Glucagon-mediated Ca2+
signaling in BHK cells expressing cloned human glucagon
receptors
Lars H.
Hansen1,
Jesper
Gromada2,
Pierre
Bouchelouche3,
Ted
Whitmore4,
Laura
Jelinek4,
Wayne
Kindsvogel4, and
Erica
Nishimura1
1 Department of Molecular
Signaling, Hagedorn Research Institute, DK-2820 Gentofte;
2 Islet Cell Physiology, Novo
Nordisk A/S, DK-2100 Copenhagen;
3 Department of Clinical
Biochemistry, Roskilde County Hospital, DK-4600 Køge, Denmark;
and 4 ZymoGenetics Inc.,
Seattle, Washington 98102
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ABSTRACT |
From video imaging of fura 2-loaded baby hamster kidney (BHK)
cells stably expressing the cloned human glucagon receptor, we found
the Ca2+ response to glucagon to
be specific, dose dependent, synchronous, sensitive to pertussis toxin,
and independent of Ca2+ influx.
Forskolin did not elicit a Ca2+
response, but treatment with a protein kinase A inhibitor, the Rp diastereomer of 8-bromoadenosine-3',5'-cyclic
monophosphothioate, resulted in a reduced glucagon-mediated
Ca2+ response as well as
Ca2+ oscillations. The specific
phospholipase C inhibitor U-73122 abolished the
Ca2+ response to glucagon, and a
modest twofold increase in inositol trisphosphate
(IP3) production could be
observed after stimulation with glucagon. In BHK cells coexpressing
glucagon and muscarinic (M1)
acetylcholine receptors, carbachol blocked the rise in intracellular free Ca2+ concentrations in
response to glucagon, whereas glucagon did not affect the
carbachol-induced increase in
Ca2+. Furthermore, carbachol, but
not glucagon, could block thapsigargin-activated increases in
intracellular free Ca2+
concentration. These results indicate that, in BHK cells, glucagon receptors can activate not only adenylate cyclase but also a second independent G protein-coupled pathway that leads to the stimulation of
phospholipase C and the release of
Ca2+ from
IP3-sensitive intracellular
Ca2+ stores. Finally, we provide
evidence to suggest that cAMP potentiates the
IP3-mediated effects on
intracellular Ca2+ handling.
phospholipase C; adenosine 3',5'-cyclic monophosphate; G proteins; inositol trisphosphate; intracellular calcium stores; baby
hamster kidney cells
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INTRODUCTION |
GLUCAGON, A PANCREATIC HORMONE that plays an important
role in maintaining glucose homeostasis, is secreted from the
-cells in response to low blood glucose levels and stimulates hepatic glucose
output by increasing glycogenolysis and gluconeogenesis while at the
same time inhibiting glycolysis (5). In addition to its metabolic
actions in the liver, glucagon is also involved in the regulation of
adipose, cardiac, renal, gastrointestinal, and pancreatic functions,
including the potentiation of glucose-induced insulin secretion (5).
These effects are mediated by specific glucagon receptors, which have
been shown to have a wide tissue distribution corresponding to the
observed multiple functions of glucagon (12).
The glucagon receptor belongs to the superfamily of seven
transmembrane-spanning receptors that couple to heterotrimeric guanine nucleotide-binding proteins (G proteins). Furthermore, on the basis of
structural homology, glucagon receptors, together with those for
glucagon-like peptide 1 (GLP-1), gastric inhibitory polypeptide (GIP), secretin, vasoactive intestinal
polypeptide (VIP), growth hormone-releasing factor,
corticotropin-releasing factor, pituitary adenylate cyclase-activating
polypeptide, parathyroid hormone, and calcitonin (5, 18), form a
subfamily of closely related receptors that is now emerging as a group
of G protein-coupled receptors able to activate multiple signaling
pathways. All these receptors are able to stimulate adenylate cyclase,
but, in addition, many have been found to activate alternative
intracellular second messengers. For example, stimulation of the
parathyroid hormone receptor leads to intracellular accumulation of
cAMP, inositol trisphosphates
(IP3), and
Ca2+ (1), and splice variants of
the pituitary adenylate cyclase-activating polypeptide receptor are
able to differentially couple to adenylate cyclase and phospholipase C
(23). The cloned receptors for calcitonin (7), GLP-1 (9, 28), GIP (29),
and glucagon (16) have been shown to stimulate cAMP production and a
rise in intracellular free Ca2+
concentration
([Ca2+]i).
It had long been postulated that the cellular effects of glucagon are
mediated not only by cAMP but also by
[Ca2+]i,
since it has been reported that, in perfused rat liver or cultured
hepatocytes, glucagon is able to increase the
[Ca2+]i
by inducing a Ca2+ influx as well
as by stimulating the release of intracellular Ca2+ stores (3, 6, 21). However,
the mediator(s) of this rise in
[Ca2+]i
remains to be determined, particularly since there appears to be some
controversy as to whether this
Ca2+ response to glucagon is a
cAMP-mediated effect or a result of a separate signaling pathway.
