From the Department of Physiology and Neuroscience,
New York University School of Medicine, New York, New York 10016, the § Departments of Physiology and Medicine, University of
Toronto, Toronto, Canada M5S 1A8, the
** Department of Physiological Chemistry and Centre for
Biomedical Genetics, University Medical Center Utrecht,
Universiteitsweg 100, 3584CG Utrecht, The Netherlands, and the
BIOLOG Life Science Institute, P. O. Box
107125, D-28071 Bremen, Germany
Received for publication, November 15, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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The second messenger cAMP exerts powerful
stimulatory effects on Ca2+ signaling and insulin
secretion in pancreatic cAMP-regulated guanine nucleotide exchange factors (referred to
here as Epac)1 link cAMP
production to the activation of the Ras-related small molecular weight
G protein Rap1 (1, 2). Two isoforms of Epac have been described (Epac1,
Epac2) (1, 2), and each is proposed to mediate the PKA-independent
signal transduction properties of cAMP. Analysis of cAMP-mediated
signaling pathways is complicated by the lack of specificity with which
cAMP acts. cAMP targets not only PKA and Epac, but also certain cAMP
phosphodiesterases and ion channels (3). Recent structure-function
analyses of cAMP action demonstrate that introduction of a 2'-methoxyl
group in place of the 2'-hydroxyl group of cAMP confers Epac
specificity to the cyclic nucleotide (4). One such analog is
8-(4-chloro-phenylthio)-2'-O-methyladenosine (8-pCPT-2'-O-Me-cAMP). Rap1 activation assays conducted
in vitro demonstrate that 8-pCPT-2'-O-Me-cAMP
binds to and activates Epac1 with higher apparent affinity
(EC50 2.2 µM) than cAMP itself
(EC50 30 µM) (4). Furthermore,
8-pCPT-2'-O-Me-cAMP is a weak activator of PKA (4). To date,
the properties of 8-pCPT-2'-O-Me-cAMP in living cells have
been evaluated only with respect to its ability to promote
Epac-mediated activation of Rap1 (4). We now demonstrate that
8-pCPT-2'-O-Me-cAMP is an effective stimulus for
Ca2+-induced Ca2+ release (CICR) and exocytosis
in human pancreatic Cell Culture--
Human islets of Langerhans were provided under
the auspices of the Juvenile Diabetes Research Foundation International
Islet Distribution Program. Single cell suspensions of human islet
cells were prepared by digestion with trypsin-EDTA, and the cells were plated onto glass coverslips (25CIR-1; Fisher) coated with 1 mg/ml concanavalin A (type V; Sigma). Cell cultures were maintained in
a humidified incubator (95% air, 5% CO2) at 37 °C in
CMRL-1066 culture medium containing 10% fetal bovine serum (FBS), 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2.0 mM
L-glutamine. INS-1 cells (passages 70-90) were maintained
in RPMI 1640 culture medium containing 10 mM HEPES, 11.1 mM glucose, 10% FBS, 100 units/ml penicillin G, 100 µg/ml streptomycin, 2.0 mM L-glutamine, 1.0 mM sodium pyruvate, and 50 µM
2-mercaptoethanol (5). INS-1 cells were passaged by trypsinization and
subcultured once a week. All reagents for cell culture were obtained
from Invitrogen.
Plasmid and Adenovirus Constructs--
A plasmid in which
expression of enhanced yellow fluorescent protein (EYFP) was placed
under the control of the rat insulin II gene promoter (RIP2) was
constructed by fusing a Immunocytochemistry--
Pancreatic islets isolated from male
Wistar rats (250 g) were plated on rat tail collagen and infected for
48 h with AdRIP2EYFP. Islets were stained for insulin using guinea
pig anti-insulin and a secondary anti-guinea pig IgG conjugated with
rhodamine TRITC (Jackson ImmunoResearch Laboratories, West Grove, PA).
Islets were stained for glucagon using rabbit anti-glucagon, 08-0064, (Zymed Laboratories Inc., San Francisco, CA) and a
secondary anti-rabbit IgG-conjugated with rhodamine TRITC. Laser
scanning confocal microscopy was performed (Carl Zeiss, LSM
410), and images were obtained using a 63× oil immersion objective.
