(Received for publication, September 13, 1995; and in revised form, October 18, 1995)
From the
Cyclic ADP-ribose (cADPR) is generated in pancreatic islets by
glucose stimulation, serving as a second messenger for Ca mobilization from the endoplasmic reticulum for insulin secretion
(Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H.(1993) Science 259, 370-373). In the present study, we observed
that the addition of calmodulin (CaM) to rat islet microsomes
sensitized and activated the cADPR-mediated Ca
release. Inhibitors for CaM-dependent protein kinase II (CaM
kinase II) completely abolished the glucose-induced insulin secretion
as well as the cADPR-mediated and CaM-activated Ca
mobilization. Western blot analysis revealed that the microsomes
contain the
isoform of CaM kinase II but do not contain CaM. When
the active 30-kDa chymotryptic fragment of CaM kinase II was added to
the microsomes, fully activated cADPR-mediated Ca
release was observed in the absence of CaM. These results along
with available evidence strongly suggest that CaM kinase II is required
to phosphorylate and activate the ryanodine-like receptor, a
Ca
channel for cADPR as an endogenous activator, for
the cADPR-mediated Ca
release.
Glucose is the primary stimulus of insulin secretion and
synthesis in the pancreatic islets of
Langerhans(1, 2, 3) . Cyclic ADP-ribose
(cADPR) ()is generated by glucose stimulation, serving as a
second messenger for Ca
mobilization from the
endoplasmic reticulum to secrete
insulin(4, 5, 6) . cADPR has been thought to
activate or enhance the ryanodine-like receptor (RyR) of a variety of
cells to release Ca
from the intracellular stores (4, 5, 6, 7, 8, 9, 10, 11, 12, 13) .
In sea urchin eggs, it was recently suggested that calmodulin (CaM)
directly interacts with RyR to enhance the cADPR-mediated
Ca
release(14, 15, 16) .
In the present study, we showed that CaM-dependent protein kinase II
(CaM kinase II) is essential for the cADPR-mediated Ca mobilization for insulin secretion in islets. A possible mode of
action of cADPR and CaM kinase II on RyR is discussed.
We measured the Ca release by cADPR from
rat islet microsomes in the presence or absence of CaM (Fig. 1A). In the absence of CaM, the cADPR
responsiveness for Ca
release was restored only at
high concentrations of cADPR. On the other hand, in the presence of
CaM, islet microsomes were sensitized to cADPR at much lower
concentrations (0.1-0.3 µM), and the Ca
release by cADPR was greatly increased. The maximal response was
achieved at 3-10 µg/ml CaM. CaM was not detected in islet
microsomes by immunoblot analysis (Fig. 1B, lane 2),
but CaM was present in islet homogenates, and the CaM concentration in
islets was estimated to be about 3-10 µg/ml (Fig. 1B). Therefore, Ca
release from
the microsomes in the presence of 3-10 µg/ml CaM rather than
in its absence is physiological. The activation of cADPR-mediated
Ca
release by CaM was also observed with rat
cerebellar microsomes (data not shown). These results suggest that CaM
is a positive modulator for cADPR-mediated Ca
release
not only in sea urchin egg microsomes (14, 15, 16) but also in mammalian
microsomes.
Figure 1:
cADPR sensitivity-conferring activity
of CaM. A, islet microsomes were incubated with or without 7
µg/ml CaM and challenged with the indicated concentrations of
cADPR. The Ca release measurement was a peak value
estimated from Fluo-3 fluorescence. B, CaM content was
estimated in islet homogenate and in islet microsomes by immunoblot
analysis. Islet homogenate (1 µg of protein, lane 1),
islet microsome (1 µg of protein, lane 2), and bovine CaM
(0.01, 0.1, 1 µg; lanes 3-5, respectively) were
chromatographed on 15% SDS-polyacrylamide gel electrophoresis and
transferred to Immobilon-P. The membrane was incubated at room
temperature for 1 h with a monoclonal antibody against CaM. The
antibody was diluted at 1 µg/ml with 5% milk powder solution. After
rinsing, the membrane was further incubated at room temperature for 1 h
with a secondary antibody labeled with horseradish peroxidase and
developed using an ECL detection system (Amersham
Corp.).
We then examined the effects of CaM and CaM kinase II
inhibitors on the cADPR/CaM-induced Ca release from
islet microsomes. A CaM inhibitor, W-7 (50 µM), and CaM
kinase II inhibitors such as KN-62 (10 µM) and KN-93 (10
µM) completely inhibited the cADPR/CaM-induced
Ca
release, but non-inhibitory analogues, W-5 (50
µM), KN-04 (10 µM), and KN-92 (10
µM), did not (Fig. 2A). In addition,
AIP(18, 19) , which is a more specific CaM kinase II
peptide inhibitor, also inhibited the activation of cADPR-mediated
Ca
mobilization by CaM, and the dose-inhibition curve
of Ca
mobilization by AIP (Fig. 2B)
was well fitted to that of CaM kinase II activity by AIP(19) .
These results strongly suggest that the activation of cADPR-mediated
Ca
mobilization by CaM is achieved by activation of
CaM kinase II but not by the direct interaction of CaM with the
Ca
release machinery.
