G Model
YCECA-1235;
No. of Pages 11
ARTICLE IN PRESS
Cell Calcium xxx (2011) xxx–xxx
Contents lists available at ScienceDirect
Cell Calcium
journal homepage: www.elsevier.com/locate/ceca
cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein
kinase A in mouse pancreatic beta cells
Maša Skelin a , Marjan Rupnik a,b,∗
a
b
Faculty of Medicine University of Maribor, Slomskov trg 15, 2000 Maribor, Slovenia
CIPKeBiP, Jamova 39,1000 Ljubljana, Slovenia
a r t i c l e
i n f o
Article history:
Received 5 October 2010
Received in revised form
20 December 2010
Accepted 21 December 2010
Available online xxx
Keywords:
Ca2+ -dependent exocytosis
Pancreatic beta cells
Ca2+ sensitivity
a b s t r a c t
Cyclic AMP regulates the late step of Ca2+ -dependent exocytosis in many secretory cells through two
major mechanisms: a protein kinase A-dependent and a cAMP-GEF/Epac-dependent pathway. We
designed a protocol to characterize the role of these two cAMP-dependent pathways on the Ca2+ sensitivity and kinetics of regulated exocytosis in mouse pancreatic beta cells, using a whole-cell patch-clamp
based capacitance measurements. A train of depolarizing pulses or slow photo-release of caged Ca2+ were
stimuli for the exocytotic activity. In controls, due to exocytosis after slow photo-release, the Cm change
had typically two phases. We observed that the Ca2+ -dependency of the rate of the first Cm change follows saturation kinetics with high cooperativity and half-maximal rate at 2.9 ± 0.2 ␮M. The intracellular
depletion of cAMP did not change amp1, while rate1 and amp2 were strongly reduced. This manipulation pushed the Ca2+ -dependency of the exocytotic burst to significantly lower [Ca2+ ]i . To address the
question of which of the cAMP-dependent mechanisms regulates the observed shifts in Ca2+ dependency
we included regulators of PKA and Epac2 activity in the pipette solution. PKA activation with 100 ␮M 6Phe-cAMP or inhibition with 500 ␮M Rp-cAMPs in beta cells significantly shifted the EC50 in the opposite
directions. Specific activation of Epac2 did not change Ca2+ sensitivity. Our findings suggest that cAMP
modulates Ca2+ -dependent exocytosis in mouse beta cells mainly through a PKA-dependent mechanism
by sensitizing the insulin releasing machinery to [Ca2+ ]i ; Epac2 may contribute to enhance the rates of
secretory vesicle fusion.
© 2011 Published by Elsevier Ltd.
1. Introduction
Glucose is the main physiological stimulus for pancreatic beta
cells to release insulin. It is currently believed that an increase in
extracellular glucose concentration and increased uptake of glucose through GLUT2 increases the metabolism of glucose to form
ATP. ATP closes ATP-dependent potassium channels (KATP channels) and membranes of the beta cell syncytium depolarize, which
increases the opening of voltage-activated Ca2+ channels [1]. Glucose increases cytosolic Ca2+ activity, which is a major trigger to
promote the release of secretory granules. As in the synapse, exocytosis is managed by SNARE (soluble N-ethylmaleimide-sensitive
Abbreviations: 6-Phe-cAMP, N6 -phenyladenosine-3 ,5 -cyclic monophosphate;
8-CPT-2 -O-Me-cAMP, 8-(4-chlorophenylthio)-2 -O-methyl-adenosine 3 ,5 -cyclic
monophosphate; Cm , membrane capacitance; CSP, cysteine string protein; GEFII,
guanidine nucleotide exchange factor 2; GIP, gastric inhibitory polypeptide; GLP-1,
glucagon-like peptide-1; LV, large vesicles; NSF, N-ethylmaleimide-sensitive factor;
PKA, protein kinase A; PLC, phospholiase C.
∗ Corresponding author at: University of Maribor, Slomskov trg 15, 2000 Maribor,
Slovenia. Tel.: +386 2 330 5854; fax: +386 2 330 5853.
E-mail address: [email protected] (M. Rupnik).
factor attachment protein receptor) fusion machinery [2,3]. The
insulin release process was found to be modulated by second messengers like cAMP or phospholipase C (PLC) depending on which
signalling pathway has been activated by physiological ligands
[4,5].
cAMP is one of the central signalling molecules that has been
identified to regulate release of various neurotransmitters and
hormones, including insulin from pancreatic beta cells [4,6,7].
Two cAMP-dependent pathways have so far been reported to
directly control Ca2+ -dependent exocytosis, either through activation of PKA or a cAMP–guanidine nucleotide exchange factor
2 (GEFII)/Epac2-dependent pathway [6,8,9]. The action of cAMP
through PKA is believed to be a dominant pathway acting in
synapses [10], chromaffin cells [11,12], pituitary cells [13,14] and
was also suggested to be a major pathway in pancreatic beta cells
[4,15,16]. However, some other studies, including our own, provide evidence that cAMP is also acting through an Epac-dependent
pathway [17–21].
Hormones like incretins, including glucagon-like peptide-1
(GLP-1) and gastric inhibitory polypeptide (GIP), are known to
potentiate glucose-induced insulin secretion by generating cAMP
in pancreatic beta cells [8]. The significantly increased insulin
0143-4160/$ – see front matter © 2011 Published by Elsevier Ltd.
doi:10.1016/j.ceca.2010.12.005
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005
G Model
YCECA-1235;
No. of Pages 11
2
ARTICLE IN PRESS
M. Skelin, M. Rupnik / Cell Calcium xxx (2011) xxx–xxx
release due to incretins has a major contribution in the feedback mechanism by which insulin keeps plasma glucose levels
within physiological limits. GLP-1 and GIP were described to exert
cAMP actions through activation of PKA in pancreatic beta cells
[22]. Furthermore, actions of GLP-1 mediated by PKA and Epac2
were described to include the recruitment and priming of vesicles, thereby increasing the number of vesicles available for release.
Besides the direct effect on secretory vesicles, GLP-1 also promotes
Ca2+ influx and mobilizes an intracellular source of Ca2+ [6,23].
Moreover, it has been reported that cAMP enhances translocation
of granules and increases the size of the ready releasable pool and
the rate of replenishment [24].
The precise mechanism of PKA action in beta cells is not
fully understood. A plethora of proteins involved in the secretory
machinery have been found to be phosphorylated by PKA in vitro
and in vivo in different cell types. PKA phosphorylation has been
described for several proteins including cysteine string protein
(CSP), snapin, Rim1, SNAP-25, syntaphilin and synapsin [9]. The
phosphorylation of CSPs by PKA has been reported to modify exocytotic kinetics [25]; phosphorylation of snapyn by PKA caused
a larger initial exocytotic burst in chromaffin cells [26]. In addition to a larger number of available secretory vesicles and increase
in the rate of their release, PKA was found to promote insulin
secretion by increasing the number of vesicles that are highly sensitive to Ca2+ and thereby sensitizing the secretory machinery to
Ca2+ [16]. More classic effects of PKA activation include larger Ltype voltage-activated Ca2+ currents that upon activation result in
higher cytosolic Ca2+ and therefore potentiate exocytosis from pancreatic beta cells [4,9] as well from pituitary cells [17]. As well as
the effect on Ca2+ channels, PKA phosphorylation has been shown
to partially increase membrane permeability to Na+ [9].
cAMP also potentiates Ca2+ -dependent exocytosis by a PKAindependent mechanism. This mechanism involving the cAMPbinding protein Epac2 has been shown to increase the number of
ready releasable vesicles [6,20]. Another hypothesis was recently
presented where activation of Rap1 by cAMP through Epac2 mediates insulin secretion by increasing the size of the non-docked
granule pool and by facilitating the recruitment of the granules
to the plasma membrane [27]. Some actions of Epac2 are not
mediated by Rap1 because it was reported that Epac2 interacts
directly with insulin granule-associated proteins, Rim2 and Piccolo
[28,29]. Epac2 was also identified as a molecule interacting with
the sulfonylurea receptor SUR1 [21] and thereby directly promoting insulin secretion. An Epac2-dependent pathway has also been
described to be involved in increase of membrane permeability to
Na+ [9].
