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Am J Physiol Gastrointest Liver Physiol 290: G1138-G1148, 2006. First published January 19, 2006; doi:10.1152/ajpgi.00357.2005
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MUCOSAL BIOLOGY

cGMP modulation of ACh-stimulated exocytosis in guinea pig antral mucous cells

Adel H. Saad,1 Chikao Shimamoto,2,3 Takashi Nakahari,3,4 Shoko Fujiwara,3,4 Ken-ichi Katsu,2,3 and Yoshinori Marunaka1,5

Departments of 1Molecular Cell Physiology and 5Respiratory Molecular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto; and Departments of 4Physiology and 2Internal Medicine and 3Central Research Laboratory (Nakahari Project), Osaka Medical College, Takatsuki, Japan

Submitted 28 July 2005 ; accepted in final form 14 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In guinea pig antral mucous cells, ACh stimulates the Ca2+-regulated exocytosis, which has a characteristics feature: an initial transient phase followed by a sustained phase. The effects of cGMP on ACh-stimulated exocytosis were studied in guinea pig antral mucous cells using video microscopy. cGMP enhanced the frequency of ACh-stimulated exocytotic events, whereas cGMP alone did not induce any exocytotic events under the ACh-unstimulated condition. cGMP did not stimulate either Ca2+ mobilization or cAMP accumulation. The Ca2+ dose-response studies demonstrated that cGMP shifted the dose-response curve upward with no shift to the lower concentration. This indicates that cGMP increased maximal responsiveness of the Ca2+-regulated exocytosis, but not the Ca2+ sensitivity. Moreover, under a condition of ATP depletion by dinitrophenol (DNP) or anoxia (N2 bubbling), ACh evoked only a sustained phase in exocytotic events with no initial transient phase. However, ACh evoked an initial transient phase followed by a sustained phase with addition of cGMP before ATP depletion, whereas only a sustained phase was evoked in a case of cGMP addition after ATP depletion. Thus cGMP-induced enhancement in ACh-stimulated exocytotic events requires ATP, suggesting that cGMP modulates ATP-dependent priming of Ca2+-regulated exocytosis in antral mucous cells. In conclusion, cGMP increases the number of primed granules via acceleration of the ATP-dependent priming, which enhances the Ca2+-regulated exocytosis stimulated by ACh.

gastric mucin secretion; guanosine 3' -cyclic monophosphate; exocytosis; acetylcholine; intracellular calcium concentration

CGMP IS AN ESTABLISHED intracellular second messenger that is accumulated in the cell by hormones, peptides, neurotransmitters, and nitric oxide (NO; see Refs. 27 and 30). The accumulation of cGMP, which is synthesized by guanylate cyclase, activates cGMP-dependent protein kinase (19), leading to the phosphorylation of target proteins. Their phosphorylation modulates many cellular functions, including ion transport, protein synthesis, cellular metabolism, and the relaxation of smooth muscle. Moreover, cGMP increases Ca2+ influx in several cell types, including pancreatic acinar cells, submandibular acinar cells, and colonic epithelial cells (1, 2, 33), and also increases cAMP content by inhibiting cAMP breakdown in platelet and vascular tissues (5, 18).

NO/cGMP protects endothelial cells from damage in the pulmonary artery (24, 25). PGE2 increases intracellular cGMP content in rabbit gastric parietal cells, protecting the cells from injuries via activation of Cl– channels (26). cGMP also increases HCO3– secretion in bullfrog duodenum, which shows a protecting function from damage (8). These observations suggest that cGMP plays an important role in gastric cytoprotection.

Gastric mucin, which is secreted by exocytosis from mucous cells and covers mucosal surfaces, protects the mucosa from gastric acid. An increase in mucin secretion is believed to be important for gastric mucosal protection. PGE2, which is a key mediator of gastric cytoprotection, stimulates mucin exocytosis in antral mucous cells (22). Moreover, ACh-stimulated PGE2 release, which leads to cAMP accumulation via the prostanoid EP4 receptor, is demonstrated to be essential for the maintenance of a large amount of mucin secretion in antral mucous cells (21, 22, 28). As mentioned above, the NO/cGMP pathway protects cells from damage in many tissues. cGMP also modulates exocytosis in salivary acinar cells, pancreatic acinar cells, HT-29 cells (human colonic mucus-secreting cells), and chromaffin cells (3, 12, 17, 34), leading us to an idea that cGMP may increase mucin exocytosis in antral mucous cells.