Experiments demonstrating that the effects of glucagon on
Ca2+ mobilization in rat liver
cells can be reproduced by cAMP analogs have led some investigators to
conclude that the glucagon-mediated rise in
[Ca2+]i
is mediated by cAMP (8, 24, 25). In contrast, others have reported
cAMP-independent effects of glucagon on intracellular Ca2+ mobilization (22, 27). On the
basis of their studies demonstrating that TH-glucagon, a biologically
active glucagon analog that is unable to activate adenylate cyclase,
could stimulate inositol phosphate production in rat hepatocytes,
Wakelam et al. (27) had proposed the possible existence of two distinct
hepatic glucagon receptors: one that couples to adenylate cyclase and
the other to the breakdown of inositol phospholipids. However, we have
since demonstrated that the cloned rat hepatic glucagon receptor is capable of mediating both a cAMP and a
Ca2+ response (16).
In the current study we have used baby hamster kidney (BHK) cells
expressing the cloned human glucagon receptor (19) to further
characterize our original observation that the glucagon receptor is
able to stimulate an increase in
[Ca2+]i.
By video imaging of fura 2-loaded cells, we have monitored the
Ca2+ response to glucagon and have
addressed the questions as to the source of the glucagon-mediated rise
in
[Ca2+]i
and the possible molecular mechanisms involved. In view of glucagon's
central role in regulating glucose metabolism and our previous findings
that a mutated glucagon receptor may in some manner contribute to the
development of non-insulin-dependent diabetes (10, 11), the
characterization of the molecular signaling pathways activated by
glucagon is not only important for understanding the normal
physiological mechanisms involved in glucagon action in target tissues
such as the liver, fat, or pancreas, but it may also lead to the
identification of defects in glucagon signal transduction that may
occur in diabetes.
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MATERIALS AND METHODS |
Cell culture and transfections.
BHK cells were cultured in DMEM supplemented with 10% heat-inactivated
FCS, 100 U/ml penicillin, 10 µg/ml streptomycin, and 2 mM
L-glutamine at
37°C in 5% CO2-95% air. The
transfection of BHK cells with the human glucagon receptor cDNA pLJ6
(19) was carried out using the Liptofectamine reagent, as previously
outlined in detail (11). The clones analyzed in the present study have been characterized previously for the level of glucagon receptor expression (~3 × 106
receptors/cell) and for their ability to bind
125I-labeled glucagon
(dissociation constant ~10 nM) as well as stimulate cAMP production
in response to glucagon (EC50 ~2
pM) (11). For the BHK cells coexpressing the glucagon and muscarinic
(M1) receptors, the glucagon
receptor-expressing BHK cells were transfected with the
M1 cholinergic receptor plasmid
pM1-R by use of the calcium-phosphate method (20).
[Ca2+]i
measurements.
BHK cells (~3 × 104),
seeded onto thin, circular coverslips (22 mm) that had been precoated
with poly-D-lysine (10 µg/ml), were cultured for 3 days. Before
Ca2+ measurements, the cells were
loaded with 7 µM fura 2-AM in the presence of the nonionic detergent
Pluronic F-127 (25%) at 37°C for 30 min, and then an equal volume
of HEPES-buffered RPMI culture medium was added. The cells were then
incubated for a further 30 min to allow complete deesterification of
fura 2. Subsequently, the cells were washed three times in NaCl-HEPES
buffer (in mM: 145 NaCl, 5 KCl, 1 Na2HPO4,
0.5 Mg2SO4,
20 HEPES, 5 glucose, 1 CaCl2, pH
7.5). Finally, the coverslips were mounted in a recording chamber and
placed on an inverted microscope (model 135 TU, Zeis axioscope,
Oberkochen, Germany). The recording chamber was continuously perfused
with NaCl-HEPES buffer supplemented with 1% BSA at 1.5-2 ml/min.
All test compounds were applied through the perfusate to give the
concentrations indicated. For experiments that required Ca2+-free medium, the
CaCl2 in the NaCl-HEPES buffer was
replaced with 1 mM EGTA.