Co-detection of EYFP and Fura-2--
Expression of EYFP was
detected through use of an inverted microscope (Eclipse TE300, Nikon
Instruments, Melville, NY) equipped with a 75-W xenon arc lamp serving
as a light source. A liquid light guide directed unfiltered excitation
light to an EYFP filter set mounted in a filter cube containing an
HQ500/20 excitation filter, a Q515LP dichroic beamsplitter, and an
HQ535/30 emission filter (HQ filter set 41028, Chroma Tech. Corp.,
Brattleboro, VT). Once an EYFP-positive cell was identified, the filter
cube was switched manually to a second filter cube containing
components of the fura-2 filter set. Excitation light provided by the
xenon arc lamp was reflected by a rotating chopper mirror through
340/20BP and 380/20BP excitation filters (Chroma) mounted in a
motorized filter wheel located at the light source. The filtered light
was then directed to the fura-2 filter set by way of the liquid light guide. The fura-2 filter cube contained a 400DCLP dichroic beamsplitter and a 510/80 excitation filter (Chroma).
Measurement of [Ca2+]i--
The fura-2
loading solution consisted of a standard extracellular saline (SES)
containing (in mM): 138 NaCl, 5.6 KCl, 2.6 CaCl2, 1.2 MgCl2, 10 HEPES, and 5.6 D-glucose. The SES was supplemented with 1 µM fura-2 acetoxymethyl ester (fura-2 AM; Molecular
Probes Inc., Eugene, OR), 2% FBS, and 0.02% Pluronic F-127 (w/v;
Molecular Probes Inc.). Cells were exposed to fura-2 AM for 20-30 min
at 22 °C. The loading solution was removed, and cells were washed and equilibrated in fresh SES for 10 min at 22 °C. Images were acquired using a 100× UVF oil immersion objective (numerical
aperature 1.3, Nikon), and dual excitation wavelength microfluorimetry
was performed ratiometrically at 0.5-s intervals using a digital video imaging system outfitted with an intensified charge-coupled
device camera (IonOptix Corp., Milton, MA).
[Ca2+]i was calculated according to methods
established (7, 8). In vitro calibration of raw
fluorescence values was performed using fura-2
(K+)5 salt dissolved in calibration buffers
from Molecular Probes Inc. (Calcium Calibration Kit 1 with
Mg2+). Values of Rmin and
Rmax were 0.20 and 7.70.
Electrochemical Detection of Exocytosis--
Cells were loaded
with serotonin (5-HT) by incubation in culture medium containing 0.6 mM 5-HT and 0.6 mM 5-hydroxytryptophan. 5-HT is
sequestered in large dense-core secretory granules by active transport,
but it is excluded from the small synaptic vesicle-like structures that
do not contain insulin (9). The release of 5-HT serves as a surrogate
marker for insulin secretion (10). Prolonged exposure of Luciferase Assay--
The activity of CRE-Luc was assessed in
lysates of INS-1 cells by use of a luciferase assay as described
previously (13-15). After a 4-h exposure to test substances, cells
were lysed and assayed for luciferase-catalyzed photoemissions using a
luciferase assay kit (Tropix, Bedford, MA) and a luminometer allowing
automated application of ATP and luciferin solutions (Model TR-717,
PerkinElmer Life Sciences). Experiments were carried out in triplicate.
Statistical analyses were performed using the analysis of variance test
combined with Fisher's Poisson least squares distribution test.
Measurement of Inositol Phosphate Accumulation--
INS-1 cells
were plated in complete growth medium in 12-well plastic culture
dishes. 3H-inositol (10 µCi/ml) was added to each well,
and the cells were incubated for 48 h. The medium was then changed
to serum-free RPMI 1640 containing 10 mM lithium chloride
and the pharmacological agents to be tested. The incubation was
continued for an additional 60 min at 37 °C. Cellular lipids and
inositol phosphates were extracted for Dowex chromatography and
quantification as described previously (16).
Sources of Reagents--
8-pCPT-2'-O-Me-cAMP,
8-CPT-cAMP, and Rp-8-Br-cAMPS were obtained from BIOLOG Life Sci.
Inst., Bremen, Germany. GLP-1, Ex-4, acetylcholine, forskolin,
8-Br-cAMP, H-89, KT 5720, and ryanodine were from Sigma. Wild type and
dominant negative pSR EYFP-based Phenotype Selection of 8-pCPT-2'-O-Me-cAMP as a Stimulus for Ca2+ Signaling in
Human 8-pCPT-2'-O-Me-cAMP as a Stimulus for Exocytosis in human
8-pCPT-2'-O-Me-cAMP as a Stimulus for CICR in INS-1 Cells--
The
action of 8-pCPT-2'-O-Me-cAMP was also evaluated in the
INS-1 insulin-secreting cell line (5). These cells exhibit Epac-mediated CICR appearing as a transient increase of
[Ca2+]i (18). 8-pCPT-2'-O-Me-cAMP
was a stimulus for CICR in INS-1 cells (Fig.
