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INSERM U-45, Hôpital E. Herriot, Lyon, France; and Unité d’Écologie et de Physiologie du Système Digestif, Institut National de la Recherche Agronomique, Jouy-en-Josas, France
Address all correspondence and requests for reprints to: Dr. Jean-Claude Cuber, INSERM U-45, Pavillon Hbis, Place d’Arsonval, 69437 Lyon Cedex 03, France. E-mail: cuber{at}lyon151.inserm.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, Gi3, and Go
immunoreactivities in STC-1 cell extracts. Additionally, peptones and
cephalexin increased the cellular content in inositol phosphates,
whereas a mild increase in cAMP content was restricted to
peptone-treated cells. Protein kinase A or C inhibition did not modify
peptone- or antibiotic drug-evoked CCK release. The extracellular
Ca2+ chelator EGTA (500 µM) and the
intracellular Ca2+ chelator BAPTA-AM
[1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetra(acetoxymethyl) ester; 20 µM] abolished the
peptone- and antibiotic drug-induced CCK release. Nifedipine and
verapamil (10 µM) reduced by about 50% the CCK secretion
evoked by these two secretagogues. In conclusion, peptones and some
cephalosporins are potent stimulants of CCK release in the STC-1 cell
line. The cellular mechanisms involve pertussis toxin-sensitive G
protein(s) and are dependent on Ca2+ availability. We
suggest that the STC-1 cell line is a useful model to study the
molecular basis of peptone-induced CCK secretion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the one hand, proteins could exert their stimulatory effect on CCK secretion via trypsin inhibition, and this has been postulated to be the major link in the feedback loop regulating the intestinal phase of pancreatic exocrine secretion (6, 7). The hypothesis that intact proteins stimulate CCK release by protecting CCK-releasing peptides from proteolytic inactivation by pancreatic trypsin was supported by the purification and sequencing of three distinct protease-sensitive CCK-releasing peptides, namely the monitor peptide from pancreatic juice (8), the luminal CCK-releasing factor from rat intestinal secretion (9), and a peptide identical to the porcine diazepam binding inhibitor, which was purified and characterized from porcine intestinal mucosa (10). Interestingly, the release of diazepam binding inhibitor was shown to be under the control of intramural cholinergic nerves (10, 11).
On the other hand, studies in the isolated vascularly perfused rat duodeno-jejunum showed that the nutrient-induced CCK release did not involve intramural nerves, as neither the axonal blocker tetrodotoxin nor atropine modified nutrient-induced CCK secretion (12). Among the individual food components, peptones (i.e. acid or enzyme hydrolysate of proteins) were the most potent in stimulating CCK release (13). Additionally, we recently showed that peptones stimulate CCK secretion and gene transcription in the intestinal CCK-producing cell line STC-1 (14), which displays many features of native intestinal CCK-producing cells (15, 16). Overall, these data indicate that peptones may at least partly stimulate CCK release via a direct mechanism. In the present study, we investigated the cellular events leading to CCK secretion. In a first step, we investigated the possibility that peptone-induced CCK release could involve a peptide transporter. As peptones contain a complex mixture of oligopeptides of variable lengths, this work was conducted in parallel with peptidomimetic antibiotics that are resistant to enzymatic degradation, thus representing a homogeneous and stable substrate over time. Additionally, it is well recognized that the peptide transporter can serve as a carrier for these peptidomimetic drugs. The roles of G proteins, protein kinase A, protein kinase C, and calcium in peptone- and antibiotic drug- stimulated release of CCK were studied as well.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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i1 and i2 (K11) or i3 and
o (K10) were obtained as described previously (17). They
were provided by Drs. J. M. Saez and D. Langlois (INSERM U-418,
Lyon, France). BAPTA-AM
[1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetra(acetoxymethyl) ester] was obtained from Calbiochem (La
Jolla, CA). All other chemicals were of analytical grade.
Cell line and culture condition
The STC-1 plurihormonal cell line is derived from an endocrine
tumor that developed in the small intestine of a double transgenic
mouse expressing the rat insulin promoter linked to the simian virus 40
large T antigen and the polyoma small t antigen, respectively (15). It
was provided by Dr. Andrew B. Leiter (Department of Medicine, New
England Medicine Center, Boston, MA). The STC-1 cell line was grown in
RPMI 1640 supplemented with 5% FCS, 2 mM glutamine,
penicillin (100 IU/ml), and streptomycin (50 µM). Cell
viability was assessed by trypan blue exclusion and measurement of
lactate dehydrogenase activity. Experiments in which cell viability was
less than 90% were rejected.