The
[Ca2+]i
measurements were performed essentially as outlined previously (4, 9,
26). Briefly, the cells were illuminated with alternating wavelengths
(340 and 380 nm) by using interference filters mounted on a filter
wheel and observed through a 510-nm emission filter. The images were
captured in real time with a low-light-level intensified charge-coupled
devise videocamera system. The system was calibrated for free
Ca2+ concentrations by use of the
formula
where
Kd is the
dissociation constant of the dye (225 nM for fura 2),
is the
proportionality coefficient (4), and R is the wavelength
ratio. Calibrations of the fura 2 fluorescence signal were
performed by dialyzing single cells with 100 µM fura 2 pentapotassium
salt and Ca2+-EGTA buffers with
free Ca2+ concentrations ranging
from 0 to 39.8 µM. Thus
R340/380 max was determined under saturating Ca2+
concentrations, whereas
R340/380 min was measured
for the same field under Ca2+-free
conditions. The ratio images
(R340/R380)
were obtained by dividing the 340-nm images by the 380-nm image on a
pixel-by-pixel basis, whereas the numeric
R340/R380
values were calculated after the pixel gray values obtained for each
wavelength were averaged. Ratio values were then converted to
Ca2+ concentrations by use of the
calibration curve, and the background (field without cells) was
subtracted.
For the experiments shown in Table 1 and
Fig. 1, the recordings were made using the Tardis imaging
software (Applied Imaging, Tyne & Wear, UK). For the toxin
sensitivity studies, the cells were pretreated with 10 µg/ml
pertussis or cholera toxin for 24 h at 37°C in the culture medium.
The recordings in Figs. 3, 4, and 6-8 were made using a
fluorescence imaging system (IonOptix, Milton, MA).
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Table 1.
Toxin sensitivity and specificity of glucagon-induced increase in
[Ca2+]i in BHK cells stably
transfected with human glucagon receptor cDNA
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Fig. 1.
Glucagon-induced mobilization of
Ca2+ in baby hamster kidney (BHK)
cells expressing cloned human glucagon receptors. Each panel represents
pseudocolor ratio images of fura 2-labeled cells obtained at time of
peak response to concentration of glucagon (in nM) indicated at
bottom left. Colors represent range of
free Ca2+ concentrations according
to scale. Original magnification, ×300.
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IP3 determinations.
BHK cells transfected with the human glucagon receptor or the human
M1 acetylcholine receptor were
seeded (5 × 104 cells) into
60-mm tissue culture dishes and allowed to grow for 3-4 days to
~90% confluency. For an estimate of cell number, the cells from two
dishes were counted for each cell line. The cells were then incubated
for
1 h in DMEM culture medium containing 10 mM
myo-inositol and for an additional 30 min in the presence of 10 mM
myo-inositol and 10 mM
LiCl2 in DMEM. Cells were
subsequently stimulated for 30 s with various concentrations of
glucagon or carbachol prepared in DMEM containing 1% BSA. The
reactions were terminated by the addition of 2 ml of ice-cold 15% TCA.
The cells were then scraped off and transferred onto ice for 20 min,
and then they were centrifuged for 15 min at 250 g at 4°C. The supernatants were
then transferred to polypropylene tubes containing 10 µl of 5 mM
EDTA. To the remaining pellets 0.5 ml of 15% TCA was added, and the
pellets were incubated for an additional 20 min on ice. The resulting
supernatant was combined with the first and extracted four times with
one volume of diethyl ether (H2O
saturated). The aqueous phase was freeze-dried, resuspended in 2 ml of
H2O, and adjusted to pH 7.5 with 1 M NaHCO3.
Amersham's
D-myo-inositol
1,4,5-trisphosphate (IP3)
3H assay system was used to
measure IP3 in the BHK cell
extracts according to the manufacturer's instructions. For each cell
extract, duplicate determinations of a 1:10 dilution were assayed. The
samples were counted for 4 min in a beta scintillation counter, and the
amount of IP3 in each sample was
determined by interpolation from the standard curve.
cAMP measurements.
Cells were seeded out in six-well plates (3 × 105 cells/well) and cultured
overnight. Before stimulation the cells were washed once in Hanks'
balanced salt solution and again in RPMI 1640 medium, and finally in 1 ml of RPMI medium supplemented with 0.5% fetal bovine serum and 0.45 mM IBMX was added to each well. The cells were stimulated with glucagon
and carbachol, and after 20 min of incubation at 37°C, 1 ml of 65%
ethanol was added to each well. The cells were scraped off and
transferred to Eppendorf tubes, which were then centrifuged at 300 g for 15 min. The resulting supernatants were dried down overnight in a Speed-Vac and stored at
20°C until they were assayed. The cAMP concentrations were determined using the cAMP 125I
scintillation proximity assay (Amersham). The dried cell extracts were
resuspended in 1 ml of assay buffer and diluted 1:150. For the assay
the acetylation protocol described by the manufacturer was followed,
and the cAMP determinations were normalized to cell number.
Reagents.
All tissue culture flasks and dishes were from Nunc (Roskilde,
Denmark). Thin (0.2-mm), circular (22-mm-diameter) glass coverslips were purchased from Struers Kebo Lab (Albertslund, Denmark). Cell culture medium and components, FCS, and Lipofectamine reagent were from
GIBCO BRL (Paisley, Scotland).