6A), and the kinetics of the
increase of [Ca2+]i matched closely that
observed in human PKA Independence of 8-pCPT-2'-O-Me-cAMP Action--
To examine the
specificity with which 8-pCPT-2'-O-Me-cAMP differentiates
between Epac and PKA, INS-1 cells were transfected with a plasmid in
which expression of luciferase was placed under the control of
multimerized cyclic AMP-response elements (CRE-Luc). This reporter is
activated by cAMP via a PKA-signaling mechanism involving CREB
(13-15). The activity of CRE-Luc was stimulated by 30-300
µM 8-CPT-cAMP (active at PKA and Epac), and was reduced by the PKA inhibitor H-89 (Fig. 7B). However,
8-pCPT-2'-O-Me-cAMP failed to stimulate CRE-Luc (Fig.
7B). Identical findings were obtained when evaluating the
actions of 8-CPT-cAMP, H-89, and 8-pCPT-2'-O-Me-cAMP in HEK
293 cells transfected with CRE-Luc (data not shown). These observations
demonstrate that when tested in vitro at a concentration of
30-300 µM, 8-pCPT-2'-O-Me-cAMP exhibits
little or no efficacy as a stimulator of the cAMP-, PKA-, and
CREB-signaling pathways in INS-1 cells.
Ca2+ Stores Targeted by 8-pCPT-2'-O-Me-cAMP--
GLP-1
or the GLP-1 receptor agonist exendin-4 (Ex-4) mobilizes
Ca2+ from ryanodine-sensitive Ca2+ stores in
INS-1 cells (18). In the present study, we found that CICR in response
to 8-pCPT-2'-O-Me-cAMP was also blocked by ryanodine (Fig.
8). However, it was recently reported
that Epac mediates stimulatory actions of cAMP on inositol
trisphosphate (IP3)-sensitive Ca2+ stores in
HEK 293 cells (19, 20). This action of cAMP is proposed to result from
Epac-mediated activation of phospholipase C-epsilon (PLC- 8-pCPT-2'-O-Me-cAMP and Implications for GLP-1 Receptor Signal Transduction in
Epac Targets Ryanodine Receptor-regulated Ca2+
Stores--
Epac mediates stimulatory actions of cAMP on phospholipase
C- Coupling of Epac to Endoplasmic Reticulum Ca2+
Stores--
Previous studies of INS-1 cells demonstrated that
cAMP-elevating agents mobilize Ca2+ from
thapsigargin-sensitive Ca2+ stores (18). These
Ca2+ stores are most likely located in the endoplasmic
reticulum (ER) where the SERCA Ca2+ ATPase targeted
by thapsigargin is found. One possible effector that links Epac
signaling to these Ca2+ stores is Rap1, a small molecular
weight G protein previously reported to form direct protein-protein
interactions with SERCA (24). In addition, an Epac/Rap1 signaling
complex in the ER might influence Ca2+ signaling by
modulating RYR gating properties, possibly through direct interactions
with the channel. Such an effect might synergize with PKA-mediated
sensitization of RYR, thereby facilitating Ca2+ release
from the ER (25). In this regard, HEK 293 cells overexpressing the
GLP-1-R exhibit ryanodine-sensitive CICR in response to GLP-1, and this
action correlates with the ability of GLP-1 to stimulate cAMP
production (26). Therefore, the targeting of RYR by GLP-1, whether
direct or indirect, is a signaling mechanism not restricted to
Functional Coupling of CICR to Exocytosis--
Here we also
demonstrate that 8-pCPT-2'-O-Me-cAMP stimulates
Ca2+-dependent exocytosis in human
The present findings are noteworthy because we demonstrate a functional
coupling of Epac-mediated CICR to exocytosis in human -cells. Previous studies of
-cells
focused on protein kinase A (PKA) as a downstream effector of cAMP
action. However, it is now apparent that cAMP also exerts its effects
by binding to cAMP-regulated guanine nucleotide exchange factors
(Epac). Although one effector of Epac is the Ras-related G protein
Rap1, it is not fully understood what the functional consequences of
Epac-mediated signal transduction are at the cellular level.