Secretion studies
Two days before the experiments, STC-1 cells were seeded into
24-well culture plates. When cells reached 85% confluence, the medium
was replaced by Krebs-Ringer bicarbonate buffer containing 0.2%
(wt/vol) BSA (KRBB; pH 7.4), with the tested agents. The osmolarity was
increased when required to 300 mosmol/kg H2O by the
addition of appropriate amounts of sodium chloride. The cells were then
incubated at 37 C for 2 h or for various periods of time as
specified. The supernatants were collected and frozen at -20 C for
subsequent RIA. The cells were extracted in 2 M
CH3COOH-20 mM HCl, sonicated, boiled for 10
min, neutralized, and frozen at -20 C before RIA of CCK, as previously
described (18). Briefly, CCK immunoreactivity was measured using
antiserum 39A that cross-reacts 100% with CCK-33 and CCK-8, 12% with
sulfated gastrin-17, 5% with unsulfated gastrin, and less than 0.1%
with unsulfated CCK-8, unsulfated CCK-7-Gly, and gastrin-34. The CCK
radioligand was CCK-10 labeled with the monoiodinated Bolton-Hunter
reagent and purified by reverse HPLC according to the method of Fourmy
et al. (19). The intra- and interassay variances were 6.7%
and 7.8%, respectively (n = 24). Sephadex G-50 chromatography of
STC-1 cell culture medium was performed as described previously (18)
and revealed a single peak coeluting with CCK-8 (data not shown). STC-1
cells were used between passages 7 and 31. Immunocytochemical analysis
revealed that more than 80% of the cells stained with the CCK
antiserum 39A. CCK cell content ranged from 1.5–4 ng/106
cells. The basal secretion rate was about 0.5–2% of the total CCK
content per h. The amount of peptide released in the medium was
expressed as a percentage of the total cell content.
CPX uptake experiments
STC-1 cells were grown to 85% confluence into
poly-D-lysine-coated 24-well plates. On the day of the
experiment, STC-1 cells were preincubated for 15 min with KRBB at pH
7.4 (20 mM HEPES) at 37 C. Plates were then incubated for
15 min at 37 C in KRBB at pH 7.4 or at pH 6.0 (20 mM MES)
containing various concentrations of CPX (0.5–25 mM). A
15-min incubation period was used because uptake was linearly related
to time for up to 20 min. After incubation, the buffer was removed, and
the cells were washed twice with ice-cold PBS. CPX was then extracted
with 1 ml distilled water adjusted to pH 3.0 with HCl. The extraction
solution was centrifuged (10,000 x g, 15 min), and the
supernatant obtained was filtered through a Millipore filter (SJGVL,
Millipore Corp., Bedford, MA; 0.22 µm). The filtrate was subjected to
HPLC with conditions as follows: column, µBondapak C18
3.9 mm x 300 mm (Waters, Milford, MA); mobile phase, 0.02
M NaH2PO4 (pH 4.5)-acetonitrile
(87:13); flow rate, 1.6 ml/min; and detector, 260 nm. Uptake was
normalized to the BSA equivalent protein content (Bio-Rad, Ivry,
France) and expressed as nanomoles per mg protein/min.
Immunoblot analysis
STC-1 cells were lysed in cold solubilization buffer containing
1% (vol/vol) Triton X-100, 50 mM HEPES (pH 7.5), 150
mM NaCl, 100 U/ml aprotinin, 20 µM leupeptin,
and 0.2 mg/ml phenylmethylsulfonylfluoride. Cell extracts were
clarified (14,000 x g, 15 min, 4 C), and aliquots of
the supernatants (50 µg) were diluted in 4 x SDS-PAGE sample
buffer (62 mM Tris-HCl, 8% SDS, 40% glycerol, 20%
2-mercaptoethanol, and 0.16% bromophenol blue), boiled for 5 min, and
resolved by electrophoresis on 6 M urea-0.1% SDS-10%
polyacrylamide. After electrophoresis, proteins were transferred to
nitrocellulose membranes. Membranes were blocked using 5% nonfat dry
milk in Tris-buffered saline containing 0.2% Nonidet P-40 and
incubated with rabbit polyclonal antiserum directed against
i1/i2 or
i3/0 overnight at 4 C. After four washes
in 5% nonfat dry milk in Tris-buffered saline containing 0.2% Nonidet
P-40, membranes were incubated for 3 h at 4 C with horseradish
peroxidase-conjugated goat antirabbit IgG (Nordic Immunological
Laboratories, Tilburg, The Netherlands). After rinsing in Tris-buffered
saline, peroxidase activity was developed using the chemiluminescent
enhanced method (Pierce Chemical Co., Rockford, IL). Electrophoresis
materials were obtained from Bio-Rad.
Analysis of total inositol phosphate
STC-1 cells were labeled with 2 µCi/ml
myo-[2-3H]inositol (16.5 Ci/mmol; Amersham, Les Ulis,
France) for 24 h at 37 C. The cells were then rinsed once and
incubated in KRBB containing 10 mM LiCl in the absence or
presence of various reagents. After a 30-min incubation at 37 C, the
reaction was stopped by adding 3 vol CHCl3/MeOH (1:2)
containing 0.1 M HCl. The contents of the wells were
transferred to glass tubes, and inositol phosphates were separated from
the phosphoinositides by adding 1 vol CHCl3 and
1 vol H2O. After vigorous vortexing for 1 min and
centrifugation (400 x g, 10 min), the total
water-soluble [3H]inositol phosphates were diluted to 3
ml in 6 mM Na2B4O7 and
separated from [3H]inositol by anion exchange
chromatography on Dowex AG-1X8 (formate form, Bio-Rad) as described by
Berridge et al. (20). The radioactive content of the
fractions was measured by liquid scintillation counting (Instagel,
Packard, Meriden, CT). Results were normalized to the control value
(without tested agents, 100%) and expressed as a percentage of the
control.
cAMP experiments
After a 30-min incubation of STC-1 cells at 37 C in 1 ml KRBB
containing 500 µM isobutylmethylxanthine and the agents
to be tested, the reaction was stopped with 100 µl perchloric acid
(11 N), and the extracts were centrifuged (400 x
g, 5 min). Measurement of the cAMP content of the
supernatants was performed by RIA (DuPont-New England Nuclear, Les
Ulis, France) according to the manufacturer’s instructions. Results
were normalized to the control value (without tested agents, 100%) and
expressed as a percentage of the control.
Calculations and statistics
Data in the figures are presented as the mean ±
SEM. Statistical significance was assessed using one-way
ANOVA, followed by the multiple comparison test of Fisher. Differences
were considered significant when P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, STC-1 basal CCK
secretion was 1.7 ± 0.1% of the total cell content after 2
h. Incubation of STC-1 cells with peptones from various origins, namely
soybean, meat, casein, and ovalbumin hydrolysates, induced a
dose-dependent increase in CCK release (0.5–1% wt/vol). Cell
treatment with di- or tripeptides (1%, wt/vol) also induced a strong
increase in CCK secretion (11.5 ± 0.5%, 5.1 ± 0.6%, and
11.1 ± 0.8% of the total cell content upon treatment with
Gly-Leu, Gly-Pro, and Gly-Pro-Ala, respectively). In contrast, STC-1
cell exposure to undigested proteins (ovalbumin or BSA; 1%, wt/vol,
each) resulted in only a mild effect on CCK release. Similarly, the
amino acid mixture HA25 (1%) or tryptophan (2.5%; 20 mM)
weakly stimulated CCK release (2.2 ± 0.1% and 2.5 ± 0.3%
of the total cell content, respectively).
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, incubation of cells with the various
cephalosporins (20 mM) stimulated CCK release with large
differences in potency, ranging from 1.5-fold (cefadroxil) to about
16-fold (cephradine) the basal CCK release. The most potent of these
antibiotics (CPX, cephalothin, and cephradine) induced a dose-dependent
increase in CCK release over the concentration range 1–20
mM. The subsequent experiments, designed to investigate the
mechanisms of CCK release, were conducted with CPX, which is one of the
preferred peptidomimetic drugs used for studying peptide
absorption.
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). CPX (20 mM)
had a dramatic effect on CCK secretion, with a plateau value at about
35% of the cell content that was observed at 30 min, whereas the same
dose of CPX or 1% AEH failed to enhance CCK release at 4 C when
exocytosis was inhibited (Fig. 2).
