Characterization of receptors mediating AVP- and OT-induced
glucagon release from the rat pancreas
Sirintorn
Yibchok-Anun,
Henrique
Cheng,
Patricia A.
Heine, and
Walter H.
Hsu
Department of Biomedical Sciences, Iowa State University, Ames, Iowa
50011
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ABSTRACT |
We characterized the receptors that mediate arginine vasopressin
(AVP)- and oxytocin (OT)-induced glucagon release by use of a number of
antagonists in the perfused rat pancreas and the fluorescence imaging
of the receptors. AVP and OT (3 pM-3 nM) increased glucagon release in
a concentration-dependent manner. The antagonist with potent
V1b receptor-blocking activity,
CL-4-84 (10 nM), abolished AVP (30 pM)-induced glucagon
release but did not alter OT (30 pM)-induced glucagon
release.
d(CH2)5[Tyr(Me)2]AVP
(10 nM), a V1a receptor
antagonist, and L-366,948 (10 nM), a highly specific OT-receptor
antagonist, failed to inhibit AVP-induced glucagon release. In
contrast, L-366,948 (10 nM) abolished OT (30 pM)-induced glucagon
release but did not change the effect of AVP. Fluorescent microscopy of
rat pancreatic sections showed that fluorescence-labeled AVP and OT
bound to their receptors in the islets of Langerhans and that the
bindings were inhibited by 1 µM of Cl-4-84 and L-366,948,
respectively. Because AVP and OT at physiological concentrations
(3-30 pM) increased glucagon release, we conclude that AVP and OT
increase glucagon release under the physiological condition through the
activation of V1b and OT
receptors, respectively.
L-366,948; V1b receptor; oxytocin receptor; perfusion; fluorescence
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INTRODUCTION |
ARGININE VASOPRESSIN (AVP) and oxytocin (OT) are
synthesized in the hypothalamus and secreted from the posterior
pituitary gland. AVP and OT are also found in various tissues,
including ovary, oviduct, follicular fluid (22), adrenal (3), testis (14), thymus (11), and pancreas (2). In addition to the regulation of
fluid homeostasis, AVP induces glycogenolysis (15), proliferation of
the pituitary gland (20) and vascular smooth muscle cells (24),
vasoconstriction (8), and the secretion of catecholamine (10),
glucagon, and insulin (6). The major physiological functions of OT are
to regulate milk ejection and uterine contractions, but it also
increases adrenocorticotropic hormone (ACTH) (23), glucagon, and
insulin release (6). A small concentration of 20 pg/ml of AVP and OT
increased glucagon release, but not insulin release, from the perfused
rat pancreas (6). Moreover, both AVP and OT elicited a
concentration-dependent stimulation of glucagon release but failed to
influence insulin release from rat islets (5). A high density of
[3H]oxytocin binding
was present in the periphery of the islets of Langerhans that
corresponded to the location of pancreatic
-cells (25). Together,
these findings suggest that AVP and OT may play a physiological role in
increasing glucagon release.
AVP receptors have been classified into
V1a,
V1b, and
V2 receptors.
V1a receptors mediate
glycogenolysis (15) and vasoconstriction (8);
V1b receptors mediate the release
of ACTH (4), catecholamines (10), insulin (17), and glucagon (29); and
V2 receptors mediate antidiuresis
(13).
A number of receptor antagonists have been used to pharmacologically
characterize the receptors that mediate the effects of AVP and OT in
many cells and tissues (18), including insulin and glucagon-secreting
cells. AVP and OT can cross-react with each other's receptors; for
example, both AVP and OT induce insulin release through
V1b receptors in the perfused rat
pancreas and the clonal
-cell line RINm5F (17). In
addition, both hormones induce ACTH release through
V1b receptors in the rat
adenohypophysis (4, 23), and lysine vasopressin stimulates
porcine myometrial contractions through OT receptors (30). Similarly,
in our previous study, AVP and OT induced glucagon release by
activating V1b receptors in clonal
-cells In-R1-G9 (29).
In this study, we characterized the receptors that mediate AVP- and
OT-induced glucagon release by using the antagonists that block
V1a,
V1b, and OT receptors,
respectively, from the perfused rat pancreas. In addition, we used
fluorescence-labeled vasopressin (VP) and OT as ligands to detect AVP
and OT receptors in the rat islets. Fluorescence-labeled peptides have
been used to study AVP receptors; for example, fluorescence-labeled AVP
analogs have been used to study
V1a receptors (12) and
V2 receptors (21). From the
results of the present study, we conclude that AVP and OT play a
physiological role in increasing glucagon release through V1b and OT receptors, respectively.
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MATERIALS AND METHODS |
Test agents.
AVP, OT,
d(CH2)5[Tyr(Me)]2AVP,
and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma
Chemical (St. Louis, MO). Phenylac1,D-Tyr(Me)2,Arg6,8,Lys9-amide-vasopressin
(Fluo-VP) and fluo-Lys8-oxytocin
(Fluo-OT) were purchased from Advanced Bioconcept (Quebec, Canada).
Pentobarbital sodium was purchased from Fort Dodge Laboratories (Fort
Dodge, IA).
4-OH-phenacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH2 (CL-4-84) was donated by Dr. Maurice Manning of Medical College of
Ohio (Toledo, OH).
Cyclo-(L-Pro-D-2-naphthyl-Ala-L-Ile-D-pipecolic acid-L-pipecolic
acid-D-His) (L-366,948) was
donated by Merck Research Laboratories (West Point, PA).
125I-labeled glucagon was
purchased from Linco Research (St. Charles, MO). Glucagon antibody was
donated by Dr. Joseph Dunbar of Wayne State University (Detroit, MI),
and glucagon standard was donated by Eli Lilly Laboratories
(Indianapolis, IN).
Pancreatic perfusion.
Male Sprague-Dawley rats weighing 500-650 g were used in the
dose-response experiments, and rats weighing 220-350 g were used in the receptor antagonism experiments. All the rats were born and
grown in our facilities (Laboratory Animal Resource). They were
maintained at 22°C, 40-60% humidity, and a 12:12-h light-dark cycle. The rats were fed ad libitum with Purina chow. The in situ rat
pancreatic perfusion with an open system was performed during the
daytime as previously described (27). Briefly, the rats were
anesthetized with pentobarbital sodium (60 mg/kg ip) and were
maintained at 37°C on a hot plate during the experiment. The celiac
arteries were cannulated with polyvinyl tubing (0.625 mm ID); then the
pancreata were immediately perfused with the Krebs-Ringer bicarbonate
buffer (KRB) supplemented with 20 mM HEPES, 5.5 mM glucose, 1%
dextran, and 0.2% BSA as a basal medium. The KRB was continuously
aerated with 95% O2-5%
CO2 at pH 7.4. The perfusion rate
was 1 ml/min, and the effluent fluid from the portal vein, which was
cannulated with a vinyl tubing (1.12 mm ID), was ~1 ml/min. The rats
were euthanized immediately after the placement of
cannulas and the beginning of the flow. After an equilibration period
of 20 min, the effluent fluid was collected every minute. For the
dose-response experiments, after the baseline period of 10 min, the
perfusate containing AVP or OT (3 pM-3 nM) was administered for 10 min
followed by a washout period with the basal medium for 10 min. For the
antagonism experiments, after the baseline period of 5 min, the
pancreas was pretreated for 10 min with the medium containing one of
the three antagonists: CL-4-84 (1, 3, or 10 nM), an antagonist
with potent V1b blocking activity
(26);
d(CH2)5[Tyr(Me)2]AVP
(10 nM), a V1a receptor antagonist
(19); and L-366,948 (1, 3, or 10 nM), a highly selective OT receptor
antagonist (27). This was followed by the medium containing AVP or OT
(30 pM) and an antagonist for 10 min, and the basal medium for another
10 min for the washout period. The perfusate containing arginine (1 mM)
was administered as a positive control for 5 min at the end of all
experiments. The effluent fractions were kept at 4°C and
subsequently assayed for glucagon by use of radioimmunoassay, following
the procedures provided by Linco Research.
Fluorescence imaging of AVP and OT receptors in pancreatic islets.