We have been studying exocytosis in isolated antral mucous cells by directly observing exocytotic events using video-enhanced microscopy. Our previous studies demonstrated that "Ca2+-regulated exocytosis" is the main mechanism for mucin release in antral mucous cells of the guinea pig (6, 7, 21, 22, 28). In the present study, we investigated whether cGMP enhances Ca2+-regulated exocytosis in antral mucous cells and clarified how cGMP modulates Ca2+-regulated exocytosis in antral mucous cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Solutions and chemicals. Solution I contained (in mM): 121 NaCl, 4.5 KCl, 25 NaHCO3, 1 MgCl2, 1.4 CaCl2, 5 Na-HEPES, 5 H-HEPES, and 5 glucose. To prepare a Ca2+-free solution, CaCl2 was excluded from solution I, and 1 mM EGTA was added. The pH values of the solutions were adjusted to 7.4 by adding 1 M HCl. The solutions were gassed with 95% O2 and 5% CO2 at 37°C. A HCO3–-free solution, in which NaHCO3 of solution I was replaced with NaCl, was gassed with 100% O2 or 100% N2. H-89 and PKI-(6–22) were purchased from Biomol (Plymouth Meeting, PA), ionomycin (IM), dibutyl-cGMP (DBcGMP), 8-bromo-cGMP (8-BrcGMP), and dinitrophenol (DNP) were purchased from Sigma (St. Louis, MO); ACh was from Daiichi Pharmaceuticals (Osaka, Japan); and collagenase (for cell dispersion, 180–220 U/mg) and BSA were from Wako (Osaka, Japan). All reagents were dissolved in DMSO and diluted to their final concentrations just before the experiments. The concentration of DMSO never exceeded 0.1%; at this concentration, DMSO does not affect exocytotic events in antral mucous cells (6, 7, 11, 21).

Cell preparations. Hartley strain male guinea pigs weighing ~250 g were purchased from Shimizu (Kyoto, Japan) and maintained on standard pellet food and water. The 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 preparation were previously described in detail (6, 7, 21, 22, 28). Briefly, the antrum was excised and the mucosal layer was stripped from the muscle layer in cooled saline (4°C), 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-µm squares 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 in the experiments within 3 h.

Observation of exocytosis. Isolated antral mucous cells were mounted on a cover slip precoated with neutralized Cell-Tak (Becton Dickinson Labware, Bedford, MA) for the firm attachment of cells. This cover slip 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; see Refs. 6, 7, 10, 11, 14, 20, 21, 28). 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. Exocytotic events, which were detected as rapid changes in the light intensity of granules (4, 6, 21, 22, 28, 29), were counted in five to six cells every 30 s and normalized to the number per cell during a unit time period (events·cell–1·30 s–1). The frequencies of exocytotic events in four to seven experiments were expressed as means ± SE. In statistically comparing the results obtained from the experiments, the initial peak frequency and sustained frequency were used. The initial peak frequency was the peak frequency within 2 min from the start of ACh stimulation, and the sustained frequency was obtained by averaging three frequencies at 3, 3.5, and 4 min after the start of ACh stimulation (21). One experiment was performed using four to seven cover slips obtained from two to four animals.

To calculate the intermediate concentration of a dose-response curve, a program for curve fitting (sigmoid curve) was used. The following is the equation used for curve fitting:

Formula
where a-d are constant, 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, and b is a constant.

Intracellular Ca2+ concentration measurements. The isolated antral mucous cells were incubated in solution I containing 2% BSA and 2.5 µM fura 2-AM (Dojindo, Kumamoto, Japan) for 25 min at room temperature (22–24°C). They were then washed with solution I containing 2% BSA three times. Fura 2-loaded cells were resuspended and stored in solution I containing 2% BSA at 4°C and then mounted on a cover slip precoated with neutralized Cell-Tak. These cover slips were set in a perfusion chamber, which was then mounted on the stage of an inverted microscope (IX70; Olympus) connected to an image analysis system (ARGUS/HiSCA; Hamamatsu Photonics; see Refs. 6, 7, 21, 22). 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. Fura 2 was excited at 340 and 380 nm, and the emission was measured at 510 nm. The fluorescence ratio [ratio of fluorescence at 340 nm to 380 nm (F340/F380)] was calculated and stored in an image analysis system. Solution II contained (in mM): 130 KCl, 20 NaCl, 2 EGTA, and 10 HEPES. To prepare the cell-free Ca2+ calibration solutions, an appropriate amount of CaCl2 (0.2–2 mM) calculated using a computer program was added to solution II. The pH was adjusted to 7.05 by adding 1 M KOH. The dissociation constant of Ca2+ and EGTA used in the present study was 214 nM (37°C, pH 7.05) (16). We measured the F340/F380 of three cells on two to three cover slips for presenting the experimental results on the intracellular Ca2+ concentration ([Ca2+]i). The results (F340/F380) shown in the present study were expressed as means ± SE.

The statistical significance of the difference 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
 GRANTS
 REFERENCES
 
In unstimulated antral mucous cells, no exocytotic events were observed. ACh (1 µM) activated exocytotic events, which consisted of two phases, an initial phase followed by a sustained phase (Fig. 1, A and C).


