Exploratory Research Department Sanofi-Synthélabo Recherche 131036 Toulouse and 267080 Strasbourg, France
Submitted 7 April 2003 ; accepted in final form 29 April 2003
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ABSTRACT |
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arginine vasopressin V1b receptors; glucagon; SSR-149415
In the present study, we investigated the effects of the selective and
orally active V1b receptor antagonist recently described,
SSR-149415 (25), on the
In-R1-G9 cell line, a model of glucagon-secreting -pancreatic cells.
For the first time, binding studies were performed in these cells with
[3H]AVP and various reference peptide and nonpeptide ligands,
including SSR-149415 and its low V1b affinity diasteroisomer
(SR-149424) to provide characterization of AVP receptors present In-R1-G9
cells. In functional studies performed in these cells, AVP induces
intracellular Ca2+ concentration
([Ca2+]i) increase, glucagon secretion, and
cell proliferation. These effects were fully antagonized by SSR-149415 with a
nanomolar potency, whereas its diasteroisomer as well as two selective
V1a and V2 receptor antagonists were much less potent.
The order of potency of AVP agonists and antagonists was in agreement with
V1b-mediated effects. The presence of V1b receptor mRNA
in these cells and in human pancreas was also confirmed by RT-PCR. Finally,
using an immunohistochemical approach, we studied the distribution of
V1b receptors in human pancreas and demonstrated by double
immunostaining that the V1b receptor was expressed in the different
cell types of islets of Langerhans. All together, these data suggest a
potential hormonal control for AVP on endocrine pancreas via V1b
receptor activation.
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MATERIALS AND METHODS |
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Biological materials. The Hamster glucagonoma In-R1-G9 cells were kindly provided by Dr. Kimberly A. Matthews (Veterans Affairs Medical Center, Huntington, WV). Human biopsy tissues (pancreas and liver) were collected in conformity with French national ethics rules. Immediately after excision, tissues without microscopic abnormalities or tumors were fixed in paraformaldehyde (4%) and then embedded in paraffin for immunohistochemical studies. This project was approved by the human subject review committees of Sanofi-Synthélabo Recherche and was carried out in collaboration with the Department of Pathological Anatomy of Strasbourg University Hospital (Strasbourg, France). Human pituitary sections embedded in paraffin were purchased from Clinisciences (Montrouge, France).
Culture of hamster pancreatic In-R1-G9 cells. In-R1-G9 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 0.03% amphotericin, 0.2% penicillin-streptomycin, and 2 mM L-glutamine. They were grown at 37°C in a humidified atmosphere of CO2. All experiments were performed using cells from passages 22 to 36.
[3H]AVP binding to In-R1-G9 membranes. Membranes from
confluent In-R1-G9 cells, cultured in 175-cm2 flasks, were prepared
as described previously (22).
Binding assays on In-R1-G9 membranes were performed in an incubation medium
containing 50 mM Tris · HCl (pH 7.4), 3 mM MgSO4, 0.1% BSA,
0.1% bacitracin, [3H]AVP (0.003-20 nM for saturation experiments or
2-3 nM for kinetic and competition studies), and increasing amounts of the
tested compound. The reaction was started by the addition of In-R1-G9
membranes (200-275 µg/assay) and lasted for 45 min at 20°C. The
reaction was stopped by adding 4 ml of ice-cold buffer followed by filtration
through GF/B Whatman glass microfi-ber filters. Filters were washed twice with
4 ml of ice-cold buffer and counted for radioactivity by liquid scintillation
in a Beta Packard 1900 TR. Nonspecific binding was determined in the presence
of 1 µM unlabeled AVP. The IC50 value was defined as the
concentration of inhibitor required to obtain 50% inhibition of the specific
binding. Inhibition constant (Ki) values were calculated
from the IC50 values by use of the Cheng and Prusoff equation
(5). Data for binding
experiments [apparent equilibrium dissociation constant
(Kd), maximum binding density (Bmax),
IC50, and Hill coeffi-cient (nH)] were analyzed using an
iterative nonlinear regression program
(15).
