0013-7227/96/$03.00/O
Endocrinology
Copyright 0 1996 by The Endocrine
Vol. 137, No. 11
Printed in U.S.A.
Society
Ca2+/Calmodulin
and Cyclic 3,5’ Adenosine
Monophosphate
Control Movement
of Secretory
Granules through
Protein Phosphorylationl
Dephosphorylation
in the Pancreatic
P-Cell*
MITSURO
HISATOMI,
HIROYOSHI
Second Department
of Internal
University
School of Medicine,
HIDAKA,
Medicine (M.H.),
65 Tsurumai-cho,
ICHIRO
AND
Department
Showa-ku,
of Pharmacology
(H.H.,
Nagoya 466, Japan
ABSTRACT
We observed the movement of insulin granules in living transformed hamster pancreatic p-cells (HIT TX) with a light microscope,
where secretory granules are moving in the cytoplasmic space. Velocity ofthe typical granule movement was approximately 1.5 pm/set.
A stimulatory concentration of glucose activated the movement of the
secretory granules. Forskolin, an activator of adenylate cyclase, increased the movement, resulting in changes in intracellular
localization of the granules. Acetylcholine also activated the granule movement, whereas high K+ and tolbutamide, which cause Ca2+ influx
through the voltage-dependent
Ca 2+ channel, had only little effect.
H
ORh4ONE secretion from the endocrine
tissue is composed of several different
processes such as hormone
biosynthesis,
secretory granule formation,
trafficking,
docking of the granules, and the final step, exocytosis. Hormonal
output is finely controlled
by various
signal transduction
systems. Increase in the intracellular
concentration
of free
Ca2+ and resultant
protein
phosphorylation
are the critical
events for the secretory machinery
of most, if not all, hormones including
insulin (1). Activation
of protein kinases A
or C potentiates
insulin secretion from the pancreatic
p-cell
(2). Thus, it seems that insulin secretion is controlled
by the
network of signal transduction
by protein kinases and phosphatases (3), but we have still limited
knowledge
on the
mechanism(s)
by which protein phosphorylation
controls the
secretory events.
In a previous
study, Lacy et al. (4) demonstrated
that glucose elicits motile events of the insulin granules
in the pancreatic P-cell. Recent progress in the microscopic
technique
enabled us to analyze movements
of individual
particles,
such as insulin
granules
in a living cell (5, 6). Here, we
assessed intracellular
distribution
and movement
of the secretory granules in a hamster insulin-secreting
cell line, HIT
T15. This cell line retains secretory sensitivity
to nutrients
and hormones,
which modulate
insulin
release, although
Science and Culture,
and 05271103)
I.N.), Nagoya
The movement was abolished by BAPTA, the intracellular
Ca2’chelator. Activation of protein lcinase C by 12-O-tetradecanoyl-phorbol 13-acetate failed to affect this movement. The motile events were
inhibited by the calmodulin antagonist, W-7, and dramatically increased by okadaic acid, an inhibitor of protein phosphatases 1 and
2A. These results suggest protein phosphorylation
by Ca2+/calmodulin- and CAMP-dependent protein kinases play a positive role in the
control of the insulin granule movements, which results in potentiation ofinsulin release from the pancreatic p-cell. (Eno!ocrinoZogy 137:
4644-4649, 1996)
dose-dependent
curve for HIT cells to glucose shifts to the
left as compared
to that for the normal
islet P-cell (7).
We investigated
involvement
of the second messenger
systems for the control of the movement
and demonstrated
that the intracellular
movement
of insulin granules
is controlled by protein
kinases depending
on Ca2+ /calmodulin
and CAMP, but not by protein kinase C. We propose that the
activation
of the movement
may be another step for protein
phosphorylation
by these kinases to enhance insulin release.
Materials
and Methods
Materials
HIT T15 S-cells (passage
numbers
70-85),
kindly
donated
by Prof. S.
Seino (Chiba University School of Medicine, Chiba, Japan). BAPTA/
AM, forskolin,
12-0-tetradecanoyl-phorbol
13-acetate
(TPA), and okadaic acid were purchased
from Wako (Tokyo,
Japan). Quinacrine
was
from
Research-Biochemicals
International
(Natick,.
MA).