Figure 2:
A,
effects of the cell-permeable CaM antagonist and CaM kinase II
antagonists on the cADPR-mediated Ca release and on
the glucose-induced insulin secretion. Ca
release was
induced by the addition of 100 nM cADPR in the presence of 7
µg/ml CaM and measured as in Fig. 1A. The
concentrations of CaM antagonists (W-7 and W-5) and CaM kinase II
antagonists (KN-62, KN-04, KM-93, and KN-92) were 50 and 10
µM, respectively. Values are expressed by percent (mean
± S.E., n = 3-4) of control without
antagonist. The average Ca
release of control was
0.327 ± 0.018 µM. For insulin secretion, the
antagonists were used at the same concentrations in KRB as for the
Ca
release. Values are expressed by percent (mean
± S.E., n = 5-6) of control without
antagonist. The average insulin secretion of control was 40.9 ±
11.3 ng/islet/h. B, inhibition of cADPR/CaM-mediated
Ca
release by AIP. cADPR/CaM-mediated Ca
release was measured in the presence of the indicated
concentrations of AIP. Ca
release was induced by the
addition of 100 nM cADPR in the presence of 7 µg/ml CaM
and measured as in Fig. 1A.
We therefore examined the
existence of CaM kinase II in the microsomes by immunoblot analysis and
revealed that the isoform of CaM kinase II but not the
isoform actually exists in islet microsomes (Fig. 3A).
To confirm the essential requirement of CaM kinase II activity for the
cADPR-mediated Ca
mobilization, we then added the
active 30-kDa chymotryptic fragment of the CaM kinase II(18) ,
which lacks the autoinhibitory domain and is therefore activated in the
absence of CaM, to islet microsomes and measured the cADPR-mediated
Ca
release. As shown in Fig. 3B, the
cADPR-mediated Ca
release was as fully and dose
dependently activated by the 30-kDa fragment of CaM kinase II as by the
addition of CaM to microsomes.
Figure 3:
Evidence that CaM kinase II is the cADPR
sensitivity-conferring factor. A, cerebral homogenate (50
µg of protein, lanes 1, 3, and 5) and islet
microsome (50 µg of protein, lanes 2, 4, and 6;
100 µg of protein, lane 7) were chromatographed on 10%
SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P.
The membrane was incubated at room temperature for 1 h with the
monoclonal antibody against the isoform (lanes 1 and 2) or
isoform (lanes 3 and 4) of CaM
kinase II and with a 1:1 mixture of the antibodies (lanes
5-7). The concentration of antibody was 1 µg/ml each
with 5% milk powder solution. After rinsing, the membrane was further
incubated with a secondary antibody at room temperature for 1 h and
developed as described in Fig. 1B. B, concentration
dependence of CaM kinase II in cADPR-mediated Ca
release from islet microsomes. For the Ca
release assay, the active 30-kDa chymotryptic fragment of CaM
kinase II at the indicated concentrations was used in the absence of
CaM. The same amounts of the 30-kDa CaM kinase II fragment previously
inactivated by 5-min boiling were used as controls. Ca
release was induced by the addition of 100 nM cADPR and
measured as in Fig. 1A.
We have proposed a model for insulin
secretion by glucose via cADPR-mediated Ca mobilization. In the process of glucose metabolism, millimolar
concentrations of ATP are generated, inducing cADPR accumulation by
inhibiting the cADPR hydrolase activity of CD38, and cADPR acts as a
second messenger for intracellular Ca
mobilization
from microsomes for insulin
secretion(4, 5, 6, 21, 23, 24) .
Recently, in sea urchin eggs, CaM has been reported to act on the
microsomes and sensitize the cADPR-mediated Ca
release without the involvement of CaM-dependent enzymes such as
CaM kinase II(14, 15, 16) . The results in
this study showed that CaM sensitized and activated the cADPR-mediated
Ca
release from islet microsomes, which contained the
isoform of CaM kinase II, and that the effect of CaM on
cADPR-mediated Ca
mobilization as well as the
glucose-induced insulin secretion were abolished by specific inhibitors
for CaM kinase II. Furthermore, the addition of the 30-kDa active CaM
kinase II fragment to the microsomes fully activated the cADPR-mediated
Ca
release in the absence of CaM. These results
suggest that the cADPR-mediated Ca
mobilization for
insulin secretion is achieved by the CaM-activated
isoform of CaM
kinase II. RyR is thought to be a Ca
channel for
cADPR as an endogenous activator, and phosphorylation and activation of
RyR by CaM kinase II were
reported(25, 26, 27) . Furthermore, in
islets, CaM kinase II was observed to be activated by glucose
stimulation(28, 29) . Therefore, as depicted in Fig. 4, when islets are stimulated by glucose, RyR can be
synergistically activated not only by cADPR but also by phosphorylation
of the Ca
channel by CaM kinase II.
Figure 4:
Model for cADPR-induced Ca mobilization via RyR for the glucose-induced insulin secretion.
Glucose stimulation induces cADPR accumulation in islets, and cADPR
acts on RyR as a second messenger for intracellular Ca
mobilization (4, 5, 6, 21, 23, 24) .
On the other hand, CaM kinase II is activated by glucose
stimulation(28, 29) , and the activated kinase
phosphorylates RyR to sensitize the Ca
channel for
the cADPR signal. Pi, phosphorylation of RyR by CaM kinase
II.