and ATP-dependent priming. In conclusion, a decrease in [Cl^–]_i accelerates ATP-dependent priming and [Ca²⁺]_i increase, which enhance Ca²⁺-regulated exocytosis in ACh-stimulated antral mucous cells.

gastric antrum; mucin exocytosis; acetylcholine; intracellular Cl^– concentration

Various agonists induce isosmotic cell shrinkage by activating K⁺ and Cl^– channels in epithelial cells, such as salivary acinar cells (5, 18), lung cells (11, 14, 16, 17, 23), sweat gland cells (30), and bronchiolar ciliary cells (26). The cell shrinkage modulates some cellular functions, such as ion transport (23, 32), apoptosis (13), ciliary beat frequency (26), and exocytosis (6). Isosmotic cell shrinkage decreases intracellular Cl^– concentration ([Cl^–]_i) (4, 14, 23, 32), which also modulates cellular functions, such as Na⁺-permeable channels in fetal lung cells (32) and salivary duct cells (3), Na⁺/K⁺/2Cl^– cotransporters (NKCCs) in tracheal epithelial cells (9) and squid giant axons (2), G proteins (10), cell cycle (29), and the exocytosis of -cells (1) and melanotrophs (24, 31).

In guinea pig antral mucous cells, ACh-induced cell shrinkage enhances Ca²⁺-regulated exocytosis, as previously reported (6). Moreover, hypoosmotic stress and bumetanide (an inhibitor of NKCC), which appear to decrease [Cl^–]_i, enhance ACh-stimulated exocytosis in antral mucous cells (6). Moreover, [Cl^–]_i is reported to modulate Ca²⁺-regulated exocytosis and granular maturation (priming) in -cells (1) and melanotrophs (31). These findings suggest that [Cl^–]_i may also modulate Ca²⁺-regulated exocytosis in antral mucous cells.

We have studied the regulation of mucin release from isolated antral mucous cells by directly observing exocytotic events using videomicroscopy (6, 7, 15, 20, 25, 26). In this study, we examined the effects of bumetanide, NO₃^– solution (a Cl^–-free solution), and a Cl^– channel blocker [5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)] on ACh-stimulated exocytotic events in antral mucous cells. Bumetanide or NO₃^– solution inhibits NKCC, which decreases [Cl^–]_i, and, moreover, NO₃^– solution may enhance decreases in [Cl^–]_i by replacing Cl^– with NO₃^– via Cl^– channels (21, 22). In contrast, NPPB may increase [Cl^–]_i by inhibiting Cl^– efflux.

In the present study, we used bumetanide, Cl^–-free solution, and NPPB as tools to control [Cl^–]_is and examined the effects of [Cl^–]_i on Ca²⁺-regulated exocytosis in antral mucous cells. The goal of this study is to confirm that an [Cl^–]_i decrease enhances Ca²⁺-regulated exocytosis in antral mucous cells. If so, which step of the exocytotic cycle, the ATP-dependent step (priming) or the Ca²⁺-dependent step (fusion), is modulated by intracellular Cl^–?

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Solutions and chemicals. Solution 1 contained (in mM) 121 NaCl, 4.5 KCl, 25 NaHCO₃, 1 MgCl₂, 1.5 CaCl₂, 5 NaHEPES, 5 HHEPES, and 5 glucose. To prepare a Ca²⁺-free solution, CaCl₂ was excluded from solution 1 and EGTA (1 mM) was added. To prepare a Cl^–-free solution, Cl^– in solution 1 was replaced with NO₃^– (NO₃^– solution) or gluconate (gluconate solution). The pHs of the solutions were adjusted to 7.4 by adding 1 M HCl, HNO₃, or gluconic acid, as appropriate. The solutions were gassed with 95% O₂ and 5% CO₂ at 37°C. A HCO₃^– -free solution, in which NaHCO₃ of solution 1 was replaced with NaCl, was gassed with 100% O₂ or 100% N₂. The following reagents were purchased: dinitrophenol (DNP) and NPPB from Sigma (St. Louis, MO); acetylcholine chloride (ACh) from Daiichi Pharmaceuticals (Osaka, Japan); and collagenase (for cell dispersion, 180–220 U/mg) and bovine serum albumin (BSA) from Wako Pure Chemical Industries (Osaka, Japan). All the reagents were dissolved in dimethyl sulfoxide (DMSO) and diluted to their final concentrations immediately before the experiments. The DMSO concentration did not exceed 0.1%. At this concentration, DMSO has no effect on cell volume and exocytotic events (6, 7, 11, 15, 19, 20, 25–28).

Cell preparation. Male guinea pigs (Hartley) weighing 250 g were purchased from SLC-Japan (Hamamatsu, Japan) and maintained on standard pellet food and water. The guinea pigs were anesthetized by an intraperitoneal injection of pentobarbital-Na (Nembutal, 60–70 mg/kg). After removal of the stomach, they were euthanized by cervical dislocation. 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 procedure for cell preparation was previously described in detail (6). Briefly, the antrum was excised and the mucosal layer was stripped from the muscle layer in cooled saline (4°C) by using glass slides. The stripped antral mucosa was minced with fine forceps and then incubated in solution 1 containing 0.1% collagenase and 2% BSA for 10 min at 37°C. The digested mucosa was then filtered through a nylon mesh with a pore size of 150-µm squares and washed three times. The cells were resuspended in solution 1 containing 2% BSA (4°C). The cells were stored at 4°C and used in the experiments within 3 h.

Observation of exocytosis. Isolated antral mucous cells were mounted on a coverslip precoated with neutralized Cell-Tak (Becton Dickinson Labware, Bedford, MA) for the firm attachment of the cells. The coverslip was set in a perfusion chamber mounted on the stage of a differential interference contrast microscope (BX50Wi, Olympus, Tokyo, Japan) connected to a video-enhanced contrast system (ARGUS-10, Hamamatsu Photonics, Hamamatsu, Japan) (6). Images were recorded continuously with a video recorder. The experiments were performed at 37°C. The volume of the perfusion chamber was 20 µl and the rate of perfusion was 200 µl/min. Exocytotic events, which were detected as rapid changes in the light intensity of granules (6), were counted in five to six cells every 30 s and normalized to the number of cells (events per cell per 30 s). The frequencies of exocytotic events in three to seven experiments were expressed as means ± SE.