Dibutyryl
CAMP (dbcAMP),
acetylcholine,
and tolbutamide
were from Sigma
Chemical Co. (St. Louis, MO). Nifedipine
ceutical
(Osaka,
Japan)
Biomedicals
(Costa Mesa,
thalenesulfonamide)
and
amide) were synthesized
were of the highest
grade
was from Fujisawa Pharma-
and Ham’s
F-12K
medium
was from
ICN
CA). W-7 IN-f6-aminohexvl)-5-chloro-l-nauhW-5 (N-(&aminohexyl)-l&phthalenesulf&as previously
described
(8). All other chemicals
available.
Cell culture
HIT T15 p-cells were cultured
at 37 C in a humidified
atmosphere
of
95% sir/5%
CO, in Ham’s F-12K medium
with kanamvcin
1303 uelmll
and 10% FCS. The cells were passaged
weekly
and harvested
by using
trypsin / EDTA, and culture
medium
was replaced
every other day. For
morphological
experiments,
HIT cells were seeded onto a glass-bottom
tissue culture
dish (35-mm
diameter,
Meridian
Instruments
Far East,
Tokyo, Japan) at a density
of 1-5 x lo5 cells/dish.
The culture medium
was replaced
with HEPES-Krebs
solution
containing
(mM): NaCl 119,
Received April 2, 1996.
Address all correspondence and requests for reprints to: Ichiro Niki,
M.D., Department of Pharmacology, Nagoya
University
School
of
Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan.
* This work was supported in part by Grants-in-Aid
for Scientific
Research
from the Ministry
of Education,
(Grants
No. 06507001,
06404019,
07670103,
NIKI
.
Japan
4644
,
\
,“.
,
INSULIN
GRANULE
MOVEMENT
KC1 4.75, NaHCO,
5, CaCl, 2.54, MgSO,
1.2, and HEPES 20 (pH 7.4 with
NaOH),
with 5 mg/ml
BSA. After 2 h preincubation
in the glucose-free
buffer,
cells were further
incubated
in the same solution
supplemented
with various
substances
as mentioned.
We need this preincubation
to
reveal the secretory
sensitivity
of HIT cells to glucose
(7). For some
experiments,
quinacrine
(0.2 PM) was loaded overnight.
For the fluorescent detection,
excitation
effected
at 450-490
nm and emitted
light
was collected
at 515-565 nm.
vents such
concentrations
Insulin
and velocity
of the secretory
Movement
of insulin
granules
was quantitatively
assessed
by a
method
originally
devised
by Lacy et al. (4) with some modification.
Briefly,
number
of the granules
which move into or out of square (3.5 X
3.5 pm) during
a 30-set period was counted
in each square. One HIT cell
involves
6-8 complete
squares.
Unless
mentioned,
the counting
was
started 2 min before and 3 min after substances were added. Changes
of the numbers between before and after the addition were compared
by paired Student’s t test. The speed was assessed by tracing the vesicle
movement
movement,
TABLE
using the image analyzer.
For assessment
of the granule
the same experiments
were repeated
at least 3 times. Sol-
1. Intracellular
movement
of secretory
granules
21
34
24
22
w-7
15 min)
mM)
TPA (162 nM)
Okadaic
acid (2
BAPTA!AM
(20
+ glucoce
(20
+ forskolin
(5
Low temperature
pM)
pM, 20 min)”
mM)e
pMr
(27 C)
or ethanol
experiments.
did not
affect
the assay
at
granules
in living
HIT
cells
cells
Granule
movement
After
challenge
% change
3.00
2.53
3.08
2.36
2.56
1.22
?
k
k
t
t
t
0.48
0.41
0.46
0.37
0.38
0.23
3.25
3.68
3.29
2.50
3.75
2.22
t
t
k
2
k
2
0.86
0.45”
0.52
0.41
0.44”
0.49”
8.3
45.5
6.8
5.9
46.5
82.0
25
28
23
26
28
28
25
42
26
2.56
2.25
3.13
3.04
2.32
1.29
2.60
2.64
3.07
t
?
