[Ca2+]i-reducing
action of cAMP in rat pancreatic
-cells: involvement of
thapsigargin-sensitive stores
Kazuro
Yaekura1,2 and
Toshihiko
Yada1,3
1 Department of Physiology and
2 First Department of Internal
Medicine, Kagoshima University School of Medicine, 8-35-1
Sakuragaoka, Kagoshima 890; and
3 Laboratory of Intracellular
Metabolism, National Institute for Physiological Sciences, Okazaki 444, Japan
 |
ABSTRACT |
In the present
study, we examined the ability of adenosine 3',5'-cyclic
monophosphate (cAMP) to reduce elevated levels of cytosolic
Ca2+ concentration
([Ca2+]i)
in pancreatic
-cells.
[Ca2+]i
and reduced pyridine nucleotide, NAD(P)H, were measured in rat single
-cells by fura 2 and autofluorescence microfluorometry. Sustained
[Ca2+]i
elevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activator
forskolin (10 µM), and an incretin glucagon-like
peptide-1-(7-36) amide (10
9 M), as well as by
glucose (16.7 mM). The
[Ca2+]i-reducing
effects of cAMP were greater at elevated glucose (8.3-16.7 mM)
than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA),
H-89, counteracted
[Ca2+]i-reducing
effects of cAMP but not those of glucose. Okadaic acid, a phosphatase
inhibitor, at 10-100 nM also reduced sustained [Ca2+]i
elevation in a concentration-dependent manner. Glucose, but not DBcAMP,
increased NAD(P)H in
-cells.
[Ca2+]i-reducing
effects of cAMP were inhibited by 0.3 µM thapsigargin, an inhibitor
of the endoplasmic reticulum (ER)
Ca2+ pump. In contrast,
[Ca2+]i-reducing
effects of cAMP were not altered by ryanodine, an ER
Ca2+-release inhibitor,
Na+-free conditions, or diazoxide,
an ATP-sensitive K+ channel
opener. In conclusion, the cAMP-PKA pathway reduces
[Ca2+]i
elevation by sequestering Ca2+ in
thapsigargin-sensitive stores. This process does not involve, but is
potentiated by, activation of
-cell metabolism. Together with the
known
[Ca2+]i-increasing
action of cAMP, our results reveal dual regulation of
-cell
[Ca2+]i
by the cAMP-signaling pathway and by a physiological incretin.
cytosolic Ca2+ concentration; Ca2+ sequestration; endoplasmic
reticulum; protein kinase A; glucagon-like peptide-1; glucose; adenosine 3',5'-cyclic monophosphate
 |
INTRODUCTION |
INSULIN RELEASE IN RESPONSE to glucose and other
secretagogues is triggered by an increase in cytosolic
Ca2+ concentration
([Ca2+]i)
in pancreatic islet
-cells (25, 33). The
[Ca2+]i
signaling in response to glucose exhibits a dynamic change: an initial
decrease, subsequent increase, and oscillation of
[Ca2+]i
(14). In the presence of stimulatory glucose, a rise in adenosine 3',5'-cyclic monophosphate (cAMP), an important regulator
of islet
-cell functions (25, 33), also elicits
[Ca2+]i
oscillation (8, 12, 14). Simultaneous measurements of [Ca2+]i
and insulin release in mouse islets revealed that
[Ca2+]i
oscillations are causally linked to oscillations of insulin release (3,
4, 10). We speculate that the time-coordinated [Ca2+]i
signaling, including oscillations, is produced by an interplay between
two mechanisms, one that increases and another that decreases [Ca2+]i.
Although the former mechanism has been well elucidated in
-cells,
the latter is yet poorly understood.
It is known that cAMP increases
[Ca2+]i
in a glucose-dependent manner by enhancing
Ca2+ influx through the L-type
Ca2+ channel (8, 12, 16, 31, 35),
in which activation of an
Na+-permeable channel has been
suggested (16). In our previous study, which was designed to determine
whether cAMP has a direct Ca2+
channel agonist action, we examined the effect of cAMP-increasing agents on sustained
[Ca2+]i
elevation induced by high KCl. cAMP not only failed to further increase
[Ca2+]i
(38), it rather, to our surprise, reduced the elevated
[Ca2+]i.