Intracellular Ca²⁺ measurement. Isolated antral mucous cells were incubated in solution 1 containing 2% BSA and 2.5 µM fura 2-acetoxymethyl ester (fura 2-AM, Dojindo, Kumamoto, Japan) for 25 min at room temperature (22–24°C). They were then washed three times with solution 1 containing 2% BSA. Fura 2-loaded cells were resuspended and stored in solution 1 containing 2% BSA at 4°C and then mounted on a coverslip precoated with neutralized Cell-Tak. These coverslips were 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 (model ARGUS/HiSCA, Hamamatsu Photonics, Hamamatsu, Japan) (6, 19). All the experiments were performed at 37°C. The volume of the perfusion chamber was 80 µl and the rate of perfusion was 500 µl/min. Fura 2 was excited at 340 and 380 nm, and emission was measured at 510 nm. Fluorescence ratio (F340/F380) was calculated and stored in an image analysis system. The calibration curve was obtained from the F340/F380 of the cell-free Ca²⁺ calibration solutions containing 10 µM fura 2. Solution 2 contained (in mM) 130 KCl, 20 NaCl, 2 EGTA, and HEPES 10. To prepare the cell-free Ca²⁺ calibration solutions, an appropriate amount of CaCl₂ (0.2–2 mM) calculated by a computer program was added to solution 2. The pH was adjusted to 7.05 by adding 1 M KOH. The dissociation constant of Ca²⁺ and EGTA used was 214 nM (37°C, pH 7.05) (12). One experiment used five to six coverslips, and the F340/F380s of seven cells on two to three coverslips were expressed as means ± SE.

[Cl^–]_i measurement. Isolated antral mucous cells were incubated in solution 1 containing 2% BSA and 5 mM N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE, Dojindo, Kumamoto, Japan) at 30°C for 50 min. They were then washed three times with solution 1 containing 2% BSA. MQAE-loaded cells were resuspended and stored in solution 1 containing 2% BSA at room temperature (22–24°C), and then mounted on a coverslip precoated with neutralized Cell-Tak. MQAE was excited at 355 nm, and emission was measured at 510 nm. The fluorescence intensity measured (F) was stored in an image analysis system. To compare among experiments, the ratio (F/F₀) was calculated, where F₀ is the fluorescence intensity at time 0. One experiment used five to six coverslips from two to three guinea pigs. The F/F₀s of seven cells on two to three coverslips were expressed as means ± SE.

To calculate the intermediate concentration (IC₅₀) of a dose-response curve, a program for curve fitting (Delta Graph 4.5, SPSS) was used. The following is the equation used for curve fitting: y = (a – d)/[1 + (x/c)^b] + d (1) where a–d are constants, x is a concentration used, and y is the frequency of exocytotic events; a is the maximum value, d is the minimum value, c is the intermediate concentration, b is a constant.

After linearization of Eq. 1 by logarithm, the significance of the difference in IC₅₀s was assessed by Student's t-test, and that between means was assessed using paired or unpaired Student's t-test, as appropriate. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In unstimulated antral mucous cells, no exocytotic events were observed. ACh (1 µM) induced a biphasic increase in the frequency of exocytotic events: an initial phase followed by a sustained phase (Figs. 1, A and B). In comparing the experiments, we used the initial peak frequency, which was the maximum frequency within 2 min from the start of ACh stimulation. The ACh concentration used was 1 µM or 10 µM.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Effect of bumetanide (Bum) on ACh-stimulated exocytotic events in antral mucous cells. The ACh concentration was 1 µM. In the control experiment, the cells were stimulated with ACh (1 µM) alone. ACh (1 µM) evoked a biphasic increase in the frequency of exocytotic events: an initial phase was followed by a sustained phase. A: bumetanide (20 µM) alone induced no exocytotic events. However, the addition of bumetanide 3 min before ACh stimulation enhanced the initial and sustained phases of ACh-stimulated exocytotic events. *Significantly different from control (1 µM ACh alone), P < 0.05. B: addition of bumetanide (20 µM) 2 min after the ACh stimulation increased the sustained phase transiently, and the frequency was sustained. The frequency of the sustained phase was approximately twice higher than that of ACh alone. *Significantly different from control (1 µM ACh alone), P < 0.05.

Similar experiments were performed using Cl^–-free solutions, in which Cl^– was replaced with NO₃^– or gluconate. NO₃^– solution added for 3 min activated no exocytotic events in most of antral mucous cells, however, in some cells (3/14 experiments), a longer exposure of NO₃^– solution, such as 5 min, activated 0.5–1 events·cell^–1·min^–1. The further addition of ACh induced an enhanced frequency of exocytotic events (approximately by 200%) (Fig. 2A). NO₃^– solution added in the sustained phase induced a biphasic increase in the sustained frequency, similarly to that of bumetanide (Fig. 2B). Experiments were also performed using gluconate solution. Gluconate solution also enhanced ACh-stimulated exocytotic events by 100% (Fig. 2C). A similar enhancement was observed when glutamate was used instead of gluconate (data not shown). In this study, we used NO₃^– solution for the Cl^–-free experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effect of Cl–-free solution on ACh-stimulated exocytotic events in antral mucous cells. A: NO3– solution. NO3– solution alone induced no exocytotic events. However, the addition of NO3– solution 3 min before ACh stimulation enhanced the initial and sustained phases of ACh-stimulated exocytotic events. B: addition of NO3– solution 2 min after the ACh stimulation increased the sustained frequency transiently, and the frequency was sustained. The sustained frequency was approximately twice higher than that of ACh alone. C: gluconate solution. Gluconate solution also enhanced the frequency of ACh-stimulated exocytotic events. *Significantly different from control (1 µM ACh alone), P < 0.05.

