cAMP modulation of Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

Takashi Nakahari1, Shoko Fujiwara2, Chikao Shimamoto2, Kumiko Kojima2, Ken-Ichi Katsu2, and Yusuke Imai1

1 Department of Physiology and 2 Second Department of Internal Medicine, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of cAMP accumulation on ATP-dependent priming and Ca2+-dependent fusion in Ca2+-regulated exocytosis were examined in antral mucous cells of guinea pigs by using video-enhanced contrast microscopy. The Ca2+-regulated exocytosis activated by 1 µM ACh consisted of two phases, an initial transient phase followed by a sustained phase, which were potentiated by cAMP accumulation. Depletion of ATP by 100 µM dinitrophenol (uncoupler of oxidative phosphorylation) or anoxia induced the sustained phase without the initial transient phase in Ca2+-regulated exocytosis. However, accumulation of cAMP before depletion of ATP induced and potentiated an initial transient phase followed by a sustained phase in Ca2+-regulated exocytosis. This suggests that the initial transient phase of Ca2+-regulated exocytosis is induced by fusion of all primed granules maintained by ATP and that accumulation of cAMP accelerates ATP-dependent priming of the exocytotic cycle. Moreover, ACh and Ca2+ dose-response studies showed that accumulation of cAMP shifted the dose-response curves to the low concentration side, suggesting that it increases Ca2+ sensitivity in the fusion of the exocytotic cycle. In conclusion, cAMP accumulation increases the number of primed granules and Ca2+ sensitivity of the fusion, which potentiates Ca2+-regulated exocytosis in antral mucous cells.

acetylcholine; adenosine 3',5'-cyclic monophosphate; gastric mucin secretion; beta -adrenergic agonist; ATP; fusion; priming


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GASTRIC MUCINS, WHICH ARE high-molecular-weight glycoproteins, are released into the lumen by exocytosis. Exocytosis is reported to be regulated by exocytosis-related proteins, activities of which are regulated by intracellular Ca2+ concentration ([Ca2+]i), protein kinase A (PKA), protein kinase C, G proteins, and ATP (14, 16, 20). In antral mucous cells, mucin secretion has been reported to be maintained by two basic mechanisms, Ca2+-regulated exocytosis and cAMP-regulated exocytosis (5, 12, 13). Our previous reports showed that ACh activated Ca2+-regulated exocytosis mediated via the cholinergic receptor (5, 12) and that PGE2 activated cAMP-regulated exocytosis mediated via the prostanoid EP4 receptor (13). With maximum stimulation, the frequency of cAMP-regulated exocytosis was 5-10% of Ca2+-regulated exocytosis. Thus Ca2+-regulated exocytosis is a major mechanism for mucin release from the antral mucous cells.

The Ca2+-regulated exocytosis in antral mucous cells consisted of two phases: an initial transient increase followed by a sustained increase. The biphasic increase in Ca2+-regulated exocytosis is not induced by a biphasic change in [Ca2+]i, since [Ca2+]i is sustained at a high level during ACh stimulation. Similar observations were reported in parotid and submandibular acinar cells during muscarinic stimulation (11, 22). In pancreatic acinar cells, the granules released their contents via three steps: the first is called docking, the second, priming, and the last, fusion. The priming step is regulated by ATP, and the fusion step is regulated by Ca2+ (14, 16, 20). This indicates that the frequency of exocytosis depends on the number of primed granules, maintaining [Ca2+]i at a high value. In parotid acinar cells, the biphasic responses are suggested to be caused by changes in the number of granules in the prefusion state (4, 22). On the basis of these observations, it seems that a reduction in the intracellular ATP level may alter the biphasic response in Ca2+-regulated exocytosis of antral mucous cells.

Accumulation of cAMP is stimulated by many agonists in gastric mucosa, such as beta -adrenergic agonists, PGE2, gastrin, secretin, and histamine (3, 8, 13). In antral mucous cells, isoproterenol (IPR, a beta -adrenergic agonist) potentiated ACh-evoked exocytotic events, although it did not activate exocytosis. In salivary acinar cells, IPR was reported to potentiate Ca2+-regulated exocytosis mediated via cAMP accumulation (21, 22). This suggests that IPR-stimulated cAMP accumulation potentiates the frequency of Ca2+-regulated exocytotic events in antral mucous cells. The question here is why cAMP accumulation potentiates Ca2+-regulated exocytosis.

