Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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We recently isolated and characterized 86-amino
acid CCK-releasing peptide from porcine intestinal mucosa. The sequence
of this peptide is identical to that of porcine diazepam-binding inhibitor (DBI). Intraduodenal administration of DBI stimulates the CCK
release and elicits pancreatic secretion in rats. In this study we
utilized a murine tumor cell line (STC-1 cells) that contains CCK to
examine if DBI directly acts on these cells to stimulate CCK release.
We investigated the cellular mechanisms responsible for this action. We
showed that DBI33-50, a biologically active fragment of
DBI1-86, significantly
stimulated CCK secretion in STC-1 cells. This action was abolished by
Ca2+-free medium. The mean basal
intracellular Ca2+ concentration
([Ca2+]i)
was 52 nM in fura 2-loaded STC-1 cells.
DBI33-50 (1-1,000 nM)
elicited Ca2+ oscillations;
DBI33-50 (10 nM) increased
the oscillation frequency to 5 cycles/10 min and elicited a net
[Ca2+]i
increase (peak basal) to 157 nM. In contrast, bombesin and forskolin caused an initial transient
[Ca2+]i
followed by a small sustained
[Ca2+]i
plateau. Withdrawal of extracellular
Ca2+ abolished
Ca2+ oscillations stimulated by
DBI33-50. L-type
Ca2+ channel blockers nifedipine
and diltiazem (3-10 µM) markedly attenuated DBI-stimulated
Ca2+ oscillations. In other cell
types L-type Ca2+ channels are
activated by cAMP-protein kinase A. DBI33-50 failed to stimulate
cAMP formation in STC-1 cells. Similarly, DBI33-50 had no effect on
myo-inositol 1,4,5-trisphosphate concentration
([IP3]), whereas
bombesin caused an eightfold increase in
[IP3] over basal. In
addition, inhibitors of phospholipase C (U-73122), phospholipase
A2 (ONO-RS-082), and protein
tyrosine kinase (genistein) did not alter the
Ca2+ oscillations elicited by
DBI33-50. It appears that
DBI33-50 acts directly on
STC-1 cells to elicit Ca2+
oscillations via the voltage-dependent L-type
Ca2+ channels, resulting in the
secretion of CCK. Mediation of this action is by intracellular
mechanisms independent of the traditional signal transduction pathways,
including phospholipase C, phospholipase A2, protein tyrosine kinase, and
cAMP systems.
cholecystokinin-releasing peptide; calcium signal transduction; porcine intestine; stimulus-secretion coupling
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INTRODUCTION |
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CCK IS A GASTROINTESTINAL hormone that plays a key role in the digestion and assimilation of nutrients. It regulates several important gastrointestinal functions, such as pancreatic enzyme secretion, gallbladder contraction, gastric acid secretion, gastrointestinal motility, and cell growth (12, 20, 37, 40).
CCK is secreted from specific endocrine cells (I cells) in the proximal small intestine (38). In humans hydrolytic products of digestion, such as amino acids and fatty acids, release CCK (31). In dogs proteins do not stimulate pancreatic secretion, but crude enzyme digests of protein that contain peptides and amino acids effectively stimulate pancreatic enzyme secretion, presumably by releasing CCK (23, 35). Undigested fat is ineffective, but products of lipolysis (e.g., fatty acids) are potent stimulants of CCK release in dogs (34). In contrast, proteins but not amino acids, carbohydrates, or fats stimulate CCK secretion in rats (29).
The mechanism by which nutrients stimulate CCK release is not clear. Several CCK-releasing peptides that are secreted luminally have been isolated. These peptides may be intraluminal regulators of hormone release in the intestine and include luminal CCK- releasing factor (28, 36, 44), CCK monitor peptide (4, 21, 28, 54), and diazepam-binding inhibitor (DBI) (18). We have shown that DBI is involved in mediating feedback regulation of pancreatic enzyme secretion and postprandial release of CCK in rats (18, 30). DBI is a peptide that has 86 amino acids and a molecular mass of 9 kDa (8). It was originally isolated from the rat brain and subsequently found in various peripheral organs, including the duodenum (7, 14). We recently reported that intraduodenal administration of DBI stimulates CCK release and pancreatic protein secretion in rats (18). Furthermore, we demonstrated that diversion of bile pancreatic juice for 2 h causes a parallel increase in plasma CCK levels and luminal DBI immunoreactivity. Intraduodenal administration of DBI antisera, but not preimmune rabbit sera, completely abolishes an increase in plasma CCK levels after diversion of bile pancreatic juice or stimulation by intraduodenal administration of 5% peptone (26, 27). These observations suggest that DBI plays an important physiological role in mediating CCK release.
