From the Departments of Pharmacology and
§ Metabolic Medicine, and the ¶ Institute of Molecular
Embryology and Genetics, Kumamoto University School of Medicine,
Kumamoto 860-0811, Japan
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ABSTRACT |
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Synapsin I is a synaptic vesicle-associated
protein involved in neurotransmitter release. The functions of this
protein are apparently regulated by
Ca2+/calmodulin-dependent protein kinase
II (CaM kinase II). We reported evidence for CaM kinase II and a
synapsin I-like protein present in mouse insulinoma MIN6 cells
(Matsumoto, K., Fukunaga, K., Miyazaki, J., Shichiri, M., and Miyamoto,
E. (1995) Endocrinology 136, 3784-3793). Phosphorylation
of the synapsin I-like protein in these cells correlated with the
activation of CaM kinase II and insulin secretion. In the present
study, we screened the MIN6 cDNA library with the full-length
cDNA probe of rat brain synapsin Ia and obtained seven positive
clones; the largest one was then sequenced. The largest open reading
frame deduced from the cDNA sequence of 3695 base pairs encoded a
polypeptide of 670 amino acids, which exhibited significant sequence
similarity to rat synapsin Ib. The cDNA contained the same sequence
as the first exon of the mouse synapsin I gene. These results indicate
that synapsin Ib is present in MIN6 cells. Synapsin I was expressed in
normal rat islets, as determined by reverse transcriptase-polymerase
chain reaction analysis. Immunoblot analysis after subcellular
fractionation of MIN6 cells demonstrated that synapsin Ib and Intracellular Ca2+ has been reported to play a
critical role in insulin secretion. Glucose seems to elevate
intracellular Ca2+ concentrations via influx through
voltage-dependent Ca2+ channels. The
Ca2+ influx primarily occurs as a result of the membrane
depolarization secondary to closure of ATP-sensitive K+
channels (1-3). After elevation of intracellular Ca2+
levels, effector molecules are activated; these include calmodulin (CaM)1 and protein kinase C. CaM is a ubiquitous Ca2+-binding protein and may be
involved in a variety of Ca2+-mediated processes. In fact,
glucose-stimulated insulin secretion is inhibited by CaM antagonists in
rat pancreatic islet cells (4). One of the primary effectors of CaM is
Ca2+/CaM-dependent protein kinase. Myosin light
chain kinase is reported to be involved in insulin secretion (5).
Myosin light chain kinase phosphorylates the myosin light chain, and
activation of the actin-activated Mg2+-ATPase is considered
to be involved in the movement of secretory granules (1). Other
Ca2+/CaM-dependent protein kinases have been
identified in rat pancreatic islets (6, 7), and one of the kinases has
kinetic properties similar to
Ca2+/CaM-dependent protein kinase II (CaM
kinase II) (7). Indeed, the immunoreactivity of CaM kinase II was
detected in rat pancreatic islets when a specific CaM kinase II
antibody was used (8). CaM kinase II inhibitors such as KN-62 (9, 10)
and KN-93 (5) inhibit glucose-induced insulin secretion from insulinoma cells and rat pancreatic islets. Moreover, an inhibitory peptide of
this enzyme attenuated the Ca2+-induced exocytosis in
single beta cells (11). CaM kinase II in isolated islets (12) and mouse
insulinoma cells (13) was activated with glucose. The activation of CaM
kinase II was reported to occur in the initial phase and also in the
second phase of glucose-stimulated insulin secretion (14). These
findings suggest a role for CaM kinase II in glucose-induced insulin secretion.
We reported that inhibition of calcineurin by the specific inhibitor,
cyclosporin A, enhances the glucose- and tolbutamide-stimulated insulin
secretion from MIN6 cells with stimulation of synapsin I-like protein
phosphorylation (15). These findings suggested that insulin secretion
from MIN6 cells is regulated by CaM kinase II and protein phosphatase
with a concomitant phosphorylation of synapsin I-like protein.
To better understand the physiological roles of CaM kinase II in
insulin secretion, investigation should focus on the endogenous substrate(s) for CaM kinase II. One of the most attractive candidates for substrates involved in stimulus-secretion coupling is synapsin I
(16, 17). Synapsin I is a family member of proteins highly specific to
nerve terminals and comprises four homologous proteins, synapsin Ia and
Ib and synapsin IIa and IIb. Synapsin Ia and Ib were discovered as
endogenous substrates for cyclic AMP- and
Ca2+-dependent protein kinases and were
subsequently found to be synaptic vesicle-associated proteins. They
appear to play key roles in structural organization of the presynaptic
compartment and in regulation of neurotransmitter release. The
dephosphorylated form of synapsin I binds to synaptic vesicles with a
high affinity. The phosphorylation of synapsin I by CaM kinase II
results in removal of the inhibition of the interaction between
synaptic vesicles and plasma membranes and thereby induces concomitant changes in Ca2+-dependent neurotransmitter release.
We reported elsewhere the existence of CaM kinase II and its endogenous
substrate, an 84-kDa protein in mouse insulinoma MIN6 cells (13). As
the 84-kDa protein had several biochemical and immunochemical
properties in common with synapsin I, we tentatively termed it a
"synapsin I-like protein." To clarify whether or not the synapsin
I-like protein is a new isoform of synapsin I expressed in endocrine
cells, we isolated and sequenced the cDNA clones that hybridized to
rat brain synapsin I cDNAs from a cDNA library of MIN6 cells.
Sucrose density gradient fractionation of MIN6 cells revealed that the
protein and Materials--
The following chemicals and reagents were
obtained from the indicated sources: Dulbecco's modified Eagle's
medium, Nissui Pharmaceutical Co. (Tokyo, Japan); RPMI 1640 medium,
Life Technologies, Inc.; fetal bovine serum, JRH Biosciences (Lenexa,
KS); [ Cell Culture--
MIN6 cells, Northern Blot Analysis--
Total RNA was prepared from rat
brain, mouse brain (C57BL/6), and MIN6 cells as described (24). Rat
synapsin Ia and Ib cDNAs were kindly provided by Dr. P. Greengard
(Rockefeller University). For RNA transfer blots, 5, 5, and 20 µg of
total RNAs from rat and mouse brains and MIN6 cells, respectively, were
denatured with formaldehyde, electrophoresed on a 1% agarose gel, and
transferred to a nylon membrane. Following overnight hybridization, the
membranes were washed once in each of the following solutions prior to
autoradiography: (a) 2× SSC, 0.1% SDS for 10 min;
(b) 1× SSC, 0.1% SDS for 10 min; and (c) 1×
SSC, 0.1% SDS for 1 h at 65 °C and exposed to x-ray film.
Isolation of cDNA Clones--
About 1.1 × 105 independent DNA Analyses--
The nucleotide sequence was determined using
the DyeDeoxyTM Terminator Cycle Sequencing Kit or PRISM
Sequenase® Terminator Double-Stranded DNA Sequencing Kit
and model 373S DNA sequencer (Applied Biosystems Japan, Chiba, Japan).
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Analysis--
Islets were isolated by collagenase digestion from the
pancreas of Wistar rats, as described (26). Although the preparation of
islets would contain non-beta cells, they would be less than 10-20%
of all the islet cell population, as described (27, 28). Therefore,
more than 80% would be beta cells. Total cellular RNA was extracted
from isolated islets using a TriZOL Total RNA Isolation Kit (Life
Technologies, Inc.), according to the manufacturer's protocol. The
total RNA was reverse-transcribed using an oligo(dT) primer (Promega)
and Moloney murine leukemia virus transcriptase (Life Technologies,
Inc.). Primers used for the cDNA synthesis were designed to span
alternatively spliced fragments thereby allowing size discrimination
between synapsin Ia and Ib. Primers used to amplify synapsin I cDNA
were 5'-AGGCTACCCGTCAGGCATCTATCTC-3' and 5'-TCACCTCATCCTGGCTAAGG-3'
(nucleotides 1759-1783 and 2120-2139, a 380-base pair fragment of
synapsin Ia or nucleotides 1759-1783 and 2082-2101, a 342-base pair
fragment of synapsin Ib) (29). PCR amplification consisted of a 5-min
start at 95 °C, followed by 35 cycles at 95 °C for 45 s,
65 °C for 45 s, 72 °C for 2 min, and a final cycle at
72 °C for 5 min. For sequencing of PCR products, each PCR fragment
was purified by agarose gel electrophoresis and ligated into the
pCRTMII cloning vector following procedures outlined in the
original TA Cloning Kit (Invitrogen, Carlsbad, CA).
