Department of Physiology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
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ABSTRACT |
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Amplification of mRNA from a human antral cell culture preparation demonstrated the presence of two receptors of the bombesin and gastrin-releasing peptide family, GRPR-1 and BRS-3. Single cell microfluorometry demonstrated that most cells that exhibited bombesin-evoked changes in intracellular Ca2+ concentration were gastrin immunoreactive, indicating that antral G cells express the GRPR subtype. There were two components to the intracellular Ca2+ response: an initial nitrendipine-insensitive mobilization followed by a sustained phase that was inhibited by removal of extracellular Ca2+ and 20 mM caffeine and was partially inhibited by 10 µM nitrendipine. Preexposure of cells to thapsigargin and caffeine prevented the response to bombesin, indicating activation of inositol 1,4,5-trisphosphate (IP3)-sensitive stores. Gastrin release could be partially reversed by removal of extracellular Ca2+ and blockade of L-type voltage-dependent Ca2+ channels, indicating that a component of the secretory response to bombesin was dependent on Ca2+ influx. These data demonstrated that bombesin-stimulated gastrin release from human antral G cells resulted from activation of GRPRs and involved both release of intracellular Ca2+ and influx of extracellular Ca2+ through a combination of L-type voltage-gated and IP3-gated Ca2+ channels.
gastrin-releasing peptide; calcium imaging; nitrendipine; calcium-sensing receptor
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INTRODUCTION |
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INTERSPECIES DIFFERENCES in receptor expression and/or signal transduction events have complicated our understanding of the mechanisms involved in the regulation of gastrin secretion from antral G cells. One such species-dependent difference is observed in response to changes in extracellular Ca2+. Gastrin release in response to hypercalcemia in dogs remains unchanged (18), whereas in humans there is a significant increase (1). This early observation can now be explained by the expression of the extracellular Ca2+-sensing receptor (CaR) on human antral G cells (16). The CaR is activated at extracellular Ca2+ concentrations between 1.5 and 1.8 mM. All previous studies with the human G cell-enriched preparation utilized cells grown and stimulated in DMEM containing 1.8 mM Ca2+, which would have resulted in chronic activation of the CaR. It is unknown whether long-term activation of the CaR would alter the response of G cells to other stimuli such as bombesin.
In previous studies of the mechanism underlying bombesin-stimulated gastrin release, we demonstrated that secretion was unaffected by the removal of extracellular Ca2+ and suggested that elevation of intracellular Ca2+ levels was responsible for producing the increased Ca2+ levels required for exocytosis (4). These data contrasted with studies in dogs in which removal of extracellular Ca2+ decreased bombesin-stimulated gastrin release (22). In view of the chronic activation of the CaR on the human G cells, a possible explanation for this difference is that the signaling pathways activated by bombesin were modulated by concurrent activation of the CaR rather than by the expression of a different bombesin receptor subtype (BRS), as suggested in the earlier study.
Currently, there are three mammalian receptors that recognize members of the bombesin and gastrin-releasing peptide (GRP) family: the GRPR or BB1 (21); the neuromedin B receptor (NMBR) or BB2 (24); and a third bombesin receptor subtype, BRS-3 or BB3 (10), an orphan receptor that does not recognize bombesin as a ligand except at high concentrations (27). Recently, a fourth amphibian bombesin receptor has been cloned from frogs, BB4 (13). The precise receptor subtype expressed on either human or canine G cells is presently unknown.
The mobilization of stored Ca2+ is thought to be the major species-independent mechanism activated by bombesin to evoke changes in secretion. However, both canine and human G cells exhibit a refractory period after the initial response, although a subsequent response can be generated, provided that sufficient time is allowed between stimuli. The mechanism underlying the inactive period is currently uncertain. The refractory response to bombesin in Swiss 3T3 cells was not associated with intracellular Ca2+ stores or protein kinase C activity (25), whereas in canine G cells the refractory time, ~30 min, probably results from the time required for receptor recycling following the initial stimulation (19). In earlier studies with human G cells, we suggested that the ability to generate a second response was dependent on the refilling of intracellular Ca2+ stores (4).
In view of the expression of the CaR on human antral G cells, the results of the previous studies on signaling pathways activated by bombesin will have reflected the composite response to activation of both CaR and bombesin receptors rather than bombesin alone. The aim of this study was first to identify the BRS expressed on gastrin-secreting cells using RT-PCR. The second aim was to reexamine the bombesin-evoked Ca2+ signaling and secretion in a modified short-term culture of human antral G cells, in which the concentration of extracellular Ca2+ was maintained below the threshold for activation of the CaR. The cytosolic Ca2+ store accessed by bombesin and the mechanism underlying the Ca2+ influx component were also investigated.