The rat pancreas was perfused with KRB, as described in
Pancreatic perfusion, for 5 min to
eliminate the blood inside the pancreas. The perfusion rate was set at
3 ml/min. The pancreas was then collected and cut into small pieces
(~3 × 3 mm2) and frozen
in
80°C isopentane. The frozen tissue was sliced into 17- to
20-µM thickness, mounted on
poly-L-lysine-coated slides, and
kept at
20°C until use. The tissue sections were processed following a protocol provided by the manufacturer (Advanced
Bioconcept). Briefly, the frozen tissue sections were preincubated in
an incubation buffer (50 mM Tris · HCl, 10 mM
MgCl2, 1% BSA, 1 mg/ml
bacitracin, and 0.5 mM PMSF, pH 7.4) containing CL-4-84 (1 or 10 µM), L-366,948 (1 or 10 µM),
d(CH2)5[Tyr(Me)2]AVP
(10 µM), and AVP (10 µM) or OT (10 µM) at 4°C for overnight and incubated with the incubation buffer containing 30 nM of Fluo-VP or
Fluo-OT in the absence or presence of an antagonist or unlabeled AVP or
OT, as indicated in Pancreatic
perfusion, at room temperature for 1 h. After
incubation, the sections were washed 4 times for 60 s in a cold rinsing
buffer (50 mM Tris · HCl and 10 mM
MgCl2, pH 7.4) at 4°C and
air-dried in the dark under a cool stream of air. The fluorescence
bindings were visualized using a fluorescent microscope (Leica DMLB;
Leica Microscopy Systems, Heerbrugg, Switzerland), and photographs were
taken with a 20X lens using the Leica MPS 60-MPS 30 photographic system.
Data expression and statistical analysis.
The effluent concentrations of glucagon were expressed as a percentage
of the baseline level (mean of last 5 baseline values) in means ± SE.
The area under the curve (AUC) for the 10-min treatment period was
calculated using Transforms and Regressions (SigmaPlot 4.0; SPSS,
Chicago, IL). In dose-response experiments, the AUC was expressed as a
percentage of the area of the basal control group. In antagonism
experiments, the AUC was expressed as a percentage of the area of the
AVP or OT control group. Data were analyzed using analysis of variance
(ANOVA) to determine the effect of treatment. Fisher's least
significant difference test was used to determine the difference
between means for which the ANOVA indicated a significant
(P < 0.05)
F ratio.
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RESULTS |
The results in Figs. 1 and
2 show the profile of glucagon release to
AVP and OT (3 pM-3 nM), respectively, together with the basal control
profile, which was obtained by perfusion with KRB alone for 40 min. AVP
and OT (3 pM-3 nM) increased glucagon release from the perfused rat
pancreas in a concentration-dependent manner. Both peptides increased
glucagon release in a biphasic pattern: a peak followed by a sustained
phase or a second peak (for 3 nM AVP and OT), in which the peak was
initiated in <1 min and reached the maximum within 2 min. AVP (3 pM-3
nM) induced a maximum increase in glucagon release by 2.5, 8, 12, and
10-fold, respectively, over the basal control group. The sustained
glucagon release induced by AVP (3-300 pM) was ~2- to 3-fold
that of the basal control group, and the second peak of glucagon
release induced by 3 nM AVP was 9-fold over that of the basal control
group (Fig. 1). At the highest concentration of AVP studied (3 nM), the
flow rate in the portal vein was decreased by ~20%, presumably
because of vasoconstriction, but the glucagon response was not delayed
or reduced to any extent. The OT (3 pM-3 nM)-induced maximum increases in glucagon release were 3, 7, 14, and 11-fold, respectively, over that
of the basal control group. The sustained glucagon release by
3-300 pM OT was ~2-fold, and the second peak induced by 3 nM OT
was 4-fold that of the basal control group (Fig. 2). The effluent glucagon concentrations returned to the baseline on removal of AVP and
OT (during the washing period) and increased to ~5- to 14-fold of the
baseline value on administration of 1 mM arginine. By comparison of the
AUCs, there were no significant differences between AVP and OT
(3-300 pM)-induced glucagon release. At 3 nM, AVP-induced glucagon
release was significantly different from that of OT. However, the
difference was only in the sustained phase (Fig.
3). The
EC50 of OT was 8.9 ± 2.9 pM, and
the EC50 of AVP was estimated to
be 25.1 ± 11.3 pM, because the maximum glucagon release was not
acquired in the AVP dose-response experiment.

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Fig. 1.
Effect of arginine vasopressin (AVP, 3 pM-3 nM) on glucagon release
from perfused rat pancreas. In these experiments, a 20-min
equilibration period preceded time 0.
AVP was administered for 10 min (heavy line). Values are means ± SE;
n = 3. , Basal control; , AVP 3 pM; , AVP 30 pM; , AVP 300 pM; , AVP 3 nM. Range of baseline
glucagon concentrations of effluents was 24-106 pg/ml.
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Fig. 2.
Effect of oxytocin (OT, 3 pM-3 nM) on glucagon release from perfused
rat pancreas. In these experiments, a 20-min equilibration period
preceded time 0. OT was administered
for 10 min (heavy line). Values are means ± SE;
n = 3. , Basal control; , OT 3 pM ; , OT 30 pM; , OT 300 pM; , OT 3 nM. Range of baseline
glucagon concentrations of effluents was 35-182 pg/ml.