Equilibrium Sucrose Density Gradients--
MIN6 cells grown in
ten 100-mm dishes to 90% confluency were harvested by trypsinization,
pelleted by centrifugation, resuspended in 5 volumes of the
homogenizing buffer (0.25 M sucrose, 10 mM EDTA, 10 mM MES (pH 6.5), 1 mM
(p-amidinophenyl)methanesulfonyl fluoride hydrochloride, 0.1 mM leupeptin, 75 µM pepstatin A, and 0.1 mg
of aprotinin/ml), and homogenized in a Teflon glass homogenizer. The
homogenate was centrifuged at 6000 × g at 4 °C for
10 min, and the resulting supernatant was loaded onto the top of a
continuous sucrose density gradient (0.5-2 M sucrose, 10 mM EDTA, and 10 mM MES, pH 6.5) and then
centrifuged at 100,000 × g at 4 °C for 6 h in
a swing rotor (RPS55T-2, Hitachi Koki Co., Katsuta, Ibaraki, Japan)
(30). Fractions (300 µl each) were collected from the top of the
gradient and used for immunoblot analysis and the insulin assay.
Insulin content in each fraction was determined using the insulin assay
kit (Phadeseph Insulin).
Preparation of the Cytosol and Particulate Fractions from Various
Cultured Cells and Freshly Obtained Bovine Adrenal Medulla--
The
cells and tissue of bovine adrenal medulla were homogenized with 5 volumes of 20 mM Tris-HCl (pH 7.5), 4 mM EGTA,
4 mM EDTA, 1 mM dithiothreitol, 0.1 mM leupeptin, 75 µM pepstatin A, and 0.1 mg
of aprotinin/ml. The homogenate was centrifuged at 16,350 × g for 20 min at 4 °C using a desk top centrifuge. The resulting supernatant was used as the crude cytosol fraction. The
pellet was homogenized with 5 volumes of 50 mM HEPES (pH
7.5), 0.1% Triton X-100 (v/v), 4 mM EGTA, 10 mM EDTA, 30 mM sodium pyrophosphate, 200 mM Isolation and Characterization of Synapsin I cDNA from MIN6
Cells--
In foregoing work, we identified the synapsin I-like
protein in MIN6 cells (13). If the protein is an isoform of synapsin I
expressed in insulinoma cells, cDNA probes corresponding to full-length nucleotides of synapsin Ia or Ib could hybridize to the
mRNA of the protein. In fact, we noted the expression of
3.7-kilobase mRNA in MIN6 cells by Northern blot analysis with
either cDNA probe (Fig. 1). No other
mRNA bands were detected, even under conditions of low stringency
(data not shown). Because the synapsin I-like protein is the major
protein that immunoreacted with the anti-synapsin I antibody (13), it
seemed plausible that the synapsin I-like protein originated from the
3.7-kilobase mRNA. To clarify this point, we cloned the cDNA
that hybridized with rat synapsin I cDNA.
Approximately 1.1 × 105 independent
In neuronal cells, the primary transcript of the synapsin I gene was
alternatively spliced, and two isoforms, Ia and Ib, were generated
(29). These two isoforms differed in their carboxyl termini, and this
alternative splicing mechanism was reported to be conserved well among
human, rat, and bovine cells (17). In addition to the synapsin I-like
protein, an autoradiograph of the immunoblot analysis showed another
faint band at position ~88 kDa on SDS-polyacrylamide gel
electrophoresis after long exposure to the x-ray film (data not shown).
