1 Department of Pharmacology,
2 First Department of Surgery, and
3 Department of Anatomy,
Nagoya University School of Medicine, Nagoya 466;
4 Department of Thoracic
Surgery, The aim of this study was to investigate how
insulin secretion is controlled by phosphorylation of the myosin light
chain (MLC). Ca2+-evoked insulin
release from pancreatic islets permeabilized with streptolysin O was
inhibited by different monoclonal antibodies against myosin light-chain
kinase (MLCK) to an extent parallel to their inhibition of purified
MLCK. Anti-MLCK antibody also inhibited insulin release caused by the
stable GTP analog guanosine 5'-O-(3-thiodiphosphate), even
at a substimulatory concentration (0.1 µM) of
Ca2+. Free
Ca2+ increased MLC peptide
phosphorylation by
calcium; calmodulin; myosin light-chain kinase; pancreatic
INSULIN SECRETION is finely regulated by nutrients and
hormones at multiple steps, i.e., synthesis, translocation, docking, and priming of secretory granules followed by exocytosis, governed by
second messenger systems such as
Ca2+, cyclic nucleotides, and
phospholipid metabolites. Intracellular Ca2+ plays a critical role in
secretory events in various exocrine and endocrine cells, including the
pancreatic Intracellular Ca2+ signaling is
mediated via interactions with
Ca2+-binding proteins (20). The
ubiquitous Ca2+-binding protein
calmodulin (CaM) is one of the
Ca2+ receptors that have been
suggested to participate in stimulus-secretion coupling in the Myosin light-chain kinase (MLCK), a
Ca2+/CaM-dependent protein kinase,
has been proposed to act in positive control of insulin secretion (19,
24). Myosin itself is composed of heavy chains and two types of light
chains, one essential and the other regulatory. Heteromer complexes of
these myosin subunits exhibit actin-dependent adenosinetriphosphatase
activity, which is predominantly controlled by regulatory light-chain
phosphorylation by MLCK and other protein kinases (31). However, the
step in the secretory process where MLCK acts has yet to be elucidated.
By observation of living In the present study, we characterized Materials.
Collagenase (type V), benzamidine, fluorescein
isothiocyanate-conjugated goat anti-mouse immunoglobulin (Ig) G
antibody, monoclonal anti-MLC antibody, guanosine
5'-O-(2-thiodiphosphate)
(GDP Preparation of pancreatic islets.
Pancreatic islets were isolated from male Wistar rats by collagenase
digestion. The solution used was
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered Krebs-Ringer bicarbonate buffer containing (in
mM) 119 NaCl, 4.75 KCl, 5 NaHCO3,
2.54 CaCl2, 1.2 MgSO4, 1.2 KH2PO4,
and 20 HEPES (pH 7.4 with NaOH) supplemented with 3 mM glucose.
Cell culture.
Preparation of monoclonal antibodies against MLCK.
Monoclonal antibodies against MLCK were raised in mice by injecting
chicken gizzard MLCK (9). Antibodies released in the culture media were
concentrated by precipitation with ammonium sulfate and dialyzed
against phosphate-buffered saline (PBS). The epitopes of the antibodies
have not been identified in this study. These antibodies were confirmed
not to react with protein kinase A, protein kinase C, or CaM kinase II.
In the preliminary experiments, we found that MM17 was the most useful
for both immunoblotting and immunohistochemistry.
Western blotting of MLCK, the MLCs, and the MHC.
Soluble proteins were extracted by homogenization of pancreatic islets
and cultured
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-cell extracts in vitro. In contrast to the
phosphorylation by purified MLCK or by calmodulin (CaM) kinase II, the
activity partially remained with the
-cell under nonstimulatory
Ca2+ (0.1 µM) conditions. The
MLCK inhibitor ML-9 inhibited the activity in the
-cell with both
substimulatory and stimulatory
Ca2+, whereas KN-62, an inhibitor
of CaM kinase II, only exerted an influence in the latter case. ML-9
decreased intracellular granule movement in MIN6 cells under basal and
acetylcholine-stimulated conditions. We propose that MLC
phosphorylation may modulate translocation of secretory granules,
resulting in enhanced insulin secretion.