Intracellular Ca2+ concentration measurements. Subconfluent In-R1-G9 cells, cultured in 175-cm2 flasks as described, were collected by trypsinization (0.05% trypsin, 0.02% EDTA) and centrifugated (230 g, 5 min). The cells were suspended in culture medium to a final concentration of 5 x 106 cells/ml and then incubated with 5 µM fura 2-AM and 0.02% pluronic F-127 at 30°C for 20 min under continuous shaking. At the end of the incubation, cells were centrifugated (230 g, 5 min) and washed with culture medium. The cells were washed twice in Hanks' buffer (in mM: 137 NaCl, 5.4 KCl, 0.34 Na2HPO4, 5.5 glucose, 4.2 NaHCO3, 0.8 MgSO4, and 10 HEPES, with 0.1 mM EGTA for the first wash only, pH 7.4). The cells were resuspended in this buffer to a final concentration of 2.7 x 106 cells/ml and kept at 4°C in the dark until use. Calcium transients were measured with an SLM 8000 C spectrofluorometer after an incubation of 4 min at 37°C (excitation at 340 and 380 nm, emission at 510 nm). Cytosolic free Ca2+ determination was performed according to Grynkiewicz et al. (11).
Glucagon release. In-R1-G9 cells were plated onto 24-well plates (Corning, Oneonta, NY) at 105 cells/well and were grown for 4 days. The culture medium was then removed, and cells were preincubated (37°C, 5% CO2, 95% humidity) in a Krebs-Ringer bicarbonate buffer (KRB) for 15 min. To determine the dose responses of AVP and various AVP/OT agonists (dPal, dDAVP, and OT), cells were preincubated with successive dilutions from 10-9 to 10-6 M in the KRB buffer for 15 min. Similar experiments were also conducted in the presence of various modulators of glucagon secretion: arginine (10-3 M), theophylline (10-2 M), and somatostatin (10-6 M). For the antagonism study, the compound to be tested was administered 15 min before the addition of 10-7 M AVP, and then the plate was incubated for 15 min. The concentration of glucagon in the medium was measured by radioimmunoassay using the glucagon RIA kit provided by Linco Research (St. Charles, MO). The titration was performed in duplicate.
Proliferation assays. In-R1-G9 cells were grown for 48 h in a 96-well plate with a clear bottom (10,000 cells/well). The cell proliferation was measured using the CellTiter 96 cell proliferation assay from Promega (Madison, WI). Cells were washed with 200 µl of PBS and treated with increasing concentrations of either agonist or AVP (5.10-10) and increasing concentrations of antagonist compounds. After 18 h of incubation (37°C, 5% CO2, 80% humidity), 20 µl of dye solution were added to each well. The plate was incubated for 3 h, and absorbance was recorded at a 490-nm wavelength with a Multiskan Ex plate reader (Labsystem) (29).
V1b receptor expression in In-R1-G9 cells and human pancreas. Total Chinese hamster ovary (CHO) or In-R1-G9 cell RNA was extracted using the Qiagen RNeasy kit. Human pancreas RNA was purchased from Invitrogen. cDNAs were synthesized from 5 µg of RNA with Superscript RT II (Invitrogen), and 1/10th of the reaction product was used for PCR amplification consisting of 35 or 45 cycles of 30-s denaturation at 95°C, 30-s annealing at 56°C, and 1-min extension at 72°C. PCR products were separated by electrophoresis in a 2% agarose gel. Both primers were designed from published human, mouse, and rat V1b sequences: sense 5'-TACATGCTGCTGGCCATGAC-3'; reverse 5'-GATGGTGAAAGCCACATTGG-3'; the expected size for the amplicon was 600 bp.