Video-enhanced contrast (VEC) optical microscopy enabled us to observe exocytotic events in living cells with high time resolution (1, 5, 11, 18). VEC microscopy is used to observe exocytosis in various exocrine cells, such as salivary acinar cells (10, 11, 15), antral mucous cells (5, 13), frog esophageal pepsinogen-secreting cells (17), and pancreatic acinar cells (2, 6, 7). In the present study, we used VEC microscopy to observe the exocytosis from guinea pig antral mucous cells stimulated with ACh and IPR. The goal of this study is to answer two questions: why Ca2+-regulated exocytosis shows a biphasic response and why IPR potentiates Ca2+-regulated exocytosis in antral mucous cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Solutions and chemicals. Solution I contained (in mM): 121 NaCl, 4.5 KCl, 25 NaHCO3, 1 MgCl2, 1.5 CaCl2, 5 NaHEPES, 5 HHEPES, and 5 glucose. To prepare Ca2+-free solutions, CaCl2 was removed from solution I and 1 mM EGTA was added. To maintain the Ca2+ concentration of test solutions at <10 µM, an appropriate amount of CaCl2, calculated using a computer program, was added into the Ca2+-free solution (9). Solutions were all adjusted to pH 7.4 by adding HCl (1 M). The HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions were gassed with 95% O2 and 5% CO2 at 37°C. To prepare a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution (pH 7.4), NaHCO3 was removed from solution I and 25 mM NaCl was added (solution II). Solution II was gassed with 100% O2 or 100% N2. N-{2-[(p-bromocinnamyl)amino]ethyl}-5-isoquinolinesulfonamide 2HCl (H-89) was purchased from Calbiochem-Novabiochem (La Jolla, CA), ACh chloride, from Daiichi Pharmaceuticals (Osaka, Japan); and IPR hydrochloride, forskolin (FK), 8-bromo-cAMP sodium (8Br-cAMP), IBMX, collagenase (for cell dispersion, 180-220 U/mg), and BSA, from Wako Pure Chemical (Osaka, Japan). All of the reagents were dissolved in DMSO at 100 mM and were prepared to their final concentrations immediately before the experiments.

Cell preparations. Hartley strain male guinea pigs weighing ~250 g were purchased from Shimizu Experimental Animals (Kyoto, Japan) and were kept on standard pellet food and water. Guinea pigs were anesthetized by inhalation of ether, after which they were killed 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 procedures for cell preparations have been previously described in detail (5, 12, 13). 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 and then incubated in solution I 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 µm2 and washed three times. The cells were resuspended in solution I containing 2% BSA (4°C). The suspension was stored at 4°C and used within 3 h in the experiments.

For histological examinations, the stripped mucosa and the isolated cells were fixed in a 150-mM phospate buffered solution containing 10% formaldehyde for 1 day, and they were dehydrated and embedded in paraffin according to the standard protocol. The sections were then stained with hematoxylin-eosin (H-E) or periodic acid-Schiff (PAS) reagent.

Observation of exocytosis. Isolated antral mucous cells were mounted on a coverslip precoated with neutralized Cell-Tak (Becton Dickinson Labware, Bedford, MA) for firm attachment of the cells. The coverslip with cells was set in a perfusion chamber that was mounted on the stage of a differential interference contrast microscope (BX50Wi; Olympus, Tokyo, Japan) connected to a VEC system (ARGUS-10; Hamamatsu Photonics, Hamamatsu, Japan). Images were recorded continuously using 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. The exocytotic events, which were detected by rapid changes in the light intensity of the granules (5, 10, 11, 15, 18), were counted in 5-6 cells every 30 s and were normalized to the cell number (events · cell-1 · 30 s-1). The frequencies of exocytotic events in 3-7 experiments were expressed as means ± SE. The peak frequency observed within 2 min from the start of stimulation was used for the initial peak frequency, and the frequency observed 4 min after the start of stimulation was used for the sustained frequency (events · cell-1 · 30 s-1).

[Ca2+]i measurements. The isolated antral mucous cells were incubated in solution I containing 2% BSA and 2.5 µM fura 2-AM (Dojindo, Kumamato, Japan) for 25 min at room temperature (22-24°C), and they then were washed three times with solution I containing 2% BSA. Fura 2-loaded cells were resuspended and stored in solution I containing 2% BSA at 4°C and mounted on coverslips precoated with neutralized Cell-Tak, and each coverslip with cells was then set in a perfusion chamber, which was then mounted onto the stage of an inverted microscope (IX70; Olympus) connected to an image analysis system (ARGUS/HiSCA, Hamamatsu Photonics) (5, 12). All experiments were performed at 37°C. The volume of the perfusion chamber was ~80 µl, and the rate of perfusion was 500 µl/min. The fura 2 was excited at 340 nm and 380 nm, and emission was measured at 510 nm. The fluorescence ratios (340/380 nm) were calculated and stored in the image analysis system. In the present study, [Ca2+]i were calculated from the calibration curve, which was obtained from the fluorescence ratio (340/380 nm) of the cell-free Ca2+ calibration solutions containing 10 µM fura 2. Solution III contained (in mM): 130 KCl, 20 NaCl, 2 EGTA, and 10 HEPES, and to prepare the cell-free Ca2+-calibration solutions an appropriate amount of CaCl2, calculated using a computer program, was added into solution III (9). The pH was adjusted to 7.05 by adding KOH (1 M). The dissociation constant of Ca2+ and EGTA used was 214 nM (37°C, pH 7.05) (9). One experiment was performed on five to six coverslips, and the typical responses in [Ca2+]i, which were obtained from three cells on a coverslip, are shown in the figures.

The statistical significance of the difference between the mean values was assessed using Student's t-test. Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present experiments, the concentration of ACh used was 1 µM and that of IPR was 10 µM.