The dose effects of ACh were examined during perfusion with NO₃^– solution. ACh (0.01 µM) activated no exocytotic events during perfusion with solution 1 or NO₃^– solution. ACh (0.1 µM) increased the frequency of exocytotic events only transiently, and NO₃^– solution enhanced it (Fig. 3A). ACh (0.4 µM) induced a biphasic increase in the frequency of exocytotic events (an initial phase followed by a sustained phase) and NO₃^– solution enhanced the frequency of the initial phase by 150%, but the enhancement of the sustained phase was uncertain (Fig. 3B). ACh (1 µM) induced a biphasic increase in the frequency of exocytotic events and NO₃^– solution enhanced the frequency of exocytotic events markedly, as shown in Fig. 2A. Stimulation with 4 µM ACh (Fig. 3C) and 40 µM ACh (Fig. 3D) also induced a transient increase in the frequency of exocytotic events, and NO₃^– solution enhanced it. The initial peak frequency enhanced by NO₃^– solution was 100–150% of the control experiments. Similar experiments were performed using bumetanide (20 µM) instead of NO₃^– solution. The results are summarized in Figs. 3E. The initial peak frequency was plotted against ACh concentration (Fig. 3, E and F). In the ACh dose-response curves of the initial peak frequency, bumetanide (20 µM) and NO₃^– solution shifted the maximum responsiveness upward by 50 and 100%, respectively. The half-maximal concentrations (IC₅₀s) of the ACh dose-response curve were 5.9 µM in the control experiments and were shifted to the low concentration by bumetanide (1.9 µM) and NO₃^– solution (2 µM). The statistical analysis, however, demonstrated that bumetanide or NO₃^– solution induces no significant shift in the ACh dose-response curve (P < 0.05). In the experiments of bumetanide or NO₃^– solution, the enhancement of the sustained phase was unclear.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Dose effects of ACh in NO3– solution () and in the control solution (). A: ACh (0.1 µM). B: ACh (0.4 µM). C: ACh (4 µM). D: ACh (40 µM). NO3– solution enhanced the initial peak frequencies of ACh-stimulated exocytotic events (initial phase). E: effects of NO3– solution and bumetanide on the initial phase. NO3– solution and bumetanide shifted the ACh dose-response curve upward. IC50 were shifted to the low concentration by bumetanide or NO3– solution; however, their shifts were not significant. *Significantly different from control (ACh alone), P < 0.05.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Effects of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). A: ACh (10 µM) induced a biphasic increase in the frequency of exocytotic events. NPPB (2 µM) decreased the initial peak frequency slightly but significantly (P < 0.05). B: NO3– solution enhanced the frequency of exocytotic events at 10 µM ACh. However, NPPB did not induce any enhancement of the frequency of ACh-stimulated exocytotic events induced by NO3– solution. C: effects of NPPB on the initial peak frequencies in the control solution and NO3– solutions. NPPB eliminated the enhancement induced by NO3– solution. *Significantly different from control (10 µM ACh alone), P < 0.05.

Effects of [Cl^–]_o. The effects of extracellular Cl^– concentration ([Cl^–]_o) on ACh-stimulated exocytotic events were examined. [Cl^–]_o was varied by mixing appropriate amounts of solution 1 and NO₃^– solution. The initial peak frequencies were 42 ± 2.2 events·cell^–1·30 s^–1 (n = 9) at 0 mM [Cl^–]_o, 29 ± 1.5 events·cell^–1·30 s^–1 (n = 4) at 33 mM [Cl^–]_o (Fig. 5A), 18 ± 0.3 events·cell^–1·30 s^–1 (n = 4) at 65 mM [Cl^–]_o (Fig. 5B), 13 ± 0.8 events·cell^–1·30 s^–1 (n = 4) at 98 mM [Cl^–]_o (Fig. 5C), and 11 ± 0.7 events·cell^–1·30 s^–1 (n = 9) at 130 mM [Cl^–]_o. The initial peak frequency of ACh-stimulated exocytotic events was plotted against [Cl^–]_o (Fig. 5D). With an increase in [Cl^–]_o, the initial peak frequency of ACh-stimulated exocytotic events decreased. Thus a decrease in [Cl^–]_i, which is caused by [Cl^–]_o decrease, appears to enhance the frequency of ACh-stimulated exocytotic events.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Effects of extracellular Cl– concentration ([Cl–]o). NO3– solution ([Cl–]o = 0 mM) enhanced the frequency of ACh-stimulated exocytotic events (dotted line). A: 33 mM [Cl–]o. B: 65 mM [Cl–]o. C: 98 mM [Cl–]o. *Significantly different from corresponding value of NO3– solution experiment, P < 0.05. D: the initial peak frequency was plotted against [Cl–]o. With an increment of [Cl–]o, the initial peak frequency decreased. Significantly different from control, P < 0.05.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effects of [Cl–]o on intracellular Cl– concentration ([Cl–]i). [Cl–]i was measured as the fluorescence ratio (F/F0) of N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE; Cl–-sensitive fluorescence dye), where F0 is the fluorescence intensity at time 0 (excitation 355 nm and emission 510 nm). In this figure, the ordinate (F/F0) is reversed (the upward direction indicates increase in [Cl–]i and the downward direction indicates a decrease in [Cl–]i). To decrease [Cl–]o, an appropriate amount of NO3– solution was added into solution 1 (see text). [Cl–]os used were 131 mM, 65 mM, 33 mM, and 0 mM. As [Cl–]o decreased, [Cl–]i also decreased. Further stimulation with ACh (1 µM) decreased [Cl–]i. The F/F0s of 7 cells on 2–3 coverslips were expressed as means ± SE.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Effects of NPPB on [Cl–]i. [Cl–]i was measured as the fluorescence ratio (F/F0) of MQAE, where F0 is the fluorescence intensity at time 0 (excitation 355 nm and emission 510 nm). A: ACh. NPPB (2 µM) decreased F/F0, which reached a plateau within 3 min. Further addition of ACh (1 µM) induced no change in F/F0. B: bumetanide (20 µM). In the presence of NPPB (2 µM), bumetanide induced no increase in F/F0, and further stimulation with ACh induced no change in F/F0. C: NO3– solution. In the presence of NPPB (2 µM), NO3– solution induced no increase in F/F0, and further stimulation with ACh did not induce any change in F/F0. Thus NPPB increased [Cl–]i and eliminated the [Cl–]i decreases induced by ACh, bumetanide, and NO3– solution. The F/F0s of 7 cells on 2–3 coverslips were expressed as means ± SE.

View larger version (33K): [in this window] [in a new window]   Fig. 7. Effects of bumetanide (A and B) and NO3– solution (C and D) on [Cl–]i. [Cl–]i was measured as the fluorescence ratio (F/F0) of MQAE, where F0 is the fluorescence intensity at time 0 (excitation 355 nm and emission 510 nm). A: ACh (1 µM) increased F/F0 rapidly. The addition of bumetanide further increased F/F0. Thus ACh decreased [Cl–]i, which was enhanced by bumetanide. B: addition of bumetanide increased F/F0, which reached a plateau within 1 min. ACh further increased F/F0. Bumetanide decreased [Cl–]i and ACh further decreased [Cl–]i. C: ACh (1 µM) increased F/F0 immediately and the addition of NO3– solution further increased F/F0, which was higher than that in A. D: addition of NO3– solution increased F/F0 which reached a plateau within 2 min but still remained at a much higher than that in B. Then, ACh induced a further increase in F/F0. Thus NO3– solution decreased [Cl–]i, which is much lower than that of bumetanide experiments. The F/F0s of 7 cells on 2–3 coverslips were expressed as means ± SE.