Porcine DBI1-86 has several tryptic and chymotryptic cleavage sites (8). A trypsin-digested porcine DBI fragment, DBI33-50 (Gln-Ala-Thr-Val-Gly-Asp-Ile-Asn-Thr-Glu-Arg-Pro-Gly-Ile-Leu-Asp-Leu-Lys), was also found to stimulate CCK release and pancreatic secretion in a dose-dependent manner, although it was 100 times less potent than the whole peptide (18). Trypsin cleaves internal bonds at lysine or arginine residues and hence DBI33-50 is still sensitive to trypsin as it contains Arg-Pro. It is conceivable that the DBI fragment may still participate in the mediation of feedback regulation of CCK release. To determine if DBI acts directly to stimulate CCK release and to investigate the cellular mechanism, we studied the action of DBI33-50 using a murine tumor cell line (STC-1) that contains CCK (41).
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METHODS |
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Materials.
Porcine DBI33-50 was
synthesized by the University of Michigan Peptide Research Center (Ann
Arbor, MI). Anti-CCK octapeptide (sulfated CCK-8) antiserum OAL-656,
developed by immunizing New Zealand White rabbits with CCK-8-keyhole
limpet hemocyanin (KLH), was kindly provided by Otsuka Pharmaceutical
(Osaka, Japan). The following materials were purchased: sulfated CCK-8
iodinated with 125I-labeled
Bolton-Hunter (BH) reagent from Amersham (Arlington Heights, IL); goat
anti-rabbit IgG antiserum and normal rabbit serum from Peninsula
Laboratories (Belmont, CA); sulfated CCK-8, bombesin, pertussis toxin
(PTx), aprotinin, and soybean trypsin inhibitor from Sigma (St. Louis,
MO); forskolin
(7-acetoxy-1
,6
,9
-trihydroxy-8,13-epoxy-labd-14-en-11-one), 8-bromoadenosine-3',5'-cyclic monophosphate (8-BrcAMP),
U-73122 [1-6-17
-3-metyoxyestra-1,3,5,(10)-trine-17-yl-amino-hexyl-1H-pyrrole-2,5-diione], ONO-RS-082
[2-(p-amylcinnamoyl)amino-4-chlorobenzoic
acid], and genistein (4',5,7-trihydroxyisoflavone) from
Biomol (Plymouth Meeting, PA). Fura 2-AM
[1,2-(5'-carboxyoxazol-2'-yl)6-aminobenzofuran-5-oxy-2-(2'-amino-5'-methyl-phenoxy) ethane-N,N,N',N'tetraacetic
acid pentaacetoxymethyl ester], nifedipine, IBMX, and caffeine
were from Calbiochem-Novabiochem (San Diego, CA).
Cell culture. STC-1 cells derived from intestinal endocrine tumor cell lines developed in murine carrying the transgene for the rat insulin promotor linked to the simian virus 40 large T antigen and the polyoma virus small T antigen (41) were kindly provided by Dr. D. Hanahan (University of California, San Francisco, CA). STC-1 cells between passage 10 and 35 were grown in high glucose (HG) DMEM, containing 15% horse serum, 2.5% fetal bovine serum, 100 kU/l penicillin, and 100 mg/l streptomycin (GIBCO BRL, Grand Island, NY), and incubated at 37°C with 95% O2-5% CO2. Studies were performed in 12-well Corning culture dishes 72 h after cells were plated. Cells were at 80-90% confluence.
CCK secretion studies.