Histological examinations and video images. The thin sections of stripped antral mucosa were stained with H-E (Fig. 1A) and PAS (Fig. 1C). The PAS-positive antral mucous cells lined the apical surface of the mucosa (Fig. 1C). Glandular columns, which were isolated by collagenase treatment, used for experiments are shown in Fig. 1B (H-E staining) and Fig. 1D (PAS staining). The antral mucous cells having PAS-positive mucous granules are positioned from the middle to the top of the isolated glandular column. Thus antral mucous cells having mucous granules were easily distinguished from other cells having no granules in the isolated glandular columns (Fig. 1, B and D).


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Fig. 1.   Histological examinations of the stripped antral mucosa and the isolated antral mucous cells. Sections of stripped antral mucosa were stained with hematoxylin-eosin (H-E; A) and periodic acid-Schiff (PAS; C). PAS-positive antral mucous cells lined the apical surface (arrowhead). Sections of isolated cells were also stained with H-E (B) and PAS (D). The antral mucous cells having PAS-positive granules were located from the middle to the top of the isolated column. Bars = 100 (A and C) and 20 (B and D) µm.

A video-enhanced contrast-differential interface contrast image of antral mucous cells in the isolated column is shown in Fig. 2A. The mucous cells, in which mucous granules were densely packed, were distributed along the glandular column. A video image taken 40 s after the start of ACh stimulation is shown in Fig. 2B. ACh evoked exocytotic events, and the granules located in the apical surface of the antral mucous cells disappeared. Figure 2, C-E, which show exocytotic events on a video record, are three consecutive video images taken 24 s after the start of ACh stimulation at an interval of 1.5 s each. In Fig. 2C, a granule is visible that disappeared in Fig. 2D. Another granule also disappeared in Fig. 2, D and E. Thus the exocytotic events were detected as a rapid change in the light intensity of the granule on the video screen.


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Fig. 2.   Video-enhanced contrast-differential interface contrast images of antral mucous cells. A: unstimulated antral mucous cells. B: 40 s after the start of ACh (1 µM) stimulation. ACh stimulated exocytotic events, and granules on the apical side disappeared. C-E: three consecutive video images taken 24 s after the start of ACh stimulation at intervals of 1.5 s each. Arrows show granules. The light intensity of the granule increased immediately, and the granule disappeared (arrows in C and D). Another granule also disappeared in D and E (* and arrow). Thus exocytotic events in antral mucous cells were detected on the video screen. Bars = 2 (A and B) and 1 (C-E) µm.

The experiments were also performed using mucosal slices and cell clumps that contained 4-6 antral mucous cells. The exocytotic responses of antral mucous cells in mucosal slices and cell clumps were the same as those in the isolated glandular column.

IPR potentiation of ACh-evoked exocytosis. Isolated antral mucous cells were stimulated with 10 µM IPR or 1 µM ACh. Stimulation with IPR alone did not increase exocytotic events (Fig. 3A). Stimulation with ACh induced biphasic increases in the frequencies of exocytotic events: an initial transient phase followed by a sustained phase. The sustained phase was maintained for ~10 min during ACh stimulation (Fig. 3B). The frequencies of the initial transient phase and the sustained phase were 9.3 ± 0.82 and 2.3 ± 0.68 events · cell-1 · 30 s-1 (n = 5), respectively. The effect of IPR on the ACh-evoked exocytotic events was examined. The addition of IPR 5 min before ACh stimulation potentiated the biphasic increases in the frequency of ACh-evoked exocytotic events (Fig. 3C). The frequencies of the initial transient phase and the sustained phase were 15.2 ± 1.1 and 5.2 ± 0.78 events · cell-1 · 30 s-1 (n = 7), respectively. Thus IPR potentiated the frequencies of ACh-evoked exocytotic events; however, it did not alter their time course, that is, the biphasic response. The IPR-induced potentiation was inhibited by pretreatment with 10 µM propranolol (an inhibitor of the beta -adrenergic receptor).


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Fig. 3.   Effects of isoproterenol (IPR) and ACh on the frequency of exocytotic events in antral mucous cells. A: stimulation with IPR did not evoke any increase in the frequency of exocytotic events (n = 5). B: stimulation with ACh evoked biphasic increases in the frequency of exocytotic events (an initial transient phase followed by a sustained phase; n = 6). C: effects of IPR on the frequency of ACh-evoked exocytotic events. Cells were first perfused with IPR for 5 min and then stimulated with ACh. IPR treatment potentiated the biphasic increases in the frequency of ACh-evoked exocytotic events (n = 5).

IPR potentiated ACh-evoked exocytotic events in a dose-dependent manner. An addition of 10 nM IPR did not induce any potentiation of ACh-evoked exocytotic events. An addition of 100 µM IPR markedly potentiated the initial transient phase (25-30 events · cell-1 · 30 s-1), but it caused a gradual decrease in the sustained phase (from 6 to 3 events · cell-1 · 30 s-1), and the frequency of exocytotic events reached a plateau value (1.5-2 events · cell-1 · 30 s-1) within 6 min. On the basis of this IPR dose-response study, we used 10 µM IPR in the present study.