”
t
+
5
2
t
+
0.40
0.27
0.64
0.55
0.40
0.26
0.86
0.32
0.41
1.00
2.29
4.17
4.54
2.00
3.68
0.00
0.00
0.00
+
?
t
+
t
2
t
i2
0.21*
0.29
0.61*
0.83*
0.31
0.51’
0.00
0.00
0.00
39.1
1.7
33.2
49.3
-13.8
185.3
d
d
16
3.75
t
0.44
2.25
” 0.54”
;3”
W-5 (10 pM, 15 min)
Forskolin
(5 pM)
dbcAMP (2
as dimethylsulfoxide
used in these
Control
Glucose
(0.1 mM)
Glucose
(20 mM)
Tolbutamide
(100 pM)
K’ (50 mM)
Acetylcholine
(100 pM)
Acetylcholine
(100 pM)
+ nifedipine
(2 pM)
FM,
T15
n
Conditions
(10
in HIT
4645
Observation
of HIT T15 cells under phase-contrast
microscopy revealed that HIT cells were equipped
with many
small, round granules with 0.75 + 0.01 pm in a diameter (n =
100 from 7 experiments).
This value is slightly
larger than
that for insulin granules in islet p-cells assessed by electron
microscopy;
0.3-0.6 pm (9) or 0.4-0.5 pm (10) and is comparable to that in living
islet p-cells under the video-enhanced and differential
interference
contrast microscopy;
0.5-1.2 Frn (5). In this study, identification
of insulin granules
in living P-cells was performed
by the quinacrine
fluorescence study. The dye is accumulated
in a low pH condition,
such as secretory granules
and lysosomes,
presumably
via
protonation
(ll), and has been demonstrated
to accumulate
in fixed or living p-cells (12, 13). As shown in Fig. 1A and B,
the intracellular
distribution
of quinacrine
fluorescence
was
almost coincident
with the small granules and lysosomes in
the image of the phase-contrast
microscopy.
Lysosomes were
readily
distinguished
because they are much bigger than
secretory
granules
and lacks in motility.
The fluorescent
granules
demonstrated
a heterogeneous
distribution
in the
cytoplasmic
area. The magnitude
of granule movement
varied in individual
cells. In some cells, most of the granules
were almost still, and in others, those were moving. When we
traced the movement
for a longer period, we noticed that the
granules
cannot go into a certain area of the intracellular
space. However,
the observation
did not allow us to identify
a fixed route or direction
for the granule movement
under
Experiments
were carried
out with an inverted
light microscope
(Axiovert
135, Carl Zeiss, Germany)
equipped
with a x40 objective
lens
(Plan-Neofluar,
Carl Zeiss) and a X2.5 insertion
lens. The images were
detected
by a charge
coupled
device
camera
(DXC-930,
Sony, Japan),
displayed
on a monitor
screen (PVM-9040,
Sony) at a final magnification
of x5.500,
and recorded
by a video tape recorder
(SVO-260,
Sony).
Pictures
were reproduced
from video tape and analyzed
on the monitor
using
an
image
analyzer
(Argus-20,
Hamamatsu
Photonics,
Hamamatsu,
Japan). All these experiments
were carried out at 37 C using
a heating stage because the granule movement
became minute
at lower
temperature
(see Table 1). The thickness
of the focal plane was about 5
pm. The thickness
of a HIT cell was between
3-5 m, and its width was
20-30 pm on average.
This cell line possesses
fewer secretory
granules
than normal
p-cell (4).
of movement
P-CELL
Results
Video microscopy
Quantification
granules
IN THE
d
-40.0
After preincubation for 2 h in the absence of glucose, HIT T15 cells were incubated under conditions described above. All the experiments
were carried out at 37 C unless mentioned. Intracellular
movement of the secretory granules in HIT T15 cells were assessed as described in
Materials ancl Methods. For control, the granule
movement
was measured
for 30 set 2 min before the condition
was changed.
The movement
in the identical
square
in the microscopic
field
was reassessed
2-3
min
after
the challenge
unless
mentioned.
Data
are expressed
as the mean
2
SEM.
a P < 0.05, * P < 0.01 vs. control value assessed by paired t test.
’ Movement of the granules was assessed after BAPTA treatment for 20 min.
d The movement
was nullified
by the BAPTA
treatment.
e Glucose
or forskolin
was further
added to the medium
addition.
after
20 min
treatment
with
BAP’I’A.
The
movement
was measured
2 min
after
the
4646
INSULIN
GRANULE MOVEMENT
IN THE P-CELL
FIG. 1. Images of HIT cells under light microscopy and their fluorescent images by quinacrine.
tissue plastic dish and cultured overnight in Ham’s F-12K medium supplemented with 0.2 w
an inverted light microscope (Axiovert 135, Carl Zeiss, Germany) equipped with a x40 objective
image of a HIT p-cell. Bar, 1 pm. B, Quinacrine fluorescence from the identical cells to Fig.
