Ca2+-induced loss of
Ca2+/calmodulin-dependent
protein kinase II activity in pancreatic
-cells
Peter M.
Jones and
Shanta J.
Persaud
Cellular and Molecular Endocrinology Group, Biomedical Sciences
Division, King's College London, London W8 7AH, United Kingdom
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ABSTRACT |
Elevations in
intracellular Ca2+ in electrically
permeabilized islets of Langerhans produced rapid insulin secretory
responses from
-cells, but the
Ca2+-induced secretion was not
maintained and was irrespective of the pattern of administration of
elevated Ca2+.
Ca2+-insensitive
-cells
responded normally to activators of protein kinase C or cAMP-dependent
kinase with increased insulin secretion. The loss of secretory
responsiveness to Ca2+ was
paralleled by a reduction in
Ca2+-induced protein
phosphorylation. This was caused by a reduction in
Ca2+/calmodulin-dependent protein
kinase II (CaMK II) activity in the desensitized cells, as assessed by
measuring the phosphorylation of a CaMK II-specific exogenous
substrate, autocamtide-2. The Ca2+-induced reductions in kinase
activity and protein phosphorylation were not dependent on the
activation of Ca2+-dependent
protein kinases and were not caused by the activation of phosphoprotein
phosphatases or of Ca2+-activated
proteases. The concomitant reductions in CaMK II activity and
Ca2+-induced insulin secretion
suggest that the activation of CaMK II is required for normal insulin
secretory responses to increased intracellular
Ca2+ concentrations.
islets of Langerhans; protein phosphorylation; calcium ion
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INTRODUCTION |
CHANGES IN INTRACELLULAR
Ca2+ are important in regulating
secretion from many cell types, including insulin-secreting pancreatic
-cells. Many stimulators of insulin release, including the
physiologically important nutrients, produce elevations in
-cell
cytosolic Ca2+ (11-14).
Cytosolic Ca2+ concentrations can
be directly manipulated in permeabilized
-cells, and these have
provided a useful experimental model in which to study regulatory
mechanisms downstream of glucose entry, metabolism, and membrane
depolarization. Secretion from permeabilized
-cells is a regulated
process (18-22), and elevations in cytosolic
Ca2+ alone are sufficient to
initiate insulin secretion (17-22, 40, 46). We have demonstrated
previously that Ca2+-induced
insulin secretion from perifused, electrically permeabilized islets of
Langerhans is a transient event in which the
-cell secretory
mechanism rapidly becomes desensitized to stimulatory concentrations of
Ca2+, despite retaining the
ability to respond normally to other second messengers (19, 20).
Selective reductions in secretory responsiveness to
Ca2+ have also been noted in other
secretory tissues permeabilized using methods that produce relatively
small pores in the plasma membrane, thus enabling the cells to retain
important cellular contents and mount prolonged secretory responses
(e.g., Refs. 23 and 42).
Numerous Ca2+-sensitive elements
are expressed within secretory cells, and many of these have been
implicated in the secretory process, but it is still uncertain how
elevations in Ca2+ are sufficient
to stimulate insulin secretion. However, there is strong evidence that
the activation of
Ca2+/calmodulin-dependent protein
kinases offers an important mechanism of
Ca2+ sensing in
-cells
(reviewed in Refs. 2 and 10). Pancreatic
-cells express several
kinases that are activated by Ca2+
and calmodulin, including myosin light chain kinase (MLCK; see Refs.
10, 25, 33) and
Ca2+/calmodulin-dependent protein
kinases II (CaMK II; see Refs. 4, 5, 10, 16, 29, 31, 41, 44, 45) and
III (CaMK III; see Ref. 16). Of these three kinase activities
identified in
-cells, the multifunctional CaMK II is most likely to
subserve a role in
-cell stimulus-secretion coupling, since both
CaMK III and MLCK are thought to be dedicated to the regulation of single functions (37). The loss of secretory responsiveness of
electrically permeabilized islets to
Ca2+ is accompanied by reductions
in Ca2+-dependent phosphorylation
of endogenous islet substrates for CaMK II (20), suggestive of an
important role for protein phosphorylation as a transducer of
Ca2+-induced insulin secretion,
although the mechanism(s) underlying the reduced protein
phosphorylation in
Ca2+-desensitized islets is
unclear. In the present studies, we have addressed whether
Ca2+-induced reductions in
Ca2+-dependent protein
phosphorylation reflect reduced
Ca2+/calmodulin-dependent protein
kinase activity and whether other Ca2+-dependent enzymes such as
proteases or phosphoprotein phosphatases are involved in the
desensitization process.