Immunohistochemistry studies. Human tissue sections (5 µm) mounted on electrostatically treated slides were processed for antigen retrieval, which included deparaffinization and rehydration. Briefly, the slides were immersed in the Trilogy reagent (diluted 1:20 with distilled water) and heated to 90°Cin a microwave oven for 20 min. The slides were washed three times for 5 min with phosphate-buffered saline (PBS), pH 7.4. For immunohistochemical detection of V1b receptor, sections were incubated with 3% H2O2 diluted in PBS for 15 min. After a wash in PBS containing 0.05% Tween-20 to neutralize nonspecific binding sites, the sections were covered with PBS containing 5% NGS and 0.3% Triton X-100 for 20 min at room temperature and then drained and incubated for 120 min at room temperature with rabbit anti-rat V1b receptor antibody diluted (1:150) in PBS containing 1.5% NGS and 0.1% Triton X-100 (antibody diluting buffer). Subsequently, the slides were covered with the EnVision HRP-conjugated dextran polymer coupled to goat anti-rabbit IgG antibodies for 30 min at room temperature. Staining was completed by incubation with peroxidase substrate DAB for 4 min at room temperature. Sections were counterstained with hematoxylin, a nuclear marker, and then mounted with a coverslip. For fluorescent immunohistochemistry after the HPR treatment, the sections were treated with the fluorophore tyramide amplification reagent labeled with fluorescein.
In another set of experiments, a double fluorescent immunostaining method was applied for simultaneous localization of the V1b receptor with insulin, glucagon, or somatostatin. For double labeling, the same conditions were used for reaction with the V1b antibody and secondary labeling with EnVision-HRP. Then the sections were incubated with the tyramide amplification reagent containing fluorescein for 4 min. In the subsequent step after intensive washing, the sections were covered for 45 min at rom temperature with mouse monoclonal anti-glucagon (1:1,500), anti-insulin (1:1,500), or anti-somatostatin (1:20) antibodies. The detection was performed with goat anti-mouse antibody coupled to Alexa 594 (10 µg/ml diluted in PBS containing 1% NGS) for 30 min. Sections were counterstained with the nuclear marker DAPI, washed in distilled water, and mounted using Gel/Mount (Biomeda).
Slides were analyzed by transmission or fluorescence microscopy using a Leica microscope (Leitz DMREB) equipped with a video and a monochrome cooled charge-coupled device camera. Micrographs were made using the MetaMorph 4.6r6 image analysis system.
Immunohistochemistry control experiments. The rat immunogenic peptide sequence (located in the extracellular NH2-terminal domain of the rat V1b receptor) used to raise the antibody was very close to the corresponding 15-32 amino acid sequence of the human V1b receptor (GTL PVP NAT TPW LGR DEE and GTL SAP NAT TPW LGR DEE, respectively). The specificity of the polyclonal rabbit anti-V1b receptor antibody previously described in the literature (12) was confirmed in the present study 1) by the presence of specific immunostaining in CHO cells transfected with the human V1b receptor cDNA (but not in wild-type CHO or transfected cells with the human V1a or V2 cDNA) and 2) by the use of reference human tissues such as anterior pituitary as positive control and liver as negative control. Additionally, to validate the specificity of staining, control experiments were performed 1) by staining in the absence of the primary antibody, replaced by buffer dilution (negative control) and 2) by staining in the presence of the antigenic peptide. Rabbit anti-rat V1b receptor antibody diluted 1:150 was incubated for 30 min at 37°C with rat or human peptide antigens (10, 30, and 100 µg/ml).