-cell; protein kinase
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-cell (2). However, we still have only limited knowledge
of the relevant mechanisms.
-cell
by biochemical and pharmacological findings (18, 30). A number of
CaM-dependent protein kinases and other CaM-binding proteins have also
been identified in pancreatic
-cells and suggested to be involved in
the secretory machinery (11, 19, 35).
-cells under a phase-contrast microscope
(10), we recently demonstrated that intracellular movement of
-cell
secretory granules is regulated by
Ca2+/CaM-dependent protein
phosphorylation. These findings raise the possibility that such granule
movement may be influenced by myosin light-chain (MLC) phosphorylation.
-cell MLCK and myosin
subunits in rat pancreatic islets and mouse pancreatic
-cell lines
by biochemical and morphological approaches. We further investigated
their possible roles in the secretory pathway in the pancreatic
-cell by secretion studies using streptolysin O (STLO)-permeabilized
islets and by observation of actual granule movement.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
S) and polyclonal antibody against platelet myosin heavy chain
(MHC) were purchased from Sigma Chemical (St. Louis, MO).
[
-32P]ATP and the
enhanced chemiluminescence (ECL) kit were from Amersham Japan (Tokyo,
Japan). Alkaline-conjugated goat antibodies against rabbit IgG and
mouse IgGAM were purchased from Zymed (San Francisco, CA).
Kanamycin sulfate was from Meiji Seika (Tokyo, Japan). STLO and the
radioimmunoassay kit for insulin assays were from Eiken Chemical
(Tokyo, Japan). Bovine serum albumin (BSA), phenylmethylsulfonyl fluoride (PMSF), okadaic acid, trichloroacetic acid (TCA),
dithiothreitol (DTT), and
p-nitrophenyl phosphate were from Wako
Chemical (Tokyo, Japan). Leupeptin and the MLC peptide were from the
Peptide Institute (Osaka, Japan). Guanosine
5'-O-(3-thiophosphate) (GTP
S)
was from Boehringer (Mannheim, Germany). All other chemicals used
here were the purest grade available. CaM was purified from rat brain as described in Ref. 7. MLCK and MLC were purified from chicken gizzard
(1, 27) and CaM kinase II from rat brain (33). KN-62
{1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine} and ML-9
[1-(5-chloronaphthalenesulfonyl)-1-H-hexahydro-1,4-diazepine] were synthesized as described in Refs. 33 and 27, respectively.
TC3 and MIN6 cells were cultured in Dulbecco's modified Eagle's
medium supplemented with 66 mg/l kanamycin sulfate and 10% fetal calf
serum (6, 16) and were used for the experiments when they reached
confluence in 10-cm dishes.
-cell lines (MIN6 and
TC3) as well as other tissues
in 10 mM MES (pH 6.9) containing 0.27 M sucrose, 1 mM ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM PMSF, 1 µg/ml leupeptin, and 1 mM benzamidine and
then centrifuged (10,000 g, 10 min).
The samples were then dissolved in sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer [50
mM tris(hydroxymethyl)aminomethane (Tris) · HCl, 10%
glycerol, 2% SDS, 2% 2-mercaptoethanol, and 0.1% bromophenol blue]. Denatured proteins (50 µg protein each for Fig.
1, A and D, and 20 µg for Fig.
1C, except where otherwise mentioned)
were loaded onto SDS-PAGE, transferred to nitrocellulose membranes, blocked with 1% BSA, and treated with anti-MLCK monoclonal antibody (Fig. 1A), anti-MLC monoclonal
antibody (Fig. 1C), or anti-MHC antibody (Fig. 1D). Chicken gizzard
MLCK (10 ng protein for Fig. 1A,
lane 2) and extracts from rat
vessels (Fig. 1C,
lane 3) and human platelets (3 µg
protein for Fig. 1D,
lane 4), as well as from rat cardiac
myocytes (for Fig. 1D,
lane 5), were applied as controls.