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). Under these
conditions, the curves of accumulation of the ß-lactam antibiotic
were linear, indicating a nonsaturable process, at either pH 7.4 or pH
6.0, the latter being optimal for the H+-coupled
transporter. Overall, these results discard the possibility of a
H+-peptide transporter in CPX uptake by STC-1 cells.
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i/o subunits of serpentine receptor-coupled G proteins
are required in the response to peptones or cephalosporins. For this
purpose, cells were treated for 5 h with 200 ng/ml Ptx before a
2-h stimulation with agonists (Fig. 4).
When STC-1 cells were treated with Ptx, they exhibited a 45–70%
decrease in peptone- or cephalosporin-induced CCK release (70% and
44% of inhibition upon treatment with 1% AEH and 20 mM
CPX, respectively). In contrast, bombesin-induced CCK release was not
affected by Ptx treatment (Fig. 4). Overall, these results suggest that
CCK release induced by peptones or peptidomimetic drugs partly involved
pertussis toxin-sensitive G protein(s). Consistent with this hypothesis
was the identification by Western blot of several Ptx-sensitive G
proteins, namely o, i2, and
i3, and relatively lower amounts of the
i1 subtype, in the STC-1 cell line (Fig. 4, inset).
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, bombesin (100
nM), used as a reference, efficiently stimulated total
inositol phosphate accumulation (705 ± 57% of the control
value), while inducing a modest increase in CCK release. In contrast,
1% AEH or 20 mM CPX had a dramatic stimulatory effect on
CCK secretion, but a slight effect on total inositol phosphate
production. AEH (1%) produced a small increase in cAMP accumulation
(162 ± 9% of the control), whereas 20 mM CPX failed
to enhance cAMP production (Fig. 5). For comparison, adenylate cyclase
activation by forskolin (100 µM) resulted in a 4-fold
increase in cAMP production (425 ± 29% of the control value).
Additionally, secretion experiments with PKA or PKC inhibitors were
performed. A 30-min pretreatment of the cells with either 10
µM H89 or 1 µM staurosporine efficiently
inhibited the CCK responses to 100 µM forskolin (-80%)
or 100 nM PMA (-100%), respectively, but was without
effect on AEH- or CPX-induced CCK secretion (data not shown).
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, the Ca2+ chelator EGTA
(500 µM) markedly inhibited the stimulatory effects of
1% AEH and 20 mM CPX on CCK release. Similarly,
preincubation of STC-1 cells with the cell-permeant Ca2+
chelator BAPTA-AM (20 µM) resulted in inhibition of the
stimulatory effects of 20 mM CPX and 1% AEH by 95% and
83%, respectively (Fig. 6B). Furthermore, the L-type Ca2+
channel blockers nifedipine (10 µM) and verapamil (10
µM) reduced by 30–60% the stimulatory effects of 1%
AEH and 20 mM CPX (Fig. 6C). For comparison, control
experiments performed with bombesin (100 nM, 2 h)
showed that preincubation of cells with nifedipine or verapamil reduced
by 90% the secretory response of STC-1 cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous reports have demonstrated that the intestinal oligopeptide transport system is distinct from the transporter system involved in the uptake of amino acids and that this transporter is responsible for the uptake of oligopeptides and peptidomimetic agents, such as cephalosporins (21, 22, 23, 25). Two peptide transporters have been recently identified. One H+-peptide cotransporter, called PepT1, has been cloned from rabbit, human, and rat intestine (27, 28, 29), whereas another one, called PepT2, was isolated from human kidney (30). As both peptones and peptidomimetic antibiotics stimulate CCK release, the hypothesis that a peptide transporter could be involved in this effect was raised. As the murine intestinal peptide transporter has not yet been cloned, an indirect method was used to test the possibility that a saturable, carrier-mediated, transport process is functional in the murine endocrine cell line STC-1. CPX uptake by STC-1 cells clearly indicated a nonsaturable mechanism at both pH 7.4 and pH 6.0. Together, these data suggest that STC-1 cells are not equipped with a peptide transporter. Passive diffusion probably allows oligopeptides and cephalosporins to enter cells and activate intracellular mechanisms. However, as functional characteristics of the putative murine peptide transporter(s) remain unknown, the existence of a saturable peptide transport process cannot be completely ruled out on the sole basis of an apparent lack of such an active pathway for antibiotics.