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MATERIALS AND METHODS |
Materials. ATP, BSA (fraction V),
calmodulin, collagenase (type XI), cAMP, dimethyl sulfoxide (DMSO),
E-64, EGTA, HEPES, 3-isobutyl-1-methylxanthine (IBMX), leupeptin,
4
-phorbol myristate acetate (PMA), myosin light chains (MLC),
phenylmethylsulfonyl fluoride (PMSF) and
N
-p-tosyl-L-lysine chloromethyl ketone (TLCK) were from Sigma Chemical (Dorset, UK). 125I-labeled Na for insulin
iodination and
3H2O
were from Amersham International (Buckinghamshire, UK) and [
-32P]ATP (3,000 Ci/mmol) was from Du Pont (Hertfordshire, UK). P81 phosphocellulose
paper was from Whatman International (Kent, UK). Calyculin A (CAL),
cantharidin (CAN), cypermethrin (CM), okadaic acid (OA), a synthetic
peptide corresponding to CaMK II-(290
309), and the specific CaMK II
substrate autocamtide-2 were purchased from Calbiochem-Novabiochem
(Nottingham, UK). All other chemicals were of analytical grade from BDH
(Dorset, UK). Rats (150-200 g; Sprague-Dawley) were from the
Biological Services Unit of King's College London.
Islet isolation and permeabilization.
Islets of Langerhans were isolated from rat pancreata by collagenase
digestion (18) and were electrically permeabilized by five exposures to
an electric field of 3.4 kV/cm in a buffer ("permeation buffer")
containing 140 mM potassium glutamate, 15 mM HEPES, 5 mM glucose, 1 mM
EGTA, and 0.5 mg/ml BSA, pH 6.6, with
CaCl2 added to give a
Ca2+ concentration of 50 nM (22).
For experiments designed to measure Ca2+-induced insulin secretion
from permeabilized islets, the permeation buffer was supplemented with
5 mM ATP and 7 mM MgSO4, since
Ca2+-induced insulin secretion
from permeabilized
-cells is dependent on the presence of millimolar
amounts of MgATP (3, 18-22).
Insulin secretion from permeabilized
islets. Groups of 50-100 permeabilized islets were
transferred to Millipore Swinnex filter chambers containing 1 µm
nylon filters and perifused at a flow rate of 1 ml/min with permeation
buffer containing either 50 nM or 10 µM
Ca2+ supplemented, in some
experiments, with PMA (500 nM) or cAMP (500 µM) plus IBMX (100 µM).
PMA and IBMX were dissolved in DMSO such that the final concentration
of DMSO was <0.1% (vol/vol), which did not affect insulin secretion.
Perifusate samples were collected at 2-min intervals, and insulin
content was measured by radioimmunoassay (21). In experiments involving
rapid and frequent (2-min) switching between a perifusion medium
containing 50 nM Ca2+ and one
containing 10 µM Ca2+, the dead
space of the perifusion chamber was reduced by using an adapted
hypodermic needle hub, and the perifusate containing 10 µM
Ca2+ was supplemented with
3H2O
(~12 × 103
counts · min
1 · ml
1).
In these experiments, perifusion was at 1 ml/min, and perifusate samples were collected at 15-s intervals. The insulin and
3H contents of each sample were
measured by radioimmunoassay and liquid scintillation counting,
respectively. In all experiments, the perifusion temperature was
maintained at a constant 37°C by performing experiments in a
temperature-controlled chamber.
Ca2+/calmodulin-dependent
protein kinase activity.
Ca2+/calmodulin-dependent
phosphorylation of endogenous islet proteins and of exogenous
substrates was determined by following the transfer of
32P from
[
-32P]ATP into the
protein substrates, as described previously (34). Groups of
100-200 permeabilized islets were incubated for 30 min (37°C)
in a modified permeation buffer (no ATP, 2 mM
MgSO4) containing either 50 nM
Ca2+ or 10 µM
Ca2+ and supplemented with
protease inhibitors, protein phosphatase inhibitors, or inhibitory
peptides, as required. We have demonstrated previously that the
Ca2+-induced desensitization of
permeabilized islets to Ca2+ is
not dependent on the presence of ATP (20). ATP was therefore omitted
from the incubation buffer in these experiments to prevent the
activation of Ca2+-dependent
protein kinases and thus rule out any possible effects on substrate
depletion or on kinase autophosphorylation during this incubation
period. After incubation, islets were pelleted by brief centrifugation
(9,000 g, 15 s), the supernatant was
discarded, and islets were disrupted by sonication (3 × 15 s,
4°C) in a buffer ("assay buffer") containing 20 mM
Tris · HCl, 2 mM EDTA, 0.5 mM EGTA, 50 µg/ml
leupeptin, 1 mM PMSF, and 0.1% (vol/vol) 2-mercaptoethanol, pH 7.4. Islet extracts equivalent to 30 permeabilized islets were incubated (10 min, 30°C) in the presence of 50 µg/ml calmodulin, 0.5 mM CaCl2, 11.1 mM
magnesium acetate, 1 mg/ml MLC, and 100 µM
[
-32P]ATP (specific
radioactivity 1.8 Ci/mmol), in a final assay volume of 30 µl.