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Fig. 3.
Effects of AVP- and OT-induced glucagon release from perfused rat
pancreas. Values are means ± SE; (n = 3), obtained by calculating areas under 10-min glucagon release curve
and expressed as a percentage of control group. * P < 0.05 vs. control group.
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AVP and OT at 30 pM were used in the antagonism experiments because of
the submaximal increase in glucagon release by the two peptides. At 30 pM, AVP and OT induced about four- and twofold increases in the peak
and the sustained phases, respectively, compared with the basal control
group. CL-4-84 (1, 3, and 10 nM), an antagonist with
V1a/V1b
blocking activity, inhibited AVP (30 pM)-induced glucagon release in a
concentration-dependent manner (Fig. 4). By
comparison of the AUCs, CL-4-84 (3 and 10 nM) significantly reduced AVP-induced glucagon release with an
IC50 of 2.2 ± 0.1 nM.
Pretreatment with CL-4-84 (10 nM) abolished AVP-induced glucagon release and even lowered glucagon to the levels below the baseline. However,
d(CH2)5[Tyr(Me)2]AVP
(10 nM), a V1a receptor
antagonist, and L-366,948 (10 nM), a highly specific OT receptor
antagonist, failed to inhibit AVP-induced glucagon release (Fig.
5). In contrast, L-366,948 (1, 3, and 10 nM) inhibited OT (30 pM)-induced glucagon release in a
concentration-dependent manner (Fig. 6). By
comparison of the AUCs, 3 nM L-366,948 significantly lowered, and 10 nM
L-366,948 abolished OT-induced glucagon release. The
IC50 of L-366,948 was 3 ± 0.3 nM. CL-4-84 (10 nM), the receptor antagonist with
V1a/V1b
blocking activity, did not significantly reduce OT (30 pM)-induced
glucagon release (Fig. 7). None of the receptor antagonists alone significantly changed glucagon release.

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Fig. 4.
Effect of CL-4-84 (1, 3, and 10 nM) on AVP-induced glucagon
release from perfused rat pancreas. After baseline period of 5 min,
CL-4-84 was administered for 10 min, followed by AVP (30 pM) in
the presence of CL-4-84 for another 10 min. Heavy lines show
treatments as indicated above them. Values are means ± SE;
n = 3. , Basal control; , AVP 30 pM; , CL-4-84 1 nM + AVP 30 pM; , CL-4-84 3 nM + AVP 30 pM; , CL-4-84 10 nM + AVP 30 pM. Range of baseline glucagon
concentrations of effluents was 32-225 pg/ml.
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Fig. 5.
Effects of
d(CH2)5[Tyr(Me)2]AVP
and L-366,948 (10 nM) on AVP-induced glucagon release from perfused rat
pancreas. After baseline period of 5 min, antagonist was administered
for 10 min, followed by AVP (30 pM) in the presence of antagonist for
another 10 min. Heavy lines show treatments as indicated. Values are
means ± SE; n = 3. , Basal
control; , AVP 30 pM; ,
d(CH2)5[Tyr(Me)2]AVP
10 nM + AVP 30 pM; , L-366,948 10nM + AVP 30 pM. Range of baseline
glucagon concentrations of effluents was 32-139 pg/ml.
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Fig. 6.
Effects of L-366,948 (1, 3, and 10 nM) on OT-induced glucagon release
from perfused rat pancreas. After baseline period of 5 min, L-366,948
was administered for 10 min, followed by OT (30 pM) in the presence of
L-366,948 for another 10 min. Heavy lines show treatments as indicated.
Values are means ± SE; n = 3. ,
Basal control; , OT 30 pM; , L-366,948 1 nM + OT 30 pM; ,
L-366,948 3 nM + OT 30 pM; , L-366,948 10 nM + OT 30 pM. Range of
baseline glucagon concentrations of effluents was 32-175 pg/ml.
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Fig. 7.
Effect of CL-4-84 (10 nM) on OT-induced glucagon release from
perfused rat pancreas. After baseline period of 5 min, CL-4-84 was
administered for 10 min followed by OT (30 pM) in the presence of
CL-4-84 for another 10 min. Heavy lines show treatments as
indicated. Values are means ± SE; n = 3. , Basal control; , OT 30 pM; , CL-4-84 10 nM + OT 30 pM. Range of baseline glucagon concentrations of effluents was
32-139 pg/ml.