Figure 1
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  		Fig. 1. Effect of cGMP on ACh-stimulated exocytotic events in antral mucous cells. The ACh concentration was 1 µM. ACh (1 µM) alone-activated exocytotic events consisted of an initial phase followed by a sustained phase (n = 6). A: 8-bromo-cGMP (8-BrcGMP, 100 µM) alone did not induce exocytotic events. However, 8-BrcGMP added 5 min before ACh stimulation enhanced the initial phase and the sustained phase in the ACh-stimulated exocytotic events (n = 4). B: dibutyl-cGMP (DBcGMP, 200 µM) enhanced the frequency of ACh-stimulated exocytotic events similar to 8-BrcGMP (100 µM, n = 5). C: effect of 8-BrcGMP on the sustained phase in ACh-stimulated exocytotic events. 8-BrcGMP (100 µM) added 4 min after the ACh addition increased the sustained phase ~100% (n = 5). The removal of 8-BrcGMP returned the sustained frequency to the control level immediately. Values marked by arrows are significantly different (P < 0.05). D: cells were stimulated with both 8-BrcGMP (200 µM) and ACh (1 µM) simultaneously. 8-BrcGMP added simultaneously enhanced the ACh-stimulated exocytotic events similarly to those shown in A. E: dose effect of 8-BrcGMP on the initial peak frequency of 1 µM ACh-stimulated exocytotic events. F: dose effect of 8-BrcGMP on the sustained phase frequency of 1 µM ACh-stimulated exocytotic events. 8-BrcGMP enhanced the initial phase and sustained phase in a dose-dependent manner. At 8-BrcGMP concentrations >50 µM, the frequencies of the initial and sustained phase reached plateau levels. One experiment was performed on 4–6 cover slips from 2–3 animals. *Significantly different from the control value (1 µM ACh alone), P < 0.05.

 
Effects of cGMP on ACh-stimulated exocytotic events. 8-BrcGMP (100 µM) enhanced the both phases of the 1 µM ACh-stimulated exocytotic events in antral mucous cells, whereas 8-BrcGMP (100 µM) alone induced no exocytotic events. 8-BrcGMP (100 µM) increased the initial peak frequency and the sustained phase frequency of ACh-stimulated exocytotic events ~100 and 80%, respectively (Fig. 1A). DBcGMP (200 µM) also enhanced the frequency of the ACh-stimulated exocytotic events similar to 8-BrcGMP (Fig. 1B). 8-BrcGMP added 4 min after the start of ACh stimulation increased the frequency of exocytotic events in the sustained phase by ~100%, and 8-BrcGMP removal immediately diminished the elevated frequency to the control level with 1 µM ACh alone (Fig. 1C). 8-BrcGMP (200 µM) simultaneously added with ACh (1 µM) also enhanced the ACh-stimulated exocytotic events similarly to that added before ACh addition (Fig. 1D). The dose effects of 8-BrcGMP on 1 µM ACh-stimulated exocytotic events are shown in Fig. 1, E and F. The initial peak frequency and sustained phase frequency of the 1 µM ACh-stimulated exocytotic events increased as increment of 8-BrcGMP concentration from 5 to 50 µM, and they reached a plateau at concentrations >100 µM. Therefore, we used 200 µM 8-BrcGMP in the present study.

cGMP was reported to accumulate cAMP by inhibiting phosphodiesterase III (5, 18). In general, cAMP induces cellular actions by stimulating protein kinase A (PKA). Therefore, if cGMP accumulates cAMP, the cGMP action is blocked by inhibition of PKA. We examined the effect of PKI (a PKA inhibitor) on cGMP action. To inhibit PKA, cells were treated with PKI (1 µM) for 5 min, which eliminated the exocytotic events stimulated by 1 µM forskolin (Fig. 2C). As a control experiment, we applied 10 µM ACh, which evoked an initial phase followed by a sustained phase in the exocytotic events (Fig. 2A). The addition of 8-BrcGMP enhanced the initial peak frequency and the sustained phase frequency of ACh-stimulated exocytotic events (Fig. 2A). PKI (1 µM) added 5 min before ACh stimulation decreased the initial peak frequency of ACh-stimulated exocytotic events by ~60% (Fig. 2A). Inhibition of PKA by H-89 (another PKA inhibitor) has been reported to decrease the initial peak frequency of ACh-stimulated exocytotic events similarly by inhibition of a PGE2/cAMP-autocrine mechanism (28). 8-BrcGMP added 5 min before ACh stimulation still enhanced the initial peak frequency of ACh-stimulated exocytotic events in PKI-treated cells (Fig. 2B). The initial peak frequencies of ACh-stimulated exocytotic events with or without 8-BrcGMP were plotted for PKI-treated cells and non-PKI-treated cells (Fig. 2D). 8-BrcGMP still increased the initial peak frequency of ACh-stimulated exocytotic events in the presence of PKI. Similar results were obtained by 20 µM H-89 (data not shown). Thus 8-BrcGMP increased the frequency of ACh-stimulated exocytotic events independent of PKA (i.e., not via accumulation of cAMP).