Accumulation of cAMP during IPR stimulation. Stimulation with both IPR and ACh also potentiated the frequencies of exocytotic events similar to those shown in Fig. 3C, and the frequencies of the initial transient phase and the sustained phase were 14.7 ± 1.3 and 5.2 ± 0.87 events · cell-1 · 30 s-1 (n = 4), respectively. Thus the potentiation was also observed when both ACh and IPR were added simultaneously (Fig. 4A). The effects of H-89 (an inhibitor of PKA) on the potentiation of ACh-evoked exocytotic events caused by IPR were examined. Our previous report showed that 20 µM H-89 completely inhibited PKA actions in antral mucous cells (EC50 = 2.4 µM) (13). Cells were perfused with solution I containing 20 µM H-89 for 5 min and then stimulated with both IPR and ACh. In the presence of 20 µM H-89, the addition IPR did not potentiate the frequencies of ACh-evoked exocytotic events (Fig. 4A), and the frequencies of the initial transient phase and the sustained phase were 7.4 ± 1.2 and 1.9 ± 0.28 events · cell-1 · 30 s-1 (n = 4), respectively. Thus H-89 inhibited the potentiation of ACh-evoked exocytotic events induced by IPR (Fig. 4A). However, the frequency of the initial transient phase (7.4 ± 1.2 events · cell-1 · 30 s-1) was low compared with that shown in Fig. 3B (9.3 ± 0.82 events · cell-1 · 30 s-1). Cells were also pretreated with 20 µM H-89 for 5 min, then stimulated with ACh alone (Fig. 4B). Stimulation with ACh evoked a biphasic increase in the frequency of exocytotic events. The frequencies of the initial transient phase and the sustained phase were 6.1 ± 1.3 and 1.3 ± 0.4 events · cell-1 · 30 s-1 (n = 5), respectively. Thus 20 µM H-89 significantly decreased the initial peak frequency of ACh-evoked exocytotic events by 20-30% (P < 0.05), but it did not decrease the sustained frequency of ACh-evoked exocytotic events (Fig. 4B). Thus H-89 partially inhibited the initial phase of the ACh-evoked exocytotic events.


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Fig. 4.   Effects of H-89 (an inhibitor of protein kinase A) on the frequency of ACh-evoked exocytotic events in antral mucous cells. A: Effects of H-89 (20 µM) on IPR potentiation of ACh-evoked exocytotic events. Cells were stimulated with both IPR and ACh in the absence of H-89. Addition of IPR potentiated the frequency of ACh-evoked exocytotic events [H-89 (-); n = 6]. Cells were perfused with H-89 for 5 min and then stimulated with IPR and ACh. However, addition of IPR did not potentiate the frequency of ACh-evoked exocytotic events [H-89 (+); n = 4]. B: effects of H-89 (20 µM) on ACh-evoked exocytotic events. Cells were perfused with H-89 for 5 min and then stimulated with ACh. The initial peak frequency of ACh-evoked exocytotic events significantly decreased in H-89 treated cells (P < 0.05), but the sustained frequency did not.

The addition of 1 µM FK increased the frequency of exocytotic events (0.2-0.3 events · cell-1 · 30 s-1) in the presence and absence of extracellular Ca2+, as previously reported (13), and it potentiated biphasic increases in the frequencies of exocytotic events evoked by ACh in a dose-dependent manner (Fig. 5). FK at a concentration >1 µM potentiated the initial transient phase markedly; however, potentiation of the sustained phase was small. The small potentiation of the sustained phase is likely to be caused by a granule depletion induced by the large initial transient phase. The effects of 0.01 µM FK on ACh-evoked exocytotic events (Fig. 5C) were similar to those of IPR (Fig. 3C). In the absence of extracellular Ca2+, FK also potentiated the initial transient phase of ACh-evoked exocytotic events; however, the sustained phase was not induced (Fig. 5). Thus the effects of FK on the sustained phase were detected only in the presence of extracellular Ca2+.


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Fig. 5.   Effects of forskolin (FK) on ACh-evoked exocytotic events in antral mucous cells. Experiments were performed both in the presence and absence of extracellular Ca2+. A: FK (1 µM) markedly potentiated the frequency of exocytotic events evoked by ACh in the presence (n = 4) and absence of extracellular Ca2+ (n = 5). However, potentiation of the sustained phase caused by 1 µM FK was small in the presence of extracellular Ca2+ and it was not detected in the absence of extracellular Ca2+. B and C: FK [0.1 µM (n = 5) and 0.01 µM (n = 4)] potentiated both phases in the ACh-evoked exocytotic events in the presence of extracellular Ca2+ (n = 5). However, in the absence of extracellular Ca2+, the initial transient phase was potentiated, but the sustained phase was not detected. The effects of 0.01 µM FK on the frequency of ACh-evoked exocytotic events were similar to those of 10 µM IPR.

Effects of IBMX (an inhibitor of phosphodiesterase) and 8Br-cAMP were also examined (Fig. 6). Both 100 µM IBMX (Fig. 6A) and 500 µM 8Br-cAMP (Fig. 6B) potentiated the initial transient phase of exocytotic events activated by ACh, markedly similar to 1 µM FK. However, potentiation of the sustained phase was also small in both experiments. Potentiation of the initial transient phase induced by both IBMX and 8Br-cAMP also caused granule depletion.