STC-1 cells were washed three times with HG-DMEM (1 ml), removed from
plates, and incubated with 250 µl of physiological salt solution
(PSS) in an Eppendorf tube with or without secretagogues for various
time intervals at 37°C. PSS contained (in mM) 137 NaCl, 4.7 KCl,
0.56 MgCl2, 1.28 CaCl2, 1.0 NaH2PO4, 10.0 HEPES-NaOH, MEM with essential
amino acids (GIBCO BRL) neutralized with NaOH, 2.0 L-glutamine, and 5.5 D-glucose. PSS was adjusted to
pH 7.4 and equilibrated with 100%
O2. After incubation cell
suspensions were centrifuged at 10,000 rpm for 40 s at 4°C, and the
resultant supernatants were transferred to another Eppendorf tube. An
equal volume of chilled 99.9% ethanol was added to each supernatant, and the mixture was vortexed. The aqueous sample was evaporated at
55°C under the nitrogen stream for 30 min to completely remove the
ethanol fraction. CCK levels were measured by RIA, utilizing 125I-BH-CCK-8 and a rabbit
anti-CCK-8 (sulfated) antiserum, OAL-656, generated in New Zealand
White rabbits by immunization with CCK-8 peptide conjugate coupled with
KLH. The administration of a tolerogenic conjugate of -alanyl
tetragastrin and a copolymer of
D-glutamic acid and
D-lysine inhibited
cross-reacting antibody formation (17, 22). OAL-656 reacted with the
amino-terminal of CCK-8 but not with the nonsulfated form of CCK-8.
OAL-656 cross-reacted at 100% with sulfated CCK-8 and CCK-33, at
84.6% with CCK-39, and at less than 0.01% with gastrin I-17 (17). The
aqueous samples were evaporated and redissolved in 400 µl of 30 mM
PBS [145 mM NaCl, 6 mM
NaH2PO4,
24 mM
Na2HPO4,
25 mM EDTA, 0.1% BSA, 0.02% sodium azide, and 200 kIU/ml aprotinin,
pH 7.6], and incubated for 48 h at 4°C with
125I-BH-CCK-8 (200 µl in PBS)
and OAL-656 (200 µl in PBS; total volume 0.8 ml). Goat anti-rabbit
IgG antiserum (100 µl in PBS) and normal rabbit serum (100 µl in
PBS) were then added, and incubation was continued for 24 h at 4°C
(total volume 1 ml). Bound and free-labeled CCK were separated by
centrifugation at 3,000 rpm for 30 min at 4°C. The radioactivity of
the pellet was counted in a gamma counter. CCK levels were expressed as
picograms per well.
Measurement of [Ca2+]i. Aliquots of STC-1 cells were placed on 22-mm circular cover glasses and cultured for 72 h at 37°C. Measurement of intracellular Ca2+ concentrations ([Ca 2+]i) in single STC-1 cells was performed as described previously (51). Briefly, cells attached to the cover glass in 12-well Corning culture dishes (2 ml HG-DMEM) were loaded with 10 µM fura 2-AM at 37°C for 50 min in the dark, washed twice, and resuspended in fresh 2 ml HG-DMEM. All experiments were performed utilizing a dual excitation wavelength (340 to 380 nm emitted at 505 nm) modular fluorometer (SPEX Fluorolog 2) coupled to a Nikon Diaphot inverted microscope (×40). Cells attached to the cover glass were continuously superfused with PSS in an eight-chambered reservoir at a flow rate of 1 ml/min. Ca2+-free solution was identical to PSS except that CaCl2 was replaced with 1 mM EGTA. A fluorescence ratio was converted to [Ca2+]i according to in vitro calibration determined with an external standard (Calcium Kit II, Molecular Probes, Eugene, OR) and 50 µM fura 2 potassium salt.
Measurement of intracellular cAMP.