8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate (8-pCPT-2'-O-Me-cAMP) is a newly described
cAMP analog, and it activates Epac but not PKA. Here we demonstrate that 8-pCPT-2'-O-Me-cAMP acts in human pancreatic
-cells
and INS-1 insulin-secreting cells to mobilize Ca2+ from
intracellular Ca2+ stores via Epac-mediated
Ca2+-induced Ca2+ release (CICR). The
cAMP-dependent increase of
[Ca2+]i that accompanies CICR is shown to be
coupled to exocytosis. We propose that the interaction of cAMP and Epac
to trigger CICR explains, at least in part, the blood glucose-lowering
properties of an insulinotropic hormone (glucagon-like peptide-1, also
known as GLP-1) now under investigation for use in the treatment of type-2 diabetes mellitus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells and an insulin-secreting cell line
(INS-1). The action of 8-pCPT-2'-O-Me-cAMP is shown to be
independent of PKA but is blocked by overexpression of dominant
negative Epac2. Therefore, 8-pCPT-2'-O-Me-cAMP is likely to
serve as a specific pharmacological tool for analyses of
PKA-independent signaling properties of cAMP in the regulation of
intracellular Ca2+ signaling and exocytosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
695-bp BamH1 fragment of RIP2 to
the coding sequence of EYFP contained within the pEYFP-N1 expression
plasmid (Clontech). LipofectAMINE Plus reagent
(Invitrogen) was used to transfect INS-1 cells with this plasmid
designated as RIP2-EYFP, and clones of INS-1 cells exhibiting stable
transfection were obtained by antibiotic resistance selection using
G418. For construction of adenovirus, RIP2-EYFP was PCR-amplified inserting XhoI (5' end, primer CTC GAG ACC GCG GGC CCG GGA
TCC) and KpnI (3' end, primer GGT ACC CCT CTA CAA ATG TGG
TAT GGC TG) digestion sites on either end of the RIP2-EYFP sequence.
The PCR product was subcloned into pCR2.1, and RIP2-EYFP was then
inserted into a XhoI/KpnI site of AdLox.HTM. The
RIP2-EYFP-AdLox.HTM vector was co-transfected with psi5 vector into
CRE8 cells expressing CRE recombinase. This resulted in recombination
of the RIPYFP-AdLox.HTM vector with the psi5 vector (6). The psi5
vector acts as a donor virus to supply viral backbone. AdRIPYFP viral
particles generated in this manner were passaged three times in CRE8
cells and CsCl gradient-purified. 109 viral particles/ml
were used to infect
-cells or islets.
-cells to
high concentrations of 5-HT produces toxic effects (11). Therefore,
exposure to 5-HT was limited to 4-16 h. Carbon fiber electrodes for
amperometric detection of secreted 5-HT were prepared as described
previously (12). A +650-mV potential was applied to a
10-µM diameter carbon fiber, the tip of which was placed
adjacent to the cell of interest. The distance from the electrode tip
to the cell was ~1 µm. An HEKA EPC-9 patch clamp amplifier was used
for detection of the amperometric current resulting from oxidation of
5-HT. The signal was filtered at 200 Hz, sampled at 1 kHz, and stored
on a MacIntosh G3 computer running PULSE 8.31 software (HEKA,
Lambrecht, Germany).
plasmids for transfection and the expression
of recombinant mouse Epac2 were obtained from the laboratory of Dr. S. Seino (17).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Cells--
Methodology was
developed allowing phenotype selection of
-cells in primary cultures
of dispersed islets bathed in SES containing 7.5 mM
glucose. A
695-bp fragment of the rat insulin II gene promoter
(RIP2) was fused to the coding sequence of EYFP (18) for initial
expression of EYFP in INS-1 cells (Fig.
1, A and B). RIP2-EYFP was then incorporated into an adenoviral vector, and expression of EYFP was conferred to human
-cells by
adenovirus-mediated gene transfer using AdRIP2EYFP (Fig. 1,
C and D). Confocal microscopy in combination with
fluorescence immunocytochemistry demonstrated that expression of EYFP
was restricted to the insulin-immunoreactive
-cells and not the
glucagon-reactive
-cells in whole islets of Langerhans derived from
rat (Fig. 2). Functional studies of small
clusters of human islet cells demonstrated that the EYFP-positive
-cells exhibited an increase of [Ca2+]i
when exposed to the insulin secretagogue glyburide (Fig.
3).
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Fig. 1.