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METHODS |
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Cell isolation. Human antra were collected from 14 multiple-organ donors in collaboration with the British Columbia Transplant Society, with ethical permission from the University of British Columbia Clinical Screening Committee. There were nine male and five female donors, having an average age of 30 years. The vascular supply was perfused in situ with chilled University of Wisconsin buffer (osmolality of 355 mosmol/kgH2O and pH of 7.0) and cooled externally with crushed ice to minimize warm ischemic time.
The antral mucosa was digested in buffered collagenase and cells separated according to size by counterflow centrifugation as previously described (6). The cell fraction containing the majority of gastrin-immunoreactive (IR) cells was collected at a flow rate of 40 ml/min and a centrifuge speed of 2,100 rpm. The cells were resuspended at 1 × 106 cells/ml in growth medium [50:50 DMEM (GIBCO, Grand Island, NY) and nutrient Ham's F-10 mixture (Sigma) to give a final Ca2+ concentration of 1.0 mM] with 5 mM D-glucose. The medium was supplemented with 50 µg/ml gentamicin, 2 mM L-glutamine, 1 µg/ml hydrocortisone, 8 µg/ml insulin, and 5% heat-inactivated FCS (GIBCO). For the gastrin-release studies, cells were seeded at 0.5 ml/well on 48-well Costar plates (Costar Data Packaging, Cambridge, MA); for the Ca2+-imaging experiments, 1 ml of the cell suspension was added to 3-aminopropyltriethoxysilane (APES)-coated grid-etched coverglass (Bellco Biotechnology, Vineland, NJ) in 12-well plates. We were unable to locate a source of plastic grid-etched coverslips; therefore, the APES coating was used to alter the electrical charge of the glass to allow attachment of the cells. In neither case was an attachment matrix such as collagen utilized. There was no discernible difference in the attachment of the cells or the enrichment of the G cells. The G cell enrichment of the antral cultures varied from 20 to 40%, with the majority of the remaining cells being gastric mucous cells and with 1-4% being somatostatin-containing D cells.RT-PCR. After 48 h in culture, mRNA was isolated from the antral cells (micro mRNA purification kit; Pharmacia Biotechnology, Baie d'Urfe, PQ, Canada). Random hexamer-primed first-strand cDNA was prepared from 300 ng of mRNA per reaction. After reverse transcription, PCR amplification was performed using oligonucleotide primers previously designed to detect the presence of GRPR, NMBR, and BRS-3 in human bronchial epithelial cells (8). The sequences for the forward and reverse primers were as follows: 5'-CTCCCCGTGAACGATGACTGG-3' (nucleotides 480-500) and 5'-ATCTTCATCAGGGCATGGGAG-3' (nucleotides 848-868), respectively, for GRPR; 5'-CGAGGGGTGGGAAAGGGATTT-3' (nucleotides 205-225) and 5'-CTTCACACAGGTCCGCAGCAA-3' (nucleotides 608-628), respectively, for NMBR; and 5'-GGCTCAAAGGCAGCCTCACT-3' (nucleotides 152-172) and 5'-AGTCTTCAGGATGGCATTGG-3' (nucleotides 622-642), respectively, for BRS-3. To increase reaction stringency, the "touchdown" method of PCR was used (9). The initial annealing temperature (66°C) was dropped to 56°C for standard PCR cycling. PCR products were resolved by electrophoresis in a 1.2% agarose gel.
To verify the authenticity of the GRPR and BRS-3 products, restriction digest enzymes were chosen based on the restriction maps obtained using PC-gene software (Oxford Molecular Group, Beaverton, OR). In the case of the GRPR product, this was digested with Kpn I and BamH I (BRL Products, Burlington, ON, Canada) restriction enzymes.Single cell microfluorometry. Antral cells, plated onto APES-coated, etched-grid coverglass (Bellco), were loaded with 5 µM of the Ca2+ fluorophore fura 2-AM (Molecular Probes, Eugene, OR), by a 15-min incubation at 37°C in 1 ml of DMEM. All experiments were carried out at 20°C using an Na+-rich balanced salt solution as the standard extracellular medium, containing (in mM) 137 NaCl, 5.4 KCl, 0.5 CaCl2, 0.8 MgSO4, 0.3 Na2HPO4, 0.4 KH2PO4, 4.2 NaHCO3, 10 HEPES, 5 D-glucose, and 0.1% BSA. The pH was adjusted to 7.2 with NaOH. The osmolality of all solutions was set at 295 ± 5 mosmol/kgH2O. At the start of each experiment, the grid coordinates of the cell cluster under investigation were noted, and a digitized image of the cells was collected both with and without the overlying regions of interest boxes (Attofluor digital fluorescence microscopy system; Atto Instruments, Rockville, MD). All records have been adjusted for background fluorescence, which is the fluorescence of cell-free coverglass.