Immunopositive bands were visualized with the ECL kit for Fig. 1,
A and
C, and with the alkaline phosphatase
reaction for Fig. 1D.
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Fig. 1.
Presence of myosin light-chain kinase (MLCK), myosin light chain (MLC),
and myosin heavy chain (MHC) in pancreatic -cell.
A: Western blot analysis of MLCK.
Aliquots (50 µg) of proteins extracted from isolated islets, vessels,
and stomach (lanes 1, 3, and
4, respectively) of rats were
separated by SDS-polyacrylamide gel electrophoresis (PAGE) and blotted
onto nitrocellulose membranes, followed by treatment with monoclonal
anti-MLCK antibody (MM17). Purified chicken gizzard MLCK (10 ng) was
also applied in parallel (lane 2).
In lanes 5 and
6, extracts from islets (50 µg
protein) and purified MLCK (10 ng), respectively, were reacted with
anti-MLCK antibody preabsorbed with an excess amount of purified MLCK.
Visualization was carried out with a chemiluminescence kit.
B: immunohistochemical staining of a
rat pancreas using anti-MLCK monoclonal antibody. Frozen sections of
rat pancreas were incubated with anti-MLCK antibody (MM17) diluted
1:100. After being washed, sections were further incubated with
fluorescein isothiocyanate-conjugated goat antibody against mouse
immunoglobulin G and mounted. Observation was with a fluorescence
microscope (bar, 10 µm). C:
immunoblotting of MLC in rat islets or insulin-secreting cell line
(MIN6) and rat vessels. Homogenates containing 20 µg proteins were
separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and
treated with anti-MLC antibody (lane
1: rat islets; lane 2,
MIN6; lane 3, rat vessels). In lane
4, extracts from MIN6 cells (20 µg proteins) were
treated with anti-MLC antibody preabsorbed with purified chicken
gizzard MLC. Immunopositive bands were visualized using a
chemiluminescence kit. D: immunoblot
analysis of MHC in insulin-secreting cell lines, rat islets, human
platelets, and rat cardiac myocytes. Aliquots of proteins (50 µg
each, except human platelets of 3 µg) were separated by SDS-PAGE and
blotted onto a nitrocellurose membrane, followed by treatment with
antibodies raised against nonmuscle MHC (lane
1, MIN6; lane 2,
TC3; lane 3, rat islets;
lane 4, human platelets;
lane 5, rat cardiac myocytes).
Visualization was carried out by alkaline phosphatase.
Separation of phosphorylated MLC in permeabilized MIN6 cells. MIN6 cells (~5 × 106) were incubated for 60 min in glucose-free HEPES-buffered Krebs-Ringer solution supplemented with 5 mg/ml BSA. After two washes, the cells were further incubated in glutamate buffer (pH 7.0) containing 100 mM K-glutamate, 42 mM Na-glutamate, 16 mM HEPES, 5 mg/ml BSA, 1 mM EGTA, 3 mM MgATP, and 0.5 IU/ml STLO. CaCl2 was then added to the glutamate buffer to give an arbitrary concentration of free Ca2+. The free Ca2+ concentration was calculated by EQCAL (Biosoft) according to the stability constants from Owen (23) and Chabarek and Martell (4). The calculated concentrations of free Ca2+ in the assay buffer were verified with fura 2 measurement. Separation of phosphorylated MLC was carried out according to Ref. 25. After addition of 5% TCA and 2 mM DTT (final concentrations), the cells were left for 10 min at room temperature and scraped. The extracts were washed with acetone containing 10 mM DTT five times in a glass tube. The pellet was dissolved in 70 µl urea sample buffer (8.3 M urea, 20 mM Tris-base, 22 mM glycine, 10 mM DTT, and 0.1% bromophenol blue), and the proteins (60 µg) were separated on a polyacrylamide gel (15% polyacrylamide, 0.75% bisacrylamide, 40% glycerol, 20 mM Tris-base, and 23 mM glycine) at 450 V for 3 h. Separated MLC was transferred onto a nitrocellulose membrane and treated with anti-MLC antibody. Immunopositive bands were visualized as previously described with the ECL kit. In this separation, the phosphorylated forms of MLC immigrated faster than the nonphosphorylated form, possibly because of their differences in viscosity and/or sedimentation coefficient, as discussed in a previous paper (25). The density of each band was determined densitometrically, and the extent of MLC phosphorylation was expressed as the percentage of the total (non-, mono-, plus diphosphorylated) MLC in each lane.