Poly-D-lysine, forskolin, pertussis toxin, cholera toxin, thimerosal, EGTA,
LaCl3, carbachol, and
myo-inositol were purchased from Sigma
Chemical (St. Louis, MO). Glucagon was obtained from Novo Nordisk
(Bagsvaerd, Denmark); GLP-1-(7
36) amide and VIP were from Peninsula
Laboratories (Belmont, CA). The cAMP antagonist, an Rp
diastereomer of 8-bromoadenosine-3',5'-cyclic monophosphothioate (Rp-8-BrcAMPS), and the cAMP agonistic
Sp diastereomer of adenosine 3',5'-cyclic
monophosphothioate (Sp-8-BrcAMPS) were obtained from BIOLOG
Life Science Institute (Bremen, Germany). Ryanodine was from Alomone
Labs (Jerusalem, Israel). The phospholipase C inhibitor U-73122 and its
inactive analog U-73343 were obtained from Calbiochem (Bad Soden,
Germany). Fura 2-AM and fura 2 pentapotassium salt were from Molecular
Probes (Eugene, OR). The IP3
3H assay system and cAMP
125I scintillation proximity assay
system (dual range) from Amersham (Little Chalfont, England) were used
for IP3 and cAMP measurements.
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RESULTS |
Specific, dose-dependent rise in
[Ca2+]i
mediated by glucagon.
In BHK cells stably expressing the cloned human glucagon receptor, we
found that stimulation by glucagon results in a transient, synchronized, and dose-dependent rise in
[Ca2+]i
(Figs. 1 and 2). The response is rapid and
recovers to prestimulus levels within 2-3 min. As can be noted in
Fig. 1, it is not only the amplitude of the response to glucagon that
increases in a dose-dependent manner but also the number of responding
cells rises with increasing concentrations of glucagon, such that at high levels (
25 nM) all cells respond. Although the amplitude of the
Ca2+ response to glucagon varies
from cell to cell, it appears that all responding cells do so in a
synchronous manner. There is a delay in the response that becomes
progressively less when higher concentrations of glucagon are used;
there is a 75 ± 3 s lapse from the time 5 nM glucagon is introduced
to the time the rise in
[Ca2+]i
peaks, whereas the delay is only 53 ± 1, 38 ± 1, and 31 ± 2 s for 10, 25, and 50 nM glucagon, respectively
(n = 6). The observed effects of glucagon on the increase in
[Ca2+]i
appear to be specific, mediated by the binding and activation of
specific glucagon receptors, since the related hormones, GLP-1-(7
36) amide and VIP, were not able to elicit a rise in
[Ca2+]i
in these cells (Table 1). Finally, in nontransfected BHK cells, no
Ca2+ response to glucagon could be
observed.

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Fig. 2.
Concentration-dependent rise in intracellular free
Ca2+ concentration
([Ca2+]i)
in response to glucagon in BHK cells expressing human glucagon
receptors. Increase in
[Ca2+]i
in response to glucagon was determined as difference between peak and
baseline Ca2+ values and is
represented as means ± SD from data obtained from 24 cells from at
least 3 different experiments.
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Toxin sensitivity of the glucagon-mediated rise in
[Ca2+]i.
The glucagon-induced rise in
[Ca2+]i
was found to be mediated by the activation of a pertussis
toxin-sensitive G protein, since pretreatment of the human glucagon
receptor expressing BHK cells with pertussis toxin resulted in the near
ablation of the Ca2+ response to
glucagon (Table 1). Under identical conditions, pertussis toxin had no
effect on the carbachol-stimulated
Ca2+ response in BHK cells
expressing the M1 cholinergic
receptors. In contrast, a glucagon-mediated rise in
[Ca2+]i
was still observed in cells pretreated with cholera toxin, although the
amplitude of the response was decreased in comparison to that observed
in nontreated cells (Table 1).
Source of the glucagon-mediated rise in
[Ca2+]i.
To determine the source of the observed increase in
[Ca2+]i,
we examined the Ca2+ response to
glucagon in the absence of extracellular free
Ca2+, which was achieved by using
Ca2+-free medium to which was
added a further 1 mM EGTA. Under these conditions, glucagon was still
able to elicit a robust rise in [Ca2+]i
of the same magnitude as obtained in the presence of extracellular Ca2+ (Fig.
3, A and
B), suggesting that this transient
rise in
[Ca2+]i
is a result of the release of intracellular
Ca2+ stores. This is also
confirmed by our finding that
LaCl3, a general Ca2+ channel blocker, did not
affect the Ca2+ response to
glucagon (Fig. 3C).

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Fig. 3.