Therefore, we investigated the possibility that synapsin Ia might be
present in MIN6 cells. Using primers spanning the splice acceptor site
on the primary RNA transcript, we found that one clone designated
sim2 had 38 nucleotides that were not present in rat brain
synapsin Ib mRNA (Fig. 3B). The 38 nucleotides were the
same as those expressed in rat brain synapsin Ia. sim2 did
not contain the complete open reading frame of synapsin Ia, yet when
the 38 nucleotides were inserted into the sim5 sequence and
transferred to amino acids, the sequence homology between rat brain
synapsin Ia and the putative full-length sim2 transcript was
94.2% (data not shown). These results strongly suggest that the
mRNA of mouse synapsin Ia is also expressed in MIN6 cells.
Expression of Synapsin I mRNA in Rat Isolated Islets--
To
determine whether rat pancreatic islets express synapsin Ia and/or Ib,
we prepared total RNA from rat islets. When the total RNA from MIN6
cells and rat islets was subjected to RT-PCR, two amplified products
were obtained (Fig. 4). When PCR
amplification was performed without reverse transcriptase reaction,
these two bands were not detected. When genomic DNA from rat liver was
used as a template, an approximately 1330-base pair band was amplified. All three products hybridized with the synapsin Ib cDNA probe (data
not shown). These observations mean that the former two products were
not amplified from genomic DNA. When we subcloned the two products for
sequencing, we confirmed that they were the predicted fragments of
synapsin Ia and Ib (data not shown).
Co-localization of Synapsin I, CaM Kinase II Expression of Synapsin I in Endocrine Cells--
We then asked if
synapsin I occurs in other endocrine cells. Fig.
6 shows immunoblot analysis of synapsin I
in various rodent endocrine cell lines, bovine adrenal chromaffin
cells, and adrenal medulla. One immunoreactive protein band migrating
at the same position as rat brain synapsin Ib was observed mainly in
the particulate fractions from When we reported evidence for the existence of synapsin I-like
protein in MIN6 cells, the unsolved question was whether or not the
protein was a new isoform of synapsin I (13). Although synapsin I was
not detected in any significant amount in non-neuronal cells by
immunoblots, immunohistochemical, and Northern blot analyses and was
considered to be neuron-specific (37, 38), the antibody we used
revealed a significant amount of the protein in MIN6 cells. In previous
work, the electrophoretic mobility of the protein by SDS-polyacrylamide
gel electrophoresis differed from either synapsin Ia or Ib purified
from rat brain. These findings raised the possibility that a new
isoform of synapsin I was present in MIN6 cells. In attempts at
clarification we isolated cDNA clones of the protein from a
cDNA library of MIN6 cells. Sequence analyses of the cDNA
clones revealed the expression of synapsin I. Compared with rat brain
synapsin Ib, 3 additional amino acids (Gln421,
Pro422, and Thr601) and substitution of 11 amino acids were observed in synapsin Ib in MIN6 cells. The difference
between these amino acids may be because of species specificity and may
explain why the synapsin Ib in MIN6 cells migrated at a higher position
than did rat synapsin Ib.
Immunoblot analysis of MIN6 cells showed one major band of synapsin I,
which was considered to be synapsin Ib, based on the sequence analysis.
Another faint band was detected at a slightly higher position than the
major band when the autoradiograph was subjected to long exposure to
x-ray film. This indicated that synapsin Ia may be expressed in MIN6
cells. We carried out partial sequencing of other clones around the
possible splicing site and found that one clone designated as
sim2 had the sequence of the synapsin Ia cDNA. These
findings suggested that synapsin Ia is a minor isoform in MIN6 cells.
This preferential expression of two isoforms was in accord with
findings that they are differentially expressed among synapses (29,
39). Functional differences in the synapsin isoforms remain to be elucidated.
Synapsin I has been considered a neuron-specific phosphoprotein,
although trace positive immunoreactivity of synapsin I was noted in
some endocrine cells by immunoblot, immunohistochemical, and Northern
blot analyses (29, 33). The synapsin I gene promoter contained a
sequence motif similar to the neuron restrictive silencer element/repressor element-1 (40, 41), and the neuron-specific expression of synapsin I was reported to be under the control of a
neuron-specific silencer element and trans-activating factor (42). A zinc finger protein termed neuron restrictive silencer factor
(NRSF)/RE-1-silencing transcription factor (REST) was expressed only in
non-neuronal cells that bind to this motif and function as a
transcriptional repressor (43, 44). It was demonstrated that mRNA
coding for NRSF/REST is absent in the insulinoma cell line INS-1 and in
three other insulin- and glucagon-producing cell lines (45). In these
cells, NRSF/REST activity was absent, and transient expression of
NRSF/REST was sufficient to silence a reporter gene containing a
NRSF/REST binding site. Our data demonstrated the expression of
synapsin I in normal rat islets, endocrine cell lines, and bovine
adrenal chromaffin cells and thereby provided further support for the
NRSF/REST hypothesis. It would be important to show that REST is absent
in MIN6 cells.