Intracellular cAMP levels were measured by RIA (Amersham), using
[3H]cAMP and
cAMP-binding protein (45). Cells were washed three times with HG-DMEM
(1 ml) and removed from plates. Cell suspensions (0.25 ml each) in PSS
were incubated with or without secretagogues at 37°C in an
Eppendorf tube. After 20 min, 0.5 ml ice-cold 99.9% ethanol was added
to stop the incubation. The cells were immediately centrifuged at
10,000 rpm for 30 s at 4°C. The supernatant (0.75 ml) was stocked,
and the resultant pellet was dissolved in 0.5 ml of chilled
ethanol-distilled water (2:1). The suspension was sonicated for 30 s
(200 W), vortexed for 30 s, and allowed to settle for 10 min at
4°C. The sonicates were centrifuged at 10,000 rpm for 10 min, and
the resultant supernatants (0.5 ml) were combined with the first
supernatants (total volume 1.25 ml). Each suspension was placed in an
Eppendorf tube and evaporated at 55°C under a nitrogen stream to
remove the ethanol fraction completely. Each sample was stored at
70°C overnight and redissolved in 110 µl of 0.05 M Tris-4
mM EDTA buffer (pH 7.5). Aliquots (50 µl) of each suspension
(5-10 µg protein/cell extracts) were incubated with an antiserum
specific for cAMP (100 µl) and 0.025 µCi/0.925 kBq of
[3H]cAMP (50 µl),
both of which were solubilized in distilled water. The total
volume per incubation was 200 µl. Incubation was performed for 120 min at 7°C with gentle shaking and stopped by 100 µl of chilled
charcoal. Bound and free-labeled cAMP were separated by centrifugation
(10,000 rpm) for 3 min at 4°C. Radioactivity in the supernatant
(200 µl) was counted in a liquid scintillation counter. Protein was
measured using a Bio-Rad protein assay system (Bio-Rad, Hercules,
CA). cAMP contents were expressed as pmol/100 µg of cell extracts.
Measurement of IP3. Intracellular myo-inositol 1,4,5-trisphosphate (IP3) was measured by RIA as described previously, using the [3H]IP3 assay system (Amersham) (50). In brief, STC-1 cells were washed three times with HG-DMEM (1 ml) and removed from plates. Cell suspensions (250 µl each) in PSS were incubated with or without secretagogues at 37°C for various time intervals in an Eppendorf tube. Incubation was terminated by adding ice-cold 50% TCA (62.5 µl) to obtain a final TCA concentration of 10% wt/vol. All cell suspensions were settled for 30 min at 4°C and centrifuged at 10,000 rpm for 10 min at 4°C. The resultant supernatant (250 µl) was extracted with 3 ml of H2O-saturated ether three times, gassing with nitrogen, and titrated to pH 7.5 with NaHCO3. The extract (100 µl; 120 µg total protein) was incubated with bovine adrenal IP3-binding protein (100 µl), 0.007 µCi of [3H]IP3 (100 µl in water-ethanol, 1:1), and 100 µl of 0.1 M Tris (4 mM EDTA and 4 mg/ml BSA, pH 9) for 15 min at 4°C (total volume 0.4 ml). Bound and free-labeled IP3 were separated by centrifugation at 10,000 rpm for 3 min. Radioactivity in the pellet was measured by a liquid scintillation counter. Intracellular IP3 concentration was expressed as picomoles per total milligram protein.
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RESULTS |
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DBI33-50 causes CCK secretion from
STC-1 cells.
After 120 min of incubation of STC-1 cells without secretagogues, the
basal CCK level in the medium was 10.7 ± 1.9 pg/well (n = 20). Although significant
DBI33-50-stimulated secretion of CCK occurred following 30 min of incubation (Table
1), maximum stable basal secretion was
observed at 120 min of incubation after cells were removed from plates.
We therefore selected this incubation time and expressed our results as
a percent of basal at 120 min of incubation.
DBI33-50 at 10 and 100 nM
caused significant increases in CCK secretion over basal levels (Fig.
1A).
DBI33-50 at 100 nM stimulated
a 139 ± 15% increase of basal CCK levels. Our time course studies
showed that maximal CCK secretion was observed at 120 min after
DBI33-50 stimulation (Table
1). Maximal CCK secretion occurred at 30 min for bombesin and 120 min
for forskolin, although CCK secretion in response to these secretagogues occurred at 15 min following incubation (data not shown).
The elimination of extracellular
Ca2+ concentration
([Ca2+]o;
1 mM EGTA + zero CaCl2)
abolished the action of
DBI33-50 for all doses
tested. Bombesin (10 and 100 nM) and forskolin (100 µM) also caused
significant increases in CCK secretion (Fig.
1B). [Ca2+]o
was also required for CCK secretion stimulated by either bombesin or
forskolin.
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DBI33-50 elicits
Ca2+
oscillations.