At completion of the experiment, the coverglass was removed, fixed in Bouin's solution for 10 min, washed with PBS and 0.1% Triton X-100, and incubated overnight with a monoclonal anti-gastrin antibody (9303, provided by CURE: Gastroenteric Biology Center Antibody/RIA Core, National Institutes of Health Grant DK-41301). After overnight incubation at 4°C, the coverglass was washed with PBS and bound antibodies were localized using biotin-labeled mouse IgG (Jackson Laboratories) at a final dilution of 1:1,000 for 1 h at room temperature. After being washed three times in PBS, the coverglass was incubated in avidin-biotin peroxidase at 1:1,000 (ABC kit; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. The peroxidase reaction was developed in a solution of 25 mg diaminobenzidene, in 10 ml 0.5 M Tris buffer at pH 7.5, with the addition of 0.01% H2O2. After the correct grid coordinates were located, the regions of interest boxes matching gastrin-IR cells were determined, and the relevant ratio data were retrieved for analysis. With the use of data collected from identified gastrin cells, the peak increase in intracellular Ca2+ levels was calculated as the difference in ratio measurements from the preceding basal level in the absence of agonist to the peak of the response. For the bombesin concentration response data, only cells that were stimulated in the order lowest to highest and receiving the full range of bombesin were included in the calculation. The plateau levels of the Ca2+ response were calculated as the difference in ratio measurements between the basal preceding application of the agonist and the sustained phase of the response. The effect of antagonists on the plateau level was calculated as the difference between the preceding basal level and the plateau level observed in the presence of the antagonists. The plateau level obtained in the absence of antagonist was then compared with the level in the presence of antagonists. In both cases, the data are expressed as means ± SE.Release experiments.
Adherent cells were washed twice with release medium (F-10, with 0.5 mM
Ca2+, 4.4 mM glucose, and 0.1%
BSA) to remove dead cells and debris. The osmolality of the release
media was set at 295 ± 5 mosmol/kgH2O. Cells were incubated
in release medium with bombesin over the concentration range of 10 pM
to 10 nM for 1 h at 37°C. To determine the effect of removing
extracellular Ca2+ (release medium
plus 2 mM EGTA), 1 µM nitrendipine, 20 mM caffeine, and 1 µM
ryanodine on bombesin-stimulated gastrin release cells were
preincubated with the medium/drugs for 15 min and washed and fresh
medium containing the drug was added before bombesin for the 1-h
release period. In addition, the effect of a concentration range of ryanodine and caffeine on basal gastrin release was
determined. After 60 min at 37°C, the medium was collected and
centrifuged to remove particulate matter. The supernatants were stored
at 20°C until gastrin RIA was performed (6). Cells from
control wells were detached in 1 ml distilled water, boiled for 10 min, and centrifuged to remove cell debris: the resultant supernatant was
stored at
20°C before RIA. The data were calculated as a percentage of total cell content (TCC) and presented as means ± SE.
Statistical significance was determined by an unpaired Student's t-test; values < 0.05 were
considered significant. The n values refer to the number of individual donors.
Immunocytochemistry. At the completion of the release studies, cells were fixed in 4% paraformaldehyde for 15 min at room temperature and washed in PBS to remove excess fixative. To examine the effect of removing extracellular Ca2+, control cells and cells after addition of 2 mM EGTA or after treatment with 20 mM caffeine or 1 µM nitrendipine for 1 h were incubated in a polyclonal rabbit anti-gastrin serum (Dako, Copenhagen, Denmark) at a dilution of 1:4,000 for 18 h at 4°C. After being washed in PBS, the bound antibodies were localized using Cy3 conjugated goat anti-rabbit IgG (Jackson Laboratories).
The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) subtype found to be on the plasma membrane of cells was the IP3R3 (15); therefore, a monoclonal antibody to the IP3R3 (Transduction Laboratories, Lexington, KY) at a dilution of 1:500 for 48 h at 4°C was used to determine the localization in gastrin cells. After being washed in PBS, the bound antibodies were localized using ALEXA 568 conjugated goat anti-mouse IgG (Molecular Probes) at a dilution of 1:2,000 for 1 h at room temperature. The immunostained cells were subsequently incubated in the rabbit anti-gastrin antibody as above, and the gastrin cells were identified using ALEXA 488 conjugated goat anti-rabbit IgG at a dilution of 1:2,000 for 1 h at room temperature. Finally, cells were incubated in a monoclonal antibody to ryanodine receptors (RyR) that detects both RyR1 and RyR2 (Affinity BioReagents, Denver, CO) at dilutions ranging from 1:50 to 1:1,000 for 48 h at 4°C, followed by an incubation in ALEXA 568 conjugated goat anti-mouse IgG at 1:1,000 for 1 h at room temperature. Immunostained cells were imaged using a Bio-Rad MRC 600 confocal microscope at ×60 magnification with a z-plane step size of 300 nm. The resultant maximum intensity images from the collected image stacks were prepared using National Institutes of Health Image software, and Fig. 12 was produced using Adobe Photoshop. ![]() |
RESULTS |
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RT-PCR.