Characterization of glucagon-induced rise in
[Ca2+]i
in BHK cells expressing human glucagon receptors.
A: stimulation with 200 nM glucagon
over time period covered by horizontal line (control).
B: stimulation with 200 nM
glucagon in Ca2+-free medium
supplemented with 1 mM EGTA. C:
stimulation with 200 nM glucagon in presence of 0.6 mM
LaCl3. Each trace represents
response in a single cell and is representative of at least 30 single-cell recordings from 3 different experiments.
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Mechanisms involved in the glucagon-mediated rise in
[Ca2+]i.
Binding of glucagon to its receptors results in activation of
heterotrimeric G proteins (Gs
subtype), stimulating the production of cAMP, which ultimately
functions as the second messenger mediating many of glucagon's
cellular effects. However, because it has also been reported that
stimulation of hepatocytes with glucagon leads to activation of
phospholipase C and production of
IP3, we have examined the putative
role of this second messenger in glucagon receptor signaling. As
evident from Fig.
4A,
incubation of the cells with the phospholipase C inhibitor U-73122
completely abolished the ability of glucagon to mobilize intracellular
Ca2+. The inactive isoform of the
inhibitor U-73343 was without effect (Fig.
4B), indicating that activation of
phospholipase C is indeed involved in the glucagon-induced
Ca2+ response. Furthermore,
incubation of the cells with 50 µM ryanodine had no effect on the
Ca2+ response to subsequent
stimulation with glucagon (Fig. 4C).
These findings are consistent with our observation that glucagon can stimulate a twofold increase in
IP3 production (Fig.
5A).
This response, however, appears to be relatively modest compared with the 100-fold increase obtained when carbachol is used to stimulate BHK
cells expressing M1 cholinergic
receptors, which are known to activate phospholipase C, resulting in
the formation of IP3 and release
of IP3-sensitive intracellular
Ca2+ stores. Figure
5B also shows that stimulation with
carbachol does not lead to any concomitant increase in cAMP production, indicating that signaling by the
M1 receptors, when transfected into BHK cells, is essentially normal. The effectiveness of the phospholipase C inhibitor U-73122 is illustrated in Fig.
5C, where treatment of the cells with
this agent (15 µM) decreases the carbachol-mediated IP3 response by 67.5 ± 7.8% (n = 4) compared with
untreated cells. In addition, the specificity of U-73122 can be seen in
Fig. 5D, where this inhibitor was
found to have no effect on adenylate cyclase activity as determined by
the accumulation of cAMP in response to glucagon.

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Fig. 4.
Mechanisms involved in glucagon-stimulated increase in
[Ca2+]i.
Fura 2-loaded BHK cells expressing human glucagon receptors were
stimulated with 200 nM glucagon after incubation for 10 min with 15 µM U-73122 (A), 15 µM U-73343
(B), or 50 µM ryanodine
(C) for time periods indicated by
horizontal lines. Each trace represents change in
[Ca2+]i
within a single cell and is representative of at least 20 single-cell
recordings from 3 experiments.
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Fig. 5.
Accumulation of
D-myo-inositol
1,4,5-trisphosphate (IP3) and
cAMP in BHK cells expressing human glucagon receptors (BHK; Glu) or
muscarinic (M1) receptors (BHK;
M1). A: glucagon- and
carbachol-stimulated IP3
production. B: glucagon- and
carbachol-stimulated cAMP production. Effect of 10 min of pretreatment
with 15 µM U-73122 on carbachol-stimulated accumulation of
IP3
(C) or glucagon-stimulated cAMP
production (D) is shown.
[IP3]i
and [cAMP]i,
intracellular free IP3 and cAMP
concentration, respectively. Values are means ± SD of 4 determinations.
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Further characterization of the
Ca2+ response to glucagon was
carried out in BHK cells coexpressing glucagon and
M1 receptors. As expected,
repeated stimulation of these cells with the same agonist, glucagon
(Fig.
6A) or
carbachol (Fig. 6B), results in desensitization of the Ca2+
response. Interestingly, we found that although prior stimulation with
carbachol could prevent the Ca2+
response to a subsequent glucagon treatment (Fig.
6D), glucagon pretreatment was not
able to prevent the rise in
[Ca2+]i
elicited by a subsequent carbachol stimulation (Fig.
6C). These results indicate that
glucagon releases Ca2+ from the
IP3-sensitive intracellular stores
but that, unlike carbachol, glucagon is not capable of emptying these
stores completely. This is further substantiated by our finding that,
after depletion of thapsigargin-sensitive intracellular
Ca2+ stores, glucagon is unable to
induce a rise in
[Ca2+]i
(data not shown). In addition, unlike carbachol, which can deplete
thapsigargin-sensitive intracellular
Ca2+ stores (Fig.