Figure 2
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  		Fig. 2. Effect of PKI [1 µM, an inhibitor of protein kinase A (PKA)]. A: ACh (10 µM) increased the frequency of exocytotic events biphasically ({diamond}; n = 6). 8-BrcGMP (200 µM) added 5 min before ACh addition enhanced the frequency of ACh-stimulated exocytotic events (bullet; n = 5). B: cells were pretreated with PKI (1 µM) for 5 min before the addition of ACh (10 µM). PKI (1 µM) decreased the frequency of 10 µM ACh-stimulated exocytotic events (n = 4). 8-BrcGMP (200 µM) added 5 min before the ACh addition enhanced the frequency of ACh-stimulated exocytotic events in the presence of PKI (n = 5). C: forskolin (FK, 1 µM) increased the frequency of exocytotic events. The frequency of FK-stimulated exocytotic events was ~0.5 events·cell–1·30 s–1 (n = 5). PKI (1 µM) added before the addition of 1 µM FK eliminated the FK-stimulated exocytotic events (n = 4). D: effect of 8-BrcGMP on initial peak frequency of ACh-induced exocytotic events. In both PKI-treated cells and non-PKI-treated cells, 8-BrcGMP (200 µM) increased the initial peak frequency of ACh-stimulated exocytotic events. * and {dagger}Significantly different from the control value (10 µM ACh alone), P < 0.05.

 
Effects of Ca2+ on cGMP-induced enhancement. Antral mucous cells were stimulated with 10 µM ACh in the Ca2+-free solution. ACh induced a small initial phase without any sustained phase of exocytotic events. Further addition of 8-BrcGMP enhanced the small initial transient phase, but no sustained phase was detected (Fig. 3A). To completely chelate intracellular Ca2+, antral mucous cells were incubated with 25 µM BAPTA-AM for 30 min at room temperature (21–23°C). In the BAPTA-loaded cells perfused with the Ca2+-free solution, ACh (10 µM) induced no exocytotic events, and the further addition of 8-BrcGMP also induced no exocytotic events (Fig. 3B). In the presence of Ca2+ channel blocker (1 mM Ni2+ or 100 µM Gd3+), ACh induced only a small initial phase, which was enhanced by the addition of 8-BrcGMP (Fig. 3, C and D).


Figure 3
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  		Fig. 3. Effects of Ca2+-free solution and Ca2+ channel blockers on cGMP-induced enhancement. A: in a Ca2+-free solution, ACh (10 µM) induced a small transient increase in the frequency of exocytotic events ({diamond}; n = 4). The further addition of 8-BrcGMP (200 µM) enhanced the small transient increase, but no sustained phase was detected (bullet; n = 5). B: BAPTA-loaded cells. In BAPTA-loaded cells, ACh (10 µM) did not induce any exocytotic events in the Ca2+-free solution irrespective of the presence of 8-BrcGMP (n = 7). C and D: cells were perfused with solution I (Ca2+-containing solution) containing Ni2+ (1 mM, n = 5; C) or Gd3+ (100 µM, n = 5; D). ACh induced a small transient increase in the frequency of exocytotic events ({diamond}). The further addition of 8-BrcGMP (200 µM) enhanced the small transient increase of ACh-stimulated exocytotic events, but no sustained phase was detected (bullet; n = 5; C and D). *Significantly different from control value (P < 0.05).

 
The effects of 8-BrcGMP on the exocytotic events activated by 1 µM IM or 2 µM thapsigargin (TG) were examined. The compounds IM and TG stimulate the store-operated Ca2+ entry by depletion of the Ca2+ stores without muscarinic receptor stimulation. Cells were perfused with the Ca2+-free solution containing 1 µM IM for 5 min, and then the perfusion solution was switched to the Ca2+-containing solution (solution I), keeping the concentration of IM constant (reintroduction of Ca2+). The reintroduction of Ca2+ induced an initial phase followed by a sustained phase of the exocytotic events in IM-treated cells, similar to ACh stimulation. 8-BrcGMP enhanced the IM-stimulated exocytotic events after the reintroduction of Ca2+ (Fig. 4A). Similar experiments were performed using 2 µM TG. In TG-treated cells, the reintroduction of Ca2+ also induced an initial phase followed by a sustained phase of the exocytotic events, and 8-BrcGMP enhanced the TG-stimulated exocytotic events (Fig. 4B). Thus 8-BrcGMP enhanced the frequency of exocytotic events stimulated by [Ca2+]i increase without ACh stimulation.


Figure 4
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  		Fig. 4. Effects of 8-BrcGMP on ionomycin (IM)- and thapsigargin (TG)-activated exocytotic events. Cells were perfused with Ca2+-free solution for 5 min, and then the perfusion solution was switched to the Ca2+-containing solution (reintroduction of Ca2+). A: IM (1 µM). The reintroduction of Ca2+ activated exocytotic events in IM-treated cells ({diamond}). Treatment with 8-BrcGMP enhanced the IM-stimulated exocytotic events (bullet). B: TG (2 µM). The reintroduction of Ca2+ activated exocytotic events in TG-treated cells ({diamond}). Treatment of 8-BrcGMP enhanced the TG-stimulated exocytosis (bullet). *Significantly different from control value, P < 0.05.