Rat insulin II gene promoter-directed
expression of EYFP. Stable transfection of INS-1 cells (top
two panels) was achieved by use of RIP2-EYFP allowing antibiotic
resistance selection using G418. Expression of EYFP in single human
-cells (bottom two panels) was achieved by infection of
primary islet cell cultures with adenovirus incorporating RIP2-EYFP. In
human
-cells the subcellular localization of EYFP was either
cytosolic (bottom, left) or nuclear
(bottom, right). Calibration bars: A,
20 µm; B, 10 µm; C and D, 5 µm.
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Fig. 2.
-Cell-specific
expression of EYFP conferred by RIP2. Left panel, confocal
microscopy demonstrates co-localization of EYFP and insulin-like
immunoreactivity in a rat islet infected with adenovirus incorporating
RIP2-EYFP. A, bright field image of a rat islet;
B, detection of EYFP in this islet; C,
insulin-like immunoreactivity in this islet; D, image
overlap of B and C demonstrating co-expression of
insulin and EYFP (orange) in
-cells. Right
panel, glucagon but not EYFP was expressed in rat
-cells
infected with RIP2-EYFP. E, bright field image of an islet;
F, detection of EYFP in this islet; G,
glucagon-like immunoreactivity in this islet; H, image
overlap of F and G demonstrating that EYFP was
not expressed in
-cells.
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Fig. 3.
Expression of EYFP in human
-cells correlates with sulfonylurea-sensitivity.
A cluster of three human islet cells was evaluated for expression
of EYFP under conditions of fura-2 loading. The cell labeled 1 (top panel) exhibited an increase of
[Ca2+]i (bottom panel) in response
to a 10-s application of 100 nM glyburide applied to the
entire cluster of cells. This same cell contained EYFP (bottom
panel inset; note that the boundary surrounding the cluster of
cells is indicated by a white outline). The remaining
two cells of the cluster did not contain EYFP and failed to respond to
glyburide. Identical findings were obtained in a total of five
separate experiments.
-Cells--
The Epac-selective cAMP analog
8-pCPT-2'-O-Me-cAMP was a highly effective stimulus for
Ca2+ signaling in human
-cells. Human islet cells
infected with AdRIP2EYFP were loaded with fura-2, and EYFP-positive
-cells were identified using the EYFP filter set. Next, the filter
set was manually switched to a fura-2 filter set, thereby allowing
ratiometric measurements of [Ca2+]i imaged
using an intensified charge-coupled device camera. The responsiveness of an EYFP-positive
-cell was demonstrated by measuring the increase of [Ca2+]i that
accompanied a 10-s application of 50 µM
8-pCPT-2'-O-Me-cAMP delivered via a micropipette (Fig.
4, A1 and A2). The
phenotype of this cell was additionally confirmed by demonstrating its
responsiveness to the insulin secretagogue glyburide (Fig. 4,
A2). The increase of [Ca2+]i
measured in response to 8-pCPT-2'-O-Me-cAMP was blocked by pretreatment of human
-cells with ryanodine (Fig.
4B).
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Fig. 4.
Human
-cells are sensitive to
8-pCPT-2'-O-Me-cAMP. A, a single
EYFP-positive cell was imaged for fura-2 (A1) and
8-pCPT-2'-O-Me-cAMP (50 µM) was applied via a
micropipette for 10 s. CICR was measured as a transient increase
of [Ca2+]i (A2). The
-cell
phenotype was confirmed by demonstrating the ability of this cell to
respond to a 10-s application of glyburide (A2;
Glyb., 100 nM). Identical findings were obtained
in a total of 10 human
-cells tested. B, CICR in response
to 8-pCPT-2'-O-Me-cAMP was inhibited by treatment of a human
-cell with ryanodine. Arrows indicate a 10-s application
of 100 µM 8-pCPT-2'-O-Me-cAMP with or without
10 µM ryanodine (Ryan.). Horizontal
bar indicates the duration of pretreatment with 10 µM ryanodine applied directly to the solution bathing the
cell. Identical findings were obtained in a total of four cells
tested.
-Cells--
The increase of [Ca2+]i
measured during exposure of 5-HT-loaded human
-cells to
8-pCPT-2'-O-Me-cAMP was associated with exocytosis, as
measured by carbon fiber amperometry (Fig.
5). The increase of
[Ca2+]i (Fig. 5, A1) was
time-locked to the appearance of amperometric current spikes resulting
from the oxidation of released 5-HT (Fig. 5, A2). When
viewed on an expanded time scale, the quantal nature of the individual
secretory events was revealed (Fig. 5, A3 and A4). No secretory events were detected in the absence of an
increase of [Ca2+]i.