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Fig. 6.   Effects of IBMX and 8-bromo-cAMP (8Br-cAMP) on ACh-evoked exocytotic events. A: IBMX (n = 4). B: 8Br-cAMP (n = 5). cAMP accumulated by both IBMX and 8Br-cAMP potentiated the frequency of ACh-evoked exocytotic events similar to 1 µM FK. The frequencies of exocytotic events evoked by ACh alone were also plotted in the same panels [IBMX (-) and 8Br-cAMP (-)].

The cumulative frequencies of ACh-evoked exocytotic events in the first 2 min (the initial transient phase) were similar (~200/cell) in 1 µM FK-, 500 µM 8Br-cAMP-, or 100 µM IBMX-treated cells, and those evoked by 10 µM ACh in the first 5 min, which induced granule depletion, were also similar. This suggests that the number of granules in a cell is limited. These large transient increases in the frequency of exocytotic events induced granule depletion, which appeared to cause potentiation of the sustained phase to be insignificant (Fig. 6)

Effects of IPR on ACh-evoked exocytotic events. The effect of the ACh dose on exocytotic events was examined in the presence and the absence of IPR (Fig. 7). Stimulation with ACh evoked biphasic increases in the frequencies of exocytotic events, which depended on ACh concentration. Pretreatment with IPR potentiated both an initial transient phase and a sustained phase in the frequencies of exocytotic events at each ACh concentration tested (Fig. 7, A-D). An addition of IPR potentiated the increases in frequencies of ACh-evoked exocytotic events; however, it did not alter the time course of ACh-evoked exocytosis, that is, an initial transient phase followed by a sustained phase. The initial peak (1 min after ACh stimulation) and the sustained frequencies (4 min after ACh stimulation) of ACh-evoked exocytotic events were plotted against ACh concentrations (Fig. 7. E and F). The initial peak and the sustained frequencies increased dose dependently in the presence and absence of IPR, and IPR shifted the dose-response curve to the left in both the initial transient phase and the sustained phase. The EC50 of ACh dose-response curves in the initial and the sustained phases were 1.2 and 1.8 µM in the presence of IPR, whereas they were 5.1 and 8.4 µM in the absence of IPR (Fig. 7, E and F). Thus IPR increased the ACh efficacy to the exocytotic responses.


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Fig. 7.   Effects of ACh dose on the frequency of exocytotic events in antral mucous cells. Frequency of ACh-evoked exocytotic events in the presence (open circle ) or absence () of IPR (10 µM) are shown. Cells were stimulated with IPR for 5 min before stimulation with 10 (A; n = 5), 4 (B; n = 6), 1 (C; n = 6), or 0.4 µM ACh (D; n = 4). IPR potentiated both phases evoked by ACh. E: initial peak frequencies of ACh-evoked exocytotic events were plotted against ACh concentrations (n = 4-6). IPR shifted the dose-response curve to the left. F: sustained frequencies of ACh-evoked exocytotic events (4 min after ACh stimulation) were also plotted against ACh concentrations (n = 4-6). IPR shifted the dose-response curve to the left. *Significant difference for IPR-treated vs. -untreated cells.

ACh-evoked exocytotic events were reported to be regulated by increases in [Ca2+]i in antral mucous cells (5, 12). Therefore, the effects of [Ca2+]i on exocytotic events evoked by 1 µM ACh were investigated in the absence and the presence of IPR by changing the extracellular Ca2+ concentration ([Ca2+]o). Both the initial and sustained frequencies of ACh-evoked exocytotic events increased as [Ca2+]o increased from 1 µM to 1.5 mM (Fig. 8, A-D). The frequencies of both phases were potentiated by IPR stimulation (Fig. 8, A-D). The relationships between [Ca2+]o and the frequencies of ACh-evoked exocytotic events in the initial transient phase and the sustained phase are shown in Fig. 8, E and F. The EC50 of the [Ca2+]o dose-response curves in the initial and sustained phases were 11 and 149 µM in the presence of IPR, respectively, whereas they were 116 and 360 µM in the absence of IPR, respectively. Thus IPR appears to increase the Ca2+ sensitivity to the exocytotic responses in antral mucous cells. The initial transient phase was still activated in a nominally Ca2+-free condition, whereas the sustained phase was eliminated.


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Fig. 8.   Effects of extracellular Ca2+ concentration ([Ca2+]o) on IPR (10 µM) potentiation of ACh-evoked exocytotic events. The [Ca2+]o was changed from 1 µM to 1.5 mM. Frequency of ACh-evoked exocytotic events in the presence (open circle ) and absence () of IPR are shown. The frequencies of ACh-evoked exocytotic events were plotted against [Ca2+]o [at 1 (A; n = 5), 10 (B; n = 5), 100 (C; n = 5), and 700 µM (D; n = 4)] in the presence and the absence of IPR. Cells were pretreated with IPR for 5 min before ACh stimulation. IPR potentiated the initial transient phases in all experiments. The sustained phase was not noted when the [Ca2+]o was <50 µM; however, it was detected and IPR potentiated it when the [Ca2+]o was <100 µM. E: initial peak frequencies were plotted against ACh concentrations (n = 4-6). IPR shifted the dose-response curve to the left. F: sustained frequencies (4 min after ACh stimulation) were also plotted against ACh concentrations (n = 4-6). IPR shifted the dose response curve to the left. *Significant difference between IPR-treated and -untreated cells.