It was also previously reported that cAMP reduces the glucose-induced [Ca2+]i
increase in suspension of islet cells (28). In the present study, we
attempted to elucidate the underlying mechanism by which cAMP-increasing agents reduce
[Ca2+]i
elevation. We also examined the effect of glucagon-like peptide-1 (GLP-1), a physiological incretin that stimulates the cAMP-signaling pathway (16, 23, 32, 35).
[Ca2+]i
and reduced pyridine nucleotide, NAD(P)H, as an indicator of energy
metabolism (9, 21, 24), were measured in rat pancreatic
-cells. We
found that cAMP attenuates
[Ca2+]i
elevation by promoting Ca2+
sequestration in the endoplasmic reticulum (ER) in pancreatic
-cells
and that GLP-1 mimics the cAMP effect.
 |
MATERIALS AND METHODS |
Preparation of single islet cells and selection of
-cells.
Islets of Langerhans were isolated, by collagenase digestion, from
Wistar rats aged 8-12 wk. Animals were anesthetized by intraperitoneal injection of pentobarbital sodium at 80 mg/kg. The
abdomen was opened, and collagenase (3 mg/ml) dissolved in 6 ml of 5 mM
Ca2+-containing Krebs-Ringer
bicarbonate buffer (KRB) solution was injected into the common bile
duct at the distal end after ligation of the duct proximal to the
pancreas. The pancreas was dissected out and incubated at 37°C for
17 min. Islets were collected and immediately dispersed into single
cells in Ca2+-free KRB. The single
cells were plated on coverslips and maintained in short-term culture
for up to 3 days in Eagle's minimum essential medium containing 5.6 mM
glucose supplemented with 10% fetal bovine serum, 100 µg/ml
streptomycin, and 100 U/ml penicillin at 37°C in a 95% air-5%
CO2 atmosphere. During the culture
period, no appreciable change was observed in the frequency or the
pattern of
[Ca2+]i
responses to glucose, tolbutamide, KCl, hormones, or cAMP-increasing agents.
-Cells were selected among single islet cells according to the
previously reported procedure (34, 35). Briefly, single islet cells
that had a diameter of 12.5-17.5 µm on coverslips and responded
to glucose (8.3 or 16.7 mM) and tolbutamide (300 mM) with increases in
[Ca2+]i
were found to be immunocytochemically positive for insulin (37). Data
were taken from the cells that fulfilled these morphological and
physiological criteria for
-cells.
Solutions and chemicals.
Measurements were carried out in KRB solution composed of (in mM) 129 NaCl, 5.0 NaHCO3, 4.7 KCl, 1.2 KH2PO4,
1.0 CaCl2, 1.2 MgSO4, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid at pH 7.4 supplemented with 0.1% bovine serum albumin.
Na+-free KRB was made by
replacement of Na+ with equimolar
N-methyl-D-glucamine (NMDG; Nacalai Tesque,
Kyoto, Japan). Fura 2 and fura 2-acetoxymethyl ester (AM) were obtained from Dojin Chemical (Kumamoto, Japan), and dibutyryl cAMP (DBcAMP) was
obtained from Boehringer Mannheim (Indianapolis, IN). H-89 [N-[2-(p-bromocinnamylamine)ethyl]-5-isoquinoline-sulfonamide] was provided by Dr. H. Hidaka and also obtained from Seikagaku Kogyo
(Tokyo, Japan). All other chemicals were from Sigma (St. Louis, MO).
Measurements of
[Ca2+]i.
[Ca2+]i
was measured by dual-wavelength fura 2 microfluorometry combined with
imaging, as previously reported (36, 37). Briefly, cells on coverslips
were incubated with 1 µM fura 2-AM for 30 min at 37°C in KRB
containing 2.8 mM glucose. Cells were then mounted in a chamber and
superfused at a rate of 1 ml/min at 37°C in KRB. Cells were excited
at 340 and 380 nm alternately every 2.5 s, emission signals at 510 nm
(F340 and
F380, respectively) were detected
with an intensified charge-coupled device camera, and ratio
(F340/F380)
images were produced by an Argus-50 system (Hamamatsu Photonics,
Hamamatsu, Japan). Ratio values were converted to
[Ca2+]i
according to calibration curves.