Endo . 1996
Vol 137. No 11
HIT cells were seeded onto a 30-mm glass-bottom
quinacrine. Observations were carried out using
lens and a X2.5 insertion lens. A, Phase contrast
1A.
Square Number
Control
FIG. 2. Effects of glucose on the movement of the insulin granules. P-granules in living HIT cells were observed with an inverted light microscope
equipped with a x40 objective lens and a ~2.5 insertion lens. The images of p-cells were recorded by a video recorder and reproduced on a monitor.
Number of the granules that move into or out of square (3.5 x 3.5 pm) was counted in each square. A, Frequency of the movement before (open
column) and after (closed column) the glucose (20 mu) challenge was demonstrated for each square (No. l-34). B, Frequency after the addition
of glucose was plotted against that before the stimulation. The same data as Fig. 2A from four independent experiments.
unstimulated conditions. Two distinct types of the movement were observed. The granules were migrating with short
amplitude (-1.5 pm), and occasionally we could trace the
movement for a longer distance (5-10 pm). The velocity of
the latter movement was approximately 1.5 Frn per second
(129 mm/day), which is equivalent to those in the normal
@cell (4) and for the fast anterograde axonal transport in
neural cells; -100-400 mm/ day (14). However, the value in
these studies must be underestimated because it is unable to
follow the movement along the z-axis. The small movement
did not allow us to measure its velocity. We believe that it
does not result from Brownian nature as suggested in the islet
cell by Somers et al. (15) because the granules became completely still when Ca2+ was chelated by BAPTA (see Table 1).
Granule movement with a longer distance was observed
only in 30-40% of HIT cells, which may occur because of the
heterogeneity of the secretory sensitivity in the individual
p-cells.
Motile events of insulin
glucose stimulation
granules
in HIT
p-cells
under
When assessed by counting numbers of the granules
that move into or out of a 3.5 x 3.5 pm square per 30 set,
the granule movement was obviously increased by a stimulatory concentration (20 mM) of glucose (Fig. 2A, B and
Table 1). The velocity of the fast movement after glucose
stimulation ,was approximately
1.3-1.5 pm per second
(from four independent
experiments). It was, therefore,
suggested that the number, but not the velocity of the
moving granules, was increased by glucose stimulation.
We consider that the activation of the movement by glucose may result from a shift of the rapidly moving granules
from the other population
with small movements. The
movement by glucose was nullified when the P-cell was
pretreated by the intracellular Ca2+ chelator BAPTA/AM
at 20 PM for 20 min. A substimulatory
concentration (0.1
INSULIN
GRANULE MOVEMENT
IN THE P-CELL
4647
mM) of glucose did not cause any detectable change in the
movement (Table 1).
Ca2+ and the motile
events in the pancreatic
p-cell
The insulinotropic sulphonylurea, tolbutamide, did not
have a significant effect on the movement. Depolarization
by high K+ (50 mM) also failed to change the frequency.
Acetylcholine at 100 PM had a significant effect to increase
the movement (Fig. 3 and Table 1). The increasing effect of
acetylcholine was not influenced or rather increased by simultaneous addition of 2 PM nifedipine, a blocker of the
voltage-dependent Ca2+ channel (Table 1).
W-7, the calmodulin antagonist (16), inhibited the movement in unstimulated cells at 10 PM (Fig. 4A). W-5, the
negative control compound of W-7 (17), did not affect the
movement under the parallel condition. When the extracellular solution was depleted of Ca’+, a gradual decrease in the
granule movement was observed. The granules were in motion even 20 min after the Ca2+ depletion, which is at variance
with the early observation using islet p-cells reported by
Lacy et al. (4).
Effects of substances which
insulin granule movement
modulate
insulin
8
$
6
4
6
Control
8
10
12
release on
Forskolin (5 PM), an activator of adenylate cyclase and a
strong potentiator of insulin release, remarkably increased
the frequency of the motile movement (Fig. 5A). The frequency was also increased by the membrane-permeable
CAMP analogue, dbcAMP at 2 mM. In a few cases, we found
that these granules were directed to a certain part of the P-cell
after the addition of forskolin (Fig. 5B, lower right panel),
whereas such directed movement was not detected before
the addition (Fig. 5B, lower left panel). After the 3-min incubation with 5 PM forskolin, the granules eventually gathered
in a right lower portion of the cell (Fig. 5B, upper right panel).