Effects of dinitrophenol and anoxia. Experiments were performed in the presence of 100 µM dinitrophenol (DNP; an uncoupler of oxidative phosphorylation) to inhibit ATP formation. Cells were perfused with solution I containing 100 µM DNP for 2 min and then stimulated with ACh, which evoked only the sustained phase and did not induce any initial transient phase (Fig. 9A). Effects of IPR on ACh-evoked exocytotic events were also examined in DNP-treated cells (Fig. 9C). Cells were perfused with solution I containing 100 µM DNP for 1 min, and IPR was then added 1 min before ACh stimulation. ACh also evoked only the sustained phase, which was potentiated (Fig. 9C). Thus in DNP-treated cells, the initial transient phase in ACh-evoked exocytotic events was eliminated with or without IPR added before ACh stimulation. The frequency of the sustained phase remaining was 1.5 ± 0.3 (n = 5) and 4.6 ± 0.6 events · cell-1 · 30 s-1 (n = 5) in the absence and presence of IPR, respectively.


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Fig. 9.   Effects of dinitrophenol (DNP) and anoxia. A: cells were perfused with solution I containing DNP for 2 min and stimulated with ACh. DNP eliminated the initial transient phase of ACh-evoked exocytotic events, and only the sustained phase was detected (n = 6). B: cells were perfused with solution II (HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free) aerated with 100% N2 for 5 min and stimulated with ACh. Application of an anoxic stress also eliminated the initial transient phase of ACh-evoked exocytotic events, and only the sustained phase was detected (n = 5). C: cells were first perfused with a DNP-containing solution for 1 min; IPR was then added, and cells were perfused for a further 1 min. Stimulation with ACh did not induce any initial transient phase but induced only the sustained phase that was potentiated (n = 6). D: cells were perfused with solution II aerated with 100% N2 for 4 min; IPR was then added, and cells were perfused for a further 1 min. Stimulation with 1 µM ACh did not induce any initial transient phase but induced only the sustained phase that was potentiated (n = 5).

To examine the effects of anoxia, cells were perfused with solution II (HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free) aerated with 100% N2. Before the experiments, we confirmed that ACh evoked biphasic increases in the frequencies of exocytotic events during perfusion with solution II bubbled with 100% O2 similar to those observed during perfusion with solution I (data not shown). Cells were perfused with an N2-bubbled solution for 5 min and then stimulated with ACh, which evoked only a sustained phase in the exocytotic events (Fig. 9B). IPR was added 1 min before ACh stimulation, which also induced only the sustained phase (Fig. 9D). Infusion of the N2-bubbled solution II did not stimulate any exocytotic events. The frequencies of the sustained phases were 1.3 ± 0.3 (n = 3) and 3.6 ± 0.5 events · cell-1 · 30 s-1 (n = 4) in the absence and presence of IPR, respectively. Thus IPR potentiated the sustained phase that remained under an anoxic condition. The effect of anoxia showed the same effect as that obtained by DNP.

Similar experiments were also performed in cells stimulated with 1 µM FK. Cells were first treated with 100 µM DNP for 1 min, and then 1 µM FK was added 1 min before ACh stimulation, which induced only the sustained phase. The frequencies of the sustained phase remaining were potentiated (Fig. 10A). In turn, cells were first perfused with solution I containing 1 µM FK for 1 min, and 100 µM DNP was then added 2 min before ACh stimulation. ACh evoked biphasic increases in the frequency of exocytotic events, which was much higher than that evoked by ACh alone (Fig. 10B). These observations suggest that FK potentiates the initial transient increase of ACh-evoked exocytotic events in the absence of DNP (in the presence of ATP); however, it does not, in the presence of DNP (in the absence of ATP). DNP in concentrations of <50 µM, did not inhibit the initial transient increase in ACh-evoked exocytotic events.


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Fig. 10.   Effects of DNP on FK-evoked potentiation. A: FK (1 µM) added after DNP. Cells were first perfused with solution I containing 100 µM DNP for 1 min; FK was then added, and cells were perfused for a further 1 min. ACh did not evoke any initial transient phase, but induced the sustained phase that was potentiated (n = 6). B: FK added before DNP. Cells were first perfused with solution I containing FK for 1 min; DNP was then added, and cells were perfused for a further 2 min. ACh evoked biphasic increases in the exocytotic events that were potentiated (n = 5).

Effects of IPR on [Ca2+]i. Effects of IPR on increases in [Ca2+]i evoked by ACh were examined. IPR alone did not evoke any increase in [Ca2+]i. ACh increased [Ca2+]i rapidly and sustained it (Fig. 11A). The [Ca2+]i at 5 s, 2 min, and 10 min after ACh stimulation were 171 ± 8.9, 186 ± 4.2, and 134 ± 7.5 nM (n = 5 experiments), respectively. Addition of IPR did not potentiate increases in [Ca2+]i evoked by ACh (Fig. 11B). The [Ca2+]i at 5 s, 2 min, and 10 min after ACh stimulation were 155 ± 4.5, 168 ± 4.8, and 136 ± 7.8 nM (n = 6 experiments), respectively. In the absence of extracellular Ca2+, ACh evoked a transient increase in [Ca2+]i, but the addition of IPR did not potentiate the transient increase in [Ca2+]i evoked by ACh (data not shown). [Ca2+]i in the initial peak (5 s after ACh stimulation) were 172 ± 3.6 nM (n = 5 experiments) and 163 ± 8.1 nM (n = 4 experiments) in the absence and the presence of IPR, respectively. The [Ca2+]i were also measured in cells perfused with the N2-bubbled solution II (an anoxic condition). ACh increased [Ca2+]i rapidly and sustained it. ACh-induced increases in [Ca2+]i under anoxic conditions were similar to those observed under oxygenated conditions (Fig. 11C). The [Ca2+]i at 5 s, 2 min, and 10 min after ACh stimulation were 146 ± 13.5, 171 ± 4.2, and 143 ± 10.0 nM (n = 6 experiments), respectively.