Immunohistochemical detection of MLCK in pancreatic
-cells.
Fixation of rat pancreas and preparation of frozen sections were as
described in Ref. 29. Incubation with the monoclonal anti-MLCK antibody
(MM17) diluted at 1:100 in PBS at 4°C overnight was followed, after
washing in PBS, by exposure to a fluorescein isothiocyanate-conjugated
goat anti-mouse IgG antibody diluted at 1:100 in PBS at room
temperature for 1 h. The sections were then mounted in 90% glycerol in
PBS containing 1 mg/ml
p-phenylendiamine as an antifading
agent and were examined under a fluorescence microscope.
Permeabilization of isolated rat pancreatic islets with STLO.
Membrane permeabilization was carried out by treating islets and MIN6
cells with STLO (14, 22). For secretion experiments, groups of five
size-matched islets were preincubated at 37°C for 1 h in the
HEPES-buffered Krebs-Ringer bicarbonate buffer just described
supplemented with 5 mg/ml BSA gassed with 95%
O2-5% CO2. The islets were then washed
twice with 1 ml of the glutamate buffer detailed earlier. The islets
were permeabilized by incubation with 0.125 IU/ml STLO in 0.6 ml of the
same glutamate buffer under various additions. After 45 min of
incubation, the media were collected for measurement of released
insulin by radioimmunoassay with rat insulin as a standard. None of the
compounds or antibodies used here interfered with the assay. In another
set of experiments, islets or MIN6 cells treated with STLO were used
for assessment of phosphorylation of endogenous MLC. The STLO
concentrations selected for the treatment of islets (0.125 IU/ml) and
MIN6 cells (0.5 IU/ml) gave the maximal insulin secretion in response
to a stimulatory concentration of
Ca2+. In the preliminary
experiments, we found that most (~90%) MLCK and MLC did not leak out
of the treated -cells during 45 min of incubation with STLO (data
not shown).
MLC phosphorylation by MLCK and by CaM kinase II.
The MLCK assay was carried out basically according to Kemp et al. (12)
with minor modifications. Activity was determined by incubation in 50 µl (final volume) containing 50 µM MLC peptide substrate
(KKRAARATSNVFA, synthesized on the basis of the phosphorylation sites
of MLC by MLCK), 5.3 nM CaM, 5 mM Mg acetate, 0.5 mM
CaCl2, 0.1% Tween-80, 40 mM HEPES
(pH 7), 10 µM
[-32P]ATP and 13 nM
chicken gizzard MLCK with or without anti-MLCK antibodies (×250).
In another set of experiments, 30 nM CaM and 31 nM CaM kinase II were
added. Reactions were initiated by the addition of
[
-32P]ATP and,
after incubation for 7 min at 30°C, were terminated by spotting of
25-µl aliquots onto Whatman P81 chromatography paper, followed by
washing three times in 0.75% phosphoric acid. Radioactivity on the
filters was determined by Cerenkov counting. In some experiments, the
kinase activity of purified MLCK was measured under parallel
conditions.
MLC phosphorylating activity in the -cell extracts.