6F), glucagon only partially empties
the thapsigargin-releasable intracellular
Ca2+ stores, since treatment with
thapsigargin immediately after glucagon stimulation can still elicit a
rise in
[Ca2+]i
(Fig. 6E).

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Fig. 6.
Desensitization of glucagon- and carbachol-mediated rise in
[Ca2+]i
in BHK cells coexpressing human glucagon receptors and
M1 acetylcholine receptors. Cells
were stimulated for time intervals indicated by horizontal lines. Each
trace represents response in a single cell and is representative of at
least 30 single-cell recordings from 3 different experiments.
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To investigate whether cAMP may be involved in the glucagon-induced
rise in
[Ca2+]i,
we examined the effects of forskolin, which stimulates the accumulation
of intracellular cAMP by directly activating adenylate cyclase. As can
be seen in Fig.
7A,
forskolin was not in itself able to elicit a
Ca2+ response in these cells,
whereas the subsequent addition of glucagon was still able to stimulate
a rise in
[Ca2+]i.
Consistent with these results, the activator of protein kinase A (PKA),
Sp-8-BrcAMPS, was also unable to stimulate an increase in
[Ca2+]i
(Fig. 7C). In addition, we examined
the effects of Rp-8-BrcAMPS, a cAMP analog that specifically
inhibits PKA (8), the effectiveness of which has been demonstrated in
BHK cells by its ability to inhibit the translocation of the catalytic
subunit of PKA, as determined by video-imaging techniques (O. Thastrup,
personal communication). Here we found that Rp-8-BrcAMPS
inhibits the Ca2+ response to
glucagon, as can be seen by the diminished
[Ca2+]i
transients (Fig. 7B). Furthermore,
in some of the Rp-8-BrcAMPS-treated cells, glucagon-evoked
Ca2+ oscillations could be
observed; the amplitude of the repetitive spikes appeared to be
somewhat lower than that of the initial response (Fig.
7B). These data suggest that the
rise in
[Ca2+]i
after glucagon stimulation is most likely not mediated by cAMP itself
but that it may in some way modulate or potentiate the Ca2+ response. Further support of
this suggestion is the finding that, together with forskolin, the
IP3 receptor-sensitizing agent
thimerosal is able to elicit a
Ca2+ response (Fig.
8A),
whereas on its own, thimerosal does not affect the
[Ca2+]i
(data not shown). A Ca2+ response
of normal magnitude is observed after stimulation with glucagon, which
is then followed by a number of
Ca2+ oscillations (Fig.
8A). To further test this hypothesis
that cAMP potentiates the Ca2+
response to IP3, we examined the
effect of cAMP on the carbachol-stimulated rise in
[Ca2+]i
in BHK cells expressing M1
receptors. We found that subthreshold concentrations of carbachol could
induce a Ca2+ response in the
presence of forskolin (Fig. 8, B and
C). Finally, in BHK cells
transfected with a mutant (Gly40Ser) glucagon receptor that had
previously been identified in some diabetic patients (10), we observed
a reduction in the Ca2+ response
to glucagon (Fig. 7D), and,
interestingly in some of these cells, glucagon-induced
Ca2+ oscillations occurred that
were similar to those seen in cells treated with
Rp-8-BrcAMPS or thimerosal (Figs.
7B and
8A, respectively).

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Fig. 7.
Role of protein kinase A in glucagon-mediated
Ca2+ response. BHK cells
expressing wild-type human glucagon receptor were stimulated with 200 nM glucagon after a previous stimulation with 10 µM forskolin
(A), 100 µM Rp
diastereomer of 8-bromoadenosine 3',5'-cyclic
monophosphothioate (Rp-8-BrcAMPS,
B), or 100 µM Sp
diastereomer of 8-bromoadenosine 3',5'-cyclic
monophosphothioate (Sp-8-BrcAMPS,
C) for time period indicated by
horizontal bar. D: BHK cells
expressing mutant (Gly40Ser) glucagon receptors were stimulated with
200 nM glucagon. Each trace represents response in a single cell and is
representative of at least 20 single-cell recordings from 3 different
experiments.
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Fig. 8.
Modulation of glucagon- and carbachol-evoked
Ca2+ responses.
A: BHK cells expressing human glucagon
receptors were stimulated with 200 nM glucagon after pretreatment with
10 µM forskolin and 100 µM thimerosal. BHK cells expressing human
M1 receptors were stimulated with
1 nM and 100 nM carbachol (B) or 1 nM carbachol after previous stimulation with 10 µM forskolin
(C). Each trace represents change in
[Ca2+]i
within a single cell and is representative of at least 20 single-cell
recordings from 3 experiments.