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RESULTS |
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Effect of SSR-149415 and AVP analogs on
[Ca2+]i increase in
In-R1-G9 cells. As measured by fura 2 fluorescence, AVP induced a
dose-dependent increase in [Ca2+]i in
In-R1-G9 cells, with a dose required for a half-maximal response
(EC50) of 24 ± 20 nM (n = 7). Reference peptides
tested, dPal, dDAVP, and OT, exhibited an agonist profile in these cells,
although they were 10-fold less potent than AVP in stimulating
[Ca2+]i elevation (EC50 = 196
± 52, 121 ± 26, and 303 ± 196 nM, respectively). However,
dDAVP showed only a partial agonistic activity in these cells, with a maximal
effect 2.5-fold lower than that of AVP or other agonists
(Fig. 2A). This
observation is consistent with previous results showing that dDAVP acted as a
partial agonist on total IP production in COS-1 cells transfected with rat
V1b receptors, whereas a full agonistic activity was observed in
human V1b receptor-expressing cells
(21). Among the antagonists
tested, SSR-149415 strongly and dose-dependently inhibited 100 nM AVP-induced
[Ca2+]i increase (Ki of
0.66 ± 0.47; n = 3). This effect was stereospecific, as
demonstrated by the lower activity of its diasteroisomer SSR-149424 in this
model (Ki = 1,185 ± 936 nM; n = 4). Under
similar operating conditions, the mixed V1b/V1a
antagonist dPen and the V1a receptor antagonist SR-49059 were less
effective (Ki = 60 ± 11 and 128 ± 40 nM,
respectively; n = 3; Fig.
2B). Finally, the V2 receptor blocker
SR-121463 failed to antagonize [Ca2+]i
elevation stimulated by AVP (Table
1). None of the antagonists tested exhibited agonistic effects
when tested alone up to 1 µM.
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Effect of SSR-149415 and several AVP agonists and antagonists on glucagon release in In-R1-G9 cells. AVP induced dose-dependent secretion of glucagon in In-R1-G9 cells with an EC50 value of 5.2 ± 1.4 nM (n = 5), similar to that previously described by Yibchok-anun et al. (31). The reference AVP/OT peptide agonists tested (dPal, dDAVP, and OT) were much less potent than AVP, and their order of potency was in agreement with the affinity found for the V1b receptor identified in binding experiments (Table 1 and Fig. 3A). Interestingly, Dunning et al. (10) reported a significant effect of AVP, and to a lesser extent OT and dDAVP, on glucagon release in the perfused rat pancreas, in agreement with these in vitro data. To complete the functional identification of the AVP receptor involved in glucagon release, several specific V1b (SSR-149415), V1a (SR-49059), and V2 (SR-121463) receptor antagonists were used to counteract AVP effects. SSR-149415 exerted powerful antagonism on 100 nM AVP-induced glucagon secretion (Ki = 1.2 ± 1 nM), whereas SR-49059 was >100-fold less potent and SR-121463 was devoid of any effect up to 1 µM (Table 1 and Fig. 3B). Once again, we observed stereospecificity in the effect of SSR-149415 in antagonizing AVP-evoked glucagon production in In-R1-G9 cells (Ki of SR-149424 >1 µM). In another set of experiments, the effects of AVP were compared with those of well-known modulators of glucagon secretion. Amino acids, such as arginine, and agents that increase the intracellular cAMP concentration promote the liberation of the hyperglycemic hormone glucagon (19). As shown in Fig. 4, 100 nM AVP induced an effect comparable to 10 nM arginine on glucagon secretion (153 and 154% vs. basal value, respectively). As expected, theophylline, a nonspecific phosphodiesterase inhibitor known to increase intracellular cAMP production in In-R1-G9 cells, also stimulated glucagon secretion (175% vs. basal value). When associated with one of these agents (arginine or theophylline), AVP (100 nM) tends to enhance its action on glucagon secretion (170 and 200%, respectively). Of note, SSR-149415 was devoid of any inhibitory effect on basal and glucagon production evoked by the various stimulators except AVP (Fig. 4), demonstrating specific interaction at AVP V1b receptors. Finally, somatostatin, an inhibitor of pancreatic secretion, had no significant effect on basal or AVP-stimulated In-R1-G9 glucagon production under standard operating conditions. However, a significant inhibitory action of somatostatin was achieved on AVP-induced glucagon production after a longer preincubation time (50% inhibition at 2 h; not shown).