MLC phosphorylating activity in
-cell extracts was assayed as
described above with minor modifications. Two thousand isolated pancreatic islets or 107 MIN6
cells were washed twice with PBS and resuspended in
buffer A containing 10 mM MES (pH
6.9), 0.27 M sucrose, 1 mM EGTA, 1 mM PMSF, 1 µg/ml leupeptin, and 1 mM benzamidine. The cells were then washed twice with
buffer A and homogenized on ice in 200 µl buffer A with 1 µM okadaic acid
and 20 mM p-nitrophenylphosphate. The
presence of these phosphatase inhibitors increased the
32P radioactivity incorporated
into the peptide substrate, which implies that the cell extracts
contained MLC phosphatase activity. After centrifugation at 10,000 g for 10 min at 4°C, 10-µl
aliquots of the supernatant (containing 10 µg protein) were incubated
in 50 µl (final volume) at 30°C for 5 min with 20 µM
[
-32P]ATP, 40 mM
HEPES (pH 7.0), 5 mM Mg acetate, 0.1% Tween-80, 30 nM calmodulin, 1.2 mM EGTA, and 50 µM MLC peptide.
CaCl2 was added to the buffer to
give an arbitrary concentration of free
Ca2+, as we have described. Under
this experimental condition, the Michaelis-Menten constant, or
Km, value for CaM
was 0.2 µM. In some experiments, ML-9 or KN-62 was added as
described. Radioactivity without the peptide substrate was subtracted
from the total, and the difference was expressed as the MLC
phosphorylating activity. Radioactivity in the absence of MLC peptide
did not exceed 45% of the total count.
Intracellular granule movement in MIN6 cells.
Intracellular movement of secretory granules in -cells was assessed
using an inverted light microscope (Axiovert 135, Carl Zeiss, Göttingen, Germany) equipped with a ×63
objective lens (Plan-Neofluar, Carl Zeiss) and a ×2.5 insertion
lens. The images were produced with a charge-coupled device camera
(DXC-930, Sony, Tokyo, Japan), displayed on a monitor
screen (PVM-9040, Sony) at a final magnification of ×8.600, and
recorded with a video tape recorder (SVO-260, Sony). Pictures were
reproduced from the video tapes and analyzed on the monitor using an
image analyzer (Argus-20, Hamamatsu Photonics, Hamamatsu, Japan). All
the experiments were carried out at 37°C. The number of the
granules that moved into or out of a square (3.5 × 3.5 µm) was
counted in each square for 30 s before and after the addition of the
substances. Details of the experimental procedures have been recently
published (10).
Statistics. Statistical significance was evaluated by one-way analysis of variance except for a paired t-test for the granule movement experiments (see Fig. 6). Correlation analysis was performed by Pearson's product-moment method (see Fig. 2).
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RESULTS |
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Immunoblot analysis and light-microscopic immunohistochemistry of
pancreatic islets by use of antibodies against MLCK, MLC, and MHC.
The monoclonal anti-MLCK antibody MM17 recognized -cell MLCK with an
apparent molecular mass of 140 kDa (Fig.
1A, lane
1). The 140-kDa band was much less pronounced when
the antibody was preabsorbed with purified chicken gizzard MLCK
(lane 5). The immunopositive band at
140 kDa was not derived from vascular smooth muscle contaminant in the
islet preparation, because MLCK from rat smooth muscle was recognized
at 135 kDa by the same antibody (lanes
3 and 4). The MLCK
concentration in the islet cell was calculated to be 31 nM by use of
purified chicken gizzard MLCK as a standard, with the assumptions that
the antibody reacts with islet and chicken gizzard MLCK to the same
extent and that the volume of an islet is 3 nl (34).
Effects of different types of monoclonal antibodies against MLCK on chicken gizzard MLCK activity. As shown in Fig. 2, different batches (MM7, MM11, MM18, and MM27) of anti-MLCK monoclonal antibodies (final ×250) inhibited chicken gizzard MLCK to a different extent (from 13 to 88%). These antibodies at the same dilution rate also exhibited different potencies in terms of their inhibition of insulin release from the permeabilized islets (Fig. 2), the two activities being in parallel (correlation coefficient r = 0.87, P < 0.05 assessed by Pearson's product-moment method). The MM27 antibody demonstrated the most potent inhibition of both MLCK and Ca2+-induced insulin release and was therefore selected for subsequent secretion experiments.
|
Effects of anti-MLCK monoclonal antibodies on insulin release from
permeabilized pancreatic islets.