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DISCUSSION |
We previously demonstrated that glucagon stimulates a
concentration-dependent rise in intracellular cAMP production in BHK cells expressing human glucagon receptors (11, 19). Here we provide
evidence to indicate that activation of the cloned human glucagon
receptor expressed in BHK cells leads to a rise in
[Ca2+]i
that does not appear to be due to cAMP-mediated activation of
Ca2+ influx. This suggests that
the glucagon receptor, like many others of the secretin receptor
family, can stimulate two independent signaling pathways: one mediated
by cAMP and the other by Ca2+.
From studies aimed at characterizing the rise in
[Ca2+]i
stimulated by glucagon, we found it to be independent of extracellular Ca2+, since it was not affected by
blocking of membrane Ca2+ channels
or by removal of extracellular
Ca2+. These findings indicate that
the source of the glucagon-stimulated rise in
[Ca2+]i
is intracellular Ca2+ stores. Thus
the general features of the glucagon-mediated rise in
[Ca2+]i
appear to differ from those described in response to activation of
GLP-1 and GIP receptors, the two most closely related to the glucagon
receptor. In contrast to the Ca2+
response to glucagon, the
Ca2+-mobilizing effects of GLP-1
in pancreatic
-cells have been demonstrated to be dependent on
extracellular Ca2+ and mimicked by
stimulation with cAMP. This response has been proposed to involve
influx of extracellular Ca2+
through voltage-dependent Ca2+
channels as well as a release of
Ca2+ from intracellular stores
(15). From further studies on
-cells, it has been proposed that the
mobilization of Ca2+ from
intracellular stores in response to GLP-1 reflects
Ca2+-induced
Ca2+ release, since it was blocked
by ryanodine (9). On the other hand, the rise in
[Ca2+]i
elicited by GIP through the cloned receptor expressed in COS-7 cells
has been shown to be biphasic, where the first transient rise in
[Ca2+]i
has been attributed to the mobilization of intracellular
thapsigargin-sensitive Ca2+ stores
and the second sustained rise in
Ca2+ has been found to be a result
of Ca2+ influx through a cation
channel (29). Despite these differences among the
Ca2+ signaling mediated by the
various related receptor types, the characteristics of the
Ca2+ response that we observe
after activation of the cloned human glucagon receptor nonetheless are
supported by previous studies in which endogenous hepatic glucagon
receptors were found to have Ca2+-mobilizing effects that could
not be reproduced by cAMP (22, 27). We do recognize, however, that by
overexpressing the glucagon receptor in BHK cells we run the risk of
"forcing" the coupling to effectors that may not necessarily
occur in cells expressing the endogenous receptors. Nevertheless,
previous studies using cultured primary hepatocytes have demonstrated
that glucagon is able to activate cAMP- and
Ca2+-mediated signaling pathways,
which supports our findings with use of the BHK cells transfected with
the cloned glucagon receptor. In addition, signaling by the cloned
M1 receptors in BHK cells overexpressed to the same extent as the glucagon receptors seems to
have the same characteristics as signaling by endogenous
M1 receptors, that is, activation
of phospholipase C and release of intracellular
Ca2+ without a concomitant
increase in cAMP production (Fig. 5, A and B).
To address the question as to the mechanism of glucagon-induced
Ca2+ signaling, we have shown that
phospholipase C and IP3 are indeed involved, resulting in the release of intracellular
Ca2+ stores. This is demonstrated
by our observation that the phospholipase C inhibitor U-73122
completely abolishes the rise in
[Ca2+]i
stimulated by glucagon (Fig. 4A). In
addition, we observed a twofold increase in
IP3 in response to glucagon
stimulation, although this was small compared with the response
observed with carbachol (Fig. 5A).
This difference in the magnitude of
IP3 formation provides an
explanation for our heterologous desensitization experiments, from
which it is apparent that, even at a maximal concentration, glucagon
only partially empties the Ca2+
stores mobilized by IP3 after
activation of M1 cholinergic
receptors. The fact that stimulation with carbachol could desensitize
the Ca2+ response to a subsequent
stimulation with glucagon whereas the opposite is not the case suggests
that glucagon and carbachol mobilize
Ca2+ from the same
IP3-sensitive intracellular stores
but to varying degrees. This is further exemplified by the finding that
thapsigargin is still able to release
Ca2+ stores after stimulation by
glucagon but not by carbachol (Fig. 6,
E and
F). Finally, Hofer et al. (14) found
that the intraorganellar sequestration of
Ca2+ in BHK cells is not affected
by ryanodine, which supports our finding that this agent has no effect
on glucagon's Ca2+ response (Fig.