Whether CCK release upon peptone or peptidomimetic drug treatment involves a seven-transmembrane domain receptor was assessed. As the effector pathways for activated serpentine receptors frequently involve adenylyl cyclases and phospholipase C, the accumulation of cAMP and phosphoinositides in stimulated cells was recorded in the present study. A mild increase in cAMP was restricted to peptone-treated cells. Accumulation of phosphoinositides in peptone- or CPX-treated cells was moderate compared with that induced by bombesin. Additionally, PKA or PKC inhibition failed to modify the peptone- or CPX-evoked CCK secretion. Overall, the involvement of a seven-transmembrane domain receptor in peptone-evoked CCK release seems unlikely.
Interestingly, the present study revealed the presence of several
Ptx-sensitive G proteins in the STC-1 cell line. Additionally, peptone-
or cephalosporin-induced CCK secretion was reduced by Ptx, suggesting
the involvement of toxin-sensitive G protein(s) in CCK secretion.
Furthermore, EGTA in the external solution and L-type Ca2+
channels inhibitors markedly reduced CCK secretion, thus arguing for a
pivotal role of Ca2+ in peptone- and antibiotic-induced CCK
secretion in the neuroendocrine cell line STC-1. One attractive
hypothesis could be that activation of pertussis toxin-sensitive G
protein upon incubation with peptones leads to Ca2+ influx
through voltage-dependent Ca2+ channels and consequently
stimulates CCK release. Indeed, Ca2+ influx mediated by
L-type channels can be stimulated by receptors acting via a variety of
Ptx-sensitive G proteins in other endocrine cells (31, 32, 33, 34). On the
other hand, Ptx-sensitive heterotrimeric G protein(s) could interact
with membrane traffic systems involved in the regulated exocytosis of
secretory granules (35) and stimulate CCK release. Further experiments
aimed at blocking the various G -subunits identified in STC-1 cells
should be performed to specify the Ptx-sensitive G protein(s) mediating
Ca2+ entry and/or interacting with membrane traffic
systems.
The present data support our previous results obtained with the isolated, vascularly perfused, rat duodeno-jejunum preparation showing that peptone stimulation of duodeno-jejunal I cells was strongly dependent on the availability of extracellular calcium. Indeed, vascularly perfused EGTA abolished the CCK response to peptones, whereas complexing luminal calcium by infusing EGTA into the duodenum had no effect (36). Additionally, peptone induced CCK secretion was strongly reduced upon intraarterial infusion of blockers of L-type Ca2+ channels (36). To further test the hypothesis that intracellular Ca2+ is required for CCK secretion, we incubated the STC-1 cells with BAPTA-AM and peptone or CPX. BAPTA-AM is a membrane-permeating inactive tetra-AM calcium chelator that is desesterified and activated by intracellular esterases (37). Its calcium-chelating action is therefore exclusively intracellular, and the observed inhibitory effects strongly indicate that an increase in [Ca2+]i is involved in peptone- and peptidomimetic antibiotic-induced CCK release. Because both EGTA and Ca2+ channels inhibitors markedly reduced peptone- or cephalosporin-induced CCK secretion, it is reasonable to assume that the increase in [Ca2+]i is essentially imputable to Ca2+ entry into the cells. On the other hand, the peptone- and cephalosporin-evoked CCK secretion was accompanied by a modest increase in intracellular phosphoinositide production, suggesting that opening of inositol 1,4,5-trisphosphate-regulated channels could cooperate in the rise in [Ca2+]i. Furthermore, mobilization of calcium from intracellular stores through inostol 1,4,5-trisphosphate-independent pathways may occur. For example, synthetic peptides known as competitive substrates of metalloendoproteases have been proposed to release Ca2+ from the endoplasmic reticulum through an ion pore with affinity for hydrophobic molecules containing internal peptide bonds (38). Such structural features are present in cephalosporin antibiotics that stimulate CCK release with potencies that correlated with the degree of hydrophobicity. Another attractive hypothesis is the participation of a metabolic component in peptone-induced CCK secretion, e.g. stimulation of mitochondrial energy product. This hypothesis requires additional experiments with various mitochondrial respiratory chain regulators.
In conclusion, peptones and several peptidomimetic antibiotics are potent stimulants of CCK release. The cellular mechanisms involve Ptx-sensitive G protein(s) and require Ca2+ availability.
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Acknowledgments |
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i1/i2 and
i3/o antibodies. Received September 3, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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