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Effect of SSR-149415 and AVP analogs on In-R1-G9 cell proliferation. As shown in Table 1, AVP promoted a strong proliferative effect in In-R1-G9 cells, and this effect was dose dependent (EC50 = 0.082 ± 0.022 nM; n = 14). Interestingly, Thibonnier et al. (29) also reported a mitogenic effect of AVP, using CHO cells transfected with the human V1b receptors. dPal and OT induced In-R1-G9 cell proliferation with EC50 values of 47 ± 22 (n = 3) and 69 ± 68 nM (n = 5) (Table 1). Surprisingly, dDAVP, a well-known V2/V1b agonist, had no agonist effect on cell proliferation. To determine the AVP receptor subtype involved in this stimulation, various reference peptide and specific nonpeptide antagonists were used. SSR-149415 counteracted 0.5 nM AVP-induced cell proliferation with the highest potency (Ki = 0.71 ± 0.41 nM; n = 4), and its effect was stereospecific, since SSR-149424 failed to block the mitogenic effect of AVP up to 3 µM. dPen was much less potent and antagonized the AVP effect with a Ki of 60 ± 35 nM (n = 3). The V1a receptor antagonist SR-49059 had a weaker potency in blocking cell proliferation (Ki = 411 ± 286 nM; n = 3), and the V2 receptor antagonist SR-121463 failed to inhibit this effect, even at high concentrations (up to 3 µM).
Expression of V1b mRNA in In-R1-G9 cells and in human pancreas. By use of specific primers, V1b receptor RNA expression was studied by PCR analysis in In-R1-G9 cells and human pancreas. A single band of 600 bp was generated from both cell types, in agreement with results obtained with CHO cells expressing human or rat V1b receptors as a positive control. In human pancreas, signal intensity was low after 35-cycle amplification but was easily detectable 10 cycles later (Fig. 5). Amplification of the DNA sequence resulting from genomic contamination of RNA samples can be ruled out, because the primers were designed on two different exons, separated in the human genome by a 5,414-bp intron. Moreover, negative controls were used where RT was omitted during cDNA synthesis. Similar results were obtained with 3 other primer pairs (data not shown).
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Localization of V1b receptors in human pancreas by immunohistochemistry. In preliminary control experiments performed to validate the anti-V1b receptor antibody and to check its specificity, immunolabeling was observed only in CHO cells expressing the V1b receptor but not in wild-type CHO nor in cells transfected with V1a or V2 receptor cDNA (not shown). Further validation of the antibody was performed using reference normal human tissues. As expected, intense staining was observed in human anterior pituitary sections used as a positive control tissue, whereas in liver, an enriched V1a receptor preparation, no labeling was detected (negative control) (Fig. 6, A and B). Under similar operating conditions, strong staining intensity was observed in human pancreas, confined to the endocrine cells of islets of Langerhans (Fig. 6C). Of note, some weak and patchy staining was occasionally observed in exocrine pancreas. Immunoabsorption of the primary V1b receptor antibody with the rat immunogenic peptide sequence, used to raise the antibody, and with the corresponding human sequence peptide, abolished the immunosignal in the tissue sections of human pancreas (Fig. 6D) and pituitary (not shown). Equally, replacement of the primary antibody with buffer eliminated the presence of the immunosignal in both human pancreas and pituitary (not shown), in agreement with the specificity of labeling observed in pancreas and hypophysis.
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Double immunofluorescent detection of V1b receptors in islets of
Langerhans with glucagon, insulin, or somatostatin was performed to further
identify the cellular localization of the V1b receptor in human
endocrine pancreas. As shown in Fig.
7, these experiments revealed colocalization of the
immunofluorescent signals for V1b receptor and for each one of
these hormones (glucagon, insulin, or somatostatin); the colocalization was
evidenced by the yellow/orange color resulting from the superimposition of the
green (anti-V1b receptor antibody) and red (glucagon, insulin, or
somatostatin antibody) colors (Fig. 7,
C, F, and I, respectively). These experiments
revealed the expression of the V1b receptor protein in a
significant number of -glucagon-
(Fig. 7B),
-insulin- (Fig.
7E), and somatostatin-
(Fig. 7H) producing
cells. Similar results were obtained with seven different human pancreas
samples.