Measurements of NAD(P)H.
Autofluorescence of NAD(P)H in two to eight
-cells in cluster was
measured under superfusion conditions identical to those used for
[Ca2+]i
measurements. Cells were excited at 360 nm every 1 s, and emission signals through a 470 ± 20-nm band-pass filter were detected by a
high-sensitivity photomultiplier using a P101 system (Nikon, Tokyo,
Japan).
Criteria for
[Ca2+]i-reducing
response and determination of response amplitude.
The amplitude of
[Ca2+]i
reduction in response to cAMP-increasing agents was determined at the
time point of maximal reduction of
[Ca2+]i.
When the KCl-induced
[Ca2+]i
elevation was not flat but mildly declining or increasing at a stable
rate, an extrapolated curve was drawn based on the
[Ca2+]i
levels before addition and 5-10 min after washing out of cAMP agents, and the maximal deviation from the extrapolated curve was taken
as the amplitude of
[Ca2+]i
reduction. Only the deviation that was >0.1 ratio
(F340/F380) unit from the extrapolated curve and which took place in a clear shape
within 5 min upon exposure to cAMP agents was considered as the
response.
Statistical analyses.
All data are presented as means ± SE
(n = number of observations). The
statistical analysis was carried out by Student's
t-test. Differences were considered
statistically significant when P < 0.05.
 |
RESULTS |
KCl-induced sustained
[Ca2+]i
elevation and its attenuation by cAMP-increasing agents and GLP-1.
To examine whether cAMP has an ability to reduce
[Ca2+]i
elevation,
[Ca2+]i
was clamped at high levels and the effect of cAMP was examined. At a
basal glucose concentration of 2.8 mM, KCl at a depolarizing concentration (25 mM) induced a sharp initial peak followed by a
sustained elevation of
[Ca2+]i
in single
-cells (Fig. 1).
The level of the sustained
[Ca2+]i
elevation either stayed constant or changed slowly and mildly at a
stable rate. The KCl-induced sustained elevation of
[Ca2+]i
was reduced by membrane-permeable cAMP analogs 5 mM DBcAMP (Fig.
1A and Table
1) and 1 mM 8-bromo-cAMP (data not
shown), by 10 µM forskolin (Fig.
1B), and by 1 nM GLP-1-(7-36)
amide (GLP-1) (Fig. 1C). DBcAMP,
forskolin, and GLP-1 evoked
[Ca2+]i
reduction in ~80% of the single
-cells. When DBcAMP was combined with 0.3 mM 3-isobutyl-1-methylxanthine, the percentage of cells showing
[Ca2+]i
reduction was further increased to ~90%. The reduction of
[Ca2+]i
started 0.5-1 min after administration of the cAMP-increasing agents, peaked at 2-5 min, and then gradually declined or stayed at the peak. In the presence of glucose concentrations that are stimulatory for the
-cell
[Ca2+]i
and insulin release (8.3 and 16.7 mM) and in the absence of 25 mM KCl,
these cAMP-increasing agents and hormone evoked increases in
[Ca2+]i,
thereby exhibiting a dual effect on
[Ca2+]i
(Fig. 1).

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Fig. 1.
Adenosine 3',5'-cyclic monophosphate (cAMP)-increasing
agents reduce sustained elevation of cytosolic
Ca2+ concentration
([Ca2+]i)
induced by high K+ at basal
glucose as well as increasing
[Ca2+]i
at stimulatory glucose in single -cells. KCl (25 mM) induced a
sustained elevation of
[Ca2+]i
in single -cells. Five milimolar dibutyryl cAMP (DBcAMP)
(A), 10 µM forskolin
(B), and 1 nM glucagon-like
peptide-1 (GLP-1)-(7-36) amide
(C) attenuated the sustained
[Ca2+]i
elevation induced by high K+ at
2.8 mM glucose (G2.8). These agents also increased
[Ca2+]i
in the presence of 8.3 mM glucose (G8.3) and at normal
K+.