Table 1 shows the effects of addition of
CaM, MLCK, and anti-MLCK antibody (MM27) on insulin release from
Ca2+-evoked insulin release from
the STLO-treated islets. Under the basal condition (0.1 µM
Ca2+), insulin release from the
permeabilized pancreatic islets was not affected. However, anti-MLCK
antibody (final ×250) inhibited insulin release induced by 10 µM Ca2+ by 70%. Addition of
exogenous MLCK (20 nM) and/or CaM (2 µM) did not cause
significant changes in the release by 10 µM
Ca2+, and nonimmunized serum did
not exert any effect (data not shown). Inhibition by the anti-MLCK
antibody was also observed when insulin release was stimulated with the
stable GTP analog GTPS, even at a substimulatory concentration of
Ca2+ (0.1 µM). However, the
inhibition disappeared when the
Ca2+ concentration was decreased
to <0.01 µM. In contrast, GDP
S preferentially inhibited
GTP
S-induced release, even with
Ca2+ at <0.01 µM, but not
Ca2+-induced release (Table
2).
|
|
Ca2+-dependent
increase in phosphorylation of MLC peptides.
Figure 3 demonstrates data for
32P incorporation into MLC
peptides in the presence of increasing concentrations of
Ca2+ (1 nM-10 µM). The
threshold for activation of chicken gizzard MLCK was ~0.3 µM, and
the smooth muscle MLCK activity was progressively increased by
Ca2+ at higher concentrations
(Fig. 3B). The peptide was also
phosphorylated by CaM kinase II in a
Ca2+-dependent manner (Fig.
3C), with a threshold between 1 and
3 µM. The -cell extracts (islets and MIN6 cells, Fig.
3A), however, exhibited MLC
phosphorylating activity to some extent (30% of the maximum), even in
the presence of 3-31 nM Ca2+.
The activity under low Ca2+
conditions was nullified by addition of 20 mM EGTA. The calculated concentration of free Ca2+ in this
case was ~0.2 nM because the
Ca2+ concentration in
twice-distilled water was <10 µM. At supraphysiologial Ca2+ concentrations (>10 µM),
no further increase in MLC phosphorylation by the
-cell homogenates
was observed.
|
Inhibition by ML-9 and KN-62 of MLC phosphorylating activity of the MIN6 homonegates. Figure 4 summarizes data for the effects of the MLCK inhibitor, ML-9, and the CaM kinase II inhibitor, KN-62, on the MLC phosphorylating activity of the MIN6 homogenates. ML-9 dose dependently inhibited MLC phosphorylating activity with both stimulatory (1.7 µM) and nonstimulatory (0.07 µM) Ca2+ concentrations. Fifty percent inhibition by ML-9 was observed at 19.5 and 16 µM with high and low Ca2+, respectively. KN-62 less potently inhibited the activity at the high Ca2+ concentration (35.4% inhibition at 30 µM KN-62) and exerted minimal effects with low Ca2+.
|
Phosphorylation of endogenous MLC in permeabilized MIN6 cells. The phosphorylated form of endogenous MLC in permeabilized MIN6 cells is demonstrated in Fig. 5. In lane 1, STLO-treated MIN6 cells were incubated in the presence of nonstimulatory (0.1 µM) Ca2+. When assessed densitometrically, only 5.5% of the total MLC was monophosphorylated (MLC-P) and 1.6% was diphosphorylated (MLC-P2). As shown in lane 2, 15-min incubation of the treated cells with stimulatory (10 µM) Ca2+ increased the amounts of MLC-P and MLC-P2 (20.0 and 8.4%, respectively). Addition of exogenous CaM (2 µM) and MLCK (20 nM) with 10 µM Ca2+ further increased both phosphorylated forms (28.6% for MLC-P and 13.6% for MLC-P2, lane 3).