Reactions were terminated by addition of 30 µl of a buffer containing
4% (wt/vol) SDS, 10% (vol/vol) 2-mercaptoethanol, 20% (vol/vol)
glycerol, and 0.2% (wt/vol) bromphenol blue in 125 mM
Tris · HCl, pH 6.8, and the samples were boiled for 3 min. Proteins were fractionated by SDS-polyacrylamide gel
electrophoresis on 10 or 15% gels, and
32P incorporation was assessed by
autoradiography and densitometric scanning on a EASY 5000 system (Ultra
Violet Products, London, UK).
Islet CaMK II activity was determined by measuring the phosphorylation
of the specific CaMK II peptide substrate autocamtide-2 (9).
Permeabilized islets were incubated for various times (1-60 min,
37°C) in a modified permeation buffer (no ATP, 2 mM MgSO4) containing either 50 nM
Ca2+ or 10 µM
Ca2+, after which islet extracts
were prepared as described above. Islet extracts equivalent to 40 islets were incubated in the absence or presence of 10 µM
autocamtide-2, 50 µg/ml calmodulin, 0.5 mM CaCl2, 11.1 mM magnesium acetate,
and 50 µM
[
-32P]ATP (specific
radioactivity 3.3 Ci/mmol) in a final incubation volume of 30 µl.
After incubation (10 min, 30°C), the reaction was terminated by the
addition of trichloroacetic acid to a final concentration
of 10% (wt/vol). Samples were incubated at 4°C for 30 min after
which precipitated material was pelleted by centrifugation (10,000 g, 2 min), and the supernatants were
spotted onto discs of P81 phosphocellulose paper. The phosphocellulose
discs were washed with shaking in 500 ml of 75 mM
H3PO4
(2 × 5 min) followed by washing in distilled water (2 × 5 min). 32P-radiolabeled
autocamtide-2 adhering to the phosphocellulose discs was measured by
liquid scintillation counting, and samples of the
[
-32P]ATP used in
the assay were subjected to scintillation counting to permit estimation
of specific radioactivity. CaMK II activity was estimated by using the
specific radioactivity of the
[
-32P]ATP to
determine total phosphate transferred to the peptide substrate after
correction for nonspecific binding and converting this mass transfer
term into a rate by expressing it per islet and per minute of
incubation.
Unless stated, data are expressed as means ± SE. Differences
between treatment groups were assessed by ANOVA and/or
Bonferroni's t-test for multiple comparisons, as appropriate, and were
considered significant at P < 0.05.
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RESULTS |
Ca2+-induced
insulin secretion from permeabilized islets.
Figure 1 shows the rapid but transient
increases in insulin secretion from perifused electrically
permeabilized islets of Langerhans on increasing the
Ca2+ concentration of the
perifusion medium from 50 nM to 10 µM (Fig. 1A). Figure
1B demonstrates that both cAMP and the
protein kinase C (PKC) activator PMA stimulated insulin secretion from
the Ca2+-insensitive permeabilized
islets, demonstrating that the loss of
Ca2+-induced insulin secretion was
not due to a generalized reduction in the secretory capacity of the
permeabilized cells. The loss of secretory responsiveness to
Ca2+ was not prevented by changing
the intracellular Ca2+ in a
periodic manner, as shown in Fig. 2. Thus
the pattern of insulin secretion produced by alternating between 50 nM
Ca2+ and 10 µM
Ca2+ at 2-min intervals (Fig. 2)
was remarkably similar to that observed in response to maintained
elevations in Ca2+ (Fig. 1) with a
rapid desensitization of the secretory response to
Ca2+. Permeabilized islets
produced a rapid and reversible secretory response to the first pulse
of 10 µM Ca2+, a rapid but less
prolonged response to the second pulse of
Ca2+, and no responses to
subsequent Ca2+ pulses (Fig. 2).
The inclusion of trace amounts of
3H2O
in the perifusion buffer containing 10 µM
Ca2+ confirmed that changes in the
Ca2+ concentration of the
perifusate were essentially immediate (Fig. 2).

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Fig. 1.
Insulin secretion from perifused permeabilized islets of Langerhans.
Electrically permeabilized islets of Langerhans were perifused with
permeation buffer containing 50 nM
Ca2+ to establish a basal rate of
insulin secretion (0-10 min), after which the
Ca2+ concentration was increased
to 10 µM (arrow) and maintained at this stimulatory concentration for
the duration of the experiments.
A: rapid but transient
increase in insulin secretion ( ) from electrically permeabilized
islets in response to 10 µM
Ca2+.