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Fig. 11.   Effects of IPR on intracellular Ca2+ concentration ([Ca2+]i) increases evoked by ACh. The typical responses in [Ca2+]i, which were obtained from 3 cells on a coverslip, are shown. A: stimulation with ACh evoked a rapid increase followed by a sustained increase in [Ca2+]i. B: pretreatment with IPR did not potentiate [Ca2+]i increases evoked by ACh. C: Anoxia. Cells were perfused with solution II (HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free) aerated with 100% N2 for 5 min and then stimulated with ACh. Stimulation with ACh evoked a rapid increase followed by a sustained increase in [Ca2+]i, similar to those in A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Potentiation of Ca2+-regulated exocytosis. IPR potentiated the frequencies of ACh-evoked exocytotic events in antral mucous cells of guinea pigs. ACh was reported to increase the frequency of exocytotic events mediated by [Ca2+]i (5, 12). The Ca2+-regulated exocytosis in antral mucous cells had characteristic features in its frequency and time course, that is, high frequencies and a biphasic response: an initial transient phase followed by a sustained phase. The initial phase was evoked by Ca2+ release from intracellular stores and Ca2+ influx, from extracellular fluid and the sustained phase, only by Ca2+ influx from extracellular fluid. The accumulation of cAMP was also reported to increase the frequency of exocytotic events (13). The features of cAMP-regulated exocytosis were, however, different from those of Ca2+-regulated exocytosis, that is, low frequency and only a sustained phase (10-20% of the sustained frequency in ACh-evoked exocytotic events). The present study has shown that stimulation with both IPR and ACh evoked biphasic increases in the frequency of exocytotic events, which were higher than those evoked by ACh alone. The responses evoked by both agonists were those of Ca2+-regulated exocytosis in terms of frequency and time course. On the basis of these observations, we concluded that IPR potentiated Ca2+-regulated exocytosis evoked by ACh in antral mucous cells.

IPR-stimulated cAMP accumulation. IPR is well known to stimulate cAMP accumulation in many cell types. IPR, however, did not evoke any increase in the frequency of exocytotic events, whereas 1 µM FK, 500 µM 8Br-cAMP, and 100 µM IBMX sustained the frequency of exocytotic events (0.2-0.3 events · cell-1 · 30 s-1). Potentiation effects of IPR on ACh-evoked exocytotic events were much less than those of 1 µM FK, 500 µM 8Br-cAMP, and 100 µM IBMX; however, 0.01 µM FK mimicked the effects of 10 µM IPR. In measuring cAMP contents in antral mucosal strips, IPR increased the cAMP content from 3.8 (n = 3) to 6.7 pmol/mg dry wt (n = 3) (unpublished observations), whereas 10 µM FK and 1 mM IBMX were reported to increase the cAMP content to 61.3 and 80.8 pmol/mg dry wt, respectively (13). Moreover, 20 µM H-89 (a PKA inhibitor) eliminated the potentiation of ACh-evoked exocytotic events by IPR. These observations indicate that IPR significantly stimulates cAMP accumulation in antral mucous cells.

However, addition of H-89 also decreased the frequency of ACh-evoked exocytotic events by 30%. We have also reported that ACh-evoked exocytotic events were partially inhibited by H-89 in rat submandibular acinar cells (11). Watson et al. (19) reported that increases in [Ca2+]i stimulated IPR-stimulated cAMP accumulation in parotid acinar cells. This suggests that ACh may also stimulate cAMP accumulation in antral mucous cells.

Ca2+-dependent fusion and ATP-dependent priming. ACh and Ca2+ dose-response studies showed that the frequencies of ACh-evoked exocytotic events depended on [Ca2+]i. Increases in [Ca2+]i evoked by ionomycin or thapsigargin have been reported to activate exocytotic events in antral mucous cells, and removal of extracellular Ca2+ has been reported to inhibit ACh-evoked exocytosis (5). Similar results were also obtained in the present study. Thus the ACh-evoked exocytosis was regulated by [Ca2+]i.

On the other hand, treatment with 100 µM DNP and anoxia (N2 bubbling) eliminated the initial transient increase in the ACh-evoked exocytotic events, although they did not affect [Ca2+]i increases evoked by ACh. DNP is a well-known uncoupler of oxidative phosphorylation, which inhibits ATP formation in the inner mitochondrial membrane because the proton-motive force across the inner mitochondrial membrane is dissipated. Anoxia also inhibits oxidative phosphorylation. Thus inhibition of ATP formation eliminated the initial transient increase in the ACh-evoked exocytotic events; that is, the initial transient increase in Ca2+-regulated exocytosis depends on ATP.

