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1 Department of Physiology2 Department of Internal Medicine (Division II)3 Central Research Laboratory (Nakahari Project), Osaka Medical College, 2-7 Daigakucho, Takatsuki 569-8686, Japan
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(Received 6 October 2005;
accepted after revision 1 November 2005; first published online 1 November 2005)
Corresponding author T. Nakahari: Department of Physiology, Osaka Medical College, 2-7 Daigakucho, Takatsuki 569-8686, Japan. Email: takan{at}art.osaka-med.ac.jp
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Introduction |
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The inhibition of PGE2 synthesis by indomethacin (IDM) or acetylsalicylic acid (ASA, aspirin) decreased the frequency of ACh-stimulated exocytosis in antral mucous cells (Shimamoto et al. 2005). However, a decrease in the frequency of ACh-stimulated exocytotic events induced by ASA was greater than that of IDM, although inhibition of cyclo-oxygenase (COX) by IDM is generally known to be stronger than that by ASA. COX synthesizes PGG/H and 15R-HPETE from arachidonic acid (AA). IDM inhibits both syntheses, while ASA inhibits PGG/H synthesis but not 15R-HPETE synthesis (Holtzman et al. 1992; Maede et al. 1993). This suggests that IDM may accumulate AA in antral mucous cells.
AA is an intracellular second messenger. AA was reported to stimulate Ca2+ release from intracellular stores and non-store-operated Ca2+ channels in many cell types (Shuttleworth, 1996; Shuttleworth & Thompson, 1998; Luo et al. 2001; Moneer et al. 2003; Watson et al. 2004), and to inhibit store-operated Ca2+ channels (Alonso-Torre & Garcia-Sancho, 1997; Bamberucci et al. 1997; Luo et al. 2001). Moreover, AA metabolites have been reported to regulate inducible nitric oxide synthase (iNOS; LaPointe & Sitkins, 1998) and to increase cGMP synthesis in myocytes (Snider et al. 1984).
As shown in our previous study (Shimamoto et al. 2005), IDM increases the frequency of ACh-stimulated exocytotic events in ASA-treated antral mucous cells, while it decreases the frequency of ACh-stimulated exocytotic events by inhibiting PGE2 formation. This indicates that IDM may induce AA accumulation as well as a decrease in PGE2 release in antral mucous cells. However, AA concentrations in antral mucous cells remain uncertain during IDM treatment. In the present study, we estimated the intracellular AA concentration in IDM-treated antral mucous cells by comparing the frequency of ACh-stimulated exocytotic events increased by IDM with that increased by AA during inhibition of the PGE2–cAMP pathway. We also aimed to confirm whether IDM increases the frequency of ACh-stimulated exocytotic events via AA accumulation.
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Methods |
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Solution I contained (mM): NaCl, 121; KCl, 4.5; NaHCO3, 25; MgCl2, 1; CaCl2, 1.5; NaHepes, 5; Hhepes, 5; and glucose, 5. 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 all adjusted to 7.4 by adding HCl (1 M). The solutions were aerated with a gas mixture (95% O2 and 5% CO2) at 37°C. Ionomycin, thapsigargin, indomethacin (IDM), {N-[2-((p-bromocinnamyl)amino)ethyl-5-isoquinolinesulphonamide, hydrochloride} (H-89) and acetylsalicylic acid (aspirin, ASA) were purchased from Sigma (St Louis, MO, USA), acetylcholine chloride (ACh) from Daiichi Pharmaceuticals (Osaka, Japan), and collagenase (for cell dispersion, 180–220 units mg–1) and bovine serum albumin (BSA) from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Arachidonyltrifluoromethyl ketone (AACOCF3) and N-(p-amylcinnamoyl) anthranilic acid (ACA) were purchased from Merck Biosciences (Germany). All the reagents were dissolved in dimethyl sulphoxide (DMSO) and were prepared to their final concentrations immediately before the experiments. The concentration of DMSO did not exceed 0.1%, and this concentration of DMSO does not have any effect on cellular actions, such as cell volume and exocytotic events (Fujiwara et al. 1999; Nakahari et al. 2002; Hosoi et al. 2004; Murao et al. 2005).