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DISCUSSION |
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[3H]AVP binding, performed for the first time on In-R1-G9 cells,
was time dependent and showed high affinity (Ki = 0.27
± 0.07 nM), specificity, and saturability (25 fmol/mg protein, i.e.,
4,000 sites/cell). In competition studies using different peptides with
various selectivities for the AVP/OT receptors and selective nonpeptide
V1a (SR-49059), V1b (SSR-149415), and V2
(SR-121463) receptor antagonists, we observed a potency order consistent with
a typical V1b receptor profile
(Table 1). The
Ki/Kd values obtained are in good
agreement with those reported for the rat and human pituitary V1b
receptor (7,
14,
20,
27,
29). From a quantitative point
of view, Ki values in In-R1-G9 cells are closer to rat
than to human V1b receptors expressed in CHO cells. Of note, OT
exhibited a 20- to 50-fold higher affinity for hamster In-R1-G9 and rat
V1b receptors than for the human ones. This may reflect species
differences very common in the AVP/OT family
(17). It is important to
underline that SSR-149415, a high-affinity V1b receptor antagonist
for rat, bovine, and human
(25), also behaves as a potent
competitive antagonist at pancreatic V1b receptors in hamster
In-R1-G9 cells.
Functional characterization of the V1b receptors identified on
In-R1-G9 cells was further addressed in several in vitro models. Earlier
cellular events provoked by the occupancy of V1b receptors by AVP
include activation of phospholipase C and protein kinase C, inositol
1,4,5-trisphosphate production, and the mobilization of intracellular free
Ca2+, mainly via Gq/11 protein recruitment.
In -cells of the endocrine pancreas, it has been shown that
Ca2+ is a key signal for AVP to trigger glucagon
secretion; of note, Ca2+ transients have been studied
extensively in In-R1-G9 cells
(19,
31). Recently, in CHO cells
transfected with the human V1b receptors, other intracellular
pathways have also been described (e.g., cAMP production, stimulation of DNA
synthesis, and MAP kinase activation), clearly depending on the level of the
V1b receptor expression
(29). Thus we explored these
functions of AVP and V1b receptors in In-R1-G9 cells. Taken
together, a generally good correlation was observed between AVP and OT analog
binding to In-R1-G9 membranes and the physiological responses elicited on
[Ca2+]i increase, glucagon release, and
In-R1-G9 cell proliferation (Table
1). In addition, the use of potent and selective V1a,
V1b, and V2 blockers confirmed the involvement of
V1b receptors in these effects; whatever the model, SSR-149415 has
a powerful effect in antagonizing AVP functional effects, whereas the
V1a antagonist SR-49059 was much less effective and the
V2 blocker SR-121463 was devoid of any effect in counteracting AVP
stimulation. Moreover, SSR-149415 exerted a marked stereospecific inhibition
on both binding and AVP responses, another argument for supporting a specific
V1b-mediated action. In the different tests, AVP induced
dose-dependent stimulation (EC50 values from 0.08 to 18 nM;
Table 1). The least efficacy
was obtained on AVP-induced [Ca2+]i increase
(EC50 = 18 ± 7 nM), suggesting that a threshold occupancy of
V1b receptors is required for full
[Ca2+]i mobilization. Other explanations
involving the operating conditions specific to the test could also explain the
difference between affinity and stimulation of
[Ca2+]i efficacy. A striking finding in this
work is the subnanomolar effi-cacy of AVP in stimulating cell growth, a
property described for the first time in pancreatic In-R1-G9 cells. AVP has
been associated with cell proliferation and mitogenicity in various tissues
and cell lines. Of note, in CHO cells transfected with the human
V1b receptors, Thibonnier et al.
(29) reported stimulation of
DNA synthesis and MAP kinase activation clearly depending on the level of
V1b receptor expression. The presence of V1b receptors
has been also reported in some small-cell lung cancer tumors
(16), and the V1b
(V3) receptor gene is overexpressed in corticotropin-secreting
tumors (8). Thus a potential
role of V1b receptors in pancreatic cell growth or during pancreas
development needs to be explored further.