We demonstrated the expression of synapsin I in isolated islets from
rat pancreas by RT-PCR with RNA from islets (Fig. 4). Because
sympathetic and parasympathetic neurons innervate pancreatic islets,
there may be contamination of nerve endings in the fraction of isolated
islets. However, this is not likely because synapsin I is translated
from its mRNA in the cell body and is transported to nerve endings.
In neuronal cells, two types of vesicles have been described; one is
synaptic vesicles that contain non-peptide neurotransmitters, and the
other is large density core vesicles that contain peptide neurotransmitters. In neuroendocrine and endocrine cells, secretory granules and small synaptic-like vesicles are thought to be
counterparts of large dense core vesicles and synaptic vesicles,
respectively. In addition to secretory granules, pancreatic beta cells
have small synaptic-like vesicles that contain One of the functional roles of synapsin I in neuronal cells was
reported to be the regulation of neurotransmitter release (17).
Greengard et al. (16) hypothesized that synapsin I forms the
link between the vesicle membrane and a primarily actin-based cytoskeleton, thus preventing the vesicles from moving to the presynaptic plasma membrane. The increase in cytosolic Ca2+
levels causes phosphorylation of synapsin I by CaM kinase II, which
results in the release of vesicles from the cytoskeletal network and
transfer of the vesicles from a resting to an active pool. In
pancreatic beta cells, the actin network beneath the plasma membrane
may prevent the interaction between secretory granules and plasma
membranes (49). By analogy with neuronal cells, synapsin I in beta
cells may possibly function as a linker of insulin granules and the
cytoskeletal network.
CaM kinases were reported to be present in pancreatic beta cells,
including CaM kinase II (8), CaM kinase III (7), and myosin light chain
kinase (5). Among these, only CaM kinase II was reported to be present
in particulate fractions, which contained insulin secretory granules
(7). CaM kinase activity and endogenous substrates have been shown to
be present in insulin secretory granules from toadfish (50).
Furthermore, in other insulinoma INS-1 cells, the CaM kinase II subunit of CaM kinase II co-localized with insulin secretory granules.
By analogy concerning regulation of neurotransmitter release, our
results suggest that phosphorylation of synapsin I by CaM kinase II may
induce the release of insulin from islet cells.
INTRODUCTION
Top
Abstract
Introduction
References
subunit of CaM kinase II (CaM kinase II
)
co-localizes with insulin secretory granules. We also found that the
protein is present in other endocrine cells.
EXPERIMENTAL PROCEDURES
-32P]dCTP, Amersham Pharmacia Biotech (Tokyo,
Japan); [
-32P]ATP and 125I-protein A, NEN
Life Science Products; bovine serum albumin, the antibody against
synaptophysin (monoclonal), and mouse IgG (polyclonal), Sigma;
(p-amidinophenyl)methanesulfonyl fluoride hydrochloride,
Wako Pure Chemical Industries (Osaka, Japan); MES, Dojindo Laboratories
(Kumamoto, Japan); Phadeseph Insulin, Kabi Pharmacia Diagnostics
(Uppsala, Sweden). Polyclonal antibody (IgG fractions) against synapsin
I was prepared as described (18). The polyclonal antibody against
carboxyl-terminal peptide derived from the
1 to
4 isoforms of CaM
kinase II
was prepared. The preparation and characterization of the
antibody will be described elsewhere.2 The polyclonal
antibody against chromogranin A was a generous gift of Dr. N. Yanagihara (University of Occupational and Environmental Health) (19).
All other chemicals and reagents were of analytical grade.