It was demonstrated that Ca2+-regulated exocytosis in the pancreatic acinar cell involves at least two biochemically distinct steps (20). The first step requires ATP, but not Ca2+, and primes the exocytotic machinery (therefore called priming), whereas the second step requires Ca2+, but not ATP, and fuses the granule to the plasma membrane (therefore called fusion) (14, 16). The present study has shown that ACh-evoked exocytosis is under regulation by both Ca2+ and ATP in antral mucous cells, suggesting that similar mechanisms regulate Ca2+-regulated exocytosis in antral mucous cells, ATP-dependent priming, and Ca2+-dependent fusion.

According to these observations, depletion of ATP decreases the number of primed granules in antral mucous cells and the granules remain unprimed. Under these conditions, increases in [Ca2+]i are unlikely to trigger fusion of granules, since granules are unprimed. In turn, in the presence of ATP, increases in [Ca2+]i immediately trigger fusion of all of the primed granules, which induces the initial transient increase in exocytotic events.

However, treatment with DNP or anoxia did not eliminate the sustained increase in ACh-evoked exocytotic events. Even with inhibition of the oxidative phosphorylation, anaerobic glycolysis supplies ATP, the amount of which is small (2 molecules of ATP per 1 molecule of glucose in glycolysis, whereas 36 molecules of ATP per 1 molecule of glucose in complete oxidation). This ATP produced by glycolysis, which may be activated by ACh stimulation, supplies the primed granules, which fuse to the plasma membrane immediately. This maintains the sustained increase of ACh-evoked exocytotic events, even in the absence of oxidative phosphorylation.

cAMP modulation of fusion and priming. Accumulation of cAMP stimulated by IPR increased the Ca2+ sensitivity of exocytosis (2.5-10 times), as shown in the ACh and Ca2+ dose-response studies. However, IPR-stimulated cAMP accumulation did not potentiate increases in [Ca2+]i evoked by ACh. Thus potentiations in the frequency of ACh-evoked exocytotic events were not caused by the potentiation of ACh-evoked increases in [Ca2+]i. Yoshimura et al. (21) reported similar observations in amylase secretion in parotid acinar cells, and they concluded that cAMP accumulation increased Ca2+ sensitivity of Ca2+-dependent fusion (4, 21, 22). These observations suggest that cAMP accumulation modulates the Ca2+-dependent fusion of exocytosis in antral mucous cells by increasing Ca2+ sensitivity.

Large accumulation of cAMP, which was stimulated by FK (1 µM), 8Br-cAMP (500 µM), or IBMX (100 µM), induced marked potentiation of the initial transient increase (ATP dependent) in the ACh-evoked exocytotic events, which caused depletion of granules in antral mucous cells (Figs. 5 and 6), but it did not induce any initial transient increase in the ACh-evoked exocytotic events after depletion of ATP. This suggests that this cAMP accumulation in the presence of ATP causes most of the granules to be primed and that an increase in [Ca2+]i immediately triggers fusion of all of the primed granules at once, which evoked the large initial transient increase in the frequency of exocytotic events, as shown in Figs. 5 and 6. Moreover, FK-stimulated cAMP accumulation before ATP depletion (DNP addition) still potentiates the initial transient phase of ACh-evoked exocytosis, whereas that after ATP depletion did not activate the initial transient phase. This suggests that the granules that are primed by FK-stimulated cAMP accumulation in the presence of ATP still remain in the primed condition after depletion of ATP, which induces a small decrease in the number of primed granules. Therefore, the increases in [Ca2+]i triggered the fusion of all of the primed granules, which also induced a large transient increase in the exocytotic events, as shown in Fig. 10. Thus cAMP accumulation accelerates ATP-dependent priming in antral mucous cells.

The present study has shown that mucin secretion from the antral mucous cells was potentiated by even a small accumulation of cAMP. There are many agonists that stimulate cAMP accumulation, particularly in the gastric antrum, such as PGE2, gastrin, secretin, and IPR (3, 13). These agonists alone are unlikely to stimulate mucin secretion; however, they potentiate mucin secretion evoked by Ca2+-mobilizing agonists.

In antral mucous cells, the biphasic response in Ca2+-regulated exocytosis is induced by the change in the number of primed granules. The initial transient phase is caused by the immediate fusion of the granules that are already in the primed condition, and the sustained increase is caused by fusion of granules that are in the process of priming.

Accumulation of cAMP accelerates the priming step, resulting in an increase in the number of primed granules, and also increases the Ca2+ sensitivity of the fusion step. These modulations by IPR-stimulated cAMP accumulation potentiate the frequency of ACh-evoked exocytotic events in antral mucous cells.


    ACKNOWLEDGEMENTS

We thank Drs. H. Yoshida (Dept. of Physiology) and A. Ohnishi (Second Dept. of Internal Medicine) for their technical support.


    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.

10.1152/ajpgi.00300.2001

Received 10 July 2001; accepted in final form 15 January 2002.


    REFERENCES
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RESULTS
DISCUSSION
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