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The results in Figs. 8 and
9 show the fluorescence imaging of AVP and
OT receptors in the rat islets. Fluorescent microscopic examination of
the pancreatic sections incubated with either Fluo-VP or Fluo-OT
revealed selective fluorescence labeling of AVP and OT receptors
expressed in the rat pancreatic islets (Figs.
8B and
9B) compared with the negative
control (Figs. 8A and
9A). The binding was specific
because the fluorescence was no longer detectable when the incubation
was performed in the presence of 10 µM VP or OT (Figs.
8C and
9C). The fluorescence labeling of Fluo-VP was selective for V1b
receptors because it was blocked by preincubation of the tissue
sections with 1 µM CL-4-84 (Fig. 8D), but not by 10 µM L-366,948
(Fig. 8E) or 10 µM
d(CH2)5[Tyr(Me)2]AVP
(Fig. 8F). The fluorescence labeling
of Fluo-OT was selective for OT receptors expressed in the rat
pancreatic islets, because it was blocked by preincubation of the
tissue sections with 1 µM L-366,948 (Fig.
9D) but not by 10 µM CL-4-84
(Fig. 9E).

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Fig. 8.
Fluorescent microscopic images of islets of Langerhans in rat
pancreatic sections labeled with Fluo-VP. Photographs were taken under
a 20X lens. Sections were incubated with an incubation buffer as
negative control (A), 30 nM Fluo-VP
(B), 10 µM unlabeled AVP + 30 nM Fluo-VP (C), 1 µM
CL-4-84 + 30 nM Fluo-VP (D), 10 µM L-366,948 + 30 nM Fluo-VP (E),
and 10 µM
d(CH2)5[Tyr(Me)2]AVP + 30 nM Fluo-VP (F) for 1 h at room
temperature. Section was preincubated with buffer, AVP, or an
antagonist at 4°C for overnight. Data shown are representative of 3 rat pancreata.
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Fig. 9.
Fluorescent microscopic images of islets of Langerhans in rat
pancreatic sections labeled with Fluo-OT. Photographs were taken under
a 20X lens. Sections were incubated with an incubation buffer as
negative control (A), 30 nM Fluo-OT
(B), 10 µM unlabeled OT + 30 nM Fluo-OT (C), 1 µM
L-366,948 + 30 nM Fluo-OT (D), and
10 µM CL-4-84 + 30 nM Fluo-OT
(E) for 1 h at room temperature.
Section was preincubated with buffer, OT, or an antagonist at 4°C
for overnight. Data shown are representative of 3 rat pancreata.
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DISCUSSION |
In the present study, AVP and OT (3 pM-3 nM) evoked glucagon release
from the perfused rat pancreas in a concentration-dependent manner, in
which AVP and OT at 3 and 30 pM increased glucagon release by about
three- and eightfold, respectively. These findings indicated that AVP
and OT may physiologically have increased glucagon release, because the
concentrations of AVP and OT studied (3 and 30 pM) are similar to the
plasma concentrations of AVP (3-20 pM) (9) and OT (8-25 pM)
in the rat (16). This statement is supported by the findings that a
neural lobe extract evoked glucagon release (5) and a rise in plasma
glucagon concentrations of the rats subjected to hemorrhage that was
found to be mediated by an increase in the release of AVP and OT (7).
In addition, AVP and OT are present in human and rat pancreatic
extracts, suggesting that both peptides are synthesized in the pancreas
and thus could exert a paracrine function on pancreatic hormone release
(2).
The increase in glucagon release mediated by AVP and OT (30 pM) was
higher in the larger rats (500-650 g, ~1 yr old, used in the
dose-response experiments) than in the smaller rats (220-350 g,
2-3 mo old, used in the antagonism experiments). We speculate that
the pancreata of the larger (or older) rats express more V1b and OT receptors or have a
more active signal transduction system for these receptors than the
smaller (or younger) rats. More work is needed to find out why these
peptides evoke more glucagon release in larger (older) rats than in
smaller (younger) rats.