|
Inhibitory effects of ML-9 on insulin granule movement. Figure 6 summarizes the finding of inhibition of intracellular granule movement by the MLCK inhibitor, ML-9. Under this condition, acetylcholine at 100 µM activated the movement of the insulin granules in living MIN6 cells (Fig. 6A, 3.00 ± 0.32 times/30 s for control vs. 4.03 ± 0.41 for acetylcholine, n = 30, P < 0.01). Incubation of MIN6 cells for 10 min with ML-9 at 30 µM significantly decreased the movement (Fig. 6B, 3.19 ± 0.17 for control vs. 2.25 ± 0.16 for ML-9, n = 32, P < 0.01 by paired t-test). Acetylcholine failed to increase the motile event after the 10-min treatment with 30 µM ML-9 (Fig. 6B, 2.32 ± 0.19 for 5 min after the acetylcholine challenge, n = 29, not significant).
|
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DISCUSSION |
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Regulatory secretion of hormones occurs in a manner dependent on
intracellular Ca2+, in cooperation
with cytosolic proteins. Myosin is one candidate regulator diversely
distributed among various muscle and nonmuscle tissues (5). It exists
in several isoforms characterized by distinct heavy-chain structures.
Immunoblot analysis using anti-nonmuscle-MHC antibody in the present
study suggested that the -cell myosin is of nonmuscle myosin II type
(5). In addition to its well-studied role in muscle contraction, myosin
has been suggested to control cellular motility during cytokinesis,
cell locomotion, and membrane ruffling (31). Association of actomyosin
with chromaffin granule membranes has been shown by in vitro assays
(3), and interaction of actin with secretory granules in the anterior
pituitary gland has been demonstrated by in situ observations (28).
Participation of myosin II in the control of secretory events is
supported by the recent finding that microinjection of anti-myosin II
antibody into presynaptic regions retards synaptic transmission (17). These findings raise the possibility that myosin and its interaction with actin may regulate intracellular transport of the secretory granules in endocrine tissues. Here, we present direct evidence that
intracellular movement of the secretory granules in the
-cell is
dynamically controlled by
Ca2+/CaM-dependent phosphorylation
of MLC.
MLCK has been suggested to be involved in
Ca2+-dependent hormone release,
because secretion of various hormones, including insulin (19) and
catecholamines (21), is inhibited by selective inhibitors of MLCK, such
as ML-9 and wortmannin, although the latter more potently inhibits
phosphatidylinositol 3-kinase (8). The present results with
anti-MLCK monoclonal antibodies as well as ML-9 also point to a role of
this kinase in the -cell. Permeabilized cells offer a useful tool
for analysis of the intracellular mechanisms of the secretory events.
Treatment with STLO results in formation of pores in the plasma
membrane through which hydrophilic molecules like
Ca2+ and large proteins like
antibodies gain access to the intracellular space (14), and the
threshold of Ca2+ for insulin
release was between 0.1 and 1 µM under the STLO-treated conditions
(22). We could thus demonstrate good agreement with earlier reports for
MLCK inhibitors. In contrast to the anti-MLCK antibody case, we found
no effects of additional MLCK on
Ca2+-induced insulin secretion,
even in the presence of an excess amount of CaM, although
phosphorylation of endogenous MLC was increased by
Ca2+ and further elevated by
additions of CaM and MLCK. It could be possible that the CaM and MLCK
were relevant to a non-rate-limiting step in the
-cell secretory
cascade.
Examination of the influence of anti-MLCK antibodies on
GTPS-stimulated release, which is due to a
non-Ca2+-dependent mechanism (26),
demonstrated that MLC phosphorylation may control a prerequisite step
for both Ca2+- and GTP
S-induced
release. This has also been suggested for catecholamine release from
the chromaffin cells by work with the peptide MLCK inhibitor (SM-1) and
wortmannin (13).