RT-PCR of mRNA isolated from human antrum yielded DNA fragments of
~388 and 490 bp corresponding to GRPR and BRS-3, respectively (Fig. 1). There was no evidence
for the presence of the NMBR, although the receptor was
present in the control human cortex sample. The identification of GRPR
and BRS-3 was confirmed by restriction digest analysis. Digestion of
the 388-bp GRPR product with Kpn I
yielded the expected products of 221 and 167 bp and digestion with
BamH I yielded a product of 357 bp.
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Ca2+ imaging
studies.
Bombesin (1 nM) increased cytosolic
Ca2+ in >90% of cells
exhibiting gastrin immunoreactivity. To establish if gastrin cells were
the only cell type responding to bombesin in the antral cultures, the
ratio data from 396 regions of interest from four antral cell preparations perfused with 1 nM bombesin were examined. Of the 396 regions of interest, 23 showed a clear response to bombesin; of these,
20 were over gastrin-IR cells and 3 were over non-gastrin-IR cells,
indicating that >85% of the cells responding to bombesin were G cells.
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Gastrin release studies. The data from the intracellular Ca2+ imaging experiments indicated that bombesin stimulation resulted in two distinct Ca2+ responses: an initial release of IP3-sensitive stores followed by a sustained phase driven by the entry of extracellular Ca2+. In previous studies, we had failed to demonstrate that bombesin-stimulated gastrin release required the presence of extracellular Ca2+. The present experiments were designed to determine if elimination of concurrent stimulation of the CaR changed the sensitivity of bombesin-stimulated gastrin release to extracellular Ca2+ levels.
The first set of experiments was designed to determine whether removal of extracellular Ca2+ (release medium with 2 mM EGTA) or addition of 1 µM nitrendipine altered bombesin-stimulated gastrin release. In the cultures used for these experiments, the TCC of gastrin was 8,400 ± 500 pg/well (n = 4) with a basal release of 0.8 ± 0.2% TCC. Addition of bombesin resulted in a concentration-dependent increase in gastrin release that was reduced by up to 35% in the absence of extracellular Ca2+ (Fig. 9). However, bombesin-stimulated gastrin release remained significantly higher than basal levels in the presence of EGTA. At the completion of these experiments, the cells were washed and incubated in trypan blue to assess viability. Although there was no significant change to cell viability, the morphology of the cultured cells was significantly altered, the usual cell clusters had dissaggregated, and the majority of these cells were rounded rather than polarized.
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Immunocytochemistry.
A comparison of gastrin-IR cells from control and wells incubated in 2 mM EGTA demonstrated that, after 1 h, G cells in the absence of
extracellular Ca2+ were still
attached to the wells, but dissociation of the cell clusters resulted
in a loss of cellular polarity (Fig. 11,
A and B). Similar changes were not seen in
cells treated with either 1 µM nitrendipine or 20 mM caffeine (data
not shown).
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DISCUSSION |
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This study demonstrated that, of the three known BRSs, only GRPR and BRS-3 were expressed in the human antral epithelial cell culture preparation. Although synthetic analogs of bombesin act as high-affinity ligands for BRS-3, the orphan receptor is unresponsive to bombesin (27); therefore, the active bombesin receptor in the human cell preparation was the GRPR. This fact coupled with the data showing that the overwhelming majority of cells responding to bombesin in the preparation (> 85%) were gastrin-IR provides strong evidence that human antral G cells express the GRPR, and it is activation of these receptors that mediates bombesin-stimulated gastrin release.
Previous studies examining the mechanism of bombesin-stimulated gastrin secretion in human G cells utilized cells cultured and stimulated in a medium containing a relatively high extracellular Ca2+ concentration (1.8 mM). After the recent localization of the CaR on human G cells (16), the culture conditions in the current study were modified to prevent activation of this receptor, which could result in alterations to intracellular signaling pathways. Cells were cultured in medium containing 1 mM Ca2+, just below the threshold for activation of the CaR. Attempts to decrease the Ca2+ concentration in the culture medium further to 0.5 mM resulted in a loss of cell viability. However, 0.5 mM Ca2+ was the concentration used for the release studies to ensure that the CaR was inactivated.