TC-6 cells, and AtT-20 cells
were cultured in Dulbecco's modified Eagle's medium supplemented with
25 mM glucose, 50 IU of penicillin/ml, 50 µg of
streptomycin/ml, 5 µM 2-mercaptoethanol, and 15%
heat-inactivated fetal bovine serum, as described (20-22). Hamster
insulinoma In-R1-G9 cells and HIT-T15 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU of penicillin/ml, and 100 mg of streptomycin/ml (23). Bovine
adrenal chromaffin cells were kindly provided by Dr. N. Yanagihara
(19).
Zap phage clones of the mouse insulinoma
MIN6 cDNA library, kindly provided by Dr. H. Ishihara (University
of Tokyo) (25), were screened with the full-length fragment of the
cDNA-encoding rat brain synapsin Ia as a probe. The probe was
labeled with [
-32P]dCTP to a specific activity of
2.9 × 109 cpm/µg with the Random Primer Kit (Takara
Biomedicals), as described by the manufacturer. The plaque
hybridization assay was performed in 0.9 M NaCl, 50 mM NaH2PO4, 5 mM EDTA
(pH 7.7), 0.1% bovine serum albumin, 0.1% Ficoll 400, 0.1%
polyvinylpyrrolidone, and 0.1% SDS in the presence of 100 µg/ml
denatured herring DNA at 65 °C. The final wash of membranes was
carried out in 0.15 M NaCl, 15 mM sodium
citrate, and 0.1% SDS at 60 °C for 60 min. The pBluescript SK(
)
phagemids containing cDNA inserts were recovered from purified positive phage clones following the procedures outlined in the Rapid
Excision Kit (Stratagene, La Jolla, CA).
-glycerophosphate, 50 mM sodium fluoride,
and protease inhibitors described above and centrifuged at 16,350 × g for 20 min. The resulting supernatant served as the
crude particulate fraction.
RESULTS
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Fig. 1.
Northern blot analysis of mRNA of
synapsin I in MIN6 cells. Total RNA from MIN6 cells (20 µg) and
mouse and rat brains (5 µg each) was separated on a 1% agarose gel,
transferred to a nylon membrane, and hybridized with a
32P-labeled cDNA probe derived from rat brain synapsin
Ia or Ib. Size markers used were rat ribosomal RNAs of 18 and 28 S.
Zap phage
clones from a cDNA library of MIN6 cells were screened with the
32P-labeled synapsin Ia cDNA probe, and seven positive
clones were obtained. The cDNA inserts from these clones were
further characterized by restriction mapping analysis and partial
sequence determination of their 5'-ends. One of these clones,
designated sim5, contained a full-length open reading frame
and was further analyzed (Figs. 2 and
3). The largest open reading frame
deduced from the 3695-base pair sequence of the sim5
cDNA encoded a polypeptide of 670 amino acids, which exhibited a
significant sequence similarity to rat synapsin Ib (97.8% identity).
All seven serine residues phosphorylated in bovine (31, 32), rat, and
human synapsin I are present in the mouse sequence (Fig. 2).
Furthermore, sim5 contained a nucleotide sequence identical
to the first exon of mouse brain synapsin I gene (33) (Fig.
3A). Thus, we concluded that sim5 originated from
the gene that encodes mouse brain synapsin I. Eleven amino acids were
different from rat brain synapsin Ib, and three additional amino acids
(Gln421, Pro422, and Thr601) were
observed in the synapsin Ib of MIN6 cells.
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Fig. 2.
Comparison of the deduced amino acid sequence
of sim5 with that of rat synapsin Ib. Sequences are
given in single letter code. Residues that are identical in the rat
synapsin Ib (RatIb) (29) to those of sim5 are
indicated by asterisks. Dashes denote gaps.
Seven vertical arrows above serine residues mark
the sequenced phosphorylation sites in rat synapsin I.
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Fig. 3.
Analyses of clones of synapsin I obtained
from MIN6 cDNA library. A, comparison of the
nucleotide sequences of sim5 with those of the first exon
from mouse synapsin I (m.synI) (31). B,
comparison of the nucleotide sequence of sim2 with those of
sim5, rat synapsin Ia (Syn Ia), and rat synapsin
Ib (Syn Ib) (29). The sim2 clone contains 38-base
nucleotides deleted by alternative splicing in mRNA of rat synapsin
Ib.