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Fig. 5.
8-pCPT-2'-O-Me-cAMP as a
stimulus for CICR and exocytosis. A human -cell was loaded with
fura-2 and 5-HT for simultaneous measurements of
[Ca2+]i and exocytosis. Application of
8-pCPT-2'-O-Me-cAMP (100 µM, 10 s)
produced CICR (A1) and exocytosis of 5-HT (A2) as
depicted on a compressed time scale. When viewed on an expanded time
scale, the quantal nature of the individual secretory events (upward
amperometric current spikes due to oxidation of 5-HT) was revealed
(A3 and A4). Identical findings were obtained in
a total of 10 human
-cells tested.
-cells (cf. Fig. 4). The action of
8-pCPT-2'-O-Me-cAMP was mimicked by forskolin (Fig. 6B) and by 8-Br-cAMP (Fig. 6C). In some cells,
the fast transient increase of [Ca2+]i was
followed by a more slowly developing and sustained increase of
[Ca2+]i (Fig. 6D). Neither effect
of 8-pCPT-2'-O-Me-cAMP was blocked by the cAMP antagonist
Rp-8-Br-cAMPS (Fig. 6D and Fig.
7A). Rp-cAMPS is an inhibitor
of both PKA and Epac, but it has a low affinity for Epac compared with
8-pCPT-2'-O-Me-cAMP.2
The action of 8-pCPT-2'-O-Me-cAMP was
dose-dependent over a concentration range of 10-100
µM (Fig. 7A) and was not blocked by 10 µM of the PKA inhibitors H-89 or KT 5720 (Fig.
7A). However, the action of 8-pCPT-2'-O-Me-cAMP
was diminished by transfection of INS-1 cells with dominant negative
pSR
Epac2 (Fig. 7A). Dominant negative Epac2 does not
bind cAMP because G114E and G422D amino acid substitutions have been
introduced into the protein's two cAMP-binding domains (17).
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Fig. 6.
8-pCPT-2'-O-Me-cAMP as a
stimulus for CICR in INS-1 cells. INS-1 cells loaded with
fura-2 exhibited a transient increase of
[Ca2+]i in response to 50 µM
8-pCPT-2'-O-Me-cAMP (A). The action of
8-pCPT-2'-O-Me-cAMP was mimicked by 10 µM
forskolin (B, forsk.) and by 1 mM
8-Br-cAMP (C). Bath application of 300 µM
Rp-8-Br-cAMPS 10 min prior to and during application of the test
substance failed to block the action of 50 µM
8-pCPT-2'-O-Me-cAMP (D). Arrows
indicate a 10-s application of each test substance. To assure the
reproducibility of these findings, the action of each test substance
was confirmed in a minimum of four separate experiments using 12 or
more INS-1 cells.
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Fig. 7.
Pharmacological properties of
8-pCPT-2'-O-Me-cAMP. A, a population study
of INS-1 cells was performed and the percentage of cells exhibiting
CICR in response to 8-pCPT-2'-O-Me-cAMP was determined. The
action of 100 µM 8-pCPT-2'-O-Me-cAMP was
dose-dependent (* indicates statistical significance < 0.001 relative to 10 µM) and was not blocked by H-89
(10 µM), KT 5720 (10 µM), or Rp-8-Br-cAMPS
(300 µM). Transfection of INS-1 cells with dominant
negative Epac2 blocked the action of 8-pCPT-2'-O-Me-cAMP
(100 µM), whereas transfection with wild type
(WT) Epac2 did not (* indicates statistical
significance < 0.001 relative to WT Epac). Transfected cells were
identified by use of RIP2-EYFP as described previously (18).
B, 8-pCPT-2'-O-Me-cAMP failed to stimulate
luciferase activity in INS-1 cells transfected with CRE-Luc.
Application of the structurally related PKA activator 8-CPT-cAMP
produced a dose-dependent stimulation of CRE-Luc, and the
action of 8-CPT-cAMP was reduced by the PKA inhibitor H-89 (10 µM). (** indicates statistical significance < 0.005 relative to 30, 100, or 300 µM 8-CPT-cAMP in the absence
of H-89). This experiment was repeated twice, and the effect of each
concentration of test substance was evaluated in triplicate.
), with
concomitant stimulation of IP3 production (19, 20).