CL-4-84 is an antagonist with high affinity for both
V1a [inhibitory constant
(Ki) = 0.45 ± 0.04 nM] and V1b
(Ki = 2.2 ± 0.1 nM) receptors (26). It is also a weak OT antagonist
[antagonistic affinity
(pA2) = 7.38 ± 0.06] (19).
d(CH2)5[Tyr(Me)2]AVP
is a potent and selective V1a
receptor antagonist (pA2 = 8.62)
(18). L-366,948 is a highly selective OT receptor antagonist, which is
>400 times more selective for OT receptors than for
V1a and
V2 receptors (27). We also
confirmed the results from pancreatic perfusion by detecting these
receptors by use of fluorescence labeling with VP and OT. We found that
Fluo-VP and Fluo-OT selectively bound to
V1b and OT receptors,
respectively, in the rat islets. The labels of Fluo-VP and Fluo-OT were
seen in the entire islets, an observation suggesting that both
V1b and OT receptors are expressed in pancreatic
- and
-cells, among others. In addition, in
perfused rat pancreata, we found that 0.3 nM OT increased insulin
release about threefold over the basal insulin level and that this
increase was antagonized by 3 nM L-366,948 (unpublished data). The
present finding is different from the previous one from our laboratory, in which 100 nM OT induced insulin release from rat perfused pancreas by activating V1b receptors (17).
Moreover, in an autoradiographic binding study of the rat pancreas, a
high density of
[3H]oxytocin binding
was found in the periphery of the islets, which corresponded to the
localization of
-cells (25). Thus the lower concentration of OT (0.3 nM) may induce insulin release by activating OT receptors, whereas the
higher concentration of OT (100 nM) may induce insulin release by
activating V1b receptors (17).
Our present findings suggest that AVP evokes glucagon release by
activating V1b receptors in
-cells of the rat pancreas, which is similar to AVP-induced ACTH
release from the rat adenohypophysis (4), catecholamine release from
the rat adrenal medulla (10), and glucagon release from the hamster
glucagonoma In-R1-G9 cells (29). However, in the perfused rat pancreas,
OT evoked glucagon release by activating OT but not AVP receptors. In
the dog, OT has been shown to increase plasma levels of glucose,
insulin, and glucagon and to increase the rate of glucose production
and uptake by activating OT receptors (1). These results differ from
those of ours in In-R1-G9 cells, in which OT increased glucagon release
through V1b receptors (29). In
addition, AVP and OT increased ACTH release through
V1b receptors (4, 23). Although AVP and OT induced glucagon release by activating different receptors, our preliminary data showed that there was no synergism between these
two peptides (unpublished data). The action of AVP on glucagon release
exerted an inverse relationship with glucose concentrations; in the
presence of 1.4 mM glucose, AVP (3 pM)-induced glucagon release was
significantly higher than that in the presence of 5.5 mM glucose
(unpublished data).
By comparison of the responses at the same concentration of AVP and OT,
the potencies of both peptides were similar in the perfused rat
pancreas, with the exception that 3 nM of AVP evoked a significantly
higher increase in glucagon release than 3 nM of OT. This finding
differs from that of our previous study, in which OT-induced glucagon
release in In-R1-G9 cells was ~30-fold less potent than AVP (29).
Apparently, OT receptors are not expressed in clonal In-R1-G9
-cells. We also confirmed these findings by detecting AVP and OT
receptors in In-R1-G9 cells by use of fluorescence-labeled VP and OT.
We found that 30 nM of both Fluo-VP and Fluo-OT bound to
V1b receptors on the cell
membrane because the bindings were blocked by 30 nM
CL-4-84 but not by 300 nM L-366,948 or
d(CH2)5[Tyr(Me)2]AVP
(unpublished data). OT, therefore, increases glucagon release from
In-R1-G9 cells by activating V1b
receptors (29). Based on these findings, we conclude that In-R1-G9
cells are not an adequate model for the study of OT-induced glucagon release.
Our present findings suggest that AVP and OT increase glucagon release
under the physiological condition by activating
V1b and OT receptors,
respectively. Because specific V1b
receptor antagonists are currently unavailable for the characterization of these receptors, further studies utilizing molecular approaches are
warranted to confirm our present findings.
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ACKNOWLEDGEMENTS |
We thank the Royal Thai Government for partial support of this
study; Dr. Maurice Manning of the Medical College of Ohio (Toledo, OH)
for providing CL-4-84; Dr. Joseph Dunbar of Wayne State University (Detroit, MI) for providing antiserum against glucagon; Merck Research
Laboratories (West Point, PA) for donating L-366,948; Eli Lilly
Laboratories (Indianapolis, IN) for donating glucagon; and Cathy
Martens, Donna Lester, and Laverne Escher for technical assistance.
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FOOTNOTES |
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: W. H. Hsu, Dept. of Biomedical Sciences,
Iowa State Univ., Ames, IA 50011-1250 (E-mail:
whsu{at}iastate.edu).
Received 23 September 1998; accepted in final form 18 March 1999.
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