Such redistribution of the granules was reproduced by db-
OJ
I
I
I
I
0
5
10
15
Time (min)
FIG. 4. Effects of W-7 and W-5 on the movement of insulin granules.
Movement of P-granules was assessed by counting the frequency as
mentioned in Materials
and Methods. A, The frequency was counted
for 30 set before and 5(O) or 15 (0) min after the addition of 10 pM
W-7. The frequency of the granule movement was plotted against the
control value. B, Inhibition ofthe granule movement by W-7 and W-5.
Granule movement in the P-cell was assessed at 5 and 15 min after
addition of 10 pM W-7 (0) and W-5 (A). Values are the mean k SEM
from 25 (W-7) and 28 (W-5) observations from three independent
experiments.: *P < 0.01 as compared with the value at 0 min.
CAMP (data not shown). Forskolin, however, did not cause
any changes when added after pretreatment with 20 PM
BAPTA / AM for 20 min (Table 1). Okadaic acid (2 PM), which
inhibits protein phosphatases 1 and 2A, had an enormous
increasing effect on the granule movement (Fig. 6 and Table
1). TPA at 162 nM, which increases insulin secretion via
activating protein kinase C, failed to affect the granule movement in these experiments (Table 1).
0
2
4
6
Control
8
10
12
FIG. 3. Effects of acetylcholine on the movement of insulin granules.
p-granules in living HIT cells were observed with a magnification of
x5.500. The images of p-cells were recorded and reproduced on a
monitor. Number of the granules which move into or out of square
(3.5 X 3.5 pm) was counted in each square. Data were plotted as
mentioned in Fig. 2B.
Discussion
The present study leads us to suggest that protein phosphorylation and dephosphorylation
may control secretory
granule movement in the pancreatic P-cell. Glucose, which
causes a dramatic change in the pattern of P-cell phosphoproteins (18), evoked a moderate increase in granule movement of the pancreatic p-cell. Because the hexose activates
INSULIN
4648
4
6
8
10
GRANULE MOVEMENT
12
Control
FIG. 5. Effects of forskolin on the granule movement. A, ARer 2 h
incubation in HEPESKrebs
buffer, forskolin (5 m) was added to the
buffer and the granule movement before and after the addition of
forskolin was observed. Frequency after the addition of forskolin was
plotted against that before the stimulation. B, Translocation of the
insulin granules in a HIT P-cell by forskolin. Images before (Zeft
panels) and after (right pads) the addition of forskolin are shown. In
the bottom panels, the granule were traced for 5 set (arrows) by
Argus-20. Note that the granules are directed to the right lower
portion of the cell after forskolin stimulation.
several different second messenger systems (l), we tried to
investigate the second messenger system(s) involved in the
control of the granule movement using various pharmacological substances. Treatment with the intracellular Ca” chelator BAPTA abolished the granule movement, which was
not overcome by further addition of glucose. This indicates
that the granule movement is dependent on intracellular
Ca2+. Inhibition by W-7 of the movement suggests the motile
events may be controlled by the Ca’+-calmodulin system. It
is interesting that acetylcholine had a prominent effect on the
granule movement, whereas the movement was not much
affected by high K+ or tolbutamide, both of which cause Ca2+
influx through the voltage-dependent Ca2+ channel as a re-
IN THE P-CELL
0
2
Endo
l 1996
I’01137 . No 11
4
6
8
10
12
Control
FIG. 6. Enhancement by okadaic acid of the movement of insulin
granules. Insulin granules were observed and recorded under phasecontrast microscopy using a x40 objective lens and a x2.5 insertion
lens (final magnification,
x5.500). Movement of the granules were
assessed by reproducing the images on the monitor from the videotape. Frequency during a 30-set incubation before and after the addition of okadaic acid (2 pM) was plotted as described in Materials
and
Methods.
sult of membrane depolarization. Therefore, Ca2+ mobilization seems to be more responsible than Ca” influx for activation of the motile events. This happens presumably
because of the heterogeneous distribution of intracellular
Ca2+ maintained by buffering action of the cytosol on the free
Ca2+ concentration (19). Thus, translocated vesicles may be
prepared to be released from the P-cell by Ca’+ influx. This
idea may explain the difference in the magnitude and time
course of insulin release by Ca2’ influx and Ca2’ mobilization: Ca2+ influx causes an acute and short-lived release,
whereas Ca” mobilization enhances insulin release without
eliciting the secretory events by itself. Moreover, this could
be one of the reasons why glucose, which causes both Ca2’
influx and Ca2+ mobilization, evokes insulin release in a
biphasic manner. It is also possible that the change by acetylcholine may result from its effect on the intracellular concentration/distribution
of ATP because there is evidence
suggesting that intracellular concentrations of ATP and Ca2’
are related in this cell line (20).
Forskolin had a strong effect on the secretory granule
movement in this study. Activation of the CAMP-dependent
system enhances, but does not evoke, insulin release from the
islet /3-cell (3), although HIT cells are reported to be stimulated by cAMP-relating agents as forskolin in the absence of
any secretagogues (7). Directed movement by CAMP (Fig. 5B)
generates heterogeneous distribution of the granules, resulting in localization of the granules to a hot region (6) for an
advantageous rearrangement for exocytosis. Because forskolin failed to influence the movement when the intracellular
Ca2+ was chelated using BAPTA/ AM, substimulatory concentrations of Ca2’ are definitely required for the movement,
and CAMP potentiates the event. In the present study, we
found limited numbers of the cell showed the typical movement of the granules. It was, however, reported that many
particles were moving in monolayer culture of the islet cells
(15). This may result from the simultaneous presence of a
INSULIN
GRANULE MOVEMENT
CAMP-producing hormone, glucagon secreted from the concomitant pancreatic a-cell in their preparations. That could
explain why FACS-purified pancreatic p-cells dramatically
restore its secretory response by reaggregating them with
purified a-cells (21). Involvement of protein phosphorylation
in the control of the granule movement is further supported
by the finding that okadaic acid, the inhibitor of protein
phosphatases 1 and ZA, also dramatically enhanced the
movement, although the inhibitor suppressed insulin release
from the pancreatic islets (22). It must be noted that TPA, an
activator of protein kinase C, has little effect on the movement, although TPA as well as forskolin enhances insulin
output (2). It may be suggested that protein kinase C phosphorylates (a) distinct substrate(s) from those for protein
kinase A in the secretory machinery as suggested in synaptic
membranes (23). This may explain that simultaneous activation of protein kinase A and C results in a vast synergistic
enhancement of insulin release from the pancreatic P-cell (2,
24). Little effect of TPA also reconfirms the idea that the
increasing effect of acetylcholine was derived from Ca2+
mobilization by II’, production rather than activation of protein kinase C by diacylglycerol.
The one or more mechanisms that underlie this facilitation
by Ca’+’ calmodulin are still missing. It has been reported
that at least two calmodulin-dependent
protein kinases, calmodulin-dependent
kinase II and myosin light chain kinase
(MLCK), may be involved in the control of insulin secretion
(25, 26). Recently, we found that monoclonal antibodies
against MLCK attenuated Ca’+- or GTP+- induced insulin
release from permeabilized rat islets (submitted for publication). We suggested that MLCK may act on a proximal
step, presumably on the granule translocation, rather than on
the final step of the secretory events. Furthermore, it has been
reported that phosphorylated MLC is dephosphorylated by
type 1 protein phosphatase (27) and okadaic acid inhibits
dephosphorylation
of this protein (28). Taking the present
findings and other reports into our consideration, it could be
suggested that MLCK activated by released Ca2+ from intracellular Ca2+ stores enhance insulin release via activation
of the secretory granule movement. It may be also suggested
that protein kinase A enhances insulin release partly via
potentiating the movement of secretory granules.
Acknowledgments
The authors
thank
bution
to set up this
Yone Production
(Tokyo,
microscopic
method.
Japan)
for their
contri-
References
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M, Matschinsky
FM 1987 Ca’+, CAMP, and phospholipid-derived
messeneers
in cou~line
mechanisms
of insulin
secretion.
Phvsiol
Rev
,
67:1185:1248
’
”
2. Hughes SJ, Christie MR, Ashcroft
SJH 1987 Potentiators
of insulin secretion
modulate
Cc?+ sensitivity
in rat pancreatic
islets. Mol Cell Endocrinol
50:231-236
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