Cell preparation
Hartley strain male guinea-pigs weighing approximately 250 g were purchased from Shimizu Experimental Animals (Kyoto, Japan), and were maintained on standard pellet food and water. The guinea-pigs were anaesthetized by intraperitoneal injection of pentobarbitone sodium (60–70 mg kg–1), after which they were killed by cervical dislocation. All experimental procedures 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 have been previously described in detail (Fujiwara et al. 1999; Ohnishi et al. 2001; Nakahari et al. 2002; Shimamoto et al. 2005). Briefly, the gastric 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 µm2 and washed with solution I 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
The isolated antral mucous cells were mounted on a coverslip precoated with neutralized Cell-Tak (Becton Dickinson Labware, Bedford, MA, USA) for the firm attachment of the cells. The coverslip with the cells was set in a perfusion chamber that was mounted on the stage of a differential interference contrast (DIC) microscope (BX50Wi, Olympus, Tokyo, Japan) connected to a video-enhanced contrast (VEC) system (ARGUS-10, Hamamatsu Photonics, Hamamatsu, Japan; Fujiwara et al. 1999; Nakahari et al. 2002; Hosoi et al. 2004; Kawakami et al. 2004; Murao et al. 2005; Shimamoto et al. 2005; Hayashi et al. 2005). Images were recorded continuously using a video recorder. The experiments were performed at 37°C. The volume of the perfusion chamber was approximately 20 µl and the rate of perfusion was 200 µl min–1. Exocytotic events, which were detected as rapid changes in the light intensity of granules (Tao et al. 1998; Fujiwara et al. 1999; Campos-Toimil et al. 2000; Ohnishi et al. 2001; Nakahari et al. 2002; Shimamoto et al. 2005), were counted in five or six cells every 30 s, and were normalized to the cell number (events per cell per 30 s). The frequencies of exocytotic events in three to seven experiments were expressed as means ± S.E.M. ACh increased the frequency of exocytotic events transiently. In comparing each experiment, the initial peak frequency was used (Nakahari et al. 2002; Shimamoto et al. 2005).
[Ca2+]i measurements
The isolated antral mucous cells were incubated in solution I 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 with solution I containing 2% BSA three times. Fura-2-loaded cells were resuspended and stored in the solution I containing 2% BSA at 4°C, and then mounted on a coverslip precoated with neutralized Cell-Tak. The coverslip with cells was set in a perfusion chamber, which was then mounted on the stage of an inverted microscope (IX70, Olympus, Tokyo, Japan) connected to an image analysis system (ARGUS/HiSCA, Hamamatsu Photonics, Hamamatsu, Japan; Murao et al. 2005; Hayashi et al. 2005; Shimamoto et al. 2005). All experiments were performed at 37°C. The volume of the perfusion chamber was approximately 80 µl and the rate of perfusion was 500 µl min–1. 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 the image analysis system. One experiment was performed using five or six coverslips, and F340/F380 values of three cells on two or three coverslips were expressed as means ± S.E.M.
Measurement of PGE2
The stripped antral mucosa was weighed and then stored in solution I at 4°C for 30 min before the start of experiments. After the 2 min warm-up period, the stripped antral mucosa was incubated in solution I (10 ml) aerated with gas mixture (95% O2 and 5% CO2) for 10 min and then incubated for a further 15 min with the addition of ACh (10 µM), AA (2 µM) or ACA (10 µM). In the control experiments, DMSO (10 µl) was added instead of ACh or ionomycin. When the inhibitors (10 µM ACA) were used with ACh, the antral mucosa, after the 2 min warm-up period, was incubated with the inhibitors (10 µM ACA) prior to the ACh stimulation. A 500 µl sample of the incubation solution was transferred to a microtube immediately before and 15 min after the stimulation. The microtube with sample was immediately cooled on ice, and stored at –30°C until the PGE2 contents assay. PGE2 contents were measured using a PGE2 enzyme immunosorbent kit (Cayman Chemical Company, Ann Arbor, MI, USA) and were expressed as micrograms per gram of wet weight tissue. The amount of PGE2 over released 15 min was calculated from the difference between values before and 15 min after the stimulation.