The effects of a Cl^– channel blocker, 2 µM NPPB, were examined. The addition of NPPB decreased F/F₀ and plateaued within 3 min. Further stimulation with ACh induced no increase in F/F₀ (Fig. 8A). The effects of bumetanide (2 µM) were examined in the NPPB-treated cells. In the presence of NPPB, the addition of bumetanide induced no increase in F/F₀, and further stimulation with ACh also induced no increase in F/F₀ (Fig. 8B). Similar experiments were performed using NO₃^– solution instead of bumetanide. In the NPPB-treated cells, NO₃^– solution induced no increase in F/F₀ with or without ACh stimulation (Fig. 8C). Thus, in the presence of NPPB, bumetanide or NO₃^– solution with and without ACh stimulation induced no increase in F/F₀; NPPB increased [Cl^–]_i and inhibited the decreases in [Cl^–]_i induced by ACh, bumetanide, and NO₃^– solution.

Effects of ionomycin, BAPTA, PKI, and Rp8BrPETcGMPS. ACh actions are induced by an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). To increase [Ca²⁺]_i without ACh stimulation, ionomycin was used. The cells were first perfused with a Ca²⁺-free solution, and then ionomycin (1 µM) was added. During perfusion with the Ca²⁺-free solution, the addition of 1 µM ionomycin did not increase the frequency of exocytotic events except for a transient increase (1–2 events·cell^–1·30 s^–1) within the first minute of ionomycin addition. This small increase in the frequency of exocytotic events was induced by Ca²⁺ release from internal stores induced by ionomycin. The perfusion solution was suddenly switched from Ca²⁺-free solution to solution 1 (1.5 mM Ca²⁺), to reintroduce Ca²⁺. The reintroduction of Ca²⁺ increased the frequency of exocytotic events similarly to that of 10 µM ACh. Similar experiments were performed using NO₃^– solution. NO₃^– solution enhanced ionomycin-stimulated exocytotic events following the reintroduction of Ca²⁺ by 100% (Fig. 9A). Thus ionomycin mimicked the ACh actions, and NO₃^– solution enhanced the frequency of ionomycin-stimulated exocytotic events.

View larger version (22K): [in this window] [in a new window]   Fig. 9. Effects of ionomycin and BAPTA-AM. A: cells were first stimulated with 1 µM ionomycin in a Ca2+-free solution, and then Ca2+ (1.5 mM) was added (reintroduction of Ca2+). The reintroduction of Ca2+ induced a transient increase in the frequency of exocytotic events, similarly to that of ACh (10 µM). The same experiments were performed using NO3– solution. Exposure of the NO3– solution enhanced a transient increase in the frequency of exocytotic events induced by the reintroduction of Ca2+. B: the cells were loaded with BAPTA-AM (25 µM) for 30 min at 30°C and then were perfused with NO3– solution containing no Ca2+ for 3 min prior to 10 µM ACh stimulation. ACh stimulation increased a small transient increase in the frequency of exocytotic events in the NO3– solution. *Significantly different from corresponding value, P < 0.05.

The effects of PKI (1 µM, an inhibitor of PKA) and guanosine 3',5'-cyclic monophosphorothiate, -phenyl-1, N²-etheno-8-bromo-Rp isomer (Rp8BrPETcGMPS; 500 nM, a PKG inhibitor) were examined (Fig. 10). In antral mucous cells, ACh accumulates cAMP via PGE₂ production (34). PKI (1 µM) decreased the frequency of ACh-stimulated exocytotic events by 50%, as previously reported (8, 32, 34) (Fig. 10A). In the presence of 1 µM PKI, NO₃^– solution still enhanced the frequency of ACh-stimulated exocytotic events by 200% (Fig. 10A).

View larger version (26K): [in this window] [in a new window]   Fig. 10. Effects of PKI (A) and Rp8BrPETcGMPS (B). Cells were pretreated with 1 µM PKI (a PKA inhibitor) or 500 nM Rp8BrPETcGMPS (a PKG inhibitor) for 5 min and then the perfusion solution was switched to the NO3– solution. The ACh concentration used for stimulation was 10 µM. A: PKI decreased the initial peak frequency of ACh-stimulated exocytotic events by 50%. Exposure of NO3– solution enhanced the initial peak frequency in the presence of 1 µM PKI. B: Rp8BrPETcGMPS (500 nM) decreased the initial peak frequency of ACh-stimulated exocytotic events by 30%. Exposure of NO3– solution still enhanced the initial peak frequency of ACh-stimulated exocytotic events by 150% in the presence of 500 nM Rp8BrPETcGMPS. *Significantly different from corresponding value, P < 0.05.

Thus NO₃^– solution enhanced Ca²⁺ actions independent of PKA and PKG.

Effects of DNP and anoxia. The effects of NO₃^– solution on the ATP-dependent priming of Ca²⁺-regulated exocytosis were examined by use of DNP (an uncoupler of oxidative phosphorylation) and N₂ bubbling (anoxia). The cells were pretreated with 100 µM DNP prior to ACh stimulation. ACh activated exocytotic events, but it induced only a sustained phase with no initial transient phase (Fig. 11A). In the next experiment, cells were first treated with 100 µM DNP and then with NO₃^– solution. Further stimulation with ACh also induced only a sustained phase, the frequency of which was enhanced (Fig. 11B). In contrast, first the cells were perfused with NO₃^– solution and then 100 µM DNP was added. Under these conditions, however, ACh induced an initial transient phase followed by a sustained phase (Fig. 11C). Similar experiments were performed in the cells bubbled with 100% N₂. In this experiment, a HCO₃^–-free solution was used. During 100% N₂ bubbling, ACh induced only a sustained phase with no initial transient phase (Fig. 11D). In the next experiment, cells were perfused with HCO₃^–-free solution, which was bubbled with 100% N₂ and then with HCO₃^–-free NO₃^– solution bubbled with 100% N₂. Further stimulation with ACh also induced only a sustained phase, the frequency of which was enhanced (Fig. 10E). In contrast, the cells were first perfused with HCO₃^–-free NO₃^– solution bubbled with 100% O₂ and then the gas was switched to 100% N₂. Further stimulation with ACh evoked an initial transient phase followed by a sustained phase in the frequency of exocytotic events. Thus ATP depletion by DNP or anoxia (100% N₂ bubbling) eliminates the initial transient phase, and exposure of NO₃^– solution prior to ATP depletion maintains the initial transient phase. Exposure of the NO₃^– solution prior to ATP depletion is suggested to accelerate the ATP-dependent step (priming) and to increase the number of the primed granules. Under this condition, ATP depletion does not cause reduction of the primed granules; that is, the primed granules are maintained, which induces the initial transient phase even during the ATP depletion.

View larger version (39K): [in this window] [in a new window]   Fig. 11. Effects of dinitrophenol (DNP) and anoxia (100% N2 bubbling). ACh was used at 1 µM. A: cells were treated with DNP (100 µM) for 3 min prior to ACh stimulation. DNP treatment sustained the ACh-stimulated exocytotic events with no initial phase. B: cells were treated with DNP, and then NO3– solution was added. Further stimulation with ACh induced only a sustained phase with no initial transient phase, and the sustained frequency was enhanced. C: cells were first perfused with NO3– solution, and then DNP was added. Further stimulation with ACh induced an initial transient increase followed by a sustained increase in the frequency of exocytotic events (a biphasic response). D: anoxia (N2 bubbling). The cells were exposed to anoxia (100% N2 bubbling) for 3 min prior to ACh stimulation. Anoxia sustained the ACh-stimulated exocytotic events with no initial phase. E: cells were exposed to anoxia (100% N2 bubbling), and then NO3– solution was added. Further stimulation with ACh induced only the sustained phase with no initial transient phase, and the sustained frequency was enhanced. F: cells were first perfused with NO3– solution and then were exposed to anoxia (100% N2 bubbling). Further stimulation with ACh induced a biphasic response in the frequency of exocytotic events. *Significantly different from corresponding value, P < 0.05.

View larger version (20K): [in this window] [in a new window]   Fig. 12. Restimulation of ACh (1 µM) after a brief recovery. A: control solution. Cells were stimulated with ACh for 1 min, and then the second ACh stimulation was performed after 7-min recovery. The first stimulation induced a transient increase in the frequency of exocytotic events, and the second stimulation induced only a sustained phase with no initial phase. B: NO3– solution. Similar experiments were performed using NO3– solution. Following the first stimulation, cells were recovered with NO3– solution for 7 min. The second stimulation induced an initial phase followed by a sustained phase in the frequency of exocytotic events. *Significantly different from corresponding value, P < 0.05.

View larger version (27K): [in this window] [in a new window]   Fig. 13. Effect of extracellular Ca2+ on the initial peak frequency of ACh-stimulated exocytotic events. The ACh concentration used was 1 µM. Exposure of the NO3– solution prior to ACh stimulation increased the initial peak frequency of ACh-stimulated exocytotic events at each extracellular Ca2+ concentration ([Ca2+]o). A: 0.01 mM [Ca2+]o. B: 0.1 mM [Ca2+]o. C: 0.4 mM [Ca2+]o. D: initial peak frequency of exocytotic events plotted against [Ca2+]o. The IC50s were 0.10 mM in the control solution and 0.18 mM in NO3– solution. NO3– solution did not shift the Ca2+ dose-response curve significantly (P < 0.05). E: intracellular Ca2+ concentration ([Ca2+]i). Similar experiments were performed to measure fura 2 ratio (F340/F380). Initial peak values of F340/F380 were plotted against [Ca2+]os. F340/F380 increased with increasing [Ca2+]o. Exposure of NO3– solution shifted the F340/F380 upward at every [Ca2+]o, except at 10 µM [Ca2+]o. *Significantly different from corresponding value, P < 0.05.

[Ca²⁺]_i measurement. The effects of NO₃^– solution on [Ca²⁺]_i were examined in antral mucous cells. Stimulation with ACh (1 µM) increased fura 2 ratio (F340/F380) rapidly and sustained it in antral mucous cells perfused with solution 1 (Fig. 14A). The effects of NO₃^– solution on F340/F380 were examined in unstimulated antral mucous cells. The addition of NO₃^– solution alone increased F340/F380 gradually and the removal decreased it (Fig. 14B). The addition of NO₃^– solution during ACh stimulation increased the F340/F380 of the ACh-stimulated cells gradually and then decreased it. The removal of NO₃^– solution decreased F340/F380 gradually and then plateaued (Fig. 14C). During perfusion with NO₃^– solution, ACh increased F340/F380, which was slightly high compared with that during perfusion with control solution (Fig. 14D). Upon switching to the control solution, F340/F380 decreased and plateaued. Further addition of NO₃^– solution increased F340/F380 again.

View larger version (30K): [in this window] [in a new window]   Fig. 14. Change in [Ca2+]i with NO3– solution. [Ca2+]i was measured by fura 2 fluorescence ratio (F340/F380, excitation 340/380 nm and emission 510 nm). A: control experiment. Cells were stimulated with ACh (1 µM); F340/F380s increased immediately and then plateaued. B: effects of NO3– solution without ACh. Exposure of the NO3– solution alone increased F340/F380 gradually, the removal of NO3– solution immediately decreased F340/F380. C: cells were first stimulated with ACh and exposed with NO3– solution. Exposure of the NO3– solution further increased F340/F380 gradually and plateaued within 5 min. The removal of NO3– solution decreased F340/F380 gradually and plateaued within 5 min. D: cells were first perfused with NO3– solution and the stimulated with ACh. In NO3– solution, ACh increased [Ca2+]i rapidly and then sustained. The removal of NO3– solution decreased F340/F380 gradually and plateaued. Further exposure of NO3– solution increased F340/F380 gradually.