[Ca2+]i
responses to G8.3 and 300 µM tolbutamide (Tolb) are also shown.
Glucose concentration is indicated at
top. Bars above tracing specify period
of exposure to agents specified. Dotted lines indicate beginning of
exposure to cAMP-increasing agents. Results are representative of 17 of
21 single -cells examined in A, 20 of 28 cells in B, and 16 of 21 cells
in C.
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Table 1.
[Ca2+]i reduction in response to
DBcAMP and okadaic acid, a phosphatase inhibitor, and effect of H-89, a
PKA inhibitor
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Glucose dependence of
[Ca2+]i-reducing
effects of cAMP.
At stimulatory glucose (8.3 and 16.7 mM) concentrations, DBcAMP and
forskolin reduced KCl-induced
[Ca2+]i
elevation to a greater extent than at 2.8 mM (Fig.
2, A and B). The amplitude of
[Ca2+]i
reduction was significantly increased when glucose concentration was
raised (Fig. 3).

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Fig. 2.
cAMP-increasing agents reduce sustained
[Ca2+]i
elevation induced by high K+ at
stimulatory glucose in single -cells. In the presence of a moderate
elevation of
[Ca2+]i
induced by 8.3 and 16.7 mM glucose, 25 mM KCl produced a further
elevation of
[Ca2+]i
in a sustained manner in single -cells. Five milimolar DBcAMP
(A) and 10 µM forskolin
(B) attenuated the sustained
[Ca2+]i
elevation induced by high K+.
Magnitude of attenuation of
[Ca2+]i
elevation was greater at stimulatory glucose than at basal glucose (see
Fig. 1). Results are representative of 23 of 26 cells in
A and 28 of 30 cells in
B.
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Fig. 3.
Glucose concentration dependence of
[Ca2+]i-reducing
action of cAMP in single -cells. Amplitude of reduction of high
K+-induced
[Ca2+]i
elevation by 5 mM DBcAMP (solid bars) and 10 µM forskolin (hatched
bars) at 2.8, 8.3, and 16.7 mM glucose in single -cells.
Experimental protocol is the same as in Figs. 1 and 2. No. of cells
examined given at top of bars.
* P < 0.001 vs. DBcAMP at
G2.8,
# P < 0.005 vs. forskolin at G2.8, and
** P < 0.001 vs. DBcAMP at G2.8 and P < 0.01 vs. DBcAMP at G8.3.
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[Ca2+]i-reducing
effects of cAMP, glucose, and a phosphatase inhibitor, and selective
inhibition of cAMP effects by a PKA inhibitor.
The KCl-induced
[Ca2+]i
elevation was attenuated both by DBcAMP and by high glucose (16.7 mM)
in single
-cells (Fig.
4A).
After the same cells had been treated for 20 min with 40 µM H-89, an inhibitor of protein kinase A (PKA), the amplitude of
[Ca2+]i
reduction by DBcAMP was significantly suppressed, whereas that by
glucose was unchanged (Fig. 4A and
Table 1). H-89 at 10 µM also suppressed the effect of DBcAMP to
reduce
[Ca2+]i
to a lesser extent (data not shown). In control experiments, when
DBcAMP was successively administered two times, the cells exhibited
[Ca2+]i-reducing
responses twice without appreciable change in their amplitudes (Fig.
4B). Okadaic acid, an inhibitor of
phosphatase, at 10-100 nM also attenuated the KCl-induced
[Ca2+]i
elevation in a concentration-dependent manner (Fig. 4,
C and D, and Table 1). Okadaic acid at 100 nM, a maximal concentration, evoked
[Ca2+]i
reduction in a similar pattern and with a similar amplitude to that
induced by DBcAMP (Fig. 4D and Table
1).

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Fig. 4.
Distinct properties between cAMP- vs. glucose-induced
[Ca2+]i
reduction. A: in presence of 25 mM KCl
and sustained
[Ca2+]i
elevation, 5 mM DBcAMP and 16.7 mM glucose (G16.7) were administered in
pulses in a single -cell (left).
