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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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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 -subunit
mouse monoclonal antibody (26), or
anti-H+-K+-ATPase
-subunit polyclonal
antibody (raised in the rat in the present study by using
SDS-PAGE-purified rabbit
-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
-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.
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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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[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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We thank John H. Jennings for editing the manuscript.
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FOOTNOTES |
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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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