Altering the culture conditions did not alter the concentration response curve for bombesin; however, both the removal of extracellular Ca2+ and addition of nitrendipine decreased bombesin-stimulated gastrin release, effects that were not observed previously. The experiments utilizing EGTA to remove any extracellular Ca2+ from the release medium were designed to examine the role of entry of extracellular Ca2+ in the gastrin response. However, it is likely that the presence of EGTA over the 1-h period would have also decreased intracellular Ca2+ levels, which would in turn have reduced gastrin release. In addition, incubation of the cells with 2 mM EGTA for 1 h resulted in a significant alteration in the morphology of the cultured cells. The cell clusters disintegrated, and, although the single cells did not detach from the plastic and remained viable, their polarity was significantly reduced. Thus the observed 35% reduction in the response to bombesin in the absence of extracellular Ca2+ could be due to a combination of the changes in cell-cell coupling and reduced intracellular Ca2+ levels rather than a consequence of the lack of Ca2+ influx.
The ability of nitrendipine to inhibit 15% of bombesin-stimulated gastrin release at concentrations >1 nM indicated that Ca2+ influx through L-type VDCC played a minor role in the response to bombesin. In the previous studies (4) with 3.5× higher extracellular Ca2+ levels, the effect of nitrendipine on bombesin-stimulated gastrin release was not significant, although activation of these channels has been shown to be essential for epinephrine-stimulated gastrin release (17). These data indicated that the concurrent stimulation of the CaR on the human G cells in the initial experiments altered both the intracellular Ca2+ signaling events and the Ca2+ sensitivity of the secretory response to bombesin.
The CaR has been shown to activate a number of different signaling pathways, depending on the cell type involved, including activation of phospholipase C and both stimulation and inhibition of adenylate cyclase (7). In antral G cells, the CaR activates a nonselective cation channel (NSCC) that leads to Ca2+ influx as well as activation of L-type VDCC (16). In the previous experiments with 1.8 mM extracellular Ca2+, entry of Ca2+ through the NSCC would have obscured any effect mediated by entry via L-type VDCC; even in the present study, the effect of nitrendipine on bombesin-stimulated gastrin release, although significant, was relatively small.
The data from the Ca2+ imaging
experiments clearly demonstrated that at low concentrations (1 nM)
bombesin evoked a Ca2+ response
characterized by a single, rapid transient increase in
[Ca2+]i,
which was unaffected by the removal of extracellular
Ca2+ or addition of nitrendipine,
indicating intracellular Ca2+
mobilization. These studies, in contrast to the data obtained from the
gastrin secretion experiments, failed to consistently demonstrate
an effect of bombesin at concentrations <1 nM. The most probable
explanations for this discrepancy are the shorter time scale of the
Ca2+ imaging experiments and the
fact that these were conducted at 20°C rather than 37°C, which
would reduce the responsiveness of the cells to low levels of agonists.
To confirm the previous suggestion that the refractory response to repetitive bombesin stimulation was due to complete depletion of Ca2+ from intracellular Ca2+ stores, we examined the effect of thapsigargin following bombesin pretreatment in the absence of extracellular Ca2+. Thapsigargin selectively inhibits Ca2+-ATPase activity on IP3-sensitive Ca2+ stores and allows any stored Ca2+ to leak back into the cytosol (12). As expected, addition of thapsigargin failed to evoke a further change in [Ca2+]i after the initial bombesin response, indicating full depletion of the IP3-sensitive stores.
In our earlier study, changes in [Ca2+]i were refractory to repeated bombesin stimulation when the intervening recovery period was limited to 5 min (4). In a recent study using a similar preparation of canine G cells (19), it was suggested that this refractory period was due to recycling of internalized receptors to the plasma membrane, as a second and third full [Ca2+]i response could be evoked by bombesin after a 30-min recovery period. In the present experiments, if low bombesin concentrations were administered to the human G cells for >2 min, subsequent changes in [Ca2+]i were obtained with minimal latency and without store replenishment. These data indicated that GRPRs remained localized to the plasma membrane and that the decline in subsequent responses was the result of depletion of intracellular Ca2+ stores rather than internalization of the receptors. Consistent with this explanation, bombesin-evoked Ca2+ responses declined as intracellular stores were depleted but were regained following intracellular store replenishment by the administration of a second agonist that directly promoted the influx of Ca2+ (in this instance, 3.6 mM Ca2+). The data obtained in the present study indicated that in human G cells the refractory period between responses to low bombesin concentrations represented the interval required for the refilling of intracellular Ca2+ stores following bombesin-evoked store depletion.