The statistical significance of the difference between mean values was assessed using Student's paired and unpaired t test as appropriate. Differences were considered significant at P < 0.05.
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Results |
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In ACh-stimulated antral mucous cells, Ca2+-regulated exocytotic events are enhanced by an autocrine mechanism via the PGE2–cAMP pathway (Nakahari et al. 2002; Shimamoto et al. 2005). To inhibit the PGE2–cAMP pathway, the cells were pretreated with H-89 (20 µM, an inhibitor of PKA), ASA (10 µM) or IDM (10 µM) for 10 min prior to ACh stimulation. In H-89-treated cells, ACh induced an initial transient phase followed by a sustained phase in the frequency of exocytotic events, and the initial peak frequency decreased by approximately 60% (Fig. 1A). ASA (10 µM) also decreased the initial peak frequency of ACh-stimulated exocytotic events by 60%, as previously reported (Shimamoto et al. 2005). IDM (10 µM), however, decreased the initial peak frequency by approximately 30% (Fig. 1B). Thus, the initial peak frequencies of ACh-stimulated exocytotic events were higher in IDM-treated cells compared with those in ASA- or H-89-treated cells. When IDM was added to H-89-treated cells (cells were perfused with IDM and H-89 for 10 min prior to ACh stimulation), IDM increased the initial peak frequency of ACh-stimulated exocytotic events by approximately 50% (Fig. 1C). A similar increase in the initial peak frequency was caused by IDM in ASA-treated cells, as previously reported (Shimamoto et al. 2005). The initial peak frequency increased by IDM in ASA- or H-89-treated cells was identical to that caused by IDM in non-ASA- or non-H-89-treated cells. Thus, IDM increases the initial peak frequency of ACh-stimulated exocytotic events during inhibition of the PGE2–cAMP pathway in antral mucous cells.
Effects of AACOCF3 and ACA
COX synthesizes PGG/H and 15R-HPETE from AA. IDM inhibits the synthesis of both compounds, whereas ASA inhibits PGG/H synthesis, but not 15R-HPETE synthesis (Holtzman et al. 1992; Maede et al. 1993). This indicates that IDM may induce AA accumulation, unlike H-89 or ASA, by inhibiting both compounds synthesis. AA is synthesized via phospholipase A2 (PLA2) from membrane lipids. To examine AA accumulation by IDM, the effects of AACOCF3 (an inhibitor of PLA2) were examined. AACOCF3 (100 µM) added 10 min prior to ACh stimulation decreased the initial peak frequency of ACh-stimulated exocytotic events by approximately 60% (Fig. 2A). The initial peak frequency of ACh-stimulated exocytotic events inhibited by AACOCF3 was similar to that inhibited by H-89 or ASA. The effects of IDM were examined in AACOCF3-treated cells (cells were perfused with IDM and AACOCF3 for 10 min prior to ACh stimulation). The addition of IDM did not increase the initial peak frequencies of ACh-stimulated exocytotic events in AACOCF3-treated cells (Fig. 2B). A similar inhibition was also observed with N-(p-amylcinnamoyl) anthranilic acid (ACA, 10 µM, another inhibitor of PLA2), as shown in Fig. 2C. Thus, IDM did not incerase the initial peak frequency of ACh-stimulated exocytotic events during inhibition of PLA2.
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Cells were treated with AACOCF3 and IDM for 10 min prior to ACh stimulation. The addition of both AACOCF3 and IDM decreased the initial peak frequency of ACh-stimulated exocytotic events by approximately 60%, as shown in Fig. 2B. Thus, IDM did not increase the frequency of ACh-stimulated exocytotic events when AA synthesis was inhibited by AACOCF3. Further addition of AA (2 µM) prior to ACh stimulation increased the initial peak frequency of ACh-stimulated exocytotic events by approximately 50% (Fig. 3A), although AA (2 µM) alone stimulates no exocytotic events. An increase in the initial peak frequency induced by AA was similar to that induced by IDM in H-89-treated cells. Thus, the addition of AA in AACOCF3-treated cells mimicked the effects of IDM on ACh-stimulated exocytotic events in ASA- or H-89-treated cells.
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The initial peak frequencies of ACh-stimulated exocytotic events are summarized in Fig. 4. The initial peak frequency of ACh-stimulated exocytotic events was decreased by approximately 60% when cells were pretreated with H-89, AACOCF3, ACA or ASA. However, IDM and SC560 (an inhibitor of COX1) decreased the initial peak frequency of ACh-stimulated exocytotic events by approximately 30% (Fig. 4).
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The effects of a range of doses of AA on the initial peak frequency of ACh-stimulated exocytotic events were examined in ASA-treated cells (Fig. 7). With the increment in AA concentration from 2 to 200 nM, the initial peak frequency of ACh-stimulated exocytotic events increased. Within the range from 2 to 200 µM, the initial peak frequency of ACh-stimulated exocytotic events reached a plateau, the level of which was approximately 40 events per cell (30 s)–1. The dose effects of IDM were also examined in ASA-treated cells. With the increment of IDM concentration from 0.1 to 1 µM, the initial peak frequency of ACh-stimulated exocytotic events increased. Within the range from 1 to 100 µM, the initial peak frequency reached a plateau, the level of which was approximately 32 events per cell (30 s)–1. The results of the AA and IDM dose–response curves suggest that the concentration of AA accumulated by IDM is less than 0.1 µM.
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Increases in [Ca2+]i were examined in AA-treated antral mucous cells. The addition of AA (2 µM) did not increase the fura-2 ratio (F340/F380; Fig. 8A). Stimulation with 1 µM ACh caused the fura-2 ratio to increase rapidly and to be sustained in non-AA-treated cells, and subsequent addition of AA did not induce further increases in the fura-2 ratio (Fig. 8B). Cells were pretreated with AA (2 µM) for 10 min, and then stimulated with 1 µM ACh. ACh (1 µM) increased the fura-2 ratio in AA-treated cells in a similar manner to that in non-AA-treated cells (Fig. 8C). Thus, AA (2 µM) did not enhance the [Ca2+]i increase during ACh stimulation.
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AA is synthesized from membrane lipid via PLA2. To examine involvement of PLA2 in modulation of ACh-stimulated exocytotic events, PGE2 release was measured in antral mucosa (Fig. 9). Since PGE2 is generated from AA synthesized by PLA2, an increase in AA causes PGE2 release to increase. In unstimulated antral mucosa, PGE2 was released spontaneously (basal PGE2 release), and ACh increased PGE2 release (ACh-stimulated PGE2 release). Addition of AA (2 µM) increased the basal and the ACh-stimulated PGE2 release. Thus, an AA accumulation increases the PGE2 release. However, the addition of a PLA2 blocker (10 µM ACA) inhibited the basal and the ACh-stimulated PGE2 release. These observations suggest that PLA2 plays a key role in the AA accumulation in antral mucous cells, that is, AA is accumulated via PLA2 in antral mucous cells.