4C). Although it is thus apparent
that phospholipase C-catalyzed IP3
production is involved in the Ca2+
response to M1 and glucagon
receptor activation, the upstream signaling mechanisms leading to this
phospholipase C activation are clearly different, since we find that
pertussis toxin blocks the glucagon-mediated rise in
[Ca2+]i
(Table 1). Because M1 cholinergic
receptors couple to phospholipase C by a pertussis toxin-resistant G
protein (Gq) (2), the
glucagon-induced Ca2+ signal must
involve a G protein distinct from that used by the M1 receptors. It has been shown
that the 
-subunit of the pertussis toxin-sensitive
Gi is able to activate the
2-isoform of phospholipase C
(17), and since it has been recently shown that the luteinizing hormone
receptor is able to couple to
G
s and
G
i to activate adenylate
cyclase and phospholipase C, with the 
-subunits released from
either G protein contributing to the stimulation of phospholipase C
-isoforms (13), it is possible that this mechanism may also be
involved in the glucagon-mediated
Ca2+ response.
Our results also indicate that Gs-
and Gi-activated pathways are
involved in the Ca2+ response to
glucagon, since Rp-8-BrcAMPS, which acts as an inhibitor of
PKA, was found to reduce the glucagon-stimulated increase in [Ca2+]i
(Fig. 7B), whereas neither forskolin
nor the cAMP agonist Sp-8-BrcAMPS was able to elicit a
Ca2+ response on its own.
Furthermore, the IP3-sensitizing
agent thimerosal, although unable to elicit a
Ca2+ response itself, leads to an
increase in IP3 if the cells have previously been exposed to forskolin (Fig.
8A). Further substantiation of the
possible intereaction between cAMP and
IP3 is given by our finding that a
subthreshold concentration of carbachol (1 nM) is also able to evoke a
Ca2+ response after prior
treatment with forskolin (Fig. 8, B
and C). This suggests that although
cAMP is not responsible for the rise in
[Ca2+]i
stimulated by glucagon, cAMP can potentiate the
Ca2+ response. It is interesting
to note that the induction of Ca2+
oscillations that occurred after thimerosal (Fig.
8A) or Rp-8-BrcAMPS (Fig.
7B) treatment was similar to our
observation in BHK cells expressing the Gly40Ser mutant receptor (Fig.
7D), which we have reported to be
present in a subset of diabetic patients (10). We previously
demonstrated that this mutation results in a decreased sensitivity of
the receptor to glucagon in terms of binding and cAMP production (11).
However, the mechanisms underlying the induction of this oscillatory
response to glucagon under certain conditions and the significance of
these oscillations remain to be determined.
Thus, taking into consideration all our findings, we suggest the
following model for glucagon-mediated
Ca2+ signaling in which two
distinct signaling pathways are involved. On the basis of sensitivity
to pertussis and cholera toxins, it appears that the glucagon receptor
can couple to multiple G proteins: Gs and possibly
Gi. In addition, the
glucagon-stimulated cAMP production appears to play a permissive role
in the rise in
[Ca2+]i,
such that cAMP, although itself unable to elicit a response, in concert
with the small incremental release of
IP3 is able to mediate the
observed rise in
[Ca2+]i
in response to glucagon. Previously, we proposed that a similar mechanism underlies the GLP-1-stimulated release of
Ca2+ from intracellular stores in
pancreatic
-cells, such that a cAMP-induced phosphorylation of the
IP3 receptor (mediated by PKA) may
enhance the Ca2+ mobilization in
response to the low levels of IP3
arising from activation of phospholipase C by localized
Ca2+ influx (9). Therefore,
although we did not see an increase in
Ca2+ in response to cAMP
stimulation alone (via forskolin), the cAMP produced in response to
activation of glucagon receptors in these cells may potentiate the
effects of the rather small increase in
IP3 we observed after glucagon
stimulation. This model is supported by our observation that thimerosal
or a subthreshold concentration of carbachol is able to induce a
Ca2+ response in the presence of
forskolin. In summary, we have analyzed the general characteristics of
the glucagon-mediated rise in
[Ca2+]i
and have provided evidence to suggest that, contrary to earlier proposals of two glucagon receptor subtypes in the liver (27), the
cloned human glucagon receptor is able to couple to multiple G
proteins, thereby activating two distinct signaling pathways. It
remains a challenge, however, to elucidate the physiological role of
these cAMP-Ca2+ signaling pathways
stimulated by glucagon in its target tissues.
 |
ACKNOWLEDGEMENTS |
The technical assistance of Ane Bøjet Baunsgaard is gratefully
acknowledged. We thank Dr. Nils Billestrup for insightful comments regarding the manuscript.
 |
FOOTNOTES |
This work was supported in part by the Danish Research Academy. The
Hagedorn Research Institute is an independent research unit of Novo
Nordisk.
Address for reprint requests: E. Nishimura, Dept. of Molecular
Signaling, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820
Gentofte, Denmark.
Received 19 March 1997; accepted in final form 9 February 1998.
 |
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