 
Effects of cGMP on the ACh dose-response curve. ACh (0.01 µM) induced no exocytotic events in antral mucous cells, and the further addition of 8-BrcGMP (200 µM) did not induce any exocytotic events (data not shown). ACh of 0.1 µM increased the frequency of exocytotic events slightly (0.8 events·cell–1·30 s–1), and the addition of 8-BrcGMP enhanced the frequency of exocytotic events stimulated by 0.1 µM ACh (1.5 events·cell–1·30 s–1; Fig. 5A). ACh of 4 µM induced an initial phase followed by a sustained phase of exocytotic events. The addition of 8-BrcGMP further increased the frequencies of both by ~100%. ACh of 40 or 100 µM induced an initial phase followed by a sustained phase, and further addition of 8-BrcGMP increased the initial peak frequency by ~30%, but it suppressed the sustained phase (Fig. 5, C and D). Because a high concentration of ACh, such as 1 mM, inhibits the initial phase of exocytotic events as shown in the ACh dose-response study (6), cGMP may enhance this inhibitory effect especially in the sustained phase. Moreover, a high concentration of ACh reduces the number of granules during the initial phase. This may also cause the exocytotic events in the sustained phase to decrease, especially in 8-BrcGMP-treated cells.


Figure 5
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  		Fig. 5. Dose effects of ACh on exocytotic events in 8-BrcGMP-treated cells(bullet) and non-8-BrcGMP-treated cells ({diamond}). The concentration of 8-BrcGMP used was 200 µM. A: 0.1 µM ACh. B: 4 µM ACh. C: 40 µM ACh. D: 100 µM ACh. E: dose effects of ACh on the initial peak frequencies of the exocytotic events in non-8-BrcGMP-treated cells ({diamond}) and 8-BrcGMP-treated cells (bullet). ACh increased the initial peak frequencies of exocytotic events in a dose-dependent manner. The IC50 were 8.9 µM in the absence of 8-BrcGMP and 5.4 µM in the presence of 8-BrcGMP. F: dose effects of ACh on the sustained phase of the exocytotic events in non-8-BrcGMP-treated cells ({diamond}) and 8-BrcGMP-treated cells (bullet). ACh increased the sustained phase frequencies of exocytotic events in a dose-dependent manner. The IC50 were 1.8 µM in the absence of 8-BrcGMP and 0.7 µM in the presence of 8-BrcGMP. *Significantly different from control value (ACh alone), P < 0.05.

 
Results of the ACh dose-response study are summarized in Fig. 5, E and F. The initial peak frequency of ACh-stimulated exocytotic events increased with an increment of ACh concentration from 0.1 to 100 µM, although it decreased at the ACh concentration of 1 mM. The addition of 8-BrcGMP enhanced the initial peak frequency of the ACh-stimulated exocytotic events within the range of ACh concentration from 0.1 to 100 µM; however, the 8-BrcGMP treatment did not influence the initial peak frequency induced by 1 mM ACh (Fig. 5E). Thus 8-BrcGMP shifted the ACh dose-response curve upward within the range of ACh concentrations from 0.1 to 100 µM, whereas at a higher ACh concentration such as 1 mM, 8-BrcGMP appears to have no effects on the exocytotic responses in antral mucous cells. In the ACh dose-response curve of the initial peak frequency, the half-maximum concentrations (ED50) were 8.9 µM in the absence of 8-BrcGMP and 5.5 µM in the presence of 8-BrcGMP (Fig. 5E).

The sustained phase frequency of ACh-stimulated exocytotic events also increased with an increment in ACh concentration from 0.01 to 100 µM (Fig. 5F). However, the frequency decreased as increment of ACh concentration from 100 µM to 1 mM. 8-BrcGMP enhanced the sustained phase frequency of ACh-stimulated exocytotic events within the range of ACh concentration from 0.1 to 10 µM; however, it decreased the sustained phase frequency at concentrations of 40 and 100 µM. Thus 8-BrcGMP shifted the ACh dose-response curve upward within the range of concentrations from 0.01 to 10 µM (Fig. 5F). In the ACh dose-response curve of the sustained phase, the ED50 in the absence and presence of 8-BrcGMP were 1.8 and 0.65 µM, respectively.

Thus it remains uncertain whether or not 8-BrcGMP shifts the ACh dose-response curve toward the lower concentration side in the initial peak frequency or sustained phase frequency of exocytotic events. To confirm this, ACh (1 µM)-stimulated exocytotic events were measured at various [Ca2+]i controlled by altering extracellular Ca2+ concentration ([Ca2+]o).