View larger version (14K): [in this window] [in a new window]   Fig. 15. Changes in [Ca2+]i with bumetanide (20 µM). A: ACh initially increased F340/F380 ([Ca2+]i) rapidly and then decreased it gradually. The addition of bumetanide further increased gradually and then decreased it gradually. B: addition of bumetanide did not increase F340/F380. Further stimulation with ACh increased F340/F380 rapidly and then decreased it gradually. Bumetanide enhanced the initial peak value of F340/F380.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ca²⁺-regulated exocytosis in antral mucous cells is composed of two biochemically distinct steps, as previously reported (15, 25, 34). The first step requires ATP, but not Ca²⁺, and primes granules with the exocytotic machinery. The second step requires Ca²⁺, but not ATP, and triggers the fusion of granules (33). Depletion of ATP by DNP or anoxia inhibits priming and decreases the pool of primed granules, which results in no initial transient phase of Ca²⁺-regulated exocytosis. However, a small amount of ATP is supplied by anaerobic glycolysis, which primes granules with the exocytotic machinery; this induces a sustained phase of Ca²⁺-regulated exocytotic events. Thus the initial phase is induced by the fusion of primed granules, and the sustained phase is induced by the recruitment of granules to the apical membrane and the repriming of the exocytotic machinery (15, 25, 34). Moreover, anoxia (N₂ bubbling) has already been reported to have no effect on the ACh-stimulated [Ca²⁺]_i increase in antral mucous cells (15), indicating that ATP depletion does not affect [Ca²⁺]_i mobilization.

However, exposure of NO₃^– solution prior to ATP depletion (by DNP or N₂ bubbling) induced an initial transient phase followed by a sustained phase in the Ca²⁺-regulated exocytosis. In contrast, exposure of NO₃^– solution following the ATP depletion induced only a sustained phase without any initial phase in the Ca²⁺-regulated exocytosis. These results suggest that a decrease in [Cl^–]_i accelerates ATP-dependent priming and increases the pool of primed granules, which is maintained even during ATP depletion. Under this condition, ACh triggers the fusion of the primed granules in the pool, which induces an initial transient phase. Moreover, NO₃^– solution also enhanced the sustained phase even following the ATP depletion by DNP or anoxia. This suggests that a decrease in [Cl^–]_i still accelerates the priming maintained by ATP supplied by anaerobic glycolysis.

On the other hand, NO₃^– solution increased [Ca²⁺]_i and enhanced ACh-stimulated [Ca²⁺]_i increases. Thus a decrease in [Cl^–]_i also enhances [Ca²⁺]_i increases in antral mucous cells. Enhancement of the ACh-stimulated [Ca²⁺]_i increase appears to cause dose-response curve of ACh or Ca²⁺ to shift to the low concentration. However, no shift of the ACh dose-response curve or the Ca²⁺ dose-response curve was noted. Thus the results of dose-response studies are inconsistent with those of [Ca²⁺]_i measurements. Similar inconsistency was also shown in Fig. 13; the frequencies of ACh-stimulated exocytotic events were similar (10 events·cell^–1·30 s^–1) as increment of [Ca²⁺]_o from 0.5 to 1.5 mM, although F340/F380 ([Ca²⁺]_i) increased from 2.1 to 2.4. The fusion step is a continuous reaction following the priming step in the exocytotic cycle. A possible explanation is that the pool size of primed granules may be limited, which may cause a similar initial frequency at different [Ca²⁺]_is. The initial peak frequency is determined by the number of the primed granules (pool size). When the pool size is limited, the maximum frequency is also limited. This limitation appears to cause a similar initial peak frequency, even during acceleration of Ca²⁺-dependent fusion induced by an enhanced [Ca²⁺]_i increase. Thus, in antral mucous cells, it seems probable that an [Ca²⁺]_i increase induced by [Cl^–]_i decrease accelerates the Ca²⁺-dependent fusion in the exocytotic cycle. However, it still remains uncertain whether an [Cl^–]_i decrease increases the Ca²⁺ sensitivity of the fusion.

MQAE fluorescence measurements revealed that both bumetanide and NO₃^– solution decreased [Cl^–]_i. Bumetanide or NO₃^– solution inhibits NKCCs, which decreases [Cl^–]_i by inhibition of Cl^– entry. A decrease in [Cl^–]_i induced by NO₃^– solution, however, is much greater than that induced by bumetanide. In NO₃^– solution, intracellular Cl^– was replaced with NO₃^– via Cl^– channels, because many Cl^– channels are highly permeable to NO₃^– (21, 22). Thus NO₃^– solution more effectively decreases [Cl^–]_i than bumetanide. Moreover, ACh activates NKCC and also activates Ca²⁺-activated K⁺ and Cl^– channels and increases KCl release, which decreases [Cl^–]_i and also cell volume. Therefore, bumetanide, which inhibits Cl^– entry via NKCCs, enhances an ACh-stimulated [Cl^–]_i decrease, and NO₃^– solution, which inhibits Cl^– entry via NKCCs and accelerates replacement of Cl^– with NO₃^– via Cl^– channels, also enhances an ACh-stimulated [Cl^–]_i decrease.

In contrast, NPPB increased [Cl^–]_i and eliminated decreases in [Cl^–]_i induced by ACh, bumetanide, and NO₃^– solution. NPPB inhibits Cl^– efflux via Cl^– channels maintaining Cl^– entry via NKCCs, which increases [Cl^–]_i, and moreover, during inhibition of Cl^– efflux by NPPB, inhibition of Cl^– entry via NKCC does not cause [Cl^–]_i to decrease. [Cl^–]_is are maintained at an increased level.

Our previous report exhibited that cell shrinkage enhances Ca²⁺-regulated exocytosis in antral mucous cells. The present study demonstrated that cell shrinkage induced by ACh is coincided with [Cl^–]_i decrease by activation of KCl release. On the other hand, NPPB induced cell swelling in antral mucous cells. A similar cell swelling was reported in alveolar type II cells (33). NPPB (2 µM) also inhibited cell shrinkage induced by NO₃^– solution and ACh. NPPB, which increased [Cl^–]_i and cell volume, decreased the frequency of the initial phase as shown in Fig. 4. These observations indicate that the isosmotic cell shrinkage decreases [Cl^–]_i (14). Thus the enhancement of Ca²⁺-regulated exocytosis induced by cell shrinkage is caused by the [Cl^–]_i decrease in antral mucous cells, although we never neglect the direct effects of cell volume.