To investigate if DBI33-50
stimulates Ca2+ oscillations, we
measured
[Ca2+]i
in fura 2-loaded individual STC-1 cells. Note that some STC-1 cells
demonstrated Ca2+ oscillations in
the resting state (about 20% occurrence); these cells were not used
for measurements of
[Ca2+]i.
Basal
[Ca2+]i
was 52 ± 4 nM (54 individual cells).
DBI33-50 elicited Ca2+ oscillations at various
concentrations (Fig. 2). Stimulation with
10-1,000 nM DBI33-50
increased
[Ca2+]i
(peak
basal) and frequencies to 81-157 nM and 2.6-5
cycles/10 min, respectively (Table 2). It
appears that maximal stimulation was observed with
DBI33-50 at 10 nM. In several
cases DBI33-50 caused either
a
[Ca2+]i
transient or a sustained
[Ca2+]i
plateau (Fig. 3,
A and
C), but 80% of the cells showed
repetitive Ca2+ spikes regardless
of the DBI33-50
concentration.
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Elimination of [Ca2+]o and L-type Ca2+ channel blockers inhibit Ca2+ spiking in response to DBI33-50. Similar to the data observed with CCK secretion studies, the elimination of [Ca2+]o resulted in the abolition of Ca2+ oscillations and the [Ca2+]i plateau in response to 10 nM DBI33-50 (Fig. 3, A and B). Because endocrine cells usually have voltage-dependent L-type Ca2+ channels (10), we next examined the effects of L-type Ca2+ channel blockers on DBI33-50-stimulated Ca2+ oscillations in STC-1 cells. Nifedipine (3 µM) and diltiazem (10 µM) inhibited Ca2+ spiking elicited by 10 nM DBI33-50 (Fig. 3, C and D). In separate experiments we showed that diltiazem (10 µM) inhibited CCK secretion during 120 min of cell stimulation with 10 nM of DBI33-50. The CCK levels decreased from 23.0 ± 2.8 to 12.6 ± 0.06 pg/well (n = 4, P < 0.05).
8-BrcAMP, forskolin, and bombesin elicit an initial
Ca2+ transient
followed by a sustained
[Ca2+]i
plateau.
Because cAMP and Ca2+ signal
transduction may be involved in the mediation of secretion by STC-1
cells in response to various secretagogues (4, 6, 24, 28, 32, 43), the
effects of cAMP- and
[Ca2+]i-elevating
agents on Ca2+ signaling were
examined. The cAMP-elevating agents, such as 8-BrcAMP (100 µM) and
forskolin (1-100 µM), and the
[Ca2+]i-mobilizing
peptide bombesin (10 nM) each caused an initial [Ca2+]i
transient followed by a sustained
[Ca2+]i
plateau (Fig. 4,
A and
B). These observations illustrate
two important facts: 1) similar to
bombesin, which is coupled to heterotrimeric Gq protein and phospholipase C
(PLC) pathways (55), cAMP pathways are capable of eliciting
Ca2+ spikes and
2) oscillatory modes of
Ca2+ spiking induced by various
concentrations of DBI33-50
are different from those elicited by forskolin (and 8-BrcAMP) or
bombesin, which caused an initial
[Ca2+]i
transient followed by a sustained
[Ca2+]i
plateau.
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Forskolin, but not DBI33-50,
increases levels of [cAMP].
To determine if DBI33-50
elicits CCK secretion via cAMP pathways, intracellular cAMP
concentration ([cAMP]) was measured by RIA. In
the absence of the phosphodiesterase inhibitor IBMX basal
[cAMP] in STC-1 cells was 18.3 ± 2.5 pmol/100 µg
protein (20 min of incubation without secretagogues,
n = 8). Incubation of STC-1 cells with
10-100 nM DBI33-50 for
20 min was sufficient to elicit
Ca2+ oscillations and CCK
secretion but did not increase [cAMP] over basal for all
doses tested. In contrast, forskolin (1-100 µM) caused a 1.7- to
11-fold increase in [cAMP] in a dose-dependent manner (Fig.
5A). In
the presence of IBMX (100 µM), basal [cAMP] was 23.0 ± 5.3 pmol/100 µg protein (n = 4). Even in the presence of IBMX,
DBI33-50 did not increase
[cAMP] over basal levels, whereas forskolin (1-100
µM) elicited a dose-dependent increase in [cAMP] in
response to a 20-min cell stimulation (Fig.