The cell was then treated for 20 min with 40 µM H-89, a protein
kinase A (PKA) inhibitor (arrow), followed by the recording shown on
right. Note that
[Ca2+]i-reducing
effect of cAMP, but not that of glucose, was markedly suppressed by a
PKA inhibitor. Treatment with H-89 had no effect on subsequent
[Ca2+]i
responses to stimulatory glucose (8.3 mM) and tolbutamide (300 µM).
B: in presence of 25 mM KCl and
sustained
[Ca2+]i
elevation, successive administration of 5 mM DBcAMP evoked
[Ca2+]i-reducing
responses two times sequentially without appreciable change in their
amplitudes in a single -cell. C:
okadaic acid (OA), an inhibitor of phosphatase, at 10 and 30 nM
attenuated the KCl-induced
[Ca2+]i
elevation in a concentration-dependent manner in a single -cell.
D: OA at 100 nM attenuated the 25 mM
KCl-induced sustained
[Ca2+]i
elevation in a manner similar to 5 mM DBcAMP in a single -cell.
Treatment with OA had no effect on subsequent
[Ca2+]i
responses to stimulatory glucose and tolbutamide.
E: effects of DBcAMP and glucose on
NAD(P)H levels in -cells. NAD(P)H autofluorescence at 470 nm in 3 -cells in cluster is expressed as a percentage relative to the
signal at the resting state with 2.8 mM glucose before KCl addition.
Note that 16.7 mM glucose, but not 5 mM DBcAMP, increased fluorescence.
Glucose concentration was 2.8 mM unless otherwise indicated in
A-E. Results shown are
representative of 13 of 17 cells in A,
8 of 12 cells in B, 25 of 28 cells in
C, and 26 of 28 cells in
D, whereas they represent 4 similar
experiments in E.
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Effects of cAMP and glucose on NAD(P)H levels in
-cells.
Because the metabolic activity is known to control the level of
[Ca2+]i
(7, 25), we next measured intracellular NAD(P)H as an indicator of the
metabolic activity. The NAD(P)H autofluorescence from
-cells at
basal glucose (2.8 mM) was not significantly altered by 25 mM KCl. In
the presence of 25 mM KCl, DBcAMP produced no change, whereas 16.7 mM
glucose rapidly and markedly increased fluorescence (Fig.
4E).
Effects of inhibitors of ER
Ca2+ pump and
Ca2+-release
channel on
[Ca2+]i-reducing
action of cAMP.
In the presence of 0.3 µM thapsigargin (TG), an inhibitor of the ER
Ca2+ pump (20, 30), KCl increased
[Ca2+]i
to a level somewhat higher than that of control. cAMP-increasing agents
failed to reduce the elevated levels of
[Ca2+]i
in the majority of single
-cells (Fig.
5); thus the fraction of cells responding
to cAMP-increasing agents with
[Ca2+]i
reduction was dramatically reduced (for the response to DBcAMP, 21.7%
with TG vs. 76.6% in control) (Table 2).
The amplitude of the
[Ca2+]i
reduction in the responding cells was also markedly attenuated [19.7 ± 2.5 nM (n = 5) with
TG vs. 94.4 ± 4.1 nM (n = 23) in
control, P < 0.0001] (Table
2).
[Ca2+]i-reducing
effects of glucose (8.3 and 16.7 mM) were also inhibited by TG,
confirming previous reports (6, 13). In contrast, 20 µM ryanodine, an
inhibitor of the ryanodine-sensitive
Ca2+-release channel in ER,
affected neither the level of the KCl-induced [Ca2+]i
elevation nor the cAMP action to attenuate this level (Fig. 6A and
Table 2).

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Fig. 5.