Therefore, Ca2+ mobilized by 8-pCPT-2'-O-Me-cAMP
as a consequence of CICR in INS-1 cells might originate from
Ca2+ stores regulated not only by ryanodine receptors (RYR)
but also by inositol trisphosphate receptors (IP3-R). INS-1
cells express muscarinic cholinergic receptors, and
acetylcholine is a stimulus for inositol phosphate (IP)
production (Table I). However, a role for
IP3-R in Epac-mediated signal transduction in INS-1 cells appears unlikely because no stimulation of IP production was observed in INS-1 cells exposed to 8-pCPT-2'-O-Me-cAMP, GLP-1, or
Ex-4 (Table I).
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Fig. 8.
Ryanodine sensitivity of
8-pCPT-2'-O-Me-cAMP-induced CICR. Ryanodine (10 µM) was applied to INS-1 cells by use of a micropipette.
A second micropipette was then positioned adjacent to the ryanodine
pretreated cell to deliver a test solution containing
8-pCPT-2'-O-Me-cAMP (100 µM) and ryanodine (10 µM). In 2 of 10 cells tested, ryanodine produced a
reversible block of CICR (A), whereas in 8 of 10 cells no
recovery from the inhibitory action of ryanodine was observed
(B). Arrows indicate a 10-s application of test
substances. The duration of ryanodine pretreatment is indicated by a
horizontal bar. Similar findings were obtained using a 1 µM concentration of ryanodine (data not shown).
8-pCPT-2'-O-Me-cAMP fails to stimulate production of inositol
phosphates in INS-1 cells
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Cell Stimulus-secretion
Coupling--
Until now, no certain means by which to specifically
activate Epac was available. Here we demonstrate that a newly developed Epac-selective cAMP analog (8-pCPT-2'-O-Me-cAMP) is a
stimulator of CICR and exocytosis in human
-cells.
8-pCPT-2'-O-Me-cAMP is also demonstrated to stimulate CICR
in an insulin-secreting cell line (INS-1), and this effect is
PKA-independent because it is not blocked by H-89, KT 5720, or
Rp-8-Br-cAMPS. Overexpression of a dominant negative Epac2 abrogates
CICR in response to 8-pCPT-2'-O-Me-cAMP as expected because
there exists in INS-1 cells an Epac2-signaling pathway critical to
hormonal regulation of Ca2+ signaling (18). The specificity
with which 8-pCPT-2'-O-Me-cAMP differentiates between PKA
and Epac is emphasized by its failure to reproduce the action of
8-CPT-cAMP in an assay of CRE-Luc activity that is diagnostic of
cAMP/PKA/CREB signal transduction. Therefore, 8-pCPT-2'-O-Me-cAMP is demonstrated to posses unique
Epac-selective properties that should allow its wide spread use in
assays of multiple cellular functions.
-Cells--
One difference between the findings of the present
study and those of our previous report (18) is that H-89 and
Rp-8-Br-cAMPS fail to block the action of
8-pCPT-2'-O-Me-cAMP, whereas both compounds exert inhibitory
effects when examining the action of GLP-1 receptor agonist exendin-4
(18). These contrasting results are understandable if
8-pCPT-2'-O-Me-cAMP acts exclusively via Epac2, whereas
exendin-4 acts not only via Epac2 but also PKA. It is also noteworthy
that in some INS-1 cells a sustained increase of
[Ca2+]i is observed in response to
8-pCPT-2'-O-Me-cAMP, and it is preceded by CICR
(cf., Figs. 6D and 8B). This biphasic
response to 8-pCPT-2'-O-Me-cAMP is reminiscent of the action
of GLP-1 in
-cells (8). GLP-1 is an inhibitor of ATP-sensitive
K+ channels (K-ATP), and by doing so it produces
-cell
depolarization, activation of Ca2+ influx, and a sustained
increase of [Ca2+]i (21, 22). It will be of
interest to ascertain if a similar inhibition of K-ATP is observed in
response to 8-pCPT-2'-O-Me-cAMP acting as an Epac-selective
modulator of K+ channel function. In this regard, it is
noteworthy that Epac contains not only Rap1 recognition motifs within
its guanyl nucleotide exchange factor catalytic domain, but also
additional protein-protein interaction motifs within its DEP domain
where homologies to Disheveled, Egl-10, and Pleckstrin are
found. A physical association of Epac and the SUR1 subunit of K-ATP has
been reported on the basis of a yeast two-hybrid screen (17), so it is
reasonable to speculate that the targets of Epac action may also
include cell surface K-ATP channels.
in HEK 293 cells (19, 20). The resultant increase of
[IP3] serves as a stimulus for CICR mediated by the
IP3-R. However, we find that the Ca2+
mobilizing action of 8-pCPT-2'-O-Me-cAMP is unlikely to be a consequence of Epac-mediated IP3 production.
8-pCPT-2'-O-Me-cAMP fails to increase levels of inositol
phosphates in INS-1 cells loaded with 3H-inositol. Instead,
available evidence indicates an important role for RYR as targets of
Epac action (8, 18, 23). Indeed, pretreatment of INS-1 cells with
ryanodine blocks CICR in response to 8-pCPT-2'-O-Me-cAMP.
Furthermore, it was previously reported that treatment of INS-1 cells
with the IP3-R inhibitor Xestospongin C fails to
block CICR in response to forskolin (18). Since our methods of analysis
were restricted to use of a dominant negative Epac2, no conclusion can
yet be reached concerning what role Eapc1 may play in
-cell
Ca2+ signaling. However, 8-pCPT-2'-O-Me-cAMP is
an activator of Epac1 (4), and in the present study it failed to
stimulate inositol phosphate production. Although these findings argue
against a role for the IP3 receptor as a target of Epac1
and Epac2 action in
-cells, additional studies are necessary to
reach a firm conclusion.
-cells.
-cells.
Exocytosis is observed to be time-locked to the increase of
[Ca2+]i generated by CICR, and indeed, no
exocytosis is observed in the absence of CICR. It appears, therefore,
that an increase of [Ca2+]i is a necessary
and not simply permissive stimulus for initiation of exocytosis. Of
course this conclusion does not preclude additional Epac-mediated
signaling events important to secretion. For example, the interaction
of Epac with Rim2, an insulin granule-associated protein, might play an
important role in conferring stimulatory effects of cAMP on exocytosis
in
-cells (17, 27-29). Despite this caveat, it is clear that the
mobilization of Ca2+ by cAMP is a highly significant
feature of
-cell stimulus-secretion coupling. Indeed, we recently
reported that CICR serves to amplify exocytosis in INS-1 cells
(12).
-cells. This
coupling is revealed through the use of a newly developed
Epac-selective cAMP analog. The findings are of medical importance
because they shed light on the blood glucose-lowering properties of an
insulinotropic hormone (GLP-1) currently under investigation for use in
the treatment of type-2 diabetes mellitus (30, 31). Multiple
PKA-independent actions of GLP-1 have been reported in
-cells, and
these actions include effects of GLP-1 on Ca2+ signaling
(18, 32), K-ATP (33, 34), insulin-granule-associated Rim2 proteins (17,
27), and insulin gene expression (13, 15). Findings presented here
demonstrate that 8-pCPT-2'-O-Me-cAMP is likely to serve as a
powerful pharmacological tool for assessment of Epac-mediated signaling
events that underlie such poorly understood effects of GLP-1.
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ACKNOWLEDGEMENTS |
---|
pSR Epac2 plasmids were the gift of
Dr. S. Seino. G. G. H. also acknowledges the contribution of human
islets from the Juvenile Diabetes Research Foundation
International-funded islet transplantation centers located at the
University of Minnesota (Dr. B. J. Hering), Washington University
(Dr. B. J. Olack), and the University of Miami (Dr. C. Ricordi).
![]() |
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. Section 1734 solely to indicate this fact.
¶ Supported by a Canadian Institutes of Health Research doctoral student award.
Supported by Operating Grant MOP12898 and a salary grant from
the Canadian Institutes of Health Research.
§§ Supported by the National Institutes of Health Grants R01-DK45817 and R01-DK52166, the American Diabetes Association (Research Grant Award), and the Marine Biological Laboratory, Woods Hole, MA (Research Fellowship). To whom correspondence should be addressed. Tel.: 212-263-5434; Fax: 212-689-9060; E-mail: holzg01@ popmail.med.nyu.edu.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211682200
2 H. Rehmann and J. Bos, unpublished findings.
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ABBREVIATIONS |
---|
The abbreviations used are: Epac, exchange protein activated by cAMP; PKA, protein kinase A; CICR, Ca2+-induced Ca2+ release; FBS, fetal bovine serum; EYFP, enhanced yellow fluorescent protein; CRE, cAMP-response elements; TRITC, tetramethylrhodamine isothiocyanate; SES, standard extracellular saline; CREB, cAMP-response element-binding protein; IP3, inositol trisphosphate; RYR, ryanodine receptors; IP3-R, IP3 receptors; IP, inositol phosphate; ER, endoplasmic reticulum.
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