Effects of [Ca2+]o. [Ca2+]o used was within the range of concentrations from 10 µM to 1.5 mM. At 10 µM [Ca2+]o, 1 µM ACh induced the initial phase without any sustained phase in exocytotic events (Fig. 6A). The frequency of the initial peak and the sustained phase frequency of ACh-stimulated exocytotic events increased as an increment of [Ca2+]o from 0.2 to 1 mM (Fig. 6, B-D). 8-BrcGMP enhanced the frequencies of ACh-stimulated exocytotic events at all [Ca2+]o, except the sustained phase frequency at 10 µM [Ca2+]o. The initial peak frequency and the sustained phase frequency were plotted against [Ca2+]o (Fig. 6, E and F). 8-BrcGMP shifted the Ca2+ dose-response curves upward for the initial peak frequency and also for the sustained phase frequency. In the dose-response curve of the initial peak frequency, ED50 were 0.21 mM in the absence of 8-BrcGMP and 0.26 mM in the presence of 8-BrcGMP (Fig. 6E). In the dose-response curve of the sustained phase frequency, the ED50 in the absence and presence of 8-BrcGMP were 0.62 and 0.62 mM, respectively (Fig. 6F). Based on these observations, it is concluded that 8-BrcGMP does not affect the Ca2+ sensitivity in the ACh-stimulated exocytosis.


Figure 6
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  		Fig. 6. Effect of extracellular Ca2+ on 8-BrcGMP-induced enhancement. The ACh concentration used was 1 µM. 8-BrcGMP (200 µM) added before ACh stimulation increased the frequency of ACh-stimulated exocytotic events at each extracellular Ca2+ concentration ([Ca2+]o). A: 0.01 mM [Ca2+]o. B: 0.2 mM [Ca2+]o. C: 0.5 mM [Ca2+]o. D: 1 mM [Ca2+]o. E: The initial peak frequency of ACh-stimulated exocytotic events was plotted against [Ca2+]o. The IC50 were 0.21 mM in the absence of 8-BrcGMP and 0.26 mM in the presence of 8-BrcGMP. F: the sustained phase frequency of exocytotic events was plotted against [Ca2+]o. The IC50 were 0.62 mM in the absence of 8-BrcGMP and 0.62 mM in the presence of 8-BrcGMP. *Significantly different from control value (ACh alone), P < 0.05.

 
Effects of DNP and anoxia. The effects of cGMP on the ATP-dependent priming of Ca2+-regulated exocytosis were examined by depleting ATP. To deplete intracellular ATP, DNP (an uncoupler of oxidative phosphorylation) and N2 bubbling (anoxia) were used. In these experiments, ACh used was 1 µM. Cells were pretreated with 100 µM DNP for 3 min before ACh stimulation. Stimulation with ACh induced only the sustained phase of the exocytotic events with no initial phase (Fig. 7A). When cells were treated with DNP (100 µM) for 3 min or longer, no initial phase of ACh-stimulated exocytotic events was detected. 8-BrcGMP added before DNP addition induced an initial phase followed by a sustained phase in the ACh-stimulated exocytotic events (Fig. 7C), whereas 8-BrcGMP added after the DNP addition induced only the sustained phase of ACh-stimulated exocytotic events, the frequency of which was enhanced (Fig. 7B). However, the initial peak frequency (7.4 ± 0.7 events·cell–1·30 s–1, n = 5) was small compared with that induced by 1 µM ACh alone (10.2 ± 1.2 events·cell–1·30 s–1, n = 5). Similar experiments were performed in cells perfused with HCO3–-free solution bubbled with N2. N2 bubbling (anoxia) eliminated the initial transient phase with no effects on the sustained phase in ACh-stimulated exocytotic events (Fig. 7D). When cells were bubbled with N2 for 5 min or longer, no initial phase of ACh-stimulated exocytotic events was detected. 8-BrcGMP added before the N2 bubbling (anoxia) evoked an initial phase followed by a sustained phase in the ACh-stimulated exocytotic events (Fig. 7F), whereas 8-BrcGMP added after N2 bubbling induced only the sustained phase, the frequency of which was enhanced (Fig. 7E). The initial peak frequency (9.7 ± 1.0 events·cell–1·30 s–1, n = 5) was small compared with that induced by ACh alone (12.3 ± 1.0 events·cell–1·30 s–1, n = 5). Thus ATP depletion by DNP or anoxia eliminates the initial phase. 8-BrcGMP added before ATP depletion induces the initial phase, whereas 8-BrcGMP added after ATP depletion induced no initial phase. cGMP added in the presence of ATP appears to increase the number of the primed granules, which was maintained by cGMP even during depletion of ATP, although it decreases gradually.


Figure 7
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  		Fig. 7. Effects of dinitrophenol (DNP) and anoxia (N2 bubbling). ACh and 8-BrcGMP were used at 1 and 200 µM, respectively. A: cells were treated with DNP (100 µM) for 3 min before ACh stimulation. DNP treatment abolished the initial transient increase in the ACh-stimulated exocytotic events without any effect on the sustained phase. B: 8-BrcGMP added after the DNP addition elevated the frequency at the ACh-induced sustained phase. No initial phase was observed irrespective of the treatment by 8-BrcGMP. C: 8-BrcGMP added before the DNP addition induced the initial phase followed by a sustained phase in ACh-stimulated exocytotic events. D: anoxia (N2 bubbling). N2 bubbling abolished the initial phase in the ACh-stimulated exocytotic events without any effect on the sustained phase. E: 8-BrcGMP added during N2 bubbling enhanced the sustained phase of the ACh-stimulated exocytotic events. No initial phase was observed irrespective of the treatment by 8-BrcGMP. F: 8-BrcGMP added before N2 bubbling induced an initial phase followed by a sustained phase in the ACh-stimulated exocytotic events.