5B). These results indicate that
DBI33-50 did not increase
cAMP formation in the STC-1 cells and therefore stimulated CCK
secretion via a cAMP-independent pathway.
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Bombesin, but not DBI33-50,
increases intracellular IP3 levels.
To examine if DBI33-50 acts
via IP3-dependent pathways, we
measured intracellular IP3 concentration
([IP3]) by RIA. Basal
[IP3] was 1.2 ± 0.4 pmol/total mg protein (1-min incubation without secretagogues,
n = 14). Administration of
DBI33-50 (0.1-10,000 nM)
did not cause a significant increase in
[IP3] over basal (Fig.
6A). In
contrast, bombesin (1-1,000 nM) significantly increased
[IP3] by 4- to
6.5-fold over basal during a 1-min stimulation. Figure
6B shows a time course study.
Stimulation with 100 nM bombesin for 15 s increased
[IP3] levels to
18.3 ± 5.0 pmol/total mg protein (n = 6), with a rapid decline within 5 min. In contrast, 1 µM DBI33-50 did not
significantly increase
[IP3] over basal at
any time points examined. These results indicate that
IP3 pathways do not mediate
DBI33-50 activity.
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Inhibitors of PLC, PLA2, and PTK do not
affect Ca2+
oscillations evoked by DBI33-50.
We further examined whether CCK secretion by
DBI33-50 involves other
signal transduction pathways, such as phospholipase A2
(PLA2) and protein tyrosine
kinase (PTK) systems. We previously demonstrated that inhibitors of PLC
(U-73122), PLA2 (ONO-RS-082), and
PTK (genistein) inhibited Ca2+
spiking and amylase secretion evoked by CCK analogs in rat pancreatic acinar cells (49, 50, 52, 53). However, these inhibitors had no
significant effect on Ca2+
oscillations stimulated by 10 nM
DBI33-50 in STC-1 cells, suggesting that, in contrast to their roles in pancreatic acini, the
PLC, PLA2, and PTK pathways are
not involved in the Ca2+
oscillations elicited by
DBI33-50 (Fig.
7,
A-C).
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PTx does not affect
Ca2+
oscillations elicited by
DBI33-50.
In several cell systems PTx-sensitive
Gi-Go
proteins may mediate Ca2+ influx
from the extracellular space (2, 42). To examine if PTx-sensitive
Gi-Go
proteins are involved in mediating
Ca2+ influx in response to
DBI33-50, STC-1 cells were
pretreated with 300 ng/ml PTx for 120 min at 37°C. There were no
significant differences in Ca2+
oscillations in the presence or absence of PTx during stimulation by
100 nM DBI33-50.
DBI33-50 caused
Ca2+ oscillations with a
[Ca2+] (peak
basal) equal to 151 ± 37 nM and frequencies equal to 3.6 ± 0.9 cycles/10 min (n = 7) in the absence
of PTx. They were 145 ± 42 nM and 3.4 ± 0.4 cycles/10 min
(n = 4) in the presence of PTx,
respectively. These results, along with the observations that
DBI33-50 did not increase
[cAMP] and
[IP3], suggest that
the heterotrimeric G proteins (e.g.,
Gs,
Gq, and
Gi/o) coupled to the
seven-transmembrane-helix receptors probably are not involved in the
mediation of
DBI33-50-stimulated
Ca2+ oscillations.
Caffeine elicits and enhances
Ca2+ spiking in
response to DBI33-50.
In other cell types voltage-sensitive
Ca2+ oscillations may be regulated
by caffeine-sensitive
"Ca2+-induced
Ca2+ release (CICR)" that
occurs in the sarcoplasmic and endoplasmic reticulums (13, 25). To
investigate if CICR occurs in STC-1 cells, 20 mM caffeine was applied
during stimulation by 10 nM DBI33-50. Caffeine enhanced
the action of DBI33-50, resulting in a peak
[Ca2+]i
of 2,398 nM (n = 8), which suggests
involvement of the caffeine-sensitive CICR on
DBI33-50-stimulated
Ca2+ spiking (Fig.