We recently reported that intracellular movement of -granules is
controlled by protein phosphorylation dependent on
Ca2+/CaM or adenosine
3',5'-cyclic monophosphate, and we found that the movement
under basal conditions could be increased by muscarinic activation with
acetylcholine (10). Therefore, we carried out in vitro phosphorylation
of MLC peptide by the
-cell homogenates and also attempted to
analyze the insulin granule movement in living
-cells. Because CaM
is reported to exist in the pancreatic islets at high levels (30, 34),
the final concentration of CaM in the assay mixture for the MLC
phosphorylation was calculated to be 0.36-0.5 µM. However, we
found that 30 nM CaM was necessary to activate the phosphorylation, and
the activity was dose dependently increased by CaM up to 2 µM
(data not shown). It may result from heterogeneous distribution of CaM
in the
-cells. Otherwise, intracellular concentration of CaM in the
active form could be much lower than the assumed value as reported in
the smooth muscle by use of fluorescent CaM (15).
In contrast to purified MLCK or CaM kinase II, -cell homogenates
were here found to possess MLC phosphorylating activity even at
substimulatory concentrations of
Ca2+. ML-9 inhibited MLC
phosphorylation by the MIN6 homogenates at low µM concentrations with
high or low Ca2+. Because CaM
kinase II is known to phosphorylate MLCK and thereby decrease its
sensitivity to Ca2+ (32), we
examined the effects of KN-62 on phosphorylation of the MLC peptide
substrate. The finding that the CaM kinase II inhibitor decreased MLC
phosphorylation by the
-cell homogenate suggests to us that
phosphorylation of MLC rather than MLCK by CaM kinase II is dominant
under this condition. The inhibition by KN-62 was much weaker than that
by ML-9 if we consider that the inhibitory constant
(Ki) value for
KN-62 to inhibit purified CaM kinase II is 0.9 µM (33)
and that Ki for
ML-9 to inhibit MLCK is 4 µM (27). The fact that inhibition by KN-62
was only observed with high Ca2+,
in contrast to the ML-9 case, is in line with a conclusion that MLC
phosphorylation by MLCK in the
-cell is dominant with substimulatory or stimulatory Ca2+, and the
phosphorylation by CaM kinase II may participate when the intracellular
Ca2+ level is raised. ML-9
decreased intracellular movement of the insulin granules under the
basal condition and, moreover, nullified activation of the movement by
acetylcholine, supporting the proposed role of MLCK. We cannot preclude
the possibility, however, that contaminant kinases in the
-cell
extracts may have phosphorylated MLC at low
Ca2+ or that the copresence of
some other factors may have altered the
Ca2+ requirement of MLCK for its
activation.
In conclusion, we propose that phosphorylation of MLC may be necessary to translocate secretory granules to the vicinity of plasma membranes, where docking and exocytosis of the granules subsequently occur. We also suggest that this mechanism requires lower concentrations of Ca2+ than those necessary for actual release of the secretory granules from the cell.
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ACKNOWLEDGEMENTS |
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We are grateful to Prof. Arun R. Wakade (Wayne State University
School of Medicine) for critical reading of this manuscript and to
Prof. S. Seino (Chiba University) for the generous gift of TC cells.
We also thank Drs. H. Watanabe (Hamamatsu University School of
Medicine), K. Naruse (Nagoya University School of Medicine), and Y. Sasaki (Asahi Chemical Industry) for their help and suggestions.
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FOOTNOTES |
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This work was supported in part by Grants 06507001, 06404019, 07670103, 09273104, and 09670151 from the Ministry of Education, Science, Sports, and Culture, Japan.
Present address of J. Miyazaki: Dept. of Nutrition and Physiological Chemistry, Osaka Univ. School of Medicine, Suita 565, Osaka, Japan.
Address for reprint requests: I. Niki, Dept. of Pharmacology, Nagoya Univ. School of Medicine, 65 Tsuruma-cho, Nagoya 466, Japan.
Received 31 December 1996; accepted in final form 20 June 1997.
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