 
Another test of the hypothesis that cGMP accelerates priming was performed by examining the ability of cGMP to enhance a second stimulation by ACh. Cells were restimulated with 1 µM ACh after a brief recovery in control condition (5 min) following a brief ACh stimulation (1 min). The second ACh stimulation induced only a sustained phase without any initial transient phase (Fig. 8A). Cells were also restimulated with 1 µM ACh after a brief recovery with 200 µM 8-BrcGMP (5 min) following a brief ACh stimulation (1 min). The second stimulation, however, induced an initial transient phase followed by a sustained phase (Fig. 8B). Thus a brief recovery with 8-BrcGMP recovered the number of primed granules, which were depleted by the first ACh stimulation. These suggest that 8-BrcGMP accelerates priming.


Figure 8
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  		Fig. 8. Cells were restimulated with ACh (1 µM) after a brief recovery (5 min) and after a brief ACh stimulation (1 min). A: after a brief recovery in the control condition, the second ACh stimulation induced only a sustained phase without any initial phase. B: after a brief recovery with 200 µM 8-BrcGMP, the second ACh stimulation induced an initial phase followed by a sustained phase in the exocytotic events.

 
[Ca2+]i measurement. cGMP is reported to increase [Ca2+]i in many cell types (1, 2, 33). We measured [Ca2+]i to examine whether or not cGMP increases Ca2+ influx in antral mucous cells. In antral mucous cells, stimulation with 1 µM ACh increased the fura 2 ratio (F340/F380) rapidly and sustained it at a level higher than basal (Fig. 9A). The addition of 8-BrcGMP alone did not increase F340/F380. Further stimulation with ACh increased the ratio to a level similar to that of the non-8-BrcGMP-treated cells (Fig. 9B). In the third experiment, cells were first stimulated with ACh, and then 8-BrcGMP was added. ACh increased F340/F380 rapidly and sustained it. The further addition of 8-BrcGMP induced no significant increase in F340/F380. Thus 8-BrcGMP did not increase [Ca2+]i under the basal or the ACh-stimulated conditions.


Figure 9
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  		Fig. 9. [Ca2+]i changes in ACh-stimulated antral mucous cells. The ratio of fura 2 [ratio of fluorescence at 340 nm to 380 nm (F340/F380)] was measured in antral mucous cells. ACh and 8-BrcGMP used were 1 and 200 µM, respectively. A: ACh increased F340/F380. B: 8-BrcGMP was added before the ACh addition. ACh increased F340/F380, which was similar to that induced by ACh alone. C: 8-BrcGMP added during ACh stimulation did not induce any further increase in F340/F380.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrated that cGMP accumulation enhances the frequency of ACh-stimulated exocytotic events in antral mucous cells. ACh stimulates Ca2+ release from stores followed by Ca2+ influx from extracellular fluid, which increases [Ca2+]i. IM or TG also increases [Ca2+]i similarly to ACh without any stimulation of muscarinic receptors (31). These increases in [Ca2+]i induce an initial transient phase followed by a sustained phase in the frequency of exocytotic events in antral mucous cells. In contrast, the inhibition of Ca2+ entry using a Ca2+-free solution or Ca2+ channel blockers causes the ACh-stimulated exocytotic events to be few and transient. A small transient increase in the frequency of ACh-stimulated exocytotic events was induced by Ca2+ released from intracellular stores. Moreover, ACh evoked no exocytotic events when intracellular Ca2+ was chelated by BAPTA. Thus an initial phase followed by a sustained phase in the ACh-stimulated exocytotic events is a characteristic feature of Ca2+-regulated exocytosis in antral mucous cells. The ACh-, IM-, or TG-evoked exocytotic events enhanced by cGMP also showed an initial transient phase followed by a sustained phase in its frequency. cGMP alone caused no exocytotic events, and had no effect on the exocytotic events during ACh stimulation in BAPTA-loaded cells. These observations indicate that cGMP enhances Ca2+-regulated exocytosis in antral mucous cells.

cGMP accumulates cAMP by inhibiting phosphodiesterase III in platelets and vascular tissues (5, 13, 15, 18). The present study demonstrated that cGMP enhanced the frequency of ACh-stimulated exocytotic events independent of PKA inhibitors. Thus cGMP-induced enhancement of Ca2+-regulated exocytosis is not induced by cAMP accumulation.

In some exocrine cells, cGMP affects Ca2+ influx or Ca2+ release from stores (1, 2). However, [Ca2+]i measurements using fura 2 fluorescence demonstrated that 8-BrcGMP induces no increase in [Ca2+]i in unstimulated cells and did not enhance an increase in [Ca2+]i in ACh-stimulated antral mucous cells. These observations indicate that cGMP does not stimulate Ca2+ mobilization in antral mucous cells. Thus cGMP enhances the frequency of ACh-stimulated exocytotic events not via the accumulation of either cAMP or Ca2+.

