(Received for publication, March 31, 1995; and in revised form, May 26, 1995)
From the
Western blotting of the insulin-secreting
The clostridial neurotoxins, botulinum and tetanus, inhibit
neurotransmitter release from presynaptic nerve endings (1) by
Zn The specificity of clostridial neurotoxins
for nerve terminals, particularly the cholinergic terminals of the
neuromuscular junction, derives from a selective uptake
mechanism(12, 13) . When the uptake barrier is removed
clostridial neurotoxins inhibit secretion from not only neuronal cