A proposed mechanism for the potentiation of cAMP- mediated acid secretion by carbachol

Yuko Muto1, Taku Nagao1, Maki Yamada2,3, Katsuhiko Mikoshiba2,3, and Tetsuro Urushidani1

1 Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033; 2 Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639; and 3 Laboratory for Developmental Neurobiology, Brain Science Institute, Institute of Physical and Chemical Research, Wako, Saitama 351-0198, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Acid secretion in isolated rabbit gastric glands was monitored by the accumulation of [14C]aminopyrine. Stimulation of the glands with carbachol synergistically augmented the response to dibutyryl cAMP. The augmentation persisted even after carbachol was washed out and was resistant to chelated extracellular Ca2+ and to inhibitors of either protein kinase C or calmodulin kinase II. Cytochalasin D at 10 µM preferentially blocked the secretory effect of carbachol and its synergism with cAMP, whereas it had no effect on histamine- or cAMP-stimulated acid secretion within 15 min. Cytochalasin D inhibited the carbachol-stimulated intracellular Ca2+ concentration ([Ca2+]i) increase due to release from the Ca2+ store. Treatment of the glands with cytochalasin D redistributed type 3 inositol 1,4,5-trisphosphate receptor (the major subtype in the parietal cell) from the fraction containing membranes of large size to the microsomal fraction, suggesting a dissociation of the store from the plasma membrane. These findings suggest that intracellular Ca2+ release by cholinergic stimulation is critical for determining synergism with cAMP in parietal cell activation and that functional coupling between the Ca2+ store and the receptor is maintained by actin microfilaments.

inositol 1,4,5-trisphosphate receptor; cytoskeleton; cytochalasin D; calcium; parietal cell; rabbit


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN THE PROCESS OF ACTIVATION of gastric acid secretion, two main pathways exist, i.e., the stimulation of adenylate cyclase via the histamine H2 receptor and the activation of phospholipase C via the muscarine M3 and cholecystokinin-B (CCK-B) receptors. Intracellular signaling from these pathways is thought to cause synergistic interactions (for review, see Ref. 25). As for the second messengers, it is postulated that a synergistic interaction occurs between cAMP and Ca2+. In rabbit parietal cell, histamine elicits two signals at once, i.e., it increases cAMP as well as intracellular Ca2+ concentration ([Ca2+]i), and synergism occurs in the cell with the agonist alone. This could explain why the synergism between histamine and carbachol is relatively weak (5, 12, 15), whereas a marked synergism is observed between N6,2'-O-dibutyryl cAMP (DBcAMP) and carbachol (1, 15) in this species. In the case of canine parietal cells, in which histamine fails to cause [Ca2+]i elevation (6), the synergism between histamine and carbachol is quite obvious (17). At present, the source as well as the target of Ca2+ utilized for the potentiation is still unclear.

[Ca2+]i increase via M3 and CCK-B receptors is elicited by inositol 1,4,5-trisphosphate (IP3), which is the product of phospholipase C. IP3 has been known to have three types of specific receptors, type 1, 2, and 3 IP3 receptors, which share 60-70% identity in amino acid sequences with each other (9, 28). Founded on many lines of evidence, all of these are thought to form calcium channels through membranes of intracellular calcium stores and work to raise [Ca2+]i. The intracellular Ca2+ release by IP3 receptor has been also shown to be essential for the Ca2+ influx through plasma membranes of B cells (19), and this can hold true for other cell types, including parietal cells (14).

Recently, it was postulated that the intracellular Ca2+ store is not only functionally but also physically coupled with the receptor (23) and that cytoskeletal components are involved in the connection (16). In the present study, it was revealed that release of intracellular Ca2+ plays an important role in the potentiating effect of carbachol on DBcAMP-stimulated acid secretion and that cytochalasin D, the microfilament-disrupting agent, works as an effective inhibitor of synergism by interfering with the functional connection between the muscarinic receptor and the Ca2+ store.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Isolation of rabbit gastric glands, measurement of [14C]aminopyrine accumulation, and [Ca2+]i mobilization. Isolated gastric glands were prepared from Japanese White rabbits (Shiraishi, Tokyo, Japan) by a combination of high-pressure perfusion and collagenase digestion (2). Acid secretion of the glands was monitored by the accumulation of a weak base, [14C]aminopyrine (2). A 1-ml aliquot of isolated glands, suspended in 20× volume of medium, was stimulated in a 1.5-ml Eppendorf tube, and each data point is the mean of the duplicate measurements. Usually, resting control was included every 18 tubes (9 treatments), and the effect of the stimulants was expressed as the aminopyrine ratio above the resting value. The same treatment was never repeated for the same gland preparation from each rabbit. Therefore, the number of experiments (n) appearing in the present study also means the number of rabbits used. Drugs were all dissolved in DMSO so that the final concentration of the vehicle was less than 0.5% vol/vol. To exclude a possible involvement of endogenous histamine, 100 µM cimetidine was always included except for the case of stimulation by histamine. For the treatment with cytochalasin D, isolated glands were incubated with the drug for 10 min at room temperature before agonist stimulation. A Ca2+-free condition was achieved by the elimination of CaCl2 and the addition of 2 mM EGTA in the medium. One milliliter of isolated glands, suspended in 20× volume of medium, was stimulated in a 1.5-ml Eppendorf tube, and each data point consists of the mean of the duplicate measurements, which were never repeated for the same gland preparation from one rabbit so that, as explained above, n also represents the number of rabbits used.

The measurement of [Ca2+]i in the single parietal cell of the isolated glands was performed as previously described (13). Briefly, the glands, incubated with 5 µM fura 2-acetoxymethyl ester for 30 min at 37°C, were positioned in a temperature-controlled chamber and continuously superfused at 1 ml/min with a buffer containing 137 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.0 mM CaCl2, 1.0 mM NaH2PO4, 10 mM HEPES, 5.5 mM D-glucose, and 1 mg/ml BSA, pH 7.4. The changes in [Ca2+]i in a parietal cell were measured using the dual-wavelength excitation ratio technique [fluorescence ratio of 340- to 380-nm excitation (F340/F380), 510-nm emission] by a digital imaging system (Argus 50; Hamamatsu Photonics, Japan). The digitized images were recorded every 10 s, and seven parietal cells in the field were selected, quantified, averaged, and employed as a single measurement.

Subcellular fractionation of the glands. Subcellular fractions were prepared from the homogenate as described (24). Isolated glands were incubated at room temperature for 10 min with or without 10 µM cytochalasin D in 100 µM cimetidine-containing medium and then incubated at 37°C for 5 min with cytochalasin D plus 100 µM carbachol or in cimetidine alone. The glands were then pelleted at 1,000 rpm for 1 min and homogenized in a buffer containing 113 mM mannitol, 37 mM sucrose, 0.5 mM EDTA, and 5 mM PIPES at pH 6.7. After cell debris was removed (40 g, for 5 min), the homogenate was sequentially fractionated into three pellets: P1 (4,000 g, for 10 min), P2 (14,500 g, for 10 min), and P3 (100,000 g, for 45 min). The low-speed pellet, P1, was suspended in 18% Ficoll and centrifuged (100,000 g, for 120 min) to obtain floating membranes, which are designated as plasma membranes. The microsomal fraction, P3, was layered on 27% sucrose and centrifuged (100,000 g, for 120 min) to obtain light (top of the sucrose layer) and heavy (pellet) microsomes.

Isolation of IP3-sensitive Ca2+ stores from gastric mucosa and measurement of Ca2+ release. The procedure was based on that described for the cerebellum (18), with modifications (13). Rabbit gastric mucosa was homogenized with 120 mM KCl, 10 mM NaCl, 1 mM KH2PO4, 0.2 mM MgSO4, 20 mM HEPES-KOH (pH 7.4), 0.1 mM phenylmethylsulfonyl fluoride, and 10 µM pepstatin, and the supernatant, after being centrifuged (20,000 g, for 15 min), was incubated with 50 mg/ml Chelex-100 on ice. After 10 min, the supernatant was transferred to a cuvette containing 2 mM MgATP, 10 mM creatine phosphate, 5 U/ml creatine kinase, 1.2 µM fura 2, 10 µM omeprazole, and 5 µg/ml oligomycin. The sample was excited with dual wavelength, and the fluorescence ratio (F340/F380) at 510-nm emission was recorded by using an intracellular Ca2+ analyzer (CAF-110; JASCO, Tokyo, Japan). At the beginning of the experiment, 2.5 µM of CaCl2 was added, and the decrease of extravesicular Ca2+ concentration was monitored by measuring the Ca2+ uptake by the vesicles. When the ratio approached a constant value, test drugs were added to the cuvette.

Cell staining and immunological analysis. For immunostaining, isolated gastric glands were fixed with 10% Formalin and permeabilized with 0.5% Triton X-100. The glands were incubated with anti-type 1 (18A10, rat monoclonal) or anti-type 3 (KM1082, mouse monoclonal) IP3 receptor antibody (8), anti-H+-K+-ATPase alpha -subunit mouse monoclonal antibody (26), or anti-H+-K+-ATPase alpha -subunit polyclonal antibody (raised in the rat in the present study by using SDS-PAGE-purified rabbit alpha -subunit as an antigen) at 4°C for 18 h. The glands were washed and then incubated with FITC-anti-rat or -anti-mouse IgG (Sigma) at 4°C for 18 h. F-actin was made visible by staining with FITC-phalloidin (20 U/ml; Molecular Probes). For double staining (types 1 and 3 or IP3 receptor and H+-K+-ATPase), the mouse or rat IgG was visualized by Cy3-anti-mouse IgG, FITC-anti-mouse IgG, tetramethylrhodamine isothiocyanate (TRITC)-anti-rat IgG, or FITC-anti-rat IgG (Sigma). The glands were then examined by microscope (Nikon Eclipse TE300) with a ×60 water-immersion objective (MTB Plan Apo 60×WI) by using a confocal laser scanning system (µRadiance; Bio-Rad). FITC was excited at 488 nm (Argon Ion Laser) and detected with a HQ515/30 filter, and Cy3 and TRITC were excited at 543 nm (Green HeNe Laser) and detected with E570LP. For double staining, a sequential acquisition mode was employed to avoid "bleed through" of the staining. The fractions of gastric glands were separated by 6% SDS-PAGE, blotted on a polyvinylidene difluoride membrane, and then incubated with anti-type 3 IP3 receptor antibody or anti H+-K+-ATPase alpha -subunit antibody. The membrane was visualized by chemiluminescence (Renaissance Western Blot Chemiluminescence Reagent; NEN) for IP3 receptor or by 3,3'-diaminobenzidine for H+-K+-ATPase with the use of horseradish peroxidase-anti-mouse or -anti-rat IgG as a second antibody. For quantification, the chemiluminescent membranes were exposed to films for at least three different lengths of time and scanned by an Epson GT-8000, and the film darkening of the band was quantified by a computer program, NIH Image 1.61. The values of each band with different exposure time were plotted, and the relative density was expressed as the ratio to the control value.

Statistical analysis. Parametric data are expressed as means ± SE. Multiple comparisons were analyzed by ANOVA and Dunnet's post hoc test with the use of a computer program (Super ANOVA; Abacus Concepts, Berkeley, CA). The level of significance was uniformly set at P < 0.05, and no further calculation of P value was performed.


    RESULTS
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ABSTRACT
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RESULTS
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Potentiating interaction between carbachol and DBcAMP in aminopyrine accumulation. Isolated rabbit gastric glands were stimulated with 100 µM carbachol, 100 µM DBcAMP, or their combination, and the aminopyrine ratios were measured 5, 10, and 30 min after stimulation (Fig. 1). As widely observed (1, 4, 15), stimulation with carbachol elicited a transient increase in aminopyrine ratio with the peak around 5 or 10 min, which returned to the resting level after 30 min of stimulation. Stimulation with DBcAMP caused an elevation of the ratio similar to that for carbachol up to 10 min, but it kept increasing until 30 min of stimulation. When the glands were stimulated with carbachol plus DBcAMP, a large increase was observed; a potentiating interaction was evident at the end of 30 min of stimulation. Namely, the stimulatory effect of the stimulants when combined was much larger than the sum of the effects taken separately.


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Fig. 1.   Potentiating interaction between carbachol (CCh) and N6,2'-O-dibutyryl cAMP (DBcAMP) in aminopyrine accumulation. Isolated rabbit gastric glands were stimulated with 100 µM CCh, 100 µM DBcAMP, or their combination, and the aminopyrine ratios were measured 5, 10, and 30 min after stimulation (open symbols). The glands were also stimulated with CCh alone for the first 20 min and then DBcAMP was added, or they were stimulated with DBcAMP alone for the first 20 min and then CCh was added, and the aminopyrine ratios were measured at 30 min (filled symbols). The aminopyrine ratio above resting control (10.0 ± 1.3; mean ± SE, n = 4) was calculated and expressed as the mean ± SE of 4 independent experiments performed in duplicate.

When carbachol was added 20 min after stimulation with DBcAMP alone, the final value (30 min after the beginning of stimulation) was observed to be about the same as that for the combination of the two agonists from the beginning. However, when DBcAMP was added 20 min after the stimulation with carbachol, the final value (at 30 min) was much less than that obtained by combined stimulation from the beginning (Fig. 1), suggesting that the potentiating effect of carbachol disappears after 20 min at 37°C.

To confirm that the potentiating interaction occurred via muscarine receptor, we examined the effects of atropine (1 µM) on DBcAMP plus carbachol-stimulated aminopyrine accumulation. Summarizing the data for four separate experiments (mean ± SE), stimulation by 100 µM DBcAMP alone for 30 min gave a ratio of 23.8 ± 0.6 above the resting value, and stimulation by DBcAMP plus carbachol (100 µM each) showed a ratio of 67.0 ± 13.2 above the resting value. When 1 µM atropine was included at the beginning of the stimulation by DBcAMP plus carbachol, the aminopyrine ratio was reduced to the same level as that by DBcAMP alone, 21.8 ± 2.0 (P < 0.05 vs. without atropine). Interestingly, when 1 µM atropine was added 5 min after the stimulation started, the aminopyrine ratio was 62.5 ± 10.0 above resting; namely, no inhibition was observed.

From the experiments of time course and the effect of atropine, the response to carbachol was determined to be prompt. We then examined whether the augmented response to DBcAMP was maintained even after carbachol was washed out shortly after stimulation. Figure 2 shows the results of this type of experiment. In experiment 1, the glands were incubated with vehicle for 5 min at 37°C and washed three times at room temperature. It was confirmed that this procedure effectively eliminated the effects of several secretagogues and inhibitors (data not shown). The glands were then stimulated for 30 min at 37°C, and the aminopyrine ratio above the resting control value was taken as 100%. In experiment 2, the glands were stimulated with 100 µM carbachol plus 100 µM DBcAMP for 5 min, washed three times, and stimulated again with carbachol plus DBcAMP for 30 min. The aminopyrine ratio in experiment 2 was ~200% of the control value in experiment 1, confirming the potentiating interaction between these agonists. When the glands were treated with 100 µM carbachol for 5 min, washed out, and stimulated with 100 µM DBcAMP alone for 30 min, the aminopyrine ratio was also ~200% of control, showing that the potentiation by carbachol on DBcAMP-stimulated secretion was still active after the washing out (experiment 3). We call this the "priming effect" of carbachol.


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Fig. 2.   "Priming effect" of CCh on the aminopyrine accumulation in rabbit isolated gastric glands. Isolated rabbit gastric glands were treated for 5 min at 37°C with 100 µM CCh or indicated agents (pretreatment). The glands were then washed 3 times at room temperature (this procedure took ~20 min) and then stimulated for 30 min at 37°C with 100 µM DBcAMP or DBcAMP plus 100 µM CCh as indicated (stimulants). Aminopyrine ratios above resting control were calculated and expressed as a percentage of the control value (obtained from experiment 1). Atr, atropine sulfate; OPZ, omeprazole. Values are means ± SE of 4 independent experiments performed in duplicate. *P < 0.05 vs. experiment 3.

The priming effect of carbachol disappeared in the presence of atropine (experiment 4). Omeprazole, included in the first 5 min, did not change the priming effect, indicating that actual acid secretion is not necessary for establishment of priming (experiment 5). To examine the role of Ca2+, 5-min stimulation by carbachol was performed in medium containing 2 mM EGTA instead of Ca2+, and the potentiation was the same as in the normal medium (experiment 6), suggesting that extracellular Ca2+ is not necessary for the priming effect.

Figure 3 shows the effect of various treatments on stimulation with a combination of DBcAMP and carbachol (100 µM each). Even when extracellular Ca2+ was eliminated by EGTA throughout stimulation, the potentiating effect was not attenuated. This effect was in contrast to the transient effect of carbachol alone, which was abolished by the elimination of extracellular Ca2+ (1, 15). Next, 10 µM bisindolylmaleimide I, a protein kinase C inhibitor, was applied, but no significant effect was observed. We already reported that this concentration of the compound did not affect stimulation by DBcAMP alone (20) but inhibited the stimulatory effect of phorbol ester (1). Therefore, we conclude that the involvement of protein kinase C is minimal in the potentiating interaction between DBcAMP and carbachol. We also checked a calmodulin kinase II inhibitor, KN-62, at 60 µM, which was enough to inhibit the stimulatory effect of carbachol alone (22) but produced no inhibitory effect (Fig. 3). We then screened various drugs to identify specific agents affecting the potentiating interaction and found that cytochalasin D, an actin-depolymerizing agent, abolished the stimulatory effect of DBcAMP plus carbachol (Fig. 3).


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Fig. 3.   Effects of cytochalasin D (cytoD), bisindolylmaleimide I (BIM), EGTA, and KN-62 on aminopyrine accumulation in isolated rabbit gastric glands stimulated by CCh plus DBcAMP. The gastric glands were stimulated with 100 µM CCh, 100 µM DBcAMP, or their combination, and the aminopyrine ratios were measured 30 min after the stimulation. R, resting control. Values are means ± SE of 4-8 separate experiments performed in duplicate. *P < 0.05 vs. control (CCh + DBcAMP).

Figure 4 shows the effects of cytochalasin D on the acid secretion stimulated by DBcAMP, carbachol, and histamine. When agonist stimulation was performed for 15 min (Fig. 4A), cytochalasin D selectively inhibited carbachol- or carbachol plus DBcAMP-stimulated aminopyrine accumulation, whereas it showed no effect on the stimulation by DBcAMP or histamine. At the point of 30 min of stimulation (Fig. 4B), cytochalasin D tended to inhibit DBcAMP- and histamine-induced aminopyrine accumulation to 60-70% of control, consistent with the report by Forte et al. (7), although these effects were not statistically significant in the present study.


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Fig. 4.   Effects of 10 µM cytoD on CCh-, DBcAMP-, or histamine (His)-stimulated aminopyrine accumulation in isolated gastric glands. Glands were stimulated for 15 min (A) or 30 min (B) in the absence (control) or presence of 10 µM cytoD, and aminopyrine ratios were measured. Values are means ± SE of 3-5 separate experiments performed in duplicate. *P < 0.05 vs. corresponding control.

[Ca2+]i mobilization in the parietal cells. From the experiments under the Ca2+-free condition, it was suggested that release of intracellular Ca2+ was involved in the potentiating interaction between DBcAMP and carbachol. We thus examined intracellular Ca2+ mobilization. As widely observed (6, 13, 15), carbachol elicited a biphasic increase in [Ca2+]i, indicated as a single sharp peak followed by a sustained plateau. As shown in Fig. 5A, cytochalasin D at 10 µM strongly inhibited the response to carbachol. Dose-response curves were obtained by quantification of the first peak of Ca2+ rise (Fig. 5B), and the increase in [Ca2+]i elicited by carbachol was demonstrated to be sensitive to cytochalasin D. 


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Fig. 5.   Effects of cytoD on the Ca2+ transient in the single parietal cell induced by CCh. A: typical intracellular Ca2+ concentration ([Ca2+]i) responses to 100 µM CCh in the absence (control) or presence of 10 µM cytoD. Isolated glands, loaded with fura 2, were placed on a perfusion chamber, and the changes in [Ca2+]i in a single parietal cell were measured using the dual-wavelength excitation ratio technique [fluorescence ratio of 340- to 380-nm excitation (F340/F380), 510 nm emission] by a digital imaging system (Argus 50). B: changes in [Ca2+] were expressed as the net changes in the ratio (Delta ratio) caused by agonist relative to the pretreatment value. Effects of 10 µM cytoD on the dose-response curve of CCh are shown as means ± SE of 3-4 measurements at each point. *P < 0.05 vs. corresponding control.

It was practically impossible to distinguish whether inhibition occurred on release from the store or on Ca2+ influx on the basis of the shape of the curve. To distinguish these cases, carbachol was added in the Ca2+-free condition. As shown in Fig. 6A, addition of carbachol in the Ca2+-free condition caused a transient [Ca2+]i rise without plateau phase, although the size of this rise was somewhat smaller than that in normal conditions. When this experiment was performed in the presence of cytochalasin D, the Ca2+ rise was almost abolished. The inhibitory effect of cytochalasin D is evident from the summarized data shown in Fig. 6B. This finding suggested that the site of action of cytochalasin D was the release of Ca2+ from the intracellular store.


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Fig. 6.   Effects of cytoD on intracellular Ca2+ release in a single parietal cell measured as explained in Fig. 5 legend. A: fura 2-loaded glands were perfused with Ca2+-free solution (2 mM EGTA) with or without (control) cytoD, and the perfusate was switched to the solution containing CCh. Data are representative of 3 experiments. B: the changes in [Ca2+] were expressed as Delta ratio caused by CCh relative to the pretreatment value and are the means ± SE of 3 measurements. *P < 0.05 vs. corresponding control.

To examine whether the inhibitory effect of cytochalasin D on intracellular Ca2+ release was due to its effect on IP3 receptor in the Ca2+-store membrane, we checked the direct effect of cytochalasin D on the Ca2+ store in vitro. Figure 7 shows typical results. IP3 was added to the Ca2+ store isolated from rabbit gastric mucosa, and dose-dependent release of Ca2+ was observed. This effect of IP3 was not affected by 10 µM cytochalasin D, suggesting that inhibition of cytochalasin D was not on the IP3 receptor.


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Fig. 7.   Effect of cytoD on Ca2+ release from the isolated Ca2+ store by inositol 1,4,5-trisphosphate (IP3). Ca2+ store isolated from gastric mucosa was transferred to a cuvette containing 2 mM MgATP, 10 mM creatine phosphate, 5 U/ml creatine kinase, 1.2 µM fura 2, 10 µM omeprazole, and 5 µg/ml oligomycin. The cuvette was excited with dual wavelength, and the fluorescence ratio (F340/F380) at 510-nm emission was recorded. The store was loaded with 2.5 µM of CaCl2, increasing doses of IP3 were added as indicated by boxes, and a dose-dependent Ca2+ release was observed. This release was unaffected by 10 µM cytoD. Data are representative of at least 3 experiments with similar results.

F-actin staining in the isolated gastric glands. In the previous experiments, cytochalasin D selectively affected some of the physiological functions in the rabbit gastric glands. We then examined the intracellular alignment of actin filaments under several conditions. Figure 8 depicts the observation by a confocal microscope of F-actin staining with FITC-phalloidin in gastric glands. In the resting control glands (Fig. 8A), phalloidin made visible the fine intracellular canalicular structure and lining of the basolateral membranes in the parietal cells. In the glands pretreated with 10 µM cytochalasin D for 10 min at room temperature and further incubated with carbachol for 5 min at 37°C (corresponding to the time when Ca2+ transient is observed), the alignment of F-actin was still similar to that of resting control, although a little decrease in the staining intensity was noted (Fig. 8B). When the incubation time at 37°C was prolonged to 15 min, fading of the intracellular structure occurred in some parietal cells, possibly reflecting the depolymerization of actin filaments supporting the microvilli of the apical surface (Fig. 8C). After the treatment with cytochalasin D for 30 min at 37°C, marked disturbance of actin filament occurred and no structures corresponding to intracellular canaliculi were visible (Fig. 8D). It is noteworthy that some physiological function, e.g., aminopyrine accumulation stimulated by histamine or DBcAMP, was little affected even in the condition shown in Fig. 8, C or D.


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Fig. 8.   F-actin staining by FITC-phalloidin in isolated gastric glands. Rabbit gastric glands, incubated with 100 µM cimetidine for 10 min at room temperature and 15 min at 37°C (A), with 10 µM cytoD for 10 min at room temperature and stimulated by 100 µM CCh for 5 min (B), 15 min, (C), and 30 min (D) at 37°C, were fixed with Formalin, permeabilized with Triton X-100, and incubated with FITC-phalloidin (20 U/ml). Bars: 10 µm.

Localization of IP3 receptors. It is reasonable to suppose that the inhibitory effect of cytochalasin D on Ca2+ release was due to the depolymerization of actin. One might expect that there would be a functional coupling between Ca2+ store and drug receptor on the plasma membrane and that their connection would be maintained by actin filaments. Although it was reported that both type 1 and 3 receptors of IP3 are present and enriched in the murine gastric parietal cell (11), it was unclear whether this was also the case in rabbit. We thus stained rabbit gastric glands with specific antibodies against type 1 and 3 IP3 receptors.

In the immunohistochemistry analysis performed using the antibodies, it was revealed that type 3 IP3 receptor was relatively rich in parietal cells, as was reported for murine gastric mucosa (11), whereas type 1 IP3 receptor was enriched in chief cells within rabbit gastric gland. Figure 9A shows the staining of type 1 IP3 receptor in red (TRITC), and Fig. 9B shows H+-K+-ATPase stained in green (FITC). It is obvious from the merged image (Fig. 9C) that type 1 IP3 receptor is much more enriched in the small, H+-K+-ATPase-negative cells (possibly chief cells) than in the larger, H+-K+-ATPase-positive parietal cells. Within the parietal cell, type 1 IP3 receptor diffusely distributed to the cytosol without any definite structures. In contrast, type 3 IP3 receptor is enriched in the cells with large size (Fig. 9D), which could be identified as parietal cell by the positive staining with anti-H+-K+-ATPase (Fig. 9E) and their merged images (Fig. 9F). Although we were interested in the difference in the intracellular distribution of these proteins, the space resolution was not enough in the present system. This point is clarified in the latter part of the present work.


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Fig. 9.   Localization of IP3 receptors and H+-K+-ATPase alpha -subunit. Rabbit gastric glands, incubated with 100 µM cimetidine at 37°C for 15 min, were fixed with Formalin and permeabilized with Triton X-100. The glands were then incubated with the anti-H+-K+-ATPase alpha -subunit monoclonal antibody (1:1,000 dilution of ascites fluid), anti-H+-K+-ATPase alpha -subunit polyclonal antibody (1:100 dilution of rat serum), anti-type 1 IP3 receptor monoclonal antibody (50 µg/ml rat IgG), or anti-type 3 IP3 receptor monoclonal antibody (10 µg/ml mouse IgG). The first antibodies were visualized with TRITC-anti-rat IgG (A and C), FITC-anti-rat IgG (E and F), Cy3-anti-mouse IgG (D and F), or FITC-anti-mouse IgG (B and C) at 1:100 dilution each. A: type 1 IP3 receptor staining. B: anti-H+-K+-ATPase staining. C: merged image of A and B. D: type 3 IP3 receptor staining. E: anti-H+-K+-ATPase staining. F: merged image of D and E. Bars: 70 µm (A-C); 10 µm (D-F).

To estimate nonspecific staining, isolated glands were treated with 50 µg/ml normal rat IgG or 10 µg/ml normal mouse IgG followed by FITC-anti-rat, FITC-anti-mouse, TRITC-anti-rat, and Cy3-anti-mouse as second antibody. Essentially no signal was detected when TRITC- or Cy3-labeled antibody was used and excited at 543 nm (Green HeNe Laser). Although very faint fluorescence was noted in FITC-labeled second antibody was excited at 488 nm (Argon Ion Laser), its intensity was low enough to detect the specific staining under the same condition for the observation. This fluorescence did not appear to be background staining but, rather, autofluorescence, possibly due to the mitochondria in the parietal cell, because it was also observed in the glands without any antibody treatments (data not shown).

It was quite interesting to test whether cytochalasin D affects the intracellular distribution of the IP3 receptor, especially type 3. However, treatment with 100 µM carbachol and 10 µM cytochalasin D did not cause any detectable changes in the distribution of both types of IP3 receptor from observation with an optical microscope (data not shown).

We then tried to detect the effect of cytochalasin D on the distribution of the IP3 receptor by using a biochemical technique. Isolated gastric glands were homogenized and fractionated into pellets P1 (4,000 g, for 10 min), P2 (14,500 g, for 10 min), and P3 (100,000 g, for 45 min) and supernatant. In preliminary Western blotting, type 3 IP3 receptor was absent in the supernatant and detectable in P2, but the majority of the protein was found in P3. It was hard to detect in P1. Because the plasma membrane was expected to be harvested in the low-speed pellet, P1, this fraction was suspended in 18% Ficoll and centrifuged (100,000 g, for 120 min) to obtain floating membranes. It was then found that type 3 IP3 receptor became detectable. The microsomal fraction, P3, was further layered on 27% sucrose and centrifuged (100,000 g, for 120 min) to obtain light (top of the sucrose layer) and heavy (pellet) microsomes. We designated the microsomal fraction as intracellular vesicles and the 18% Ficoll fraction as the plasma membrane. Figure 10 depicts the results of immunoblotting of these fractions. In the control glands, type 3 IP3 receptor was found in the plasma membrane fraction as well as in the microsomes (Fig. 10A). Within the microsomal fraction, the IP3 receptor distributed not to the light but to the heavy membranes (Fig. 10A), whereas the amount of H+-K+-ATPase was inverse, i.e., it was rich in the light and scarce in the heavy microsomes (Fig. 10B). This suggests that the Ca2+ store membranes containing IP3 receptors are different from tubulovesicles containing proton pump. When the glands were treated with carbachol plus cytochalasin D, the content of type 3 IP3 receptor in the plasma membrane fraction decreased and redistributed to the heavy microsomes in the microsomal fraction (Fig. 10B). The relative density value for type 3 IP3 receptor in the plasma membrane fraction from cytochalasin D-treated glands was significantly decreased to 53.6 ± 8.3% of control (mean ± SE, n = 3; P < 0.05), whereas that in the heavy microsomal fraction was significantly increased to 140.0 ± 6.4% of control (mean ± SE, n = 3; P < 0.05). These changes were not due to a simple breakdown of the structure of intracellular membranes because the content of H+-K+-ATPase in the microsomes or the plasma membrane fraction was not affected by this treatment (Fig. 10B). Similar changes were observed in glands treated with cytochalasin D alone but not with carbachol alone (data not shown).


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Fig. 10.   Distribution of type 3 IP3 receptor and H+-K+-ATPase in the fractions of gastric gland homogenate. Isolated glands were incubated at room temperature for 10 min with or without 10 µM cytoD in 100 µM cimetidine-containing medium and then incubated at 37°C for 5 min with cimetidine alone (R) or with cytoD plus 100 µM CCh (Cy). The glands were homogenized and fractionated into pellets P1 (4,000 g, for 10 min), P2 (14,500 g, for 10 min), and P3 (100,000 g, for 45 min). P1 was then suspended in 18% Ficoll and centrifuged (100,000 g, for 120 min) to obtain floating membranes, which are designated as plasma membranes (PM). P3 was further layered on 27% sucrose and centrifuged (100,000 g, for 120 min) to obtain light microsomes (LM; top of the sucrose layer) and heavy microsomes (HM; pellet). A: each fraction (50 µg protein/lane) was separated by 6% SDS-PAGE, blotted on a polyvinylidene difluoride membrane, incubated with anti-type 3 monoclonal antibody (0.4 µg/ml mouse IgG), and visualized by chemiluminescence using horseradish peroxidase (HRP)-anti-mouse IgG (1:4,000 dilution) as second antibody. B: a membrane equivalent to that in A was incubated with anti-H+-K+-ATPase alpha -subunit monoclonal antibody (1:50,000 dilution of mouse ascites fluid) and visualized by 3,3'-diaminobenzidine using HRP-anti-mouse IgG (1:5,000 dilution) as second antibody. Arrows indicate the position of prestained molecular size marker (kDa).

Type 1 IP3 receptor was barely detectable in both the heavy microsomes and the plasma membrane fraction but was dispersed much more toward the heavy microsomes than was the case for type 3 IP3 receptor. We did not perform further experiments because this type of IP3 receptor is enriched in chief cells rather than parietal cells, as shown in Fig. 9.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is well known that a potentiating interaction occurs among histamine, acetylcholine, and gastrin in the activation of parietal cells (17). The potentiation is thought not to be in the area of the drug receptors but in the intracellular signal transduction, although the precise sites are presently unknown. The consensus is that activation of histamine H2 receptor leads to the elevation of intracellular cAMP, and the activation of either acetylcholine or gastrin receptor leads to an increase of intracellular Ca2+ induced by the elevation of IP3, as well as activation of protein kinase C. Theoretically, the potentiation should occur between cAMP-dependent protein kinase and Ca2+ or between cAMP-dependent protein kinase and protein kinase C. However, there have been many contradictory results so far (for review, see Ref. 25).

Li et al. (12) claim that the augmentation of the cAMP pathway by carbachol or gastrin is due to the elevation of [Ca2+]i. They showed that secretion stimulated by DBcAMP in the isolated rat parietal cell was augmented by both carbachol and gastrin and that those augmentations were inhibited by the intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. They also observed that the augmentation by carbachol and gastrin was surrogated by a Ca2+ ionophore, A-23187. They considered the effect of Ca2+ as the essential component for the morphological changes that are characteristic of the stimulated parietal cell. To support this hypothesis, they showed that carbachol and gastrin caused morphological changes in the parietal cell without large increase in the aminopyrine accumulation, while the addition of DBcAMP increased the accumulation. However, in the rabbit isolated glands, in which normal morphology is better preserved than in isolated cells, only poor morphological changes could be observed in response to cholinergic stimulation (24). Furthermore, there is little knowledge about the source of Ca2+ and the molecular entity responsible for the potentiating interaction.

In the present study, we confirmed that carbachol augmented acid secretion stimulated by DBcAMP in rabbit gastric glands. This effect of carbachol occurred within a short time (5 min) and persisted even after the carbachol was washed out at the room temperature, but it disappeared at 37°C within 20 min. This means that once a parietal cell gets an episode of cholinergic stimulation, it shifts to a condition sensitive to stimulation by DBcAMP for a short period of time and then returns to the preprimed state by a metabolism-dependent pathway. This "priming" effect of carbachol does not involve endogenous histamine but is the result of direct activation of the muscarinic receptor on the parietal cell. Removal of extracellular Ca2+ affected neither the priming effect of carbachol nor the augmented secretion by carbachol plus DBcAMP. This strongly suggests that the Ca2+ influx is not involved in the potentiating effect of carbachol. Therefore, we deduced that activation of protein kinase C and release of intracellular Ca2+ by IP3 were the remaining possibilities.

The dominant opinion concerning the role of protein kinase C in the acid secretion is that it is inhibitory (25). In certain cases, however, protein kinase C is able to work as an accelerator for acid secretion. For example, when intracellular cAMP is elevated (1, 4), or when extracellular K+ is elevated (1), phorbol esters apparently increase aminopyrine accumulation in rabbit gastric glands, effects that are suppressed by protein kinase C inhibitors. This means that protein kinase C could also be a candidate for the mediator for potentiation, at least between carbachol and DBcAMP.

It is difficult to use "protein kinase C inhibitors" for analyzing the involvement of protein kinase C in acid secretion (4, 20). We chose bisindolylmaleimide I because this drug (at 10 µM) does not affect the secretion stimulated by DBcAMP alone (20) but inhibits phorbol ester-stimulated secretion (1). We found that 10 µM bisindolylmaleimide I failed to affect the augmented secretion stimulated by DBcAMP plus carbachol. This finding suggests that the priming effect of carbachol involves the release of intracellular Ca2+ and not protein kinase C.

The importance of the role of intracellular Ca2+ release was also supported by another significant fact. We screened several agents that inhibited potentiation between cAMP and carbachol and finally found that cytochalasin D fit this purpose. The inhibitory effect of cytochalasin D has been considered to operate by disrupting the microvilli, which are essential for acid secretion (7). Cytochalasin D tended to inhibit both histamine- and DBcAMP-stimulated acid secretion by ~40% at the end of stimulation for 30 min, whereas inhibition was negligible at 15 min of stimulation. In the case of secretion stimulated by carbachol alone (15 min of stimulation) and carbachol plus DBcAMP (15 and 30 min of stimulation), the inhibition was almost 100%. This clearly indicated that the inhibitory effect of cytochalasin D was more specifically directed to the effects of carbachol alone and its augmentation on DBcAMP.

Because the action of intracellular Ca2+-release was considered to be important in the stimulatory effect of carbachol, we examined the effect of cytochalasin D on [Ca2+]i mobilization in parietal cells. It was clearly shown that cytochalasin D suppressed the increase in [Ca2+]i by carbachol. On the basis of analysis of the Ca2+-free condition, its inhibition was considered to be on the intracellular Ca2+ release rather than Ca2+ influx. Cytochalasin D did not change the effect of IP3 on the isolated Ca2+-stores from the gastric mucosa. Therefore, we conclude that the inhibitory effect of cytochalasin D on intracellular Ca2+ release is indirect, possibly due to the uncoupling between the receptor and the effector. On the basis of these observations, we consider it reasonable to suppose that the M3 receptor and the Ca2+ store are functionally connected via actin microfilaments in the parietal cell, that depolymerization of F-actin by cytochalasin D disrupts this functional connection, and thus that it inhibits the potentiating interaction between carbachol and cAMP. Alternatively, cytochalasin D may block the putative actin-binding site on the M3 and/or IP3 receptors, considering the observation that uncoupling of M3 receptor to Ca2+ release occurred even when F-actin staining on the apical membrane of parietal cell was apparently not disturbed.

Tsunoda (21) reported that intracellular Ca2+ release and acid secretion induced by gastrin were inhibited not only by microtubule- but also microfilament-disrupting agents. More recently, receptors producing IP3 and Ca2+ stores were shown to be physically and functionally coupled via cytoskeletal components in the fibroblast cell line NIH/3T3 (16). In their case, however, treatment with cytochalasin D was so intense that most of the microfilament in the cell was completely disrupted and many physiological functions might be distorted. In the present study, we were able to show that cytochalasin D selectively abolished the physiological function mediated by receptor-coupled intracellular Ca2+ release when other functions, e.g., cAMP-mediated acid secretion, as well as the morphology of the cell were normal. This is thus the first example that Ca2+ signaling involved in the physiological function is maintained by microfilaments in the normal cell.

We have no idea at present about the target for the released Ca2+. Li et al. (12) discussed a possible involvement of calmodulin kinase II. In the present study, however, 60 µM KN-62, a calmodulin kinase II inhibitor, failed to inhibit the augmented secretion by DBcAMP plus carbachol, suggesting that the priming effect of carbachol mediated by intracellular Ca2+ release does not involve this enzyme activation. Further work is clearly necessary to find out the target for Ca2+ focusing on the calmodulin-independent pathway as well.

Recently, the subtypes of the IP3 receptor in the gastric parietal cell were identified, and they were mainly types 1 and 3, but not type 2 (11), in the murine stomach. It is interesting that there might be subtype-specific regulation in the acid secretion, e.g., type 1 has calmodulin-binding domain but type 3 does not (27). In the present study, staining of each receptor with the corresponding antibody revealed the characteristic distribution in rabbit gastric gland. In contrast to findings in the murine stomach, type 1 IP3 receptor was scarce in rabbit parietal cell. Within the parietal cell, type 3 IP3 receptor was diffusely distributed in the cytosol. Because IP3 receptor is definitely membrane protein, the cytosolic staining reflects its presence in the intracellular membranes, which was confirmed by Western blotting showing that the type 3 IP3 receptor was found not in the cytosol but in the microsomal fraction. The tubulovesicles of the parietal cell were harvested from the light microsomes, and the amount of H+-K+- ATPase was inversely proportional to the density of the microsome, consistent with a previous report (10). In contrast, the type 3 IP3 receptor was harvested from the heavy membranes in the microsome, suggesting that the membrane population of these two proteins is clearly different. In the endocrine or neuroendocrine cells, an interesting hypothesis has been proposed stating that the secretory granule itself has type 3 IP3 receptors on its membrane and utilizes them for the exocytosis or membrane traffic (3). However, this type of regulation might not be working in the case of the parietal cell, because tubulovesicles do not appear to contain IP3 receptors.

A considerable amount of type 3 IP3 receptor was found in the plasma membrane fraction in addition to the heavy microsomal fraction. This suggests that the Ca2+ store-containing type 3 IP3 receptor either consists of both large and small membrane vesicles or consists of only small vesicles but has a physical connection to the large plasma membrane. After the cytochalasin D treatment, the content of the IP3 receptor in the larger size membranes drastically decreased and that in the heavy microsomes increased, as judged by Western blotting. This suggests that type 3 IP3 receptor resides not on the larger membranes but on the small vesicles, which are physically connected to the larger membrane, possibly via cytoskeletal components. This strongly supports our hypothesis mentioned above that cytochalasin D preferentially inhibits the priming effect of carbachol by disrupting the physical and functional connection between M3 receptor and Ca2+ store via actin filaments. We could not find any detectable changes in the immunostaining of type 3 IP3 receptor in the cytochalasin D-treated cells. This seems to be a limitation of observation by a light microscope, and study using an electron microscope is necessary in future work.

As for the physical connection between M3 receptor and IP3 receptor, the present data only show that the connection was maintained by the actin microfilament. It was reported that group 1 metabotropic glutamate receptor and IP3 receptor coimmunoprecipitated as a complex with Homer protein. It was also shown that the Ca2+ response via the glutamate receptor was modulated by the peptide fragments of Homer (23). The NH2-terminal region of Homer has homology to EVH [enabled/vasodilator-stimulated phosphoprotein (VASP) homology] family proteins, which have been postulated to interact with cytoskeletal components. Although this mechanism is proposed in the neuronal cells, it is possible that a similar mechanism also exists in the parietal cell.

Judging from the distribution of the IP3 receptor within rabbit gastric gland, type 1 IP3 receptor might have a role in pepsinogen secretion rather than acid secretion, at least in this species. It was observed that the type 1 IP3 receptor knockout mouse showed a normal acid secretory response to carbachol (Yamada M, Horie S, Watanabe K, and Mikoshiba T, unpublished observation), possibly because the type 3 IP3 receptor surrogated the specific physiological role, if any, of the type 1 IP3 receptor. It was also suggested that type 1 and 3 may form a heterotetrameric structure (11) in the parietal cell. The possible type-specific function of IP3 receptor is thus left for future study.

In conclusion, cholinergic stimulation in the parietal cell works synergistically with the cAMP pathway and is mediated by the release of intracellular Ca2+ from the store, physically and functionally connected to the muscarinic receptor. The coupling between type 3 IP3 receptor and the store requires a cytoskeletal connection consisting of F-actin.


    ACKNOWLEDGEMENTS

We thank John H. Jennings for editing the manuscript.


    FOOTNOTES

This work was supported in part by Japanese Ministry of Education, Science, Sports, and Culture Grants 10557219 and 11672160.

Address for reprint requests and other correspondence: T. Urushidani, Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Univ. of Tokyo, Tokyo 113-0033, Japan (E-mail: urushi{at}mol.f.u-tokyo.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.

Received 17 May 2000; accepted in final form 8 August 2000.


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ABSTRACT
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DISCUSSION
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