At higher concentrations of bombesin (1 nmol/l), a sustained elevated
plateau of
[Ca2+]i
was observed following the initial signaling event. Removal of
extracellular Ca2+ and addition of
20 mM caffeine completely reversed this, whereas addition of
nitrendipine partially inhibited the second phase, indicating that the
response was maintained by Ca2+
influx, through both IP3R and
L-type VDCCs. Recent evidence from other cell systems suggests that
depletion of intracellular Ca2+
stores activates Ca2+-conducting
cation currents (11, 26). This store-dependent "capacitative"
Ca2+ influx may be mediated via
IP3Rs located on the plasma
membrane, specifically IP3R3 (2,
15). In the present study, low bombesin concentrations were incapable
of generating an influx component probably due to the generation of
insufficient IP3 to mobilize capacitative Ca2+ influx. At
higher bombesin concentrations,
IP3 generation would be increased,
thus activating IP3-dependent
Ca2+ entry across the plasma
membrane. The suggested entry of extracellular Ca2+ through
IP3R was supported by the finding
of IP3R3 immunoreactivity on the
plasma membrane of G cells. In this situation, the majority of the
Ca2+ influx would occur via
capacitative entry, with a minor component due to
nitrendipine-sensitive L-type VDCC opened due to the depolarizing charge associated with the influx of cations. The data from the intracellular Ca2+ imaging
experiments, which demonstrated that removal of extracellular Ca2+ and caffeine perfusion
completely eliminated the sustained plateau phase but nitrendipine only
partially reversed the influx component, are consistent with this suggestion.
Interestingly, although it would be expected that the influx component should be sufficient to refill intracellular Ca2+ stores, thus allowing repetitive responses to 10 nM bombesin, this was not observed possibly because of the increased IP3 levels blocking store refilling. An alternative explanation would be internalization or desensitization of the receptors by high bombesin concentrations such that activation of intracellular signaling systems was prevented. The shorter latency (5 min compared with 30 min in canine cells) observed in the human cell preparation suggests that desensitization rather than receptor recycling was occurring.
In neuronal cultures, repetitive Ca2+ signaling events can be evoked following repeated administration of bombesin, although the initial transient rise in [Ca2+]i decreased with successive applications of bombesin, a finding consistent with the gradual depletion of cytosolic stores (20). Interestingly, the amplitude of the sustained second phase was unaffected and was maintained for extended periods following the removal of the agonist. In the context of the present study, these results would be consistent with the mobilization of intracellular IP3-sensitive Ca2+ stores being responsible for the first phase of the response and that these were exhausted in a manner similar to those in the G cells. However, repetitive receptor stimulation of the neuronal cells was still capable of initiating Ca2+ influx, indicating that capacitative Ca2+ entry through IP3-gated channels was not involved in the influx component in the neuronal cells.
High concentrations (mM) of caffeine are known to rapidly and reversibly inhibit the release of Ca2+ from IP3-gated cytoplasmic stores (3, 23). The concentration of caffeine used (20 mM) was chosen because this had previously been shown to completely block inositol phosphate production in smooth muscle cells stimulated with carbachol (14). Both the latter studies and studies of pancreatic acinar cells (5) demonstrated that the effects of caffeine on intracellular Ca2+ levels and inositol phosphate generation could be mimicked by increasing intracellular levels of cAMP alone (smooth muscle cells) or both cAMP and cGMP (acinar cells). These data suggest that caffeine may not directly inhibit phospholipase activity but may act indirectly by inhibiting phosphodiesterase activity to increase cyclic nucleotide concentrations that in turn inactivate phospholipase. The effects of caffeine on intracellular Ca2+ levels are rapidly reversible; caffeine levels are below levels of detection within seconds of removal of the drug (23). Unfortunately, the only other way of inhibiting IP3Rs would be to use heparin, which is cell impermeant and would have to be microinjected into the G cells. This is not possible with our present cell preparation because we have no way of determining (before fixation and immunostaining) which cell is a G cell. In the present study, preapplication of 20 mM caffeine prevented the initial increase in [Ca2+]i after bombesin stimulation, supporting the proposed role of IP3-gated Ca2+ stores. In the continued presence of caffeine, no increase in [Ca2+]i was observed; however, this was regained immediately after washout of caffeine, indicating that reactivation of phosphodiesterase rapidly decreased the levels of cyclic nucleotides, thus removing the inhibition of inositol phosphate production.
At low concentrations (µM), caffeine activates a second ryanodine-gated intracellular Ca2+ store. The ability of 20 mM caffeine to increase both basal and bombesin-stimulated gastrin release suggested that either RyR may be expressed in addition to the IP3-sensitive stores or that the increase in cAMP due to inhibition of phosphodiesterase (14) was increasing gastrin release.