8). Similar to
Ca2+ spiking modes in response to
DBI33-50, the action of
caffeine was totally dependent on the presence of
[Ca2+]o
(data not shown). It is interesting to note that caffeine, in turn,
inhibits voltage-independent and intracellular
Ca2+ oscillations induced by CCK
analogs in pancreatic acinar cells (49, 53).
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DISCUSSION |
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This study examined the intracellular mechanisms by which DBI33-50 stimulates the secretion of CCK from STC-1 cells. Using a perfusion system containing isolated mucosal cells from the proximal intestine of rats, we showed that DBI1-86 and DBI33-50 dose dependently stimulated CCK secretion (18). In this study we demonstrated that DBI33-50 evoked CCK secretion in STC-1 cells. As the elimination of [Ca2+]o abolished CCK secretion in response to DBI33-50 the presence of [Ca2+]o is required for stimulus-secretion coupling in STC-1 cells.
DBI33-50
(109 to
10
6 M) elicited
Ca2+ oscillations in a manner
dependent on the presence of [Ca
2+]o. A
reduction in the amplitude and frequency of
Ca2+ oscillations elicited by a
supramaximal concentration of
DBI33-50 (1 µM) is
consistent with the observation that 1 µM
DBI33-50 reduced CCK
secretion. The L-type Ca2+ channel
blockers nifedipine and diltiazem decreased the
DBI33-50-stimulated Ca2+ spike amplitude and
frequency; therefore, it appears that L-type Ca2+ channels are involved in
Ca2+ entry from the extracellular
space in response to DBI33-50 stimulation. This is not unexpected because endocrine cells usually possess voltage-dependent L-type
Ca2+ channels (10). Consistent
with our
[Ca2+]i
data, blockade of voltage-dependent L-type
Ca2+ channels also abolished CCK
secretion stimulated by
DBI33-50.
The receptors that mediate the action of DBI to stimulate CCK secretion have not been clearly identified. DBI has multiple biological actions; benzodiazepine (BZD)-binding sites mediate some of these actions, whereas others are unrelated to BZD-binding sites. In animal studies DBI induces anxiety and proconflict responses (14). In primary cultures of mouse spinal cord neurons, DBI reduces GABA-induced chloride channel opening (3). These activities are related to the actions of DBI in the "central" BZD-binding site. In addition, DBI stimulates mitochondrial steroid biosynthesis in adrenocortical, Leydig, and glial cells by a "peripheral" BZD-binding site-dependent mechanism (8, 9). DBI has been shown to be a paracrine and an autocrine stimulator of Leydig cell proliferation, acting via a plasma membrane peripheral BZD-binding site independent of mitochondrial function (15). Some reported DBI actions are unrelated to known BZD-binding sites. Porcine and rat DBI markedly decrease the late phase of glucose-induced insulin release from perfused rat pancreas independently of any known BZD-binding sites (7, 39). In this study, using STC-1 cells, we have shown that DBI33-50 can act directly to stimulate CCK release. The receptors responsible for this action remain to be elucidated. Because we did not perform binding studies, we cannot conclusively establish that DBI is acting via a receptor-mediated mechanism. Furthermore, it remains to be determined whether DBI1-86 and its trypsin-digested DBI fragment DBI33-50 act on the same or different receptor.
In our studies we clearly demonstrate that secretagogues, such as
8-BrcAMP and forskolin, which mimic and increase [cAMP], respectively, and bombesin which increases
[Ca2+]i,
are capable of stimulating Ca2+
spiking and
[Ca2+]o-dependent
CCK secretion in STC-1 cells. However, the mechanism by which
DBI33-50 stimulates
[Ca2+]o-dependent
oscillations and CCK secretion seems to be different from that used by
cAMP-elevating reagents and bombesin.
DBI33-50 at concentrations
sufficient to elicit Ca2+
oscillations and CCK secretion, did not increase [cAMP] and
[IP3] over basal. In
the same system we showed that forskolin
(106-10
4
M) and bombesin
(10
9-10
6
M) significantly increased [cAMP] and
[IP3], respectively,
over basal. Therefore, it appears that
DBI33-50 stimulated CCK secretion from STC-1 cells via a mechanism independent of cAMP and
IP3.