[Ca2+]i-reducing
action of cAMP is inhibited by an endoplasmic reticulum (ER)
Ca2+ pump blocker in single
-cells. Effect of 5 mM DBcAMP on the 25 mM KCl-induced
[Ca2+]i
elevation in absence and presence of 0.3 µM thapsigargin (TG) in
single -cells. At time when administration of DBcAMP was started
(indicated by dashed lines),
[Ca2+]i
level was lower in the test (with TG) than in control
(A), whereas it was higher in the
test than in control in B. Note that
[Ca2+]i-reducing
effect was inhibited by TG, irrespective of whether the
[Ca2+]i
level at beginning of DBcAMP administration in the test was lower or
higher than in control. After treatment with TG, cells responded to 8.3 mM glucose and 300 µM tolbutamide with increases in
[Ca2+]i.
These results, showing inhibition of
[Ca2+]i-reducing
effect of cAMP by TG, are representative of 18 of 23 cells.
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Table 2.
Percentage of cells responding to cAMP with
[Ca2+]i reduction and mean amplitude
of [Ca2+]i reduction in single
-cells under conditions that alter endoplasmic
reticulum and plasma membrane Ca2+ transport
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Fig. 6.
Effects of conditions that modify ER
Ca2+ release, plasma membrane
Na+/Ca2+
exchange, and ATP-sensitive K+
channel (KATP) on
[Ca2+]i-reducing
action of cAMP in single -cells. A:
in presence of 20 µM ryanodine, 10 µM forskolin reduced sustained
[Ca2+]i
elevation induced by 25 mM KCl in a single -cell.
B and
C: under
Na+-free conditions, in which
Na+ was replaced with
N-methyl-D-glucamine, 5 mM DBcAMP
reduced the sustained
[Ca2+]i
elevation induced by 25 mM KCl in a manner similar to control with
normal Na+ at both 2.8 mM
(B) and 16.7 mM glucose
(C) in single -cells.
D: in presence of 0.4 mM diazoxide
(DZ), a KATP opener, 5 mM DBcAMP
reduced the sustained
[Ca2+]i
elevation induced by 25 mM KCl in a single -cell.
E: sustained
[Ca2+]i
elevation induced by 1 mM tolbutamide, a
KATP blocker, was also reduced by
5 mM DBcAMP in a single -cell. Glucose concentration was 2.8 mM in
A, B,
D, and
E, whereas it was 16.7 mM in
C. Results shown are representative of
16 of 18 cells in A, 21 of 27 cells in
B, 17 of 21 cells in
C, 20 of 28 cells in
D, and 11 of 17 cells in
E.
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Effects of inhibition or activation of plasma membrane
Na+/Ca2+
exchange and KATP on
[Ca2+]i-reducing
action of cAMP.
It has been shown that the plasma membrane
Na+/Ca2+
exchange serves as a Ca2+
extrusion mechanism in
-cells (15) and plays a greater role at
elevated glucose than at basal glucose in the regulation of [Ca2+]i
(13, 38). Therefore, we assessed an involvement of
Na+/Ca2+
exchange in the cAMP-induced
[Ca2+]i
reduction at basal and elevated glucose concentrations by examining the
effect of Na+-free conditions that
inhibit
Na+/Ca2+
exchange. Na+-free conditions were
achieved by replacement of extracellular Na+ with NMDG. Under
Na+-free conditions, high
K+-induced sustained
[Ca2+]i
elevation was attenuated by DBcAMP in a manner similar to control conditions, and this was observed at both basal and elevated glucose concentrations (Fig. 6, B and
C, and Table 2).
It is also possible that cAMP reduces
[Ca2+]i
by influencing the ATP-sensitive
K+ channel
(KATP), which is the major
determinant of the
-cell resting membrane potential (2). This
possibility was examined by testing the effect of cAMP under conditions
in which KATP was fixed in either
a maximally open or closed state. A sustained [Ca2+]i
elevation induced by KCl plus the
KATP opener diazoxide at 0.4 mM, a
concentration that fully opens this channel (2, 10), was attenuated by
DBcAMP (Fig. 6D and Table 2) and by
forskolin (data not shown). The
KATP blocker tolbutamide at 1 mM,
a supramaximal concentration for complete closure of this channel (2,
9), induced sustained elevation of
[Ca2+]i,
which was attenuated by DBcAMP (Fig.