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Discussion |
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The COX catalyses the synthesis of PGG/H and 15R-HPETE from AA. ASA, which acetylates COX, is known to inhibit PGG/H synthesis but not that of 15R-HPETE (Holzman et al. 1992; Maede et al. 1993). This indicates that the acetylated COX1 still consumes AA to synthesize 15R-HPETE in antral mucous cells despite the inhibition of PGG/H synthesis. This suggests that ASA does not accumulate AA, unlike IDM. Nor does H-89, which inhibits PKA, accumulate AA, since it does not inhibit COX1.
PGE2 is synthesized from AA, which is produced by PLA2 from membrane lipid. The present study demonstrated that the inhibitors of PLA2 (AACOCF3 and ACA) also decrease the frequency of ACh-stimulated exocytotic events by approximately 60% and inhibit PGE2 release. Moreover, in the presence of AACOCF3, IDM did not increase the frequency of ACh-stimulated exocytotic events. This indicates that AACOCF3 or ACA inhibits the AA synthesis in ACh-stimulated antral mucous cells.
We also examined the effects of AA on ACh-stimulated exocytotic events in H-89- and AACOCF3-treated cells. The addition of AA increased the frequency of ACh-stimulated exocytotic events in H-89- and ASA-treated cells, similar to the addition of IDM. Thus, the effects of IDM during inhibition of the PGE2–cAMP pathway were mimicked by the addition of AA. Moreover, the addition of AA also increased the frequency of ACh-stimulated exocytotic events in AACOCF3-treated cells, although IDM did not. These observations suggest that IDM accumulates AA by inhibiting COX1 in antral mucous cells.
On the basis of these observations, IDM has two opposite effects on the ACh-stimulated exocytotic events in antral mucous cells. First, IDM decreases the frequency of exocytotic events by inhibiting PGE2 production; and second, IDM increases the frequency of ACh-stimulated exocytotic events by accumulating AA.
The effects of 15R-HPETE on the ACh-stimulated exocytotic events still remain uncertain. The ACh-stimulated exocytotic events in ASA-treated cells were similar to those in AACOCF3- or ASA-treated cells. The addition of ASA during ACh stimulation has no effects on exocytotic events in IDM-treated cells. This appears to suggest that 15R-HPETE has no effects on ACh-stimulated exocytotic events in antral mucous cells.
AA has already been established as a second messenger. In many cell types, AA stimulates non-store-operated Ca2+ entry channels (Shuttleworth, 1996; Shuttleworth & Thompson, 1998; Luo et al. 2001; Moneer et al. 2003; Watson et al. 2004), as well as stimulating Ca2+ release from stores followed by Ca2+ entry (Dettbarn & Palade, 1993; Fleming & Mellow, 1995; Krump et al. 1995). However, the present study demonstrated that AA (2 µM) induces no increase in [Ca2+]i. A previous report showed that 10 µM IDM also did not increase [Ca2+]i (Shimamoto et al. 2005). In many reports, the concentrations of AA used for Ca2+ mobilization were more than 5 µM (Luo et al. 2001; Shuttleworth, 1996; Watson et al. 2004). These observations indicate that a low concentration of AA, such as 2 µM, does not stimulate the Ca2+ mobilization in antral mucous cells.
The present study demonstrated that 2 µM AA alone did not activate exocytotic events in antral mucous cells and that an extremely low concentration of AA, such as 20–200 nM, still increased the frequency of ACh-stimulated exocytotic events in H-89- or ASA-treated cells. The concentration of AA accumulated by IDM is estimated to be less than 100 nM. A low concentration of AA, such as 100 nM, is unlikely to stimulate Ca2+ mobilization. The present study also demonstrated that AA stimulates both cAMP- and non-cAMP-signalling pathways. In previous reports, AA and AA metabolites have been reported to accumulate cGMP in myocytes (Snider et al. 1984; Lapointe & Sitkins, 1998). Moreover, 5,8,11,14-eicosatetraynoic acid (ETYA) an anologue of AA, has been reported to accumulate cGMP in vascular smooth muscle cells (Moneer et al. 2003). ETYA is also an agonist of peroxisome proliferator-activated receptors (PPARs) 
-subtype. These findings suggest that AA may also increase the frequency of ACh-stimulated exocytotic events via non-Ca2+ and non-cAMP pathways. At present, it remains uncertain how AA modulates Ca2+-regulated exocytosis. Futher studies are needed to clarify this.
The enhancement of exocytotic events induced by 2 µM AA was larger during stimulation with 1 µM ACh than during stimulation with 10 µM ACh, especially in the initial peak frequency. ACh at 10 µM induces a large amount of Ca2+ release from stores, which stimulates Ca2+-regulated exocytotic events maximally. Further addition of AA may slightly enhance the initial peak frequency stimulated by 10 µM ACh.
AA is continuously synthesized from membrane lipids by PLA2. Moreover, PLA2 is a well-known Ca2+-regulated enzyme. In antral mucous cells, ACh increases [Ca2+]i, which activates various cellular actions, including mucin exocytosis. This suggests that ACh also stimulates AA synthesis via PLA2. The present study demonstrated that the ACh-stimulated PGE2 release is inhibited by a PLA2 inhibitor (ACA), suggesting that ACh stimulates AA synthesis. This suggests that AA accumulation during ACh stimulation may modulate the Ca2+-regulated exocytosis directly. However, the AA-induced modulation of Ca2+-regulated exocytosis appears to be small, since an intracellular AA concentration increased by ACh is low in antral mucous cells, as shown in the present results. The AA-induced modulation of Ca2+-regulated exocytosis may play an important role in some pathological conditions, such as inflammation, which increases AA production.
The present study demonstrated that PGE2 is released from unstimulated antral mucosa, and that the addition of AA increases PGE2 release. This seems to be inconsistent with the previous result, namely that PGE2 stimulates cAMP-regulated exocytotic events (Ohnishi et al. 2001). However, we never observed exocytotic events in isolated antral mucous cells without any stimulation. In ACh-stimulated antral mucous cells, however, PGE2 released via COX1 enhances Ca2+-regulated exocytotic events (Shimamoto et al. 2005). This suggests that COX1 activities are low in unstimulated antral mucous cells. In the present study, we measured PGE2 release from antral mucosa, which contains epithelial cells and interstitial cells. PGE2 released from unstimulated antral mucosa may be produced by the interstitial cells rather than antral mucous cells.
In conclusion, the IDM-induced modulation of Ca2+-regulated exocytosis is shown in Fig. 10. ACh stimulation increases [Ca2+]i, which stimulates AA synthesis from membrane lipid via PLA2. IDM inhibits COX1 in antral mucous cells, which accumulates AA, and the AA accumulation induced by IDM may be enhanced by ACh. The AA accumulated increases the frequency of Ca2+-regulated exocytosis in antral mucous cells.
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A. H. Saad, C. Shimamoto, T. Nakahari, S. Fujiwara, K.-i. Katsu, and Y. Marunaka cGMP modulation of ACh-stimulated exocytosis in guinea pig antral mucous cells Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1138 - G1148. [Abstract] [Full Text] [PDF]
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). Cells were treated with AACOCF3, IDM and AA for 10 min prior to ACh stimulation. Further addition of 2 µM AA increased the initial peak frequency of ACh-stimulated exocytotic events (•). B, cells were treated with 10 µM ASA for 10 min prior to ACh stimulation. ASA decreased the initial peak frequency of ACh-stimulated exocytotic events by approximately 60%, similar to H-89. The addition of AA increased the initial peak frequency by approximately 80% in ASA-treated cells. Cells were also treated with ASA and IDM for 10 min prior to ACh and AA stimulation. In the presence of 2 µM AA, IDM did not increase the initial peak frequency of ACh-stimulated exocytotic events. *Significantly different from the corresponding value (P < 0.05).
are significantly different from those marked 