Three different sets of experiments were undertaken to determine if the G cells expressed RyR. The Ca2+ imaging studies failed to show an increase in [Ca2+]i in response to ryanodine perifusion, gastrin release from the cultured cells was insensitive to a range of ryanodine concentrations, and finally immunocytochemistry with an antibody to RyR1 and -2 failed to demonstrate any positive immunostaining. The lack of RyR in the G cells would be consistent with the proposal that 20 mM caffeine increased intracellular cAMP levels, leading to the increased gastrin release. The fact that addition of the phosphodiesterase inhibitor, IBMX, to the gastrin cell cultures also increased both basal and bombesin-stimulated gastrin release in a manner similar to 20 mM caffeine would support the latter suggestion.
In conclusion, RT-PCR indicated the presence of GRPR and BRS-3 in the human antral cell culture preparation. Single cell microfluorometry, demonstrating that the majority of cells exhibiting bombesin-evoked changes in intracellular Ca2+ were gastrin immunoreactive, indicated that antral G cells express the GRPR subtype. Changes in [Ca2+]i were observed immediately following bombesin receptor activation and represented mobilization of IP3-sensitive Ca2+ stores. Low bombesin concentrations depleted IP3-sensitive Ca2+ stores, and, without store replenishment, the cells required a recovery period to obtain repetitive [Ca2+]i responses. High bombesin concentrations recruit both capacitative Ca2+ entry through IP3R3 and activate L-type VDCC, resulting in Ca2+ influx and a sustained plateau level of increased [Ca2+]i. Although the entry of extracellular Ca2+ through L-type VDCCs played a minor role in bombesin-stimulated gastrin release, the contribution of the mobilization of intracellular Ca2+ stores and entry through the capacitative Ca2+ channels could not be determined due the effects of caffeine on phosphodiesterase activity.
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ACKNOWLEDGEMENTS |
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The technical assistance of Dolar Sidpra is gratefully acknowledged.
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FOOTNOTES |
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This study was supported by a Medical Research Council grant to A. M. J. Buchan.
P. E. Squires, now working at King's College (London), was a Killam Postdoctoral Research Fellow.
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. §1734 solely to indicate this fact.
Address for reprint requests: A. M. J. Buchan, Dept. of Physiology, Faculty of Medicine, 2146 Health Sciences Mall, Univ. of British Columbia, Vancouver, BC, Canada V6T 1Z3.
Received 16 June 1998; accepted in final form 14 September 1998.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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1.
Barreras, R. F.,
and
R. M. Donaldson, Jr.
Calcium stimulation of gastrin and gastric acid secretion: effect of small doses of calcium carbonate.
Gut
18:
442-448,
1967[Abstract].
2.
Bennett, D. L.,
M. D. Bootman,
M. J. Berridge,
and
T. R. Cheek.
Ca2+ entry into PC12 cells initiated by ryanodine receptors or inositol 1,4,5-trisphosphate receptors.
Biochem. J.
329:
349-357,
1998[Medline].
3.
Berridge, M. J.
Caffeine inhibits inositol trisphosphate-induced membrane potential oscillations in Xenopus oocytes.
Proc. R. Soc. Lond. B Biol. Sci.
244:
57-62,
1991[Medline].
4.
Buchan, A. M. J.,
and
R. M. Meloche.
Signal transduction events involved in bombesin-stimulated gastrin release from human G cells in culture.
Can. J. Physiol. Pharmacol.
72:
1060-1065,
1994[Medline].
5.
Camello, P. J.,
O. H. Petersen,
and
E. C. Toescu.
Simultaneous presence of cAMP and cGMP exert a coordinated inhibitory effect on the agonist evoked Ca2+ signal in pancreatic acinar cells.
Eur. J. Physiol.
432:
775-781,
1996[Medline].
6.
Campos, R. V.,
A. M. J. Buchan,
R. M. Meloche,
R. A. Pederson,
Y. N. Kwok,
and
D. H. Coy.
Gastrin secretion from human antral G cells in culture.
Gastroenterology
99:
36-44,
1990[Medline].
7.
Chattopadhyay, N.,
A. Mithal,
and
E. M. Brown.
The calcium-sensing receptor: a window into the physiology and pathophysiology of mineral ion metabolism.
Endocr. Rev.
17:
289-307,
1996[Abstract].
8.
DeMichele, M. A. A.,
A. L. Gaither,
L. Davis,
J. D. Hunt,
R. J. Landreneau,
and
J. M. Siegfried.
Expression of mRNA for three bombesin receptor subtypes in human bronchial epithelial cells.
Am. J. Respir. Cell Mol. Biol.
11:
66-74,
1994[Abstract].
9.
Don, R. H.,
P. T. Cox,
B. J. Wainwright,
K. Baker,
and
J. C. Mattic.
Touchdown PCR to circumvent spurious priming during gene amplification.
Nucleic Acids Res.
19:
4008-4012,
1991[Medline].
10.