This study also showed that a decrease in [Cl^–]_i increases [Ca²⁺]_i and enhances the ACh-stimulated [Ca²⁺]_i increase, as mentioned above. However, the addition of bumetanide or NO₃^– solution during ACh stimulation increased [Ca²⁺]_i gradually and plateaued within 5 min, whereas the addition of bumetanide or NO₃^– solution in the sustained phase immediately induced a transient increase in the frequency of ACh-stimulated exocytotic events (within 1 min). The transient enhancement of the sustained phase is unlikely to be induced by the gradual [Ca²⁺]_i increase following [Cl^–]_i decrease, and it appears to be caused by an acceleration of priming, which increases in the number of the primed granules and induces a transient increase in the frequency of exocytotic events.

Intracellular Cl^– modulates many cellular functions, such as nonselective cation channels in fetal lung cells (32) and salivary duct cells (3), NKCCs in squid axons and tracheal cells (2, 9), cell proliferation in human gastric cancer cells (29), G proteins (10), and exocytosis. In insulin-secreting pancreatic cells, the priming of granules requires granular Cl^– uptake, suggesting that Cl^– modulates the priming (1). In melanotrophs, an increase in [Cl^–]_i increase exocytosis via G proteins and intracellular Cl^– plays an important role in granule maturation (24, 31). Moreover, in rat lactotrophs, a low [Cl^–]_o decreases Ca²⁺ entry via voltage-gated Ca²⁺ channels by modulating G proteins (8). Thus intracellular Cl^– also modulates exocytosis in endocrine cells. However, the effects of intracellular Cl^– on exocytosis and Ca²⁺ channels in antral mucous cells (exocrine cells) are opposite to those in endocrine cells (such as insulin-secreting pancreatic cells, lactotrophs, and melanotrophs); a decrease in [Cl^–]_i enhances exocytosis and Ca²⁺ channels in antral mucous cells, whereas an increase in [Cl^–]_i enhances them in endocrine cells. The modulation mechanisms of Cl^– in antral mucous cells may be different from those in endocrine cells; for example, Cl^– modulates stimulatory G proteins in endocrine cells, whereas it modulates inhibitory G proteins in antral mucous cells.

Figure 16 shows the Cl^– modulation of Ca²⁺-regulated exocytosis in antral mucous cells. Intracellular Cl^– modulates ATP-dependent priming; that is, an [Cl^–]_i decrease accelerates priming, which increases primed granules. Moreover, intracellular Cl^– also modulates [Ca²⁺]_i; that is, an [Cl^–]_i decrease increases [Ca²⁺]_i, which accelerates fusion of the primed granules. Both actions enhance Ca²⁺-regulated exocytosis in antral mucous cells.

View larger version (14K): [in this window] [in a new window]   Fig. 16. Intracellular Cl– modulation of Ca2+-regulated exocytosis in guinea pig antral mucous cells.

	ACKNOWLEDGMENTS

 
We thank Prof. Y. Nishimura (Department of Mathematics, Osaka Medical College) for advice in the statistical analysis. We also thank Prof. Y. Marunaka (Department of Physiology, Kyoto Prefectural University of Medicine) for useful advices in experiments and for discussion. This work was performed as part of the Nakahari Project of the Central Research Laboratory (Osaka Medical College).

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Nakahari, Dept. of Physiology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, Japan (e-mail: takan{at}art.osaka-med.ac.jp)