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Fig. 4.
Expression of synapsin I mRNA in normal
rat islets. Total RNA extracted from MIN6 cells or isolated rat
islets was subjected to RT-PCR as described under "Experimental
Procedures." The genomic DNA from rat liver was subjected to PCR. The
PCR products were separated on a 0.8% agarose gel and were visualized
by the use of ethidium bromide. SynIs, synapsins I.
, and Secretory
Granules in MIN6 Cells--
In neuronal cells, peptide and nonpeptide
neurotransmitters are packed in large density core vesicles and
synaptic vesicles, respectively, and synapsin I was reported to be
associated with synaptic vesicles rather than large density core
vesicles (34). Large density core vesicles were considered to be
counterparts of the secretory granules in endocrine cells. In addition
to insulin secretory granules, islet beta cells were seen to have
synaptic vesicle-like microvesicles, which contain
-aminobutyric
acid and glutamic acid decarboxylase (35). From these data, we
considered the possibility that synapsin I in MIN6 cells is associated
with microvesicles but not with insulin secretory granules. We then carried out equilibrium sedimentation through a linear 0.5-2.0 M sucrose gradient using the MIN6 cell extracts (Fig.
5). Individual fractions were collected,
and immunoblot analysis was done with antibodies against synapsin I,
synaptophysin, chromogranin A, and CaM kinase II
. The insulin
secretory granule fraction was identified based on measurements of
immunoreactive insulin. Fig. 5 summarizes the results of one of four
independent experiments. Synapsin I was present in relatively dense
fractions, and synaptophysin, a marker for endosomes and small density
vesicles, was mainly observed in distinct membrane fractions from
synapsin I. A minor peak of synaptophysin was also observed in the
insulin secretory granule fraction. Moreover, most of the
immunoreactive insulin (Fig. 5B) and the immunoreactivity of
chromogranin A, a marker for large secretory granules (36) (data not
shown) and CaM kinase II
, were detected in the same fractions as
synapsin I. Thus, synapsin I was co-fractionated with insulin secretory
granules in MIN6 cells. Co-localization of synapsin I with insulin
secretory granules was observed in an independent beta cell line,
HIT-T15 cells (data not shown).
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Fig. 5.
Co-fractionation of synapsin I and CaM kinase
II with secretory granules. A, equal volumes of
fraction of MIN6 cells were blotted with polyclonal anti-synapsin I
(1:200), monoclonal anti-synaptophysin (1:200), and polyclonal anti-CaM
kinase II
(1:200) antibodies. The positions of synapsin I
(Syn I), synaptophysin (Synapt), and CaM kinase
II
(KII
) are indicated. B, top
panel, plots of synapsin I (
), synaptophysin
(
), and CaM kinase II
(
)
immunoreactivity (A) for each fraction. Bottom
panel, plots of immunoreactive insulin (IRI)
(
) determined by radioimmunoassay and protein
concentrations (
) for each fraction. Immunoreactivity was
determined using a Bio-image analyzer (BAS 1000, Fuji Film Co.) and
normalized to total immunoreactivity summed over all fractions.
Fractions were collected from the top of the tube with the first
fraction representing the lightest sucrose fraction.
TC cells and AtT-20 cells. More than
four proteins were detected in both cytosol and particulate fractions
from In-R1-G9 cells; one migrated at the same position as synapsin Ib,
and properties of other immunoreactive proteins were not clear. Protein
bands of lower molecular masses were considered to be degraded
products. In bovine adrenal chromaffin cells, the two immunoreactive
bands observed around the position of synapsin Ib were exclusively
present in the particulate fraction. Two immunoreactive bands were also observed in the particulate fraction when freshly isolated bovine adrenal medulla was examined by immunoblot analysis (Fig.
6B). Synapsin I was observed in the cytosol fractions of
TC cells, In-R1-G9 cells, AtT-20 cells, and bovine adrenal medulla.