According to the literature, the role and the mechanism of action of AVP
and OT in the pancreas are a matter of debate; the two hormones AVP and OT and
their receptors are found in the pancreas, and OT (like AVP) has also been
associated with glucagon and insulin release in the perfused rat pancreas
model. It has been suggested that pancreatic -cells may possess AVP
(V1-like/V1b) receptors or that OT receptors could
cross-react with pancreatic AVP receptors. Recent data showed that, in
physiological situations, AVP and OT induced glucagon secretion in the
perfused rat pancreas through activation of V1b and OT receptors,
respectively (32). Experiments
by fluorescent labeling with OT and AVP and [3H]OT in rat pancreas
sections also support the presence of specific OT receptors in
glucagon-producing
-cells of islets of Langerhans in rats
(26). In the present work, by
combining binding with selective ligands, various functional studies using a
specific blocker, and a molecular approach with specific primers directed
against the V1b receptor cDNA, we demonstrated that the
V1b receptor is present in hamster In-R1-G9
-cells and is
responsible for Ca2+ transients, cell proliferation, and
glucagon secretion and that OT has a much lower efficacy than AVP on these
cells. Thus the OT effects observed in In-R1-G9 are mediated by V1b
receptor, and not by OT receptor, activation.
Obviously, the absence of human pancreatic endocrine cell lines available
for cell culture is a major drawback in evaluating the role of V1b
receptors in pancreatic functions in humans, even though we confirmed in this
study the presence of V1b mRNA in both animal and human pancreas.
Therefore, to complete this work, we addressed the expression pattern of the
V1b receptors in normal human pancreas by use of an
immunohistochemistry approach. Colocalization was evidenced in a number of
glucagon, insulin, and somatostatin cells, indicating a wide and specific
distribution of the V1b receptor in endocrine pancreas. Among
various regulatory hormones, insulin and glucagon are the primary agents
responsible for controlling glucose levels in the blood. Diabetes is a
polygenic disease characterized by insulin deficiency and/or insensitivity,
with elevated glucose plasma levels. Of note, the existence of type 1 or type
2 diabetes has been correlated with high plasma levels of glucagon, which
contributes to the hyperglycemia of diabetes by inappropriately stimulating
hepatic glucose secretion (6).
Moreover, in this disease, the glucagon effect is no longer counterbalanced by
the opposing action of insulin. Several studies have also reported that plasma
AVP is elevated in patients with type 1 or 2 diabetes as well as in various
animal models (experimental or genetic) of diabetes, even if this AVP increase
is poorly understood (3). On
the basis of this latter observation and the results of the present report,
one can speculate that, in the diabetic state, AVP could be involved (at least
in part) in the abnormal glucagon secretion by stimulating V1b
receptors present in pancreatic -cells. Interestingly, glucagon
receptor antagonists, which act downstream on the AVP/V1b receptor
pathway, dropped plasma glucose levels in diabetic rats
(6). Obviously, further in vivo
studies need to be performed to support this hypothesis. Similarly, the role
of V1b receptors on insulin secretion needs to be explored. Of
note, it could be of interest to address V1b receptor expression
level and activity, using SSR-149415 in particular, in various animal models
of diabetes such as ob/ob mice, Zucker rats, or specific rat strains
such AVP-deficient Bratlleboro rats to further explore the regulation of
pancreatic function by AVP and V1b receptors.
In conclusion, the pancreatic receptors involved in intracellular Ca2+ increase, cell proliferation, and glucagon release in In-R1-G9 cells fulfill all the criteria of V1b AVP receptors. These receptors are functional and display high affinity for AVP. All these effects are blocked by a selective V1b receptor antagonist such as SSR-149415. The present work also provides new insight into the expression pattern of V1b receptors in human pancreas and indicates a wide distribution of the V1b receptor protein in cells of islets of Langerhans, suggesting a potential control of AVP on endocrine pancreas.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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