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

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Barg S, Huang P, Eliasson L, Nelson DJ, Obermüller S, Rorsman P, Thévenod F, Renström E. Priming of insulin granules for exocytosis by granular Cl^– uptake and acidification. J Cell Sci 114: 2145–2154, 2001.[Abstract/Free Full Text]
2. Breitwieser GE, Altamirano AA, Russell JM. Elevated [Cl^–]_i and [Na⁺]_i inhibit Na⁺,K⁺,Cl^– cotransport by different mechanisms in squid giant axons. J Gen Physiol 107: 261–270, 1996.[Abstract/Free Full Text]
3. Dinudom A, Young JA, Cook DI. Na⁺ and Cl^– conductances are controlled by cytosolic Cl^– concentration in the intralobular duct cells of mouse mandibular glands. J Membr Biol 135: 289–295, 1993.[Web of Science][Medline]
4. Foskett JK. [Ca²⁺]_i modulation of Cl^– content controls cell volume in single salivary acinar cells during fluid secretion. Am J Physiol Cell Physiol 259: C998–C1004, 1990.[Abstract/Free Full Text]
5. Foskett JK, Melvin JE. Activation of salivary secretion: coupling of cell volume and [Ca²⁺]_i in single cells. Science 244: 1582–1585, 1989.[Abstract/Free Full Text]
6. Fujiwara S, Shimamoto C, Katsu K, Imai Y, Nakahari T. Isosmotic modulation of Ca²⁺-regulated exocytosis in guinea-pig antral mucous cells: role of cell volume. J Physiol 516: 85–100, 1999.[Abstract/Free Full Text]
7. Fujiwara S, Shimamoto C, Nakanishi Y, Katsu K, Kato M, Nakahari T. Enhancement of Ca²⁺-regulated exocytosis by indomethacin in guinea-pig antral mucous cells: arachidonic acid accumulation. Exp Physiol 91: 249–256, 2006.[Abstract/Free Full Text]
8. Garcia L, Couderc B, Odessa MF, Dufy-Barbe L, Sartor P. Effects of Cl^– substitution on electrophysiological properties, Ca²⁺ influx and prolactin secretion of rat lactotrophs in vitro. Neuroendocrinology 70: 332–342, 1999.[CrossRef][Web of Science][Medline]
9. Haas M, McBrayer D, Lytle C. [Cl^–]_i-dependent phosphorylation of the Na-K-Cl cotransport protein of dog tracheal epithelial cells. J Biol Chem 270: 28955–28961, 1995.[Abstract/Free Full Text]
10. Higashijima T, Ferguson KM, Sternweis PC. Regulation of hormone-sensitive GTP-dependent regulatory proteins by chloride. J Biol Chem 262: 3597–3602, 1987.[Abstract/Free Full Text]
11. Hosoi K, Min KY, Iwagaki A, Murao H, Hanafusa T, Shimamoto C, Katsu K, Kato M, Fujiwara S, Nakahari T. Delayed shrinkage triggered by the Na⁺-K⁺ pump in terbutaline-stimulated rat alveolar type II cells. Exp Physiol 89: 373–385, 2004.[Abstract/Free Full Text]
12. Konishi M, Olson A, Hollingworth S, Baylor SM. Myoplasmic binding of fura-2 investigated by steady state fluorescence and absorbance measurements. Biophys J 54: 1089–1104, 1988.[Web of Science][Medline]
13. Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y. Normotonic cell shrinkage because of disordered volume regulation is early prerequisite to apoptosis. Proc Natl Acad Sci USA 97: 9487–9492, 2000.[Abstract/Free Full Text]
14. Marunaka Y. Hormonal and osmotic regulation of NaCl transport in renal distal nephron epithelium. Jpn J Physiol 47: 499–511.
15. Nakahari T, Fujiwara S, Shimamoto C, Kojima K, Katsu K, Imai Y. cAMP modulation of Ca²⁺-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig. Am J Physiol Gastrointest Liver Physiol 282: G844–G856, 2002.[Abstract/Free Full Text]
16. Nakahari T, Marunaka Y. Regulation of cell volume by ₂-adrenergic stimulation in rat fetal distal lung epithelial cells. J Membr Biol 151: 91–100, 1996.[CrossRef][Web of Science][Medline]
17. Nakahari T, Marunaka Y. -Agonist-induced activation of Na⁺ absorption and KCl release in rat fetal distal lung epithelium: a study of cell volume regulation. Exp Physiol 82: 521–536, 1997.[Abstract]
18. Nakahari T, Murakami M, Yoshida H, Miyamoto M, Sohma Y, Imai Y. Decrease in rat submandibular acinar cell volume during ACh stimulation. Am J Physiol Gastrointest Liver Physiol 258: G878–G886, 1990.[Abstract/Free Full Text]
19. Nakahari T, Yoshida H, Imai Y, Fujiwara S, Ohnishi A, Shimamoto C, Katsu K. Inhibition of Ca²⁺ entry caused by depolarization in acetylcholine-stimulated antral mucous cells of guinea-pig: G protein regulation of Ca²⁺-permeable channels. Jpn J Physiol 49: 545–550, 1999.[CrossRef][Web of Science][Medline]
20. Ohnishi A, Shimamoto C, Katsu K, Ito S, Imai Y, Nakahari T. EP1 and EP4 receptors mediate exocytosis evoked by prostaglandin E₂ in guinea-pig antral mucous cells. Exp Physiol 86: 451–460, 2001.[Abstract]
21. Palmer LG, Frindt G. Cl^– channels of the distal nephron. Am J Physiol Renal Physiol 291: F1157–F1168, 2006.[Abstract/Free Full Text]
22. Perez-Cornejo P, Santiago JA, Arreola J. Permeant anion control gating of calcium-dependent chloride channels. J Membr Biol 198: 125–133, 2004.[CrossRef][Web of Science][Medline]
23. Robertson MA, Foskett JK. Na⁺ transport pathway in secretory acinar cells: membrane cross talk mediated by [Cl^–]_i. Am J Physiol Cell Physiol 267: C146–C156, 1994.[Abstract/Free Full Text]
24. Rupnik M, Zorec R. Intracellular Cl^– modulates Ca²⁺-induced exocytosis from rat melanotrophs through GTP-binding proteins. Pflügers Arch 431: 76–83, 1995.[CrossRef][Web of Science][Medline]
25. Saad AH, Shimamoto C, Nakahari T, Fujiwara S, Katsu K, Marunaka Y. cGMP modulation of ACh-stimulated exocytosis in guinea pig antral mucous cells. Am J Physiol Gastrointest Liver Physiol 290: G1138–G1148, 2006.[Abstract/Free Full Text]
26. Shiima-Kinoshita C, Min KY, Hanafusa T, Mori H, Nakahari T. ₂-Adrenergic regulation of ciliary beat frequency in rat bronchiolar epithelium: potentiation by isosmotic cell shrinkage. J Physiol 554: 403–416, 2004.[Abstract/Free Full Text]
27. Shimamoto C, Fujiwara S, Kato M, Ito S, Katsu K, Mori H, Nakahari T. Inhibition of ACh-stimulated exocytosis by NSAIDs in guinea pig antral mucous cells: autocrine regulation of mucin secretion by PGE₂. Am J Physiol Gastrointest Liver Physiol 288: G39–G47, 2005.[Abstract/Free Full Text]
28. Shimamoto C, Nakanishi Y, Katsu K, Nakano T, Kubota T, Mori H, Nakahari T. Prostaglandin E₂ release in gastric antral mucosa of guinea-pigs: basal PGE₂ release by cyclo-oxygenase 2 and ACh-stimulated PGE2 release by cyclo-oxygenase 1. Exp Physiol 91: 1015–1024, 2006.[Abstract/Free Full Text]
29. Shiozaki A, Miyazaki H, Niisato N, Nakahari T, Iwasaki Y, Itoi H, Ueda Y, Yamagishi H, Marunaka Y. Furosemide, a blocker of Na⁺/K⁺/2Cl^– cotransporter, diminishes proliferation of poorly differentiated human gastric cancer cells by affecting G₀/G₁ state. J Physiol Sci 56: 401–406, 2006.[CrossRef][Web of Science][Medline]
30. Takemura T, Sato F, Saga K, Suzuki Y, Sato K. Intracellular ion concentrations and cell volume during cholinergic stimulation of eccrine secretory coil cells. J Membr Biol 119: 211–219, 1991.[CrossRef][Web of Science][Medline]
31. Turner JE, Sedej S, Rupnik M. Cytosolic Cl^– ions in the regulation of secretory and endocytotic activity in melanotrophs from mouse pituitary tissue slice. J Physiol 566: 443–453, 2005.[Abstract/Free Full Text]
32. Tohda H, Foskett JK, O'Brodovich H, Marunaka Y. Cl^– regulation of a Ca²⁺-activated nonselective cation channel in -agonist-treated fetal distal lung epithelium. Am J Physiol Cell Physiol 266: C104–C109, 1994.[Abstract/Free Full Text]
33. Tokuda S, Shimamoto C, Yoshida H, Murao H, Kishima G, Ito S, Kubota T, Hanafusa T, Sugimoto T, Niisato N, Marunaka Y, Nakahari T. HCO₃^–-dependent pH_i recovery and overacidification induced by NH₄⁺ pulse in rat lung alveolar type II cells: HCO₃^–-dependent NH₃ excretion from lungs? Pflügers Arch 2007 Jun 12 [Epub ahead of print].
34. Williams JA, Groblewski GE, Ohnishi H, Yule DI. Stimulus-secretion coupling of pancreatic digestive enzyme secretion. Digestion 58, Suppl 1: 42–45, 1997.[CrossRef][Web of Science][Medline]

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