6E). These results indicate that
neither
Na+/Ca2+
exchange nor KATP is involved in
the
[Ca2+]i-reducing
action of cAMP.
 |
DISCUSSION |
The present study has shown that membrane-permeable cAMP analogs,
forskolin and GLP-1, agents that increase cAMP, all reduce sustained
[Ca2+]i
elevation induced by high K+ and
tolbutamide, thereby revealing the
[Ca2+]i-reducing
action of cAMP in pancreatic
-cells. The
[Ca2+]i
reduction by cAMP was abolished or markedly inhibited by TG at the
concentration that specifically inhibits glucose-induced [Ca2+]i
decrease, a process mediated by
Ca2+ sequestration in ER (13). It
is well established that TG inhibits the ER and sarcoplasmic reticulum
(SR) Ca2+-ATPase
(Ca2+ pump) activity without
influencing the plasma membrane
Ca2+-ATPase and
Na+-K+-ATPase
in a variety of preparations (20, 30). In contrast, [Ca2+]i-reducing
effects of cAMP were observed similarly in the absence and presence of
ryanodine. Therefore, it is unlikely that cAMP attenuates
[Ca2+]i
elevation by inhibiting the ryanodine-sensitive
Ca2+ release from ER. The
[Ca2+]i
reduction could, alternatively, be due to alteration of ion transport
systems in the
-cell plasma membrane.
Na+/Ca2+
exchange functions as a Ca2+
extrusion mechanism in
-cells (15, 38). However,
[Ca2+]i-reducing
effects of cAMP were unaltered under
Na+-free conditions, irrespective
of whether the glucose concentration was basal or stimulatory.
Therefore, the cAMP-induced
[Ca2+]i
reduction is not accounted for by stimulation of
Na+/Ca2+
exchange at the plasma membrane. Another possibility is that cAMP
reduces
[Ca2+]i
via influencing KATP, the channel
that mainly determines the resting membrane potential of
-cells. In
the present study, however, in the presence of the
KATP opener diazoxide at 0.4 mM, a
supermaximal concentration that fully opens this channel (2), the
KCl-induced [Ca2+]i
elevation was also reduced by cAMP. Likewise, the
KATP blocker tolbutamide at 1 mM,
a concentration that fixes this channel at the completely closed state
(13), induced sustained
[Ca2+]i
elevation, and it was also attenuated by cAMP. Thus
KATP appears not to be involved in
[Ca2+]i-reducing
effects of cAMP. Taken together, it is concluded that [Ca2+]i-reducing
effects of cAMP are due primarily to stimulation of Ca2+ sequestration in the
TG-sensitive ER in
-cells.
Glucose also stimulates Ca2+
sequestration in the TG-sensitive ER in
-cells, accounting for the
glucose-induced initial decrease in
[Ca2+]i
(6, 11, 13, 26, 27, 36). It is likely that the glucose-induced
[Ca2+]i
reduction is due to activation of
-cell metabolism for the following
reasons. Glucose is metabolized by
-cells, resulting in production
of ATP from ADP (2); the ATP/ADP ratio is a regulatory factor of the
Ca2+-pumping ATPase; and the
steady-state
[Ca2+]i
varies inversely with the ATP/ADP ratio in the permeabilized insulinoma
cells (7). It is possible that activation of
-cell metabolism is
also involved in the cAMP-induced
[Ca2+]i
reduction. In the present study, however, the NAD(P)H level in
-cells, an indicator of metabolism, was not significantly altered by
cAMP, whereas it was markedly increased by high glucose, as previously
reported (9, 24). In contrast, the
[Ca2+]i-reducing
effect of cAMP, but not that of high glucose, was inhibited by
pretreatment with the PKA inhibitor H-89. Furthermore, okadaic acid, an
inhibitor of phosphatase, mimicked cAMP-increasing agents to reduce the
KCl-induced
[Ca2+]i
elevation. These findings indicate that cAMP and glucose reduce [Ca2+]i
elevation by distinct mechanisms and that PKA-mediated phosphorylation may be involved in the
[Ca2+]i-reducing
action of cAMP.
Although the
[Ca2+]i-reducing
effect of cAMP does not involve activation of
-cell metabolism, it
was substantially enhanced as the glucose concentration was increased.