Fathi, Z.,
M. H. Corjay,
H. Shapira,
E. Wada,
R. Benya,
R. Jensen,
J. Viallet,
E. A. Sausville,
and
J. F. Battey.
A novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells.
J. Biol. Chem.
268:
5979-5984,
1993
11.
Krause, E.,
F. Pfeiffer,
A. Schmid,
and
I. Schulz.
Depletion of intracellular stores activates a calcium conducting nonselective cation current in mouse pancreatic acinar cells.
J. Biol. Chem.
271:
32523-32528,
1996
12.
Lytton, J.,
M. Westlin,
and
M. R. Hanley.
Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.
J. Biol. Chem.
266:
17067-17071,
1991
13.
Nagalla, S. R.,
B. J. Barry,
K. C. Creswick,
P. Eden,
J. T. Taylor,
and
E. R. Spindel.
Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4).
Proc. Natl. Acad. Sci. USA
92:
6205-6209,
1995
14.
Prestwich, S. A.,
and
T. B. Bolton.
Inhibition of muscarinic receptor-induced inosital phospholipid hydrolysis by caffeine, -adrenoceptors and protein kinase C in intestinal smooth muscle.
Br. J. Pharmacol.
114:
602-611,
1995[Abstract].
15.
Putney, J. W.
Type 3 inositol 1,4,5-trisphosphate receptor and capacitative calcium entry.
Cell Calcium
21:
257-261,
1997[Medline].
16.
Ray, J. M.,
P. E. Squires,
S. B. Curtis,
R. M. Meloche,
and
A. M. J. Buchan.
Expression of the calcium-sensing receptor on human antral gastrin cells in culture.
J. Clin. Invest.
99:
2328-2333,
1997
17.
Ray, J. M.,
P. E. Squires,
R. M. Meloche,
D. W. Nelson,
T. P. Snutch,
and
A. M. J. Buchan.
L-type calcium channels regulate gastrin release from human antral G cells.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G281-G288,
1997
18.
Reeder, D. D.,
B. M. Jackson,
J. Ban,
B. G. Clendinnen,
W. D. Davidson,
and
J. C. Thompson.
Influence of hypercalcemia on gastric secretion and serum concentration in man.
Ann. Surg.
172:
540-546,
1970[Medline].
19.
Seensalu, R.,
D. Avedian,
R. Barbuti,
M. Song,
L. Slice,
and
J. H. Walsh.
Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not protein kinase C, and is enhanced by disruption of Rho/cytoskeletal pathways.
J. Clin. Invest.
100:
1037-1046,
1997
20.
Simeone, D. M.,
C. Kimball,
and
M. W. Mulholland.
Bombesin-mediated calcium fluxes in myenteric plexus neurons.
Peptides
16:
1307-1311,
1995[Medline].
21.
Spindel, E. R.,
W. W. Chin,
J. Price,
L. H. Rees,
G. M. Besse,
and
J. F. Habener.
Cloning and characterization of cDNAs encoding human gastrin-releasing peptide.
Proc. Natl. Acad. Sci. USA
81:
5699-5703,
1984[Abstract].
22.
Sugano, K.,
J. Park,
A. H. Soll,
and
T. Yamada.
Stimulation of gastrin release by bombesin and canine gastrin-releasing peptides. Studies with isolated canine G-cells in primary culture.
J. Clin. Invest.
79:
935-942,
1987[Medline].
23.
Toescu, E. C.,
S. C. O'Neill,
O. H. Petersen,
and
D. A. Eisner.
Caffeine inhibits the agonist-evoked cytosolic Ca2+ signal in mouse pancreatic acinar cells by blocking inositol trisphosphate production.
J. Biol. Chem.
267:
23467-23470,
1992
24.
Wada, E.,
J. Way,
H. Shapira,
K. Kusano,
A. M. Lebacq-Verheyden,
D. H. Coy,
D. Jensen,
and
J. Batty.
cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor.
Neuron
6:
421-430,
1991[Medline].
25.
Walsh, J. H.,
M. Bouzyk,
and
E. Rozengurt.
Homologous desensitization of bombesin-induced increases in intracellular calcium in quiescent Swiss 3T3 cells involves a protein kinase C-independent mechanism.
J. Cell. Physiol.
156:
333-340,
1993[Medline].
26.
Wayman, C. P.,
A. Gibson,
and
I. McFadzean.
Depletion of either ryanodine- or IP3-sensitive calcium stores activates capacitative calcium entry in mouse anococcygeus smooth muscle cells.
Pflügers Arch.
435:
231-239,
1998[Medline].
27.
Wu, J. M.,
D. E. Nitecki,
S. Biancalana,
and
R. I. Feldman.
Discovery of high affinity bombesin receptor subtype 3 agonist.
Mol. Pharmacol.
50:
1355-1363,
1996[Abstract].