The Ca2+-regulated exocytosis in antral mucous cells consisted of two phases, as mentioned above. Similar biphasic responses in exocytosis were reported in pancreatic acinar cells, salivary acinar cells, and bullfrog peptic cells (4, 29, 35). In pancreatic acinar cells, exocytosis is composed of two biochemically distinct steps (32). The first step requires ATP, but not Ca2+, and primes granules with exocytotic machinery. The second step requires Ca2+, but not ATP, and triggers fusion of the granules to the apical membrane (23).

The present study demonstrated that depletion of ATP by DNP or anoxia abolished the initial phase in ACh-stimulated exocytotic events without any effect on the sustained phase. These observations are explained as follows: depletion of ATP inhibits priming and decreases the pool of primed granules, which results in no initial phase of ACh-stimulated exocytotic events. However, a small amount of ATP is supplied from the anaerobic glycolysis, and it primes granules with exocytotic machinery, which maintains the exocytotic events at the ACh-stimulated sustained phase. The anoxia (N2 bubbling) has already been reported not to affect [Ca2+]i in non-ACh-stimulated and ACh-stimulated antral mucous cells (21). Thus the initial phase is induced by the fusion of the pooled primed granules, and the sustained phase is induced by the recruitment of granules to the apical membrane and the repriming of the exocytotic apparatus, as previously reported for pancreatic acinar cells and antral mucous cells (21, 23). However, cGMP added before ATP depletion (by DNP or anoxia) induced an initial transient phase followed by a sustained phase in the ACh-stimulated exocytotic events. In contrast, cGMP added after ATP depletion induced only a sustained phase with no initial transient phase in the ACh-stimulated exocytotic events. These results suggest that 1) cGMP accelerates the ATP-dependent priming and increases the number of primed granules, and 2) cGMP maintains a pool of these primed granules, even in the absence of ATP. Under this condition, ACh triggers the fusion of the pooled primed granules, which evokes the initial phase. However, cGMP also enhanced the sustained phase even after the ATP depletion by DNP or anoxia. This suggests that cGMP still accelerates the priming step, which might be maintained by ATP supplied from the anaerobic glycolysis.

cGMP did not shift the Ca2+ dose-response curve toward the lower concentration. These observations indicate that cGMP did not increase the Ca2+ sensitivity of ACh-stimulated exocytotic events, that is, cGMP is unlikely to enhance Ca2+-dependent fusion of Ca2+-regulated exocytosis in antral mucous cells. Our previous study has demonstrated that cAMP increases the Ca2+ sensitivity of ACh-stimulated exocytosis in antral mucous cells, suggesting that cAMP enhances fusion (21). This indicates that cGMP modulates ACh-stimulated exocytosis differently from cAMP.

NO is reported to accumulate cGMP in gastric parietal cells and duodenal cells, and an increase in [Ca2+]i stimulates NO synthase in many cell types. In gastric mucosal cells, the NO/cGMP pathway is known to play an important role in mucosal protection. In antral mucous cells, NO/cGMP pathway may enhance Ca2+-regulated exocytosis, which may play an important role in mucosal protection. Further studies will be needed to clarify what stimulates the NO/cGMP pathway in antral mucous cells.

Figure 10 shows a possible model of the cGMP modulation of Ca2+-regulated exocytosis in antral mucous cells. The accumulation of cGMP accelerates priming of the Ca2+-regulated exocytosis, but not fusion. On the other hand, cAMP modulates both priming and fusion (21). The modulation by cAMP has already been reported to be essential in maintaining Ca2+-regulated exocytosis in antral mucous cells during massive mucous secretion, such as during a meal (21, 28). Although the cGMP-induced enhancement of Ca2+-regulated exocytosis is small compared with the cAMP-induced one, cGMP continuously increases the frequency of ACh-stimulated exocytotic events in the sustained phase. This continuous secretion appears to be of particular importance in mucosal protection.


Figure 10
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  		Fig. 10. A possible model of cGMP modulation of Ca2+-regulated exocytosis in guinea pig antral mucous cells.

 
In conclusion, cGMP enhances Ca2+-regulated exocytosis by accelerating the priming step in antral mucous cells.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was partly supported by Grants-in Aid for Scientific Research from the Japan Society of The Promotion of Science to C. Shimamoto (no. 15590698), T. Nakahari (no. 16590169), and Y. Marunaka (no. 17390057) and was performed as a part of 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
 GRANTS
 REFERENCES
 

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C. Shimamoto, E. Umegaki, K.-i. Katsu, M. Kato, S. Fujiwara, T. Kubota, and T. Nakahari
[Cl ]i modulation of Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig
Am J Physiol Gastrointest Liver Physiol, October 1, 2007; 293(4): G824 - G837.
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