B: insulin secretory response to 10 µM Ca2+ (first arrow, 10 min),
followed by responses to cAMP ( , 500 µM) or to 4 -phorbol
myristate acetate (PMA, , 500 nM) in the continued presence of 10 µM Ca2+ (second arrow,
40-70 min). Points show the mean values for 3 separate perifusion
chambers. Error bars have been omitted for the sake of clarity, and SE
ranged from 7-16% of the mean value for all points
shown.
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Fig. 2.
Insulin secretory responses to pulsatile changes in
Ca2+ concentration. Insulin
secretion ( , left axis) from
perifused permeabilized islets in response to 2-min pulses of buffer
containing 10 µM Ca2+
alternating with 2-min pulses of buffer containing 50 nM
Ca2+ is shown. Trace amounts of
3H2O
were added to the perifusion buffer containing 10 µM
Ca2+, such that subsequent
measurement of 3H in the
perifusate samples (15-s intervals) could be used to assess the rate of
change of the Ca2+ concentrations
when switching between buffers containing 50 nM
Ca2+ or 10 µM
Ca2+ (dotted line,
right axis). Points show means
(n = 2).
A: pattern of insulin secretion in
response to pulsatile Ca2+ was not
maintained after the second 2-min pulse of 10 µM
Ca2+.
B: expanding the time scale clearly
demonstrated a rapid and reversible secretory response to the first
pulse of 10 µM Ca2+ and a less
prolonged response to the second pulse of
Ca2+.
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Ca2+-induced
reductions in
Ca2+/calmodulin-dependent
kinase activity in permeabilized islets.
Extracts prepared from permeabilized islets that had been incubated for
1 h in the presence of a substimulatory
Ca2+ concentration (50 nM)
contained protein kinase activity that, in the presence of
Ca2+ and exogenous calmodulin,
transferred 32P from
[
-32P]ATP to
endogenous islet proteins and to exogenous MLC, as shown in Fig.
3. Major endogenous substrates for
Ca2+-dependent phosphorylation
migrated with apparent molecular masses of 54-57 kDa, although
several other phosphorylation events were also detectable. The
54/57-kDa phosphorylated substrates in islets have been detected
previously (6), and proteins of similar molecular weights have been
identified as autophosphorylated CaMK II by immunoblotting (29),
immunoprecipitation (44), and protein purification (31). In accordance
with this identification, the Ca2+-dependent phosphorylation of
the 54/57-kDa proteins was inhibited by a synthetic peptide
corresponding to amino acid residues 290-309 from the sequence of
CaMK II [CaMK-(290
309)], which is a selective, substrate
site-directed inhibitor of CaMK II (Ref. 32; Fig. 3,
lane 3). Note, however, that the
phosphorylation of exogenous MLC was only partially inhibited by
CaMK-(290
309), suggesting that, although this peptide fully inhibits
islet CaMK II activity, it only has small inhibitory effects on
endogenous islet MLCK activity (Fig. 3, lane
3).

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Fig. 3.
Ca2+/calmodulin-dependent
32P incorporation in islet
extracts: effects of the autoinhibitory peptide sequence
CaMK-(290 309). Autoradiographs of
32P incorporation into islet
proteins and exogenous myosin light chain (MLC) separated by SDS-PAGE
on a 10% gel are shown. The 54/57-kDa endogenous substrates may be
autophosphorylated
Ca2+/calmodulin-dependent kinase
II (CaMK II), whereas the identity of the 32-kDa endogenous substrate
is unknown. Lane 1 shows
32P incorporation in the absence
of Ca2+ and calmodulin, whereas
lane 2 shows the
Ca2+/calmodulin-dependent
phosphorylation of endogenous proteins and exogenous MLC. The presence
of the CaMK autoinhibitory peptide sequence CaMK-(290 309) (100 µM,
lane 3) greatly reduced the
Ca2+/calmodulin-dependent
phosphorylation of endogenous substrates. Results shown are from 1 experiment from 3 similar experiments.
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The Ca2+-induced inhibition of
Ca2+/calmodulin-dependent protein
phosphorylation in islet extracts is shown in Fig.
4. Extracts prepared from permeabilized
islets incubated for 1 h in a substimulatory concentration of
Ca2+ (50 nM) showed the expected
Ca2+/calmodulin-dependent
phosphorylation of the major islet substrate of 54 kDa (Fig. 4,
lanes 1 and
2), but this was markedly reduced in
extracts prepared from permeabilized islets incubated in the presence
of a stimulatory concentration of
Ca2+ (10 µM; Fig. 4,
lanes 3 and
4). In five separate experiments of this type, the presence of Ca2+
and calmodulin enhanced the phosphorylation of the 54-kDa substrate by
419 ± 29% (mean ± SE, P < 0.001 vs. absence of Ca2+ and
calmodulin) in control extracts (50 nM
Ca2+ incubation) and by 131 ± 33% in Ca2+-desensitized extracts
(10 µM Ca2+ incubation,
P > 0.2 vs. absence of
Ca2+ and calmodulin;
P < 0.001 vs. control extracts).

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Fig. 4.
CaMK-(290 309) does not prevent
Ca2+-induced reduction in protein
kinase activity. Autoradiograph of
32P incorporation in islet
proteins in the absence (lanes 1,
3, and
5) or presence
(lanes 2,
4, and
6) of
Ca2+ and calmodulin is shown.
Extracts of permeabilized islets that had been incubated for 30 min in
a buffer containing a substimulatory concentration (50 nM) of
Ca2+ contained protein kinase
activity that was independent of
Ca2+ and calmodulin
(lane 1) and
Ca2+/calmodulin-dependent kinase
activity that enhanced 32P
incorporation into a major islet substrate of 54 kDa
(lane 2). This
Ca2+/calmodulin-stimulated protein
phosphorylation was much reduced in extracts of permeabilized islets
that had been desensitized to Ca2+
by incubation in a buffer containing a stimulatory concentration (10 µM) of Ca2+
(lanes 3 and
4). The presence of CaMK-(290 309)
(100 µM) during the incubation with 10 µM
Ca2+ did not prevent the
Ca2+-dependent reduction in
Ca2+/calmodulin-dependent protein
kinase activity in the islet extract (lanes
5 and 6). Proteins
were separated on a 15% gel, and the autoradiograph is typical of
effects seen in 2 separate experiments.
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Similar results were obtained when CaMK II activity was measured by the
incorporation of 32P into the
specific exogenous substrate autocamtide-2. Initial experiments
demonstrated that incubation of permeabilized islets for 1 h in the
presence of 10 µM Ca2+ caused a
marked reduction in the CaMK II activity measured in subsequent islet
extracts (10 µM Ca2+, 21.1 ± 0.9% activity of controls incubated in 50 nM
Ca2+,
P < 0.01, n = 4). In subsequent experiments, we measured islet CaMK II activity in
extracts of permeabilized islets after various times of exposure to
substimulatory (50 nM) or stimulatory (10 µM) concentrations of
Ca2+. Figure
5 shows that incubating the permeabilized
islets in 50 nM Ca2+ for up to 30 min had no effect on the levels of CaMK II activity but that 10 µM
Ca2+ caused a rapid and marked
reduction in the enzyme activity in subsequent islet extracts. CaMK II
activity showed a small but significant
(P < 0.05) reduction within 1 min of
exposure of the permeabilized islets to 10 µM
Ca2+; maximum reduction in
activity was achieved within 15-30 min, and the ~80% reduction
in activity at these times is in accordance with the initial data
obtained after a 1-h incubation in the presence of 10 µM
Ca2+ and with the marked, but not
total, reduction in Ca2+-dependent
phosphorylation of endogenous substrates seen in Fig. 4. The time
course of the Ca2+-induced
reduction of CaMK II activity in permeabilized islets (Fig. 5) is
similar to that of the transient
Ca2+-induced insulin secretory
response from permeabilized islets (Figs. 1 and 2).

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Fig. 5.
Time course of Ca2+-induced loss
of islet CaMK II activity. Electrically permeabilized islets were
incubated for 1-30 min in the presence of 50 nM
Ca2+ ( ) or 10 µM
Ca2+ ( ), and CaMK II activity
of islet extracts was estimated by phosphorylation of the exogenous
substrate autocamtide-2. Islets incubated in the presence of 50 nM
Ca2+ showed sustained CaMK II
activity (P > 0.8 by ANOVA), whereas
there was a time-dependent decrease in CaMK II activity in islets
exposed to 10 µM Ca2+
(P < 0.001 by ANOVA). Points show
means ± SE; n = 4.
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The Ca2+-induced reduction in
Ca2+/calmodulin-dependent protein
kinase activity was independent of the activation of CaMK II during the
preincubation period, since it also occurred when the CaMK II inhibitor
CaMK-(290
309) was present during the preincubation period (Fig. 4,
lanes 5 and
6). These observations suggest that the reductions in
Ca2+/calmodulin-dependent protein
phosphorylation in
Ca2+-desensitized islets reflect a
loss of CaMK II activity, and this reduction in activity cannot be
explained by the activation, autophosphorylation, and consequent
Ca2+ independence of CaMK II
during the preincubation period.
Effects of inhibitors of phosphoprotein phosphatases
and proteases. In further experiments, we investigated
whether the Ca2+-induced
inhibition of
Ca2+/calmodulin-dependent protein
phosphorylation could be accounted for by the activation of
phosphoprotein phosphatases 1 (PP1), 2A (PP2A), or 2B (PP2B) or by the
proteolytic inactivation of Ca2+/calmodulin-dependent kinase.
Inhibition of islet phosphoserine/threonine phosphatase activities
using a combination of inhibitors of PP1 and PP2A (1 µM each of CAL,
CAN, and OA) and an inhibitor of PP2B (1 µM CM) enhanced the overall
levels of 32P incorporation in
islet extracts but had no effect on
Ca2+/calmodulin-dependent protein
kinase activity in islet extracts. Thus, as shown in Table
1,
Ca2+/calmodulin-dependent
32P incorporation into the 54-kDa
substrate was greatly reduced in extracts prepared from
Ca2+-desensitized islets
(incubated with 10 µM Ca2+ for
30 min), and this was not prevented by inhibition of phosphoprotein phosphatase activity in permeabilized islets during the incubation period and in the subsequent assay of
Ca2+/calmodulin-dependent protein
kinase activity. Similarly, the presence of a cocktail of protease
inhibitors (20 µM each of E-64, PMSF, and TLCK; 50 µg/ml leupeptin;
100 µg/ml E-64C) during the incubation, extraction, and kinase assay
did not prevent the Ca2+-induced
loss of Ca2+/calmodulin-dependent
32P incorporation, as shown in
Table 1.
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Table 1.
Inhibitors of phosphoprotein phosphatases or proteases do not
prevent the Ca2+-dependent reduction in
Ca2+/calmodulin-dependent kinase activity in islet
extracts
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DISCUSSION |
Ca2+ is generally considered to be
the most important intracellular regulator of nutrient-induced insulin
secretion from pancreatic
-cells. Glucose causes a rapid increase in
-cell cytosolic Ca2+ by
promoting Ca2+ influx through
voltage-operated Ca2+ channels,
and glucose-induced insulin secretion is largely dependent on the
presence of extracellular Ca2+
(reviewed in Refs. 11, 14, 36). However, although secretory responses
to glucose and other nutrients are maintained for the duration of the
stimulus, secretory responses to experimental elevations in
intracellular Ca2+ are transient,
whether studied in intact cells using
Ca2+ ionophores (47) or in
permeabilized
-cell preparations (17, 19, 20). Our measurements of
Ca2+-induced insulin release from
electrically permeabilized islets of Langerhans in the present studies
have confirmed our previous observations of the pattern of secretion
(19, 20), and similar Ca2+-induced
reductions in secretory responsiveness to
Ca2+ have been reported in
electrically permeabilized chromaffin cells (23, 24),
-toxin
permeabilized gonadotrophs (42), and insulin-secreting HIT-T15
-cells (17). As we have discussed in detail elsewhere (19, 20), the
loss of
-cell secretory responsiveness to
Ca2+ is not caused by
time-dependent or Ca2+-dependent
resealing of the permeabilized membranes or by a decrease in the
overall secretory capacity of the cells, as confirmed in the present
study by the secretory responses of
Ca2+-insensitive
-cells to the
activation of PKC or cAMP-dependent protein kinase by PMA
or cAMP, respectively.
Glucose-induced elevations in
-cell cytosolic
Ca2+ show an oscillatory pattern
with rapid, small-amplitude oscillations (2-5/min) superimposed on
larger-amplitude oscillations with a frequency of 0.2-0.5
oscillations/min (reviewed in Refs. 13 and 14). The physiological
relevance of the slower, large-amplitude oscillations in
-cell
Ca2+ is unclear, but work in other
tissues suggests that an oscillatory pattern of intracellular
Ca2+ may facilitate an efficient
exocytotic response, and it has also been suggested that oscillations
in intracellular Ca2+ may prevent
desensitization of intracellular targets to
Ca2+ (12, 38). However, our
measurements of insulin secretion from permeabilized islets do not
provide any evidence for enhanced secretory responses to oscillatory
patterns of intracellular Ca2+.
Furthermore, our results suggest that the loss of responsiveness to
Ca2+ was not a direct consequence
of the pattern of change in cytosolic Ca2+, since essentially similar
responses and time courses were observed in response to 2-min
oscillations in Ca2+ and to
prolonged elevations in Ca2+.
These results extend our previous studies in which desensitization of
Ca2+-induced insulin secretion was
observed when the Ca2+ was
presented as pulses of 10-min duration (20) and confirm earlier
observations in
-toxin permeabilized HIT-T15 cells in which the
secretory response to Ca2+ was not
maintained by 1-min pulses of Ca2+
(17). The observation that the appearance of glucose-induced oscillations in
-cell Ca2+
varies with species (39) may also raise questions as to the importance
of the Ca2+ oscillations in the
maintenance of normal secretory responses. Whatever the physiological
significance of glucose-induced oscillations in
-cell
Ca2+, the transience of
Ca2+-induced insulin secretion
appears to be due to a selective and rapid desensitization of the
secretory apparatus to Ca2+, which
is unrelated to the pattern of changes in intracellular Ca2+.
Although there is no doubt about the importance of an influx of
extracellular Ca2+ in the
initiation of insulin secretion, the mechanisms through which
elevations in intracellular Ca2+
initiate exocytosis in the
-cell remain unclear. There is a body of
circumstantial evidence that the
Ca2+-dependent activation of
protein kinases is an important initiator of nutrient-induced insulin
secretion. Thus increases in Ca2+
cause rapid increases in protein phosphorylation in intact
-cells and in broken cell preparations (reviewed in Refs. 2 and 10); glucose
and other insulin secretagogues activate CaMK II in islets of
Langerhans and in insulin-secreting cell lines (5, 31, 44), and
inhibitors of CaMK inhibit depolarization-induced increases in membrane
capacitance in single
-cells (1) and inhibit glucose-induced insulin
secretion from intact islets of Langerhans (29, 45). More recently, a
temporal correlation between glucose-induced CaMK II activation and
insulin secretion has been established and presented as evidence of a
role for CaMK II in transducing secretory responses to
Ca2+-mobilizing stimuli (5).
In accordance with an important role for kinase activation in
-cell
secretory responses to Ca2+, we
have demonstrated previously that elevations in cytosolic Ca2+ enhanced
32P incorporation into several
proteins in electrically permeabilized islets (21) and that this
Ca2+-dependent protein
phosphorylation was much reduced by prior exposure of the permeabilized
islets to stimulatory concentrations of
Ca2+ (20). These studies suggested
that the desensitization of the secretory process to
Ca2+ may be caused by reduced
Ca2+-dependent protein
phosphorylation in desensitized
-cells but did not define
the underlying cellular event. In the present studies, we have
demonstrated that the Ca2+-induced
reduction in Ca2+-dependent
phosphorylation of endogenous substrates seen in our previous studies
reflects a loss of endogenous
Ca2+/calmodulin-dependent kinase
activity. Furthermore, the use of a peptide substrate specific for CaMK
II (9) indicates that the loss of
Ca2+-induced phosphorylation is
primarily due to a reduction in the phosphorylating activity of CaMK
II, an enzyme widely implicated in
Ca2+-induced secretory responses
(5, 29, 31, 44, 45). The time course of the
Ca2+-induced reductions in CaMK II
activity were remarkably similar to the transient patterns of insulin
secretion induced by elevations in intracellular
Ca2+, with both CaMK II activity
and Ca2+-induced insulin secretion
being markedly reduced after 15 min exposure to a stimulatory
concentration of Ca2+. These
observations are fully consistent with CaMK II activity being required
for insulin secretion from
-cells.
The mechanism(s) underlying the
Ca2+-induced reduction in CaMK II
activity is not clear. It cannot be accounted for by a
Ca2+-dependent loss of endogenous
calmodulin, since the pores formed in electrically permeabilized cells
are of insufficient dimensions to allow loss of endogenous proteins of
the size of calmodulin (20). In any case, the concentration of
calmodulin was not rate limiting in the in vitro assays for CaMK II,
since an excess of exogenous calmodulin was supplied in the reaction
mixture.
CaMK II is well known to autophosphorylate upon activation, reducing
the Ca2+ dependence of its
catalytic activity (5, 31, 37, 44), and autophosphorylated CaMK II is
almost certainly one of the major endogenous substrates detected in the
present studies. There are a number of reasons why the
Ca2+-induced reduction in CaMK II
activity is unlikely to be caused by autophosphorylation of the kinase.
First, we have previously demonstrated that the loss of activity is not
dependent on the presence of ATP during the preincubation period (20),
whereas autophosphorylation is ATP dependent. Second, there was no
corresponding increase in
Ca2+-independent phosphorylations
or kinase activity in
Ca2+-desensitized islets, which
would be expected if CaMK II were autophosphorylated and hence
Ca2+ independent (44). Third, the
loss of CaMK II activity was not prevented by the presence of a
selective peptide inhibitor of CaMK II (32). Electrically permeabilized
islets are a useful experimental model for using peptide inhibitors,
and we have shown that autoinhibitory peptides will enter the
permeabilized cells and selectively inhibit the appropriate kinase (3).
In the present study, the autoinhibitory sequence CaMK-(290
309) was shown to inhibit CaMK activity in islet extracts but was without effect
on the process of Ca2+-induced
reduction of Ca2+-dependent kinase
activity. These observations are supported by the report that
Ca2+ desensitization of insulin
secretion from permeabilized HIT-T15
-cells was not inhibited by
KN-62, an inhibitor of CaMK II (17). Finally, the peptide substrate
used in our in vitro assays, autocamtide-2, is a substrate for both the
unphosphorylated,
Ca2+/CaM-dependent form of CaMK II
and for the autophosphorylated Ca2+-independent form of CaMK II,
which is generated in islets in response to
Ca2+-mobilizing stimuli (5, 39).
The observed reductions in autocamtide-2 phosphorylation by extracts
prepared from permeabilized islets after exposure to micromolar amounts
of Ca2+ cannot therefore be
explained by Ca2+-induced
autophosphorylation of CaMK II, and there must exist another
Ca2+-dependent mechanism through
which CaMK II activity is decreased.
Pancreatic
-cells express a number of phosphoprotein phosphatase
activities, including PP1, PP2A, and PP2B (28), and there is some
evidence that these enzymes may be involved in the regulation of
insulin secretion by modifying the phosphorylation state of as yet
unidentified proteins (7, 28). Our results suggest that the
Ca2+-induced inhibition of
Ca2+-dependent protein
phosphorylation and of CaMK II activity was not dependent on the
activation of PP1, PP2A, or PP2B in the permeabilized islets, since the
loss of Ca2+/calmodulin-dependent
kinase activity was not prevented by the presence of inhibitors of
these enzymes during the desensitization period and the subsequent
kinase assay. An additional class of phosphoprotein phosphatase,
phosphoprotein phosphatase 2C (PP2C), has been reported to
dephosphorylate recombinant autophosphorylated CaMK II, thus restoring
its Ca2+ dependency (8). However,
as discussed above, the
Ca2+-induced loss of CaMK II
activity cannot be accounted for by changes in the phosphorylation
state and thus Ca2+ dependence of
the enzyme. Furthermore, although we cannot rule out a role for PP2C in
the generation of Ca2+
desensitization in
-cells, PP2C expression in pancreatic
-cells has not yet been confirmed, and the ionic dependence of PP2C on Mg2+ rather than
Ca2+ makes it an unlikely
candidate for a Ca2+-dependent
process.
An alternative Ca2+-dependent but
calmodulin- and ATP-independent mechanism for the reduction in
Ca2+/calmodulin-dependent kinase
activity could be through the activation of
-cell proteases, and
there is some evidence that this mechanism is operational for other
protein kinase activities in
-cells. Thus the family of
diacylglycerol-sensitive,
Ca2+/phospholipid-dependent PKC
isoenzymes is susceptible to proteolytic cleavage by
membrane-associated Ca2+-activated
proteases (30). This may be the basis for the reduction in
-cell PKC
activity by prolonged activation of the enzyme (15), since the loss of
enzyme activity in permeabilized islets was reduced by the presence of
the protease inhibitors leupeptin and PMSF (35). The calpains are
Ca2+-dependent cysteine proteases,
which are ubiquitously expressed in mammalian cells (reviewed in Ref.
27) and which are known to proteolyse many calmodulin-binding proteins,
including several protein kinases (reviewed in Ref. 43). However, in
our study, the Ca2+-induced
reduction in
Ca2+/calmodulin-dependent kinase
activity was not prevented by the presence of a mixture of protease
inhibitors, which are known to inhibit calpain activity (26) and which
would have ready access to the enzyme in the electrically permeabilized
islets used in our experiments. These results suggest that the
reduction in
Ca2+/calmodulin-dependent kinase
activity was not caused by the activation of
Ca2+-dependent proteases, and the
mechanism underlying the desensitization process in
-cells remains
unclear.
The importance of elevations in intracellular
Ca2+ in the generation and
maintenance of physiologically appropriate insulin secretory responses
is well established (reviewed in Refs. 11, 13, 14, 36). It is not yet
clear whether the Ca2+
desensitization observed in the current studies occurs under more
physiological conditions, although it has been suggested that
disturbances in
-cell Ca2+
handling may be involved in the secretory defects seen in type 2 diabetes mellitus (12). Irrespective of its function in pathological conditions, our experimental observations support a role for CaMK II in
the regulation of insulin secretion. Thus
Ca2+-mobilizing insulin
secretagogues promote rapid increases in CaMK II activity in islets
(5), and insulin secretion is inhibited by inhibitors of CaMK II (29,
45). The present demonstration of reduced CaMK II activity concomitant
with reduced secretory responsiveness to
Ca2+ provides further evidence
that the activation of
Ca2+/calmodulin-dependent
kinase(s) is important in the generation of appropriate insulin
secretory responses to the elevations in intracellular
Ca2+, which are induced by
physiologically important nutrient secretagogues, such as glucose.
 |
ACKNOWLEDGEMENTS |
Financial support from the Medical Research Council and the
Wellcome Trust is gratefully acknowledged.
 |
FOOTNOTES |
S. J. Persaud was a Wellcome Trust Research Fellow (Grant no. 039057).
Address for reprint requests: P. Jones, Biomedical Sciences Division,
King's College London, Campden Hill Rd., London W8 7AH, UK.
Received 11 June 1997; accepted in final form 18 December 1997.
 |
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