The IP3-independent Ca2+ oscillation mechanism elicited by DBI33-50 was further confirmed by the demonstration that the PLC inhibitor U-73122, which has been shown to inhibit Ca2+ oscillations and amylase secretion evoked by CCK-8 in rat pancreatic acinar cells (49), did not affect DBI33-50-stimulated Ca2+ oscillations in STC-1 cells. Similarly, the PLA2 inhibitor ONO-RS-082 and PTK inhibitor genistein did not affect DBI33-50-stimulated Ca2+ oscillations in STC-1 cells. We previously demonstrated that ONO-RS-082 (10 µM) abolished arachidonic acid production, Ca2+ oscillations, and pancreatic amylase secretion evoked by the high-affinity CCK receptor agonists JMV-180 and CCK-OPE (50). It has been shown that genistein (50-100 µM) inhibited protein tyrosine phosphorylation, PTK activities, sustained Ca2+ entry, and pancreatic amylase secretion in response to CCK-8 (52). Therefore, our data suggest that DBI33-50 stimulates the opening of L-type Ca2+ channels without involving intracellular second messengers such as PLC, PLA2, and PTK.
In several cell types the stimulatory GTP binding protein
(Gs) regulates L-type
Ca2+ channels. For example, in
cardiac muscle cells, Gs and
the
1-subunit of L-type
Ca2+ channels are closely
associated with the T tube plasma membrane. Gs
and cAMP-dependent protein
kinase A differentially phosphorylate L-Ca2+ channels to regulate the
opening of the channels (5, 56, 57). Furthermore, in pancreatic
-cells and adrenal chromaffin cells, cAMP has been shown to modify
the gating properties of the L-type
Ca2+ channels (11, 16, 19). STC-1
cells may possess these cAMP-dependent Ca2+ channels, as forskolin
elicited Ca2+ spiking and
[Ca2+]o-dependent
CCK secretion; however, this is not the pathway used by
DBI33-50 as it did not
increase cAMP over basal. Similar observations have been made in smooth
muscle cells, neurons, and endocrine cells, where L-type
Ca2+ channels are not coupled to
the cAMP pathways (48). Our studies also suggest that PTx-sensitive
Gi-Go
proteins are probably not involved in
DBI33-50-stimulated
Ca2+ spiking, as PTx did not
affect Ca2+ oscillations in
response to DBI33-50.
The mechanism by which voltage-dependent Ca2+ oscillations occur in secretory cells has not been elucidated (46-48). Currently, there are two hypothetic models to explain Ca2+ oscillations in the plasma membrane (1). The first model proposes that voltage-dependent and repetitive Ca2+ influx and efflux may occur at the plasma membrane, coupled with changeable K+ conductance. The second model suggests that voltage-dependent Ca2+ entry triggers the release of Ca2+ through the caffeine-sensitive Ca2+ channels (ryanodine receptor) in the sarcoplasmic reticulum. Ca2+-ATPase then takes up the released Ca2+ in the sarcoplasmic reticulum. The second model proposes that the Ca2+ oscillation occurs between the cytoplasm and the sarcoplasmic reticulum, but it is still dependent on the presence of [Ca2+]o because the initial trigger responsible for this mechanism is Ca2+ entry from the extracellular space. This phenomenon is called "calcium-induced calcium release," which initially was shown to occur in skeletal and cardiac muscle cells and neurons (13, 25). Our studies show that caffeine enhanced Ca2+ spiking in response to DBI33-50, which suggests that the CICR mechanism may be involved in Ca2+ oscillations in the plasma membrane of STC-1 cells.
In conclusion, we have demonstrated that DBI33-50 directly acts on STC-1 cells to elicit Ca2+ oscillations via voltage-dependent L-type Ca2+ channels, resulting in secretion of CCK. This is mediated by intracellular mechanisms independent of the traditional signal transduction pathways, including the PLC, PLA2, PTK, and cAMP systems.
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ACKNOWLEDGEMENTS |
---|
We thank Otsuka Pharmaceutical (Japan) for providing the anti-CCK-8 (sulfated) antiserum OAL-656.
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FOOTNOTES |
---|
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-32830 and 5D 30-DK-34933.
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 and other correspondence: C. Owyang, 3912 Taubman Center, Univ. of Michigan, Ann Arbor, MI 48109-0362.
Received 17 July 1998; accepted in final form 17 November 1998.
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