Thus interactive effects of cAMP and glucose were demonstrated. This
interaction may be accounted for by the following hypothetical
mechanism. The cAMP-dependent phosphorylation pathway stimulates
Ca2+ sequestration in ER by
upregulating the active form of
Ca2+-ATPase
(Ca2+ pump); because
Ca2+-ATPase is an ATP-dependent
enzyme, higher ATP levels at higher glucose concentrations make more
Ca2+-ATPase in the active form,
thereby allowing a larger potentiation of the
Ca2+-ATPase activity by cAMP.
However, precise mechanisms for the interaction between cAMP and
glucose in reducing
[Ca2+]i
are yet to be elucidated.
Because the
[Ca2+]i-reducing
action of cAMP is blocked by an ER
Ca2+ pump inhibitor and a PKA
inhibitor and mimicked by a phosphatase inhibitor, a possible
involvement of PKA-mediated phosphorylation in the TG-sensitive
Ca2+ sequestration in ER is
suggested. It has been shown that glucagon stimulates
Ca2+ uptake in ER of liver cells
(1, 5), cAMP mimics this effect (1, 29), and
Ca2+-pumping activity is enhanced
by PKA (22). In cardiac muscles, phospholamban, a membrane-associated
protein in SR, is a substrate of PKA, and the ratio of phosphorylated
to unphosphorylated phospholamban correlates with the
Ca2+ transport enzyme activity and
the rate of Ca2+ uptake in SR (17,
18, 22). However, phospholamban or related proteins have not yet been
demonstrated in pancreas. The mechanisms that link PKA to the ER
Ca2+ sequestration in pancreatic
-cells remain to be investigated.
It should be noted that administration of cAMP-increasing agents
induced large transients of
[Ca2+]i
in 6.6% of
-cells (data not shown). These
[Ca2+]i
transients took place usually before, and occasionally during, the
[Ca2+]i
reduction. The duration of a transient was 30-120 s. A previous study (19) reported that cAMP induces the pronounced transients of
[Ca2+]i
in glucose- or KCl-stimulated
-cells and suggested a mechanism by
which cAMP sensitizes the inositol 1,4,5-trisphosphate
(IP3) receptor to stimulate
mobilization of intracellular
Ca2+. Therefore, our
[Ca2+]i
transients could reflect the
IP3-mediated
Ca2+ mobilization. However, the
time resolution of our
[Ca2+]i
measurements may not be fast enough to detect short transients evoked
by IP3. It should be emphasized
that
[Ca2+]i-reducing
effects of cAMP were observed irrespective of whether the
[Ca2+]i
transients occurred. A link between the cAMP-induced
[Ca2+]i
reduction and
[Ca2+]i
transients remains unknown.
The
[Ca2+]i-reducing
action of cAMP may play a role in optimizing the amplitude of
[Ca2+]i
signals, thereby finely controlling
Ca2+-dependent functions in
-cells. It is also suggested that the [Ca2+]i-reducing
action of cAMP takes part in buffering an excessive rise in
[Ca2+]i,
a putative cytotoxic signal which leads to
-cell dysfunction and
death. On the other hand, it is well established that cAMP increases
[Ca2+]i
in a glucose-dependent manner in rat
-cells. Thus the present study
reveals a dual action of cAMP on
[Ca2+]i.
The dual function of the cAMP-signaling pathway may play a role in
producing frequency-coded
[Ca2+]i
signals, such as oscillations of
[Ca2+]i,
in pancreatic
-cells.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Geoffrey W. G. Sharp for carefully reading the
manuscript. We are grateful to Dr. H. Hidaka at Nagoya University for
kindly providing H-89 and to Drs. M. Kakei and H. Tanaka at Kagoshima
University for encouragement during this study.
 |
FOOTNOTES |
This research was supported in part by grants from the Ministry of
Education, Science, Sports and Culture of Japan (to T. Yada).
Address for reprint requests: T. Yada, Dept. of Physiology, Kagoshima
Univ. School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890, Japan.
Received 25 June 1997; accepted in final form 27 October 1997.
 |
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