Although small particles are not included in the crude cytosol fraction by the preparation method, the hypotonic conditions would result in the
inclusion of the loosely bound proteins of the particulate fraction in
the cytosol fraction.
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Fig. 6.
Synapsin I in various endocrine cells.
Crude cytosol (C) and particulate (P) fractions
(12 µg of protein each) from mouse glucagonoma TC-6 cells, hamster
insulinoma In-R1-G9 cells, mouse anterior pituitary AtT-20 cells,
cultured bovine adrenal chromaffin cells (BACC)
(A), and freshly obtained bovine adrenal medulla
(BAM) (B) were analyzed by Western blot using the
polyclonal anti-synapsin I antibody. The entire homogenates of rat
brain (2 µg) (A) and MIN6 cells (20 µg) (B)
were used as the control. SynI, synapsin I.
DISCUSSION
-aminobutyric acid (35). Although synapsin I has been reported to be associated only with
synaptic vesicles or synaptic-like microvesicles in rat and bovine
neurohypophysis (46), we found that synapsin I was not present in
fractions that contained synaptic-like microvesicles, as determined by
immunoblot analysis using the anti-synaptophysin antibody but was
associated with secretory granules. In agreement with our observation,
synaptic-like microvesicles in pinealocytes were not found to interact
with synapsin I, as demonstrated immunohistochemically (47). In
endocrine and neuroendocrine cells, synapsin I can be absent on
synaptic-like microvesicles. In this context, it is important that
ribbon synapses in cone, rod, and bipolar cells of the retina with
synaptic-like microvesicles do not contain synapsin I (48).
was
seen to be associated with insulin secretory granules (51). We also
confirmed the association of CaM kinase II
with insulin secretory
granules in MIN6 cells by sucrose gradient subcellular fractionation
and immunoblot analysis with an antibody specific to CaM kinase II
(Fig. 5). Taken together, it is plausible that CaM kinase II is present
on the surface of insulin secretory granules. In the present work, we
observed that synapsin I co-localizes with secretory granules. In the
brain, synapsin I has been reported to be complexed with CaM kinase II
on the synaptic vesicle surface (52). We suggest that synapsin I
may be associated with CaM kinase II
on insulin secretory granules.
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ACKNOWLEDGEMENTS |
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We thank Dr. H. Ishihara (Third Department of
Internal Medicine, University of Tokyo) for providing the MIN6
cDNAs library, Dr. P. Greengard (Rockefeller University) for
providing synapsin Ia and Ib cDNA, Dr. J. Miyazaki (Department of
Nutrition and Physiological Chemistry, Osaka University School of
Medicine) for providing MIN6 cells, Dr. N. Yanagihara (Department of
Pharmacology, University of Occupational and Environmental Health,
School of Medicine) for providing polyclonal antibody against rat
chromogranin A and for preparation of bovine adrenal chromaffin cells
and adrenal medulla, Dr. R. Takaki and Dr. K. Hamaguchi (First
Department of Medicine, Oita Medical University) for the In-R1-G9 cells
and TC clone 6 cells, Dr. H. Higashida (Department of Biophysics Neuroinformation Research Institute, Kanazawa University School of
Medicine) for valuable discussion, and M. Ohara for critical comments
on the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and by a research grant from the Human Frontier Science Program (to H. Y., K. F., and E. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF085809.
To whom correspondence should be addressed: Dept. of
Pharmacology, Kumamoto University School of Medicine, 2-2-1 Honjo,
Kumamoto 860-0811, Japan. Tel.: 81-96-373-5076; Fax: 81-96-373-5078;
E-mail: emiyamot{at}gpo.kumamoto-u.ac.jp.
The abbreviations used are: CaM, calmodulin; CaM kinase, Ca2+/calmodulin-dependent protein kinase; RT, reverse transcriptase; PCR, polymerase chain reaction; NRSF, neuron restrictive silencer factor; REST, RE-1-silencing transcription factor; MES, 2-(N-morpholino) ethanesulfonic acid.
2 Y. Takeuchi, H. Yamamoto, K. Matsumoto, T. Kimura, S. Katsuragi, T. Miyakawa, and E. Miyamoto, J. Neurochem., in press.
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