In the present paper we assessed the role of cAMP during
Ca2+ -dependent exocytosis using electrophysiological approach.
The addition of cAMP into cytosol of beta cells triggered exocytosis
of the secretory vesicles at lower intracellular Ca2+ concentration
([Ca2+ ]i ) and the half-maximal rate of the release was achieved
at lower [Ca2+ ]i . This suggests that cAMP sensitizes the secretory
machinery to Ca2+ . A direct activation of PKA, but not Epac2, also
significantly shifted Ca2+ sensitivity. The present work suggests
that the mechanism of action underlying such a change in the sensitivity to Ca2+ should be destabilization of the fusion pore.
2. Methods
2.1. Isolation of primary beta cells
Adult male NMRI mice were killed by cervical dislocation.
Liberase (0.3 mg ml−1 ; Roche, USA) dissolved in Hank’s buffered
salt solution (Invitrogen, USA) was injected into the pancreas
through the bile duct. The pancreas was removed and digested for
10–15 min at 37 ◦ C. After digestion, 10 ml of ice cold Hanks’ bal-
anced salt solution supplemented with 10 mM HEPES, 50 IU ml−1
penicillin G and 0.05 mg ml−1 streptomycin was added to the suspension to stop the reaction. Islets were collected by centrifugation,
trypsinized into single cells and cultured on coverslips in RPMI 1640
medium (Invitrogen, USA) supplemented with 10% fetal bovine
serum, 2 mM l-glutamine, 100 IU ml−1 penicillin G and 0.1 mg ml−1
streptomycin in a humidified atmosphere with 5% CO2 at 37 ◦ C.
Cells were cultured 24–48 h before the electrophysiological experiments.
2.2. Preparation of mouse pancreatic tissue slices
Mouse pancreatic tissue slices were prepared as previously
described [30]. Low-gelling agarose (Seaplaque GTG agarose, Lonza,
USA; 0.475 g in 25 ml extracellular solution) was melted and kept
on 37 ◦ C. Adult male NMRI mice were killed by cervical dislocation. After opening the abdominal cavity, agarose was injected
into the pancreas through bile duct. After injection, the pancreas
was cooled with an ice-cold extracellular solution. The injected
and hardened pancreas was then extracted and placed in ice-cold
extracellular solution. Fat and connective tissue was removed from
pancreas. Small pieces (3 mm × 3 mm) were cut from the pancreas
and embedded into the agarose. Agarose-embedded pancreatic tissue cubes were glued (Super Glue, ND Industries, Troy, Mich., USA)
onto the sample plate of the vibrotome (VT 1000 S, Leica, Nussloch,
Germany). The pancreatic tissue was cut at 0.05 mm/sec at 70 Hz
into 140 ␮m-thick slices. During slicing the tissue was kept in an
ice-cold extracellular solution bubbled continuously with carbogen (95% O2 , 5% CO2 ). After slicing the tissue slices were kept in
carbogen-bubbled extracellular solution and used for electrophysiological experiments within the next 8 h.
2.3. Electrophysiology
The coverslip with cells or an agarose-embedded slice was transferred from the incubator into the perfusion chamber and fixed
with a U-shaped platinum frame. When using dispersed cells 1.5 ml
of extracellular solution consisting of 150 mM NaCl, 10 mM HEPES,
3 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2 , pH 7.2 and
osmolality 300 ± 10 mOsm was added to the chamber. For pancreatic slices, extracellular solution containing 125 mM NaCl, 2.5 mM
KCl, 26 mM NaHCO3 , 1.25 mM Na2 HPO4 , 2 mM sodium pyruvate,
0.25 mM ascorbic acid, 3 mM myo-inositol, 6 mM lactic acid, 1 mM
MgCl2 , 2 mM CaCl2 and 3 mM glucose, adjusted to pH 7.3 by gassing
with carbogen for at least 30 min before the experiment, was
continuously supplied to the perfusion chamber. When using depolarization protocol for triggering Cm change, 5 mM CaCl2 instead of
2 mM CaCl2 was included in the extracellular solution. The pipette
solution used for Ca2+ -induced capacitance and current measurements was composed of 5 mM NP-EGTA (Invitrogen, USA), 4 mM
CaCl2 , 0.1 mM Fura 6F (Invitrogen, USA) together with 125 mM CsCl,
40 mM HEPES, 2 mM MgCl2 , 20 mM TEA–Cl, 2 mM Na2 ATP at pH 7.2
and osmolality 300 ± 10 mOsm. The current measurements in isolated beta cells turned out to be a useful tool to differentiate among
the different Cm phases, particularly helpful to exclude the possibility of the contamination of the Cm changes by the endocytotic processes. The latter were almost completely absent in the beta cells
still inside the whole pancreas slice. However, the current characteristics remained the same as in beta cell in dispersed cell cultures.
The competitive antagonist of cAMP binding to PKA, Rpadenosine 3 ,5 -cyclic-monophosphorothioate triethylammonium
salt (Rp-cAMPs; Biolog Life Sci., Germany) and PKA agonist, 6-PhecAMP (Biolog Life Sci., Germany), were included in the pipette
solution at concentrations of 0.5 mM and 0.1 mM, respectively.
A selective cAMP-GEF/Epac agonist, 8-(4-chlorophenylthio)-2 -
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005
G Model
YCECA-1235;
No. of Pages 11
ARTICLE IN PRESS
M. Skelin, M. Rupnik / Cell Calcium xxx (2011) xxx–xxx
O-methyl-adenosine 3 ,5 -cyclic monophosphate (8-CPT-2 -O-MecAMP; Biolog Life Sci., Germany), was added to the pipette solution
to a final concentration of 0.1 mM. All chemicals were purchased
from Sigma–Aldrich (Germany) unless otherwise indicated.
Cells were visualized with an upright Nikon Eclipse E600 FN
microscope (Nikon, Tokyo, Japan) and a mounted CCD camera
(Cohu, San Diego, CA, USA). Patch pipettes were pulled from borosilicate glass capillaries (GC150F-15, Harvard Apparatus, USA) using
a horizontal pipette puller (P-97, Sutter Instruments, USA). The
pipette resistance was 2–3 M in Cs-based solution. Fast pipette
capacitance (Cfast ) was compensated before and slow membrane
capacitance (Cslow ) as well as series conductance (Gs) were compensated after whole-cell breakthrough. Only experiments with
Gs > 50 nS were processed. Recordings were performed in the standard whole-cell mode via a patch-clamp lock-in amplifier (SWAM
IIc, Celica, Slovenia), low-pass filtered, transferred to a PC via an
A/D converter (12 bit, Nidaq PCI-6035E, National Instruments) and
recorded on the hard disk using WinWCP V3.9.6 software (John
Dampster, University of Strathclyde, UK). The same software was
used to apply the voltage protocol for identifying beta cells by
their Na+ current inactivation pattern. A continuous sine voltage
(1600 Hz, 11 mV RMS amplitude) was applied to measure Cm , a
parameter that is proportional to membrane surface area [31]. Resting membrane potential in voltage-clamp mode was −80 mV.
In beta cells within the intact pancreatic tissue slices all currents were analyzed and presented after leak subtraction. Secretory
activity was triggered by trains of depolarization. To determine the
evoked Cm , Cm was first averaged over the 30 ms preceding the
depolarization to obtain a baseline value that was subtracted from
the value estimated after the depolarization averaged over a 40ms pulse. The first 30 ms after the depolarization were excluded
from the Cm measurement to avoid contamination by nonsecre-
3
tory capacitive transients related to gating charge movement [32].
Only experiments not displaying crosstalk between Cm and membrane conductance monitored in parallel were used for analysis.
To calculate Ca2+ charge entry, the first 2 ms of the inward current
integral were omitted to reduce contamination with Na+ current.
All electrophysiological experiments were performed at room
temperature or at 32 ◦ C when using isolated cells or tissue slices,
respectively. Control experiments (no cAMP in the patch pipette)
were performed on adult male beta cells. Signal processing and
curve fitting was done using Matview (Wise Technologies, Ljubljana, Slovenia) and Matlab (Mathworks, USA). Estimations of the
number of released vesicles are based on a value of 3.6 fF per vesicle
for adult beta cells [33,34].
2.4. Ca2+ -measurements
Fura 6F (0.1 mM in pipette solution; Molecular Probes, USA)
was used to measure the intracellular Ca2+ concentration ([Ca2+ ]i )
simultaneously with the patch-clamp recordings. Fura 6F was
excited at 380 nm with a monochromator (Polychrome IV; TILL
Photonics, Germany). A long-pass dichroic mirror reflected the
monochromatic light nm to the perfusion chamber and transmitted the emitted fluorescence above 400, which was further filtered
through a 420-nm barrier filter. The fluorescence intensity was
measured with a photodiode (TILL Photonics, Germany). [Ca2+ ]i was
calculated as described previously [35].
2.5. Statistical analysis
Statistical analysis was performed using Sigmaplot 11.0 (SPSS,
USA). Data and the results are reported as means ± S.E.M. for the
indicated number of experiments. The unpaired Student’s t-test
Fig. 1. Exocytosis during the stimulation with repetitive depolarization pulses. (A) Cumulative Cm change (middle panel) in response to a train of 50 depolarization pulses
(upper panel) of 40 ms duration from −80 mV to +10 mV at a frequency of 10 Hz in control cell (black circles) and cell with increased cAMP concentration (gray circles). In
cell treated with 200 ␮M cAMP, Cm was triggered faster compared to control, while there was no significant difference in the amplitude of current change (lower panel).
(B) Cumulative Cm change of representative control cell (black circles) and cell with increased cAMP concentration (gray circles) plotted against the cumulative Ca2+ current
integrals normalized to the cell size (normalized Q).
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005
G Model
YCECA-1235;
4
No. of Pages 11
ARTICLE IN PRESS
M. Skelin, M. Rupnik / Cell Calcium xxx (2011) xxx–xxx
Fig. 2. Slow photo release of caged Ca2+ induced a biphasic increase in Cm with maximal rate of the first phase showing a saturation kinetic with high cooperativity. (A) Slow
photo-release of Ca2+ produced a ramp-like increase in [Ca2+ ]i reaching its peak amplitude (Caamp2 ) after about 10 s. (B) After reaching the threshold value of [Ca2+ ]i (Catr )
a biphasic increase in membrane capacitance (Cm ) has been triggered, with the first phase reaching maximal amplitude within the first second after the initiation (amp1)
and the second phase reaching maximal amplitude (amp2) at Caamp2 . (C) Time derivative of Cm amplitude presented in (B), with maximal rate of the first phase (rate1) and
the maximal rate of Cm change during the second phase (rate2). (D) The rate of the Cm change shown in C shows saturation kinetics when plotted versus [Ca2+ ]i , with high
cooperativity and half-effective [Ca2+ ]i (EC50 ) at 2.9 mM; inset, curves showing a Hill function fit through the Ca2+ -dependence data in isolated cells (solid line) and pancreatic
slices (dashed line).
was used to evaluate the statistical significance, unless otherwise indicated. For multiple comparisons, one-way ANOVA and
Holm–Sidak post hoc test were used. A p value <0.05 was considered statistically significant.
3. Results
3.1. Trains of depolarization triggered Ca2+ -dependent exocytosis
at lower Ca2+ current in cAMP treated cells compared to the
control beta cells
In beta cells within the pancreatic tissue slices we used a voltage pulse protocol with 50 40-ms long depolarization pulses (from
−80 mV to +10 mV, Fig. 1A, upper panel) to mimic the glucose
induced-spiking activity and thereby elicit inward Ca2+ current
(Fig. 1A, lower panel, black line). Increase in cytosolic Ca2+ concentration ([Ca2+ ]i ) triggered Ca2+ -dependent exocytosis which we
detected as a change in Cm (Fig. 1A, middle panel, black circles). As
previously described [30] at least one third of the beta cells did not
show voltage dependent inward current during the voltage protocol. Next we included 200 ␮M cAMP in the pipette solution. Faster
exocytotic responses were observed in cAMP treated cells (Fig. 1A,
middle panel, gray circles) compared to the controls (Fig. 1A, middle panel, black circles) while there were no differences in the
amplitude of the inward current (Fig. 1A, lower panel, black and
gray lines for control and cells with elevated cAMP concentration,
respectively). Next we plotted change in Cm as a function of cumulative charge (Q) triggered with depolarization pulses [36]. In cells
treated with 200 ␮M cAMP (Fig. 1B, gray circles) Cm was triggered
at lower Ca2+ charge entry compared to the controls (Fig. 1B, black
circles).
3.2. Kinetic phases of exocytosis at increased [Ca2+ ]i in pancreatic
beta cells
Since at least one third of beta cells did not react to the depolarization protocol and because of the high variability in Ca2+
current density during voltage protocol we decided to elevate
[Ca2+ ]i uniformly through the cell using slow photo-release of
caged Ca2+ . Under control conditions, slow photo-release of Ca2+
caged to NP-EGTA buffer released Ca2+ ions in the [Ca2+ ]i range
of several ␮M (Fig. 2A) with a rate of change comparable with
global [Ca2+ ]i changes during the train of depolarizing pulses
[30]. Increased [Ca2+ ]i stimulated exocytosis change (Fig. 2B). We
perceived exocytosis as a change in Cm using high-resolution
whole-cell patch-clamp recordings. In this representative experiment, [Ca2+ ]i reached a threshold level at Catr , followed by a
biphasic membrane capacitance with maximal amplitudes amp1
(311 ± 37 fF, n = 28) and amp2 (1589 ± 225 fF, n = 26) for the first
and the second phase, respectively. The first phase reached its maximal amplitude within the first second after the start of Cm change.
The second phase saturated within the next 5 s after the onset of
Cm change (Fig. 2B). The time derivatives for the Cm traces showed
two separate kinetic exocytotic components, fast and slow phase,
with the maximal rates of Cm change within each phase (rate1 and
rate2; Fig. 2C). The maximal rate of the first phase was compa-
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005
G Model
YCECA-1235;
No. of Pages 11
ARTICLE IN PRESS
M. Skelin, M. Rupnik / Cell Calcium xxx (2011) xxx–xxx
5
Fig. 3. Types of Cm change observed during slow photo-release of caged Ca2+ . (A) Increased [Ca2+ ]i triggered Cm change in a Ca2+ -dependent manner. If [Ca2+ ]i did not reach
the threshold level, no Cm was observed (lower trace). When [Ca2+ ]i reached the Catr level but did not exceed Cai2 , only the first phase of Cm was observed. On the other
hand, [Ca2+ ]i higher than Cai2 triggered the first and the second phase of Cm change with amp2 proportional to the [Ca2+ ]i change (upper two traces). (B) In addition to
Ca2+ -dependent exocytosis, Ca2+ -dependent endocytosis was observed with amp2 proportional to the [Ca2+ ]i change.
rable with the results obtained from rat islets in pancreatic slices
where depolarization was used to trigger exocytosis [36]. When
the photo-release resulted in lower [Ca2+ ]i only the first phase may
be recorded (Fig. 2A, trace at 4 ␮M Ca2+ ). We expressed the rate
of the release in fFs−1 instead of events per seconds because there
is an unsolved dilemma regarding the involvement of small vesicles in the regulated exocytotic events in beta cells [18]. Detailed
analysis of the experimental traces revealed that the initial level of
[Ca2+ ]i (Catr ) to trigger the Cm increase was 2.6 ± 0.1 ␮M (n = 28) and
very reproducible. Expressing rate1 for the corresponding [Ca2+ ]i
showed saturation kinetics (Fig. 2D). We fitted the Hill function
and established that the half-maximal rate of the Cm change was
achieved at 2.9 ± 0.2 ␮M [Ca2+ ]i (EC50 , n = 26, Fig. 2D, inset, solid
line). The cooperativity of the dependence has been high range
(data not shown). Comparing the results from dispersed beta cells
with data obtained from beta cells in pancreatic slices, there were
no differences in half-maximal effective Ca2+ concentration or the
maximal rates of exocytotic events (Fig. 2D inset, dash line). However, there were major differences in the presence of substantial
endocytosis following the first phase of Cm change (Fig. 3B), which
seems to be completely absent in pancreatic slice preparations.
Due to the comparable characteristics between the preparations,
we performed most of the experiments in dispersed cells, because
of the ease of experimentation.
Depending on the [Ca2+ ]i reached during slow photolysis, various types of Cm traces were observed (Fig. 3A). If [Ca2+ ]i did not
reach the threshold level, there was no change in Cm . On the
other hand, when the [Ca2+ ]i just exceeded Catr only the first
phase of Cm was observed; when the [Ca2+ ]i was higher than
Cai2 (Fig. 4A), the first and second phase of Cm were observed.
Furthermore, amp2 was highly [Ca2+ ]i dependent (Fig. 3A). As
well as the exocytotic changes, the Ca2+ -dependent endocytosis
was also observed in about 25% of control cells in cell culture
(Fig. 3B).
The slow photo-release of caged Ca2+ , in addition to Cm change,
also triggered an inward (Im ) current with 2 distinguishable phases
(Fig. 4C). The [Ca2+ ]i to stimulate the first phase was 0.5 ± 0.1 ␮M
(Cai0 , n = 20), significantly lower than that required to trigger
the first phase of Cm change. The second phase of Im started at
4.2 ± 0.2 ␮M (Cai2 , n = 20, Fig. 4C) which only slightly preceded the
second phase of Cm change and may represent the time of onset of
Fig. 4. Increased [Ca2+ ]i triggered a biphasic increase in Cm and biphasic current
change. (A) Increase in [Ca2+ ]i after slow photo-release. Cai0 and Cai2 indicate the
[Ca2+ ]i where the first and the second phase of current were triggered, respectively.
(B) A biphasic increase in Cm with the first and the second phase if we extend the Cm
trace to the base line to mark the onset of the second phase (dark gray, dashed line).
(C) A biphasic current change triggered after increase in [Ca2+ ]i . In the control, the
first phase of the current change was triggered at [Ca2+ ]i , which is 2 ␮M lower than
[Ca2+ ]i at which the first phase of Cm was triggered; the second phase of current
change was triggered simultaneously with the second phase of Cm change.
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005
G Model
YCECA-1235;
6
No. of Pages 11
ARTICLE IN PRESS
M. Skelin, M. Rupnik / Cell Calcium xxx (2011) xxx–xxx
Fig. 5. cAMP increased Ca2+ sensitivity of the exocytotic burst. (A) An increase in [Ca2+ ]i (upper panel) together with Cm change (lower panel) in control cells (black line),
cells with increased cAMP concentration (dark gray line) and cells dialysed with 0 mM ATP (light gray line). (B) Bar chart on the left represents the rate of the first phase
with no significant difference in control cells, cells treated with cAMP or dialysed with 0 mM ATP. Bar chart on the right represents the half-effective [Ca2+ ]i (EC50 ). EC50 was
significantly decreased in cells treated with 200 ␮M cAMP compared with the controls and cells dialysed with 0 mM ATP. Data was analyzed using one-way ANOVA and the
results are presented as means ± S.E.M. ***p < 0.001. The number of cells tested is given on the bars. (C) Bar chart represents the [Ca2+ ]i needed for triggering a Cm change
(Catr ). Catr was significantly decreased in cells treated with 200 ␮M cAMP compared with the controls and cells dialysed with 0 mM ATP. Data was analyzed using one-way
ANOVA and the results are presented as means ± S.E.M., ***p < 0.001. The number of cells tested is given on the bars.
the second exocytotic phase, which is otherwise mostly hidden in
the initial exocytotic burst. To mark the onset of the second phase
of Cm change we extrapolated the Cm trace to the base line (Fig. 4B,
dark gray dash line).
3.3. The effect of cAMP on Ca2+ -dependent exocytosis
To assess how cAMP affects the Ca2+ -dependent exocytosis in
pancreatic ␤-cells we first included 200 ␮m cAMP in the pipette
solution to provide saturating conditions [17]. The maximal capacitance rate in cells dialysed with cAMP did not differ from the
control (Fig. 5B, left panel); the half-maximal rate was achieved
at 1.6 ± 0.2 ␮M of free Ca2+ (n = 23, p < 0.001, Fig. 5B, right panel)
which is significantly lower compared with the control. The Ca2+
concentration triggering Ca2+ -dependent exocytosis (Catr ) was significantly lower (1.5 ± 0.2 ␮M, n = 28, p < 0.001, Fig. 5C), and amp1
was indistinguishable from the control (Fig. 6A). We did not observe
any significant differences in amp2 (Fig. 6B) or rate2 (Fig. 6D).
Next we performed the wash out of ATP, which should result
in reduced cAMP production. Slow photo-release of caged Ca2+
shows the same pattern including two separate exocytotic components. This manipulation did not differ in amp1 (Fig. 5A) or in
rate1 (Fig. 5B, left panel) compared with the control. The [Ca2+ ]i
required for triggering Cm change (Catr ) was also comparable
with the control (Fig. 5C). Cells dialysed with 0 mM ATP exhibit
a shift in Ca2+ -dependency towards higher Ca2+ concentrations
(3.2 ± 0.2 ␮M, n = 10, p < 0.001; Fig. 5B, right panel) compared with
the cells with increased cAMP concentration, which could suggest
an important role of cAMP in sensitizing the secretory machinery
to Ca2+ .
Although ATP depletion did not attenuate the amplitude of
the first phase of Cm change, we expected the second phase to
be depleted as previously reported [37]. We observed a strongly
reduced amp2 (664 ± 156 fF, n = 14, p < 0.05; Fig. 6B) and rate2
(98 ± 25 fF/s, n = 12, p < 0.05; Fig. 6D) in contrast to control cells and
cells treated with cAMP; the Cai2 at which the second phase of Cm
was triggered was comparable with the control (Fig. 6C).
3.4. PKA-dependent and PKA-independent pathway of insulin
release
To assess whether cAMP acts through a PKA- or EPAC2-sensitive
mechanism we first included 100 ␮M N6 -phenyladenosine-3 ,5 cyclic monophosphate (6-Phe-cAMP) in the pipette solution.
6-Phe-cAMP is a potent site-selective activator of PKA showing a
strong preference for site A of both isozymes (type I and type II) and
at the same time does not activate Epac2 [38]. Glucose-stimulated
insulin secretion in pancreatic beta cells has been reported to be
both PKA- and Epac2-dependent [18]. The cumulative Cm change
under this condition did not significantly differ from the control.
The amp1 (Table 1) and amp2 (Table 1) were comparable with the
control as well as rate1 (Fig. 7D). On the other hand, activation
of PKA by 6-Phe-cAMP triggered an additional high Ca2+ -sensitive
phase (Fig. 7A, gray line) at [Ca2+ ]i comparable with those in cells
treated with cAMP (Fig. 7C). The half-maximal rate of Cm change
was also achieved at significantly lower EC50 (Fig. 7B, gray solid
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005
ARTICLE IN PRESS
G Model
YCECA-1235;
No. of Pages 11
M. Skelin, M. Rupnik / Cell Calcium xxx (2011) xxx–xxx
7
Fig. 6. ATP depletion attenuates amp2. (A) Bar chart represents the first amplitude of Cm change with no significant differences between controls, cAMP-treated cells and
cells dialysed with 0 mM ATP. (A) Bar chart represents the second phase amplitude of Cm change. The second amplitude was significantly reduced in cells dialysed with 0 mM
ATP compared with the controls and cells treated with 200 ␮M cAMP. (C) Bar chart represents the [Ca2+ ]i at which the second phase of Cm change was triggered (Cai2 ) with
no significant differences between controls, cAMP-treated cells and cells dialysed with 0 mM ATP. D, bar chart represents the rate of the second phase (rate2). Rate2 was
significantly reduced (one-way ANOVA), in cells dialysed with 0 mM ATP compared with the controls and cells treated with 200 ␮M cAMP. Data was analyzed using one-way
ANOVA and the results are presented as means ± S.E.M., *p < 0.05. The number of cells tested is given on the bars.
line) compared with the control conditions, suggesting that cAMP
acts through a PKA-dependent mechanism.
We next decided to remove the PKA activity by adding 0.5 mM
Rp-cAMPs, a competitive antagonist of cAMP binding to PKA, to the
pipette solution. The Cm change was triggered at Catr (Fig. 7C) which
is indistinguishable from the control. No changes were observed in
amp1 (Table 1), rate1 (Fig. 7D), amp2 (Table 1) and rate2 (Fig. 7F).
Despite unchanged amplitudes of Cm change, we observed that RpcAMPs pushed the EC50 towards a higher [Ca2+ ]i (p < 0.001; Fig. 7B,
gray dash line) compared with the cells treated with cAMP and 6Phe-cAMP, which indicates that activation of PKA is important to
determine the Ca2+ sensitivity of the fusion machinery.
Table 1
Numerical data for different experimental conditions.
Amp1 (fF)
Control
cAMP
Without ATP
6-Phe-cAMP
Rp-cAMPS
8-pCPT-O-2 -Me-cAMP
311
263
221
320
316
286
±
±
±
±
±
±
37
44
44
41
30
45
Rate1 (fF/s)
329
302
333
402
452
610
±
±
±
±
±
±
42
48
62
105
60
153
EC50 (␮M)
2.9
1.6
3.2
1.8
3.3
2.7
±
±
±
±
±
±
0.2
0.2
0.2
0.4
0.2
0.2
Amp2 (fF)
1588
1332
671
1464
1445
1536
±
±
±
±
±
±
225
184
155
158
156
197
Rate2 (fF/s)
209
225
98
334
265
404
±
±
±
±
±
±
30
35
25
69
48
43
Catr (␮M)
2.6
1.5
2.8
1.7
2.8
2.2
±
±
±
±
±
±
0.1
0.2
0.2
0.4
0.2
0.2
Cai2 (␮M)
4.2
4.1
4.6
4.5
3.9
3.6
±
±
±
±
±
±
0.2
0.3
0.2
0.5
0.3
0.1
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005
G Model
YCECA-1235;
8
No. of Pages 11
ARTICLE IN PRESS
M. Skelin, M. Rupnik / Cell Calcium xxx (2011) xxx–xxx
Fig. 7. cAMP increases the sensitivity to Ca2+ of exocytosis primarily through PKA. (A) Time derivatives of Cm rates of the first phase in control cells and 6-Phe-cAMP-treated
cells show an additional high Ca2+ -sensitive phase in PKA-activated cells. (B) Normalized Cm rate versus [Ca2+ ]i in control cells (black, dotted line), cells with increased cAMP
concentration (black, solid line), cells treated with PKA activator (light gray, solid line) and inhibitor (light gray, dashed line), and cells treated with specific Epac activator
(dark gray, solid line). The half-effective Ca2+ concentration was achieved at significantly lower [Ca2+ ]i in cells with increased cAMP concentration and cells dialysed with
PKA activator 6-Phe-cAMP compared with the control. (C) Bar chart represents the [Ca2+ ]i at which the first phase of Cm change was triggered (Catr ). The Catr was significantly
lower in cells treated with cAMP and selective PKA activator 6-Phe-cAMP compared with the control and PKA inhibitor Rp-cAMPS. (D) Bar chart represents the rate of the first
phase of Cm change (rate1). Rate1 was significantly increased in cells treated with Epac activator 8-pCPT-2 -O-Me-cAMP compared with the controls and cells with increased
cAMP concentration. (E) Bar chart represents the [Ca2+ ]i at which the second phase of Cm change was triggered (Cai2 ) with no significant differences among different pipette
solutions. (F) Bar chart represents the rate of the second phase (rate2). Rate2 was significantly increased in cells treated with Epac activator 8-pCPT-2 -O-Me-cAMP compared
with the controls and cells with increased cAMP concentration. Data was analyzed using one-way ANOVA and the results are presented as means ± S.E.M., *p < 0.05, **p < 0.01,
***p < 0.001. The number of cells tested is given on bars.
It has recently been shown [18,39] that PKA-dependent and
PKA-independent pathways regulate exocytosis in endocrine cells.
The presence of an alternative cAMP-dependent pathway through
Epac2 in secretion was tested with 8-pCPT-2 -O-Me-cAMP, a
potent, specific and membrane-permeant activator of the Epac
cAMP receptor. When 8-pCPT-2 -O-Me-cAMP was used, no significant changes were observed in amp1 (Table 1) or amp2 (Table 1).
On the other hand, rate1 (p < 0.001; Fig. 7D) and rate2 (p < 0.05;
Fig. 7F) were significantly higher compared with the controls
and cells treated with 200 ␮M cAMP. The Catr (Fig. 7C) and EC50
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005
G Model
YCECA-1235;
No. of Pages 11
ARTICLE IN PRESS
M. Skelin, M. Rupnik / Cell Calcium xxx (2011) xxx–xxx
9
(Fig. 7B, dark gray solid line) in 8-pCPT-2 -O-Me-cAMP-treated
cells were indistinguishable from the changes in controls but
significantly higher compared with the cells treated with either
200 ␮M cAMP (p < 0.02) or 6-Phe-cAMP (p < 0.05), meaning that
the activity of cAMP is not mimicked by the Epac-selective cAMP
analogue.
3.5. PKA and [Ca2+ ]i dependent Im changes in beta cells
As mentioned earlier slow photo-release of caged Ca2+ produced
changes in Cm and Im . Detailed analysis did not reveal any significant differences in the [Ca2+ ]i (Cai0 , data not shown) or [Ca2+ ]i
(Cai2 ; Fig. 7E) at which the first and the second phase of inward
current was triggered, respectively. In control cells, the first phase
of current change was not associated with a change in Cm (Fig. 8).
However, in cells treated with cAMP, the first phase of Im started
at the same time as the first phase of Cm change. Similarly, with
direct activation of PKA when 6-Phe-cAMP was included in the
pipette solution, the first phase of current change was again triggered simultaneously with the Cm change.
4. Discussion
In beta cells within the intact pancreatic tissue slice, change
in Cm was triggered using voltage depolarization protocol. Since
at least one third of beta cells did not respond to this protocol
we preferred to use slow photo-release of caged Ca2+ to trigger
Ca2+ -dependent exocytosis. Slow photo-release of caged Ca2+ is
an effective method for studying regulated exocytosis because the
ramp-like increase in [Ca2+ ]i leads to robust secretion in pancreatic beta cells [40]. Furthermore, slow photo-release of caged Ca2+
increases [Ca2+ ]i uniformly in the cell and thereby avoids the problem of loss of Ca2+ and Na+ channels during cell isolation procedure.
This approach bypasses any modulatory effects that act on electrical excitability and Ca2+ influx [41] and enables the detailed study
of Ca2+ sensitivity.
Our aim was to assess the effect of cAMP on the Ca2+ -dependent
exocytosis triggered by slow photo-release in mouse pancreatic
beta cells. The secretory rates observed in isolated pancreatic beta
cells were comparable with those observed by trains of depolarizations in pancreatic tissue slices where the maximal rate of
secretory granule fusion was around 400 fF/s This is indeed significantly slower compared to the original estimates on isolated
beta cells [42]. All subsequent studies on the rate of Cm changes
triggered by short (40–50 ms) depolarization pulses yielded comparable rates (see fig. 3B in [36,43]). Longer depolarization pulses
produced significantly slower Cm changes in the range of 50 fF/s
[43]. We believe that Ca2+ microdomains are not necessary to
explain fast release in beta cells and that uniform increase of Ca2+
is a good measure of a Ca2+ change during the physiological stimulation. In slow photo-release when the Hill function was fitted
through Ca2+ -dependent data, the half-maximal rate of the Cm
change was achieved at EC50 which is comparable with the data
obtained from pancreatic slices for beta cells (Fig. 2D, inset) and ␣cells (data not shown) but not adrenal chromaffin cells or pituitary
cells (data not shown). Furthermore, if we estimate the number of
vesicles released during the first phase of Cm change, we can see
that approximately 86 large vesicles (LV) with average diameter of
3.6 fF [34] were fused, although the first phase of Cm change may
also partially represent a group of synaptic vesicles [18]. This is the
reason why we chose to express the exocytotic process as dCm /dt
instead of a per vesicle rate. In control cells the first phase amplitude was comparable with previous reports where a Cm change
with an amplitude of 200 fF was observed with a time constant
of 1 s upon increase of [Ca2+ ]i to 3 ␮M [44,45]. These reports suggested that the first phase of Cm change represents exocytosis from
Fig. 8. Increased [Ca2+ ]i evoked biphasic Cm change as well as biphasic current
change. Increased [Ca2+ ]i (upper panel) evoked biphasic Cm change (middle panel) as
well as biphasic current change (lower panel). The first phase of current change was
triggered at the same [Ca2+ ]i in controls, cell with increased cAMP concentration and
in cells treated with PKA activator 6-Phe-cAMP. In cAMP- and 6-Phe-cAMP-treated
cells the changes in Cm were also related to change in the first phase of current
change; in controls, the first phase of current change was not associated with Cm
change. In controls, the first phase of Cm change was triggered at almost 2 ␮M higher
[Ca2+ ]i compared with [Ca2+ ]i at which the first phase of current was triggered.
a ready releasable pool. The rate of vesicle fusion during the first
phase in control cells was 327 ± 42 fF/s, which can also be expressed
as 90 LV s−1 . This is in accordance with previously published data
[46].
If cAMP stimulates insulin granule mobilization, an increase
in the size of the pool available for immediate release should be
observed. According to our results, when the intracellular cAMP
concentration was increased to 200 ␮M, the amplitude as well as
the rate of the first phase of Cm change did not differ from the con-
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005
G Model
YCECA-1235;
10
No. of Pages 11
ARTICLE IN PRESS
M. Skelin, M. Rupnik / Cell Calcium xxx (2011) xxx–xxx
trol and the cumulative Cm change was also indistinguishable from
control cells. On the other hand, the Cm change was triggered at
significantly lower [Ca2+ ]i (Fig. 5C). Furthermore, the Ca2+ dependency of the secretory rate obtained in Cm recordings from cells
treated with cAMP was pushed to significantly lower EC50 (Fig. 5B,
right panel). It is not clear whether this first phase of Cm change
represents the same pool of secretory granules as elicited in controls or the cAMP triggers the fusion of a high Ca2+ -sensitive pool of
vesicles as suggested recently [16]. The low affinity release-ready
vesicles fuse later. Our findings indicate another effect of cAMP on
regulated exocytosis in pancreatic beta cells where cAMP does not
influence the number of vesicles available for release but rather it
sensitizes these granules to Ca2+ and thus triggers Cm increase at
lower [Ca2+ ]i .
Our study also provides evidence that cAMP, through activation of PKA, regulates Ca2+ -dependent exocytosis in pancreatic beta
cells [4,9,47]. PKA activation by 6-Phe-cAMP triggered a triphasic Cm change at [Ca2+ ]i comparable with those in cells treated
with cAMP with an additional high Ca2+ -sensitive phase similar
to data obtained from cells with increased cAMP concentration
(Fig. 8, middle panel). The half-maximal rate of Cm change was
also achieved at significantly lower [Ca2+ ]i concentrations in cells
treated with 6-Phe-cAMP (Fig. 7B), which indicates that activation
of PKA is essential for altering the Ca2+ -dependency of the rate of
the exocytotic burst. That action of cAMP mediated mainly through
PKA was confirmed with the addition of Rp-cAMPs, a competitive
antagonist of cAMP binding to PKA, where the change in sensitivity was not observed because the results did not differ from the
controls (Fig. 7B). Moreover, in cells treated with Rp-cAMPs, vesicles started to fuse at Catr , which was also comparable with the
control.
Besides the PKA-dependent pathway, a PKA-independent pathway has also been described to be involved in cAMP-mediated
insulin secretion [9,20,27]. Epac2 is known to be present in pancreatic beta cells [22], therefore we used 8-pCPT-2 -O-Me-cAMP,
a potent, specific and membrane-permeant activator of the Epac
receptor, and we did not observe any significant difference in
amp1 and amp2 or in Ca2+ sensitivity compared with the control. On the other hand, the rate of fusion is increased when Epac2
is activated (Fig. 7D,F). The underlying molecular mechanism is
not yet known but we can speculate from our data that specific
Epac activation increases the number of ready releasable vesicles as previously suggested [6,20] or by increasing the size of
the non-docked granule pool and by facilitating the recruitment
of the granules to the plasma membrane [27]. If this is the case,
then the question arises why increased secretory rates are not
observed in cells with increased cAMP concentration? The possible explanation is that PKA activation by cAMP is the dominant
pathway through which cAMP is affecting insulin exocytosis and
thereby Epac activation seems to vanish. Because, in the presence of cAMP, the insulin secretion is triggered at significantly
lower [Ca2+ ]i by activation of PKA, it can be assumed that at
this [Ca2+ ]i the effect of the Epac2-dependent pathway is not yet
fully operational. From our data it is clear that the Epac-selective
analogue, 8-pCPT-O-2 -Me-cAMP, does not mimic the effect of
cAMP by changing the sensitivity of the secretory machinery to
[Ca2+ ]i .
At present the nature of the observed Im change is not clear
(Figs. 4C and 8). It may be completely independent of vesicle fusion
as it seems from the control conditions as published previously [44].
However it is well coordinated with the Cm change in cAMP stimulated conditions. One possibility is that the current represents a
current through the fusion pore that is still too tight to allow the
measurement of the vesicle capacitance. As seems from the Fig. 4,
the second phase vesicles fuse simultaneously as the Im change,
likely through the fusion pore expansion which is sufficient to allow
also the measurement of the vesicle capacitance. One of the reasons
why such isolated Im change has not been noticed before is that
flash photolysis experiments raised [Ca2+ ]i to several tens of ␮M
and at this concentration the second phase overtakes the fastest
component in slow photo-release. Why does cAMP coordinate the
earliest Im phase by the fastest Cm phase? A possible explanation for
this phenomenon is the ability of cAMP to destabilize the tight form
of fusion pore and thereby enabling secretory vesicles to fuse with
plasma membrane at lower [Ca2+ ]i . In control conditions, a stable
fusion pore complex could be formed but the physiological stimulus would not be sufficient to trigger the full fusion. As has been
reported before, the Cm measurements are sometimes less sensitive
as amperometry or current measurements [48]. In cells treated with
6-Phe-cAMP, the high Ca2+ -sensitive phase of Cm change was again
well coordinated with the change in the first phase of Im , which
could suggest that cAMP acts through a PKA-dependent mechanism.
When the second phase of Cm change was analyzed in cells
treated with cAMP, no significant differences compared with the
control were observed. The amplitude and the rate of the second phase were indistinguishable from the control and the second
phase of Cm change was triggered at the same [Ca2+ ]i (Fig. 6A–D).
These findings suggest that cAMP does not change the sensitivity
or the number of secretory granules available for release during the
second phase. On the other hand, ATP depletion caused a significant reduction in the amplitude and the rate of the second phase of
Cm change (Fig. 6A–D). This can be a consequence of reduced cAMP
production or the effect of ATP depletion because it seems that ATP
is required for a vesicle-recruitment step prior to docking as well
as for ATP-dependent priming reactions that may occur after vesicle docking [49]. Although amp2 was reduced compared with the
control, there were no differences in amp1, which represents the
pool of granules that are capable of undergoing exocytosis in an
ATP-independent manner [37].
We can conclude that cAMP stimulated exocytosis of insulin
granules in a PKA-dependent and PKA-independent manner. Our
data suggest that cAMP does not seem to increase the number
of secretory granules available for release but rather it sensitizes
the exocytotic machinery to Ca2+ and thereby promotes insulin
exocytosis at lower Ca2+ concentration. The increased Ca2+ sensitivity by cAMP is consistent with previously published data
from experiments using depolarization-evoked insulin exocytosis where cAMP enhanced the size of a pool of vesicles available
for immediate release and in addition accelerated refilling of a
releasable pool of vesicles [50]. Furthermore, cAMP acts mainly
through activation of PKA and protein kinase-dependent phosphorylation is a major method of regulation of sensitivity to glucose
in pancreatic beta cells. In contrast, Epac2 may contribute to
enhance the rates of secretory vesicle fusion. As GLP-1 and GIP
were described to exert cAMP actions through activation of PKA
in pancreatic beta cells we suggest that they modify the Ca2+ sensitivity and enhance the beta cell reaction to weak stimulus like
glucose.
Conflict of interest
No conflict of interest exists.
Author contributions
Both authors designed the study and wrote the paper. M.S. performed and analyzed the depolarization experiments and slow
photo-release experiments at the Faculty of Medicine, University
of Maribor, Slovenia.
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005
G Model
YCECA-1235;
No. of Pages 11
ARTICLE IN PRESS
M. Skelin, M. Rupnik / Cell Calcium xxx (2011) xxx–xxx
Acknowledgements
This work was supported by the Slovenian Research Agency
Programme P3-0310-2334 and Grant J3-2290-2334, MPG Partner
Group Programme.
References
[1] S. Speier, A. Gjinovci, A. Charollais, P. Meda, M. Rupnik, Cx36-mediated coupling
reduces beta-cell heterogeneity, confines the stimulating glucose concentration range, and affects insulin release kinetics, Diabetes 56 (2007) 1078–1086.
[2] J. Lang, Molecular mechanisms and regulation of insulin exocytosis as a
paradigm of endocrine secretion, Eur. J. Biochem. 259 (1999) 3–17.
[3] R.A. Easom, Beta-granule transport and exocytosis, Semin. Cell Dev. Biol. 11
(2000) 253–266.
[4] C. Ammala, F.M. Ashcroft, P. Rorsman, Calcium-independent potentiation of
insulin release by cyclic AMP in single beta-cells, Nature 363 (1993) 356–358.
[5] W.S. Zawalich, K.C. Zawalich, Regulation of insulin secretion by phospholipase
C, Am. J. Physiol. 271 (1996) 409–416.
[6] E. Renström, L. Eliasson, P. Rorsman, Protein kinase A-dependent and independent stimulation of exocytosis by cAMP in mouse pancreatic B-cells,
J. Physiol. 502 (1997) 105–118.
[7] R.D. Burgoyne, A. Morgan, Secretory granule exocytosis, Physiol. Rev. 83 (2003)
581–632.
[8] J.J. Holst, J. Gromada, Role of incretin hormones in the regulation of insulin
secretion in diabetic and nondiabetic humans, Am. J. Physiol. Endocrinol.
Metab. 287 (2004) E199–206.
[9] S. Seino, T. Shibasaki, PKA-dependent and PKA-independent pathways for
cAMP-regulated exocytosis, Physiol. Rev. 85 (2005) 1303–1342.
[10] L.E. Chavez-Noriega, C.F. Stevens, Increased transmitter release at excitatory
synapses produced by direct activation of adenylate cyclase in rat hippocampal
slices, J. Neurosci. 14 (1994) 310–317.
[11] A. Morgan, D. Roth, H. Martin, A. Aitken, R.D. Burgoyne, Identification of cytosolic protein regulators of exocytosis, Biochem. Soc. Trans. 21 (1993) 401–405.
[12] V. Carabelli, A. Giancippoli, P. Baldelli, E. Carbone, A.R. Artalejo, Distinct potentiation of L-type currents and secretion by cAMP in rat chromaffin cells, Biophys.
J. 85 (2003) 1326–1337.
[13] A.K. Lee, Dopamine, (D2) receptor regulation of intracellular calcium and membrane capacitance changes in rat melanotrophs, J. Physiol. 495 (Pt 3) (1996)
627–640.
[14] S.K. Sikdar, M. Kreft, R. Zorec, Modulation of the unitary exocytic event amplitude by cAMP in rat melanotrophs, J. Physiol. 511 (Pt 3) (1998) 851–859.
[15] L. Eliasson, X. Ma, E. Renstrom, et al., SUR1 regulates PKA-independent cAMPinduced granule priming in mouse pancreatic B-cells, J. Gen. Physiol. 121 (2003)
181–197.
[16] Q.F. Wan, Y. Dong, H. Yang, X. Lou, J. Ding, T. Xu, Protein kinase activation
increases insulin secretion by sensitizing the secretory machinery to Ca2+ , J.
Gen. Physiol. 124 (2004) 653–662.
[17] S. Sedej, T. Rose, M. Rupnik, cAMP increases Ca2+ -dependent exocytosis through
both PKA and Epac2 in mouse melanotrophs from pituitary tissue slices, J.
Physiol. 567 (2005) 799–813.
[18] H. Hatakeyama, N. Takahashi, T. Kishimoto, T. Nemoto, H. Kasai, Two cAMPdependent pathways differentially regulate exocytosis of large dense-core and
small vesicles in mouse beta-cells, J. Physiol. 582 (2007) 1087–1098.
[19] G. Kang, C.A. Leech, O.G. Chepurny, W.A. Coetzee, G.G. Holz, Role of the cAMP
sensor Epac as a determinant of KATP channel ATP sensitivity in human pancreatic beta-cells and rat INS-1 cells, J. Physiol. 586 (2008) 1307–1319.
[20] S. Seino, H. Takahashi, W. Fujimoto, T. Shibasaki, Roles of cAMP signalling in
insulin granule exocytosis, Diabetes Obes. Metab. 11 (2009) 180–188.
[21] C.-L. Zhang, M. Katoh, T. Shibasaki, et al., The cAMP sensor Epac2 is a direct
target of antidiabetic sulfonylurea drugs, Science 325 (2009) 607–610.
[22] G.G. Holz, Epac: a new cAMP-binding protein in support of glucagon-like
peptide-1 receptor-mediated signal transduction in the pancreatic B-cell, Diabetes 53 (2004) 5–13.
[23] G.G. Holz, New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells, Horm. Metab.
Res. 36 (2004) 787–794.
[24] M. Hisatomi, H. Hidaka, I. Niki, Ca2+ /calmodulin and cyclic 3,5 adenosine
monophosphate control movement of secretory granules through protein
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
11
phosphorylation/dephosphorylation in the pancreatic beta-cell, Endocrinology
137 (1996) 4644–4649.
G.J. Evans, A. Morgan, Phosphorylation-dependent interaction of the synaptic
vesicle proteins cysteine string protein and synaptotagmin I, Biochem. J. 364
(2002) 343–347.
M.G. Chheda, U. Ashery, P. Thakur, J. Rettig, Z.H. Sheng, Phosphorylation of
Snapin by PKA modulates its interaction with the SNARE complex, Nat. Cell
Biol. 3 (2001) 331–338.
T. Shibasaki, H. Takahashi, T. Miki, et al., Essential role of Epac2/Rap1 signaling
in regulation of insulin granule dynamics by cAMP, Proc. Natl. Acad. Sci. 104
(2007) 19333–19338.
Y. Kashima, T. Miki, T. Shibasaki, et al., Critical role of cAMP-GEFII·Rim2
complex in incretin-potentiated insulin secretion, J. Biol. Chem. 276 (2001)
46046–46053.
K. Fujimoto, T. Shibasaki, N. Yokoi, et al., Piccolo, a Ca2+ sensor in pancreatic
␤-cells, J. Biol. Chem. 277 (2002) 50497–50502.
S. Speier, M. Rupnik, A novel approach to in situ characterization of pancreatic
␤-cells, Pflüg. Arch. Eur. J. Physiol. 446 (2003) 553–558.
E. Neher, A. Marty, Discrete changes of cell membrane capacitance observed
under conditions of enhanced secretion in bovine adrenal chromaffin cells,
Proc. Natl. Acad. Sci. U.S.A. 79 (1982) 6712–6716.
F.T. Horrigan, R.J. Bookman, Releasable pools and the kinetics of exocytosis in
adrenal chromaffin cells, Neuron 13 (1994) 1119–1129.
P. Thomas, J.G. Wong, A.K. Lee, W. Almers, A low affinity Ca2+ receptor controls
the final steps in peptide secretion from pituitary melanotrophs, Neuron 11
(1993) 93–104.
P. Rorsman, E. Renstrom, Insulin granule dynamics in pancreatic beta cells,
Diabetologia 46 (2003) 1029–1045.
S. Sedej, T. Tsujimoto, R. Zorec, M. Rupnik, Voltage-activated Ca(2+) channels
and their role in the endocrine function of the pituitary gland in newborn and
adult mice, J. Physiol. 555 (2004) 769–782.
T. Rose, S. Efendic, M. Rupnik, Ca2+ -secretion coupling is impaired in diabetic
Goto Kakizaki rats, J. Gen. Physiol. 129 (2007) 493–508.
L. Eliasson, E. Renstrom, W.G. Ding, P. Proks, P. Rorsman, Rapid ATP-dependent
priming of secretory granules precedes Ca(2+)-induced exocytosis in mouse
pancreatic B-cells, J. Physiol. 503 (1997) 399–412.
Ř.G. Dagfinn, E. Roald, H.S. Robert, P.M. Jon, D.Ř.S. Stein Ove, Comparison of the
two classes of binding sites (A and B) of type I and type II cyclic-AMP-dependent
protein kinases by using cyclic nucleotide analogs, Eur. J. Biochem. 181 (1989)
19–31.
J.B. Sørensen, U. Matti, S.-H. Wei, et al., The SNARE protein SNAP-25 is linked
to fast calcium triggering of exocytosis, Proc. Natl. Acad. Sci. U.S.A. 99 (2002)
1627–1632.
Y. Liu, C. Schirra, D.R. Stevens, et al., CAPS facilitates filling of the rapidly
releasable pool of large dense-core vesicles, J. Neurosci. 28 (2008) 5594–5601.
E. Neher, A comparison between exocytic control mechanisms in adrenal chromaffin cells and a glutamatergic synapse, Pflüg. Arch. Eur. J. Physiol. 453 (2006)
261–268.
C. Ammala, L. Eliasson, K. Bokvist, O. Larsson, F.M. Ashcroft, P. Rorsman, Exocytosis elicited by action potentials and voltage-clamp calcium currents in
individual mouse pancreatic B-cells, J. Physiol. 472 (1993) 665–688.
S. Gopel, Q. Zhang, L. Eliasson, X.S. Ma, J. Galvanovskis, T. Kanno, A. Salehi, P.
Rorsman, Capacitance measurements of exocytosis in mouse pancreatic alpha, beta- and delta-cells within intact islets of Langerhans, J. Physiol. 556 (2004)
711–726.
N. Takahashi, T. Kadowaki, Y. Yazaki, Y. Miyashita, H. Kasai, Multiple exocytotic
pathways in pancreatic beta cells, J. Cell Biol. 138 (1997) 55–64.
S. Barg, X. Ma, L. Eliasson, et al., Fast exocytosis with few Ca(2+) channels in
insulin-secreting mouse pancreatic B cells, Biophys. J. 81 (2001) 3308–3323.
E. Neher, T. Sakaba, Multiple roles of calcium ions in the regulation of neurotransmitter release, Neuron 59 (2008) 861–872.
S.G. Straub, G.W. Sharp, Glucose-stimulated signaling pathways in biphasic
insulin secretion, Diabetes Metab. Res. Rev. 18 (2002) 451–463.
A.F. Oberhauser, I.M. Robinson, J.M. Fernandez, Simultaneous capacitance and
amperometric measurements of exocytosis: a comparison, Biophys. J. 71 (1996)
1131–1139.
T.F. Martin, Stages of regulated exocytosis, Trends Cell Biol. 7 (1997) 271–276.
E.P. Kwan, L. Xie, L. Sheu, T. Ohtsuka, H.Y. Gaisano, Interaction between
Munc13-1 and RIM is critical for glucagon-like peptide-1 mediated rescue of
exocytotic defects in Munc13-1 deficient pancreatic beta-cells, Diabetes 56
(2007) 2579–2588.
Please cite this article in press as: M. Skelin, M. Rupnik, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A
in mouse pancreatic beta cells, Cell Calcium (2011), doi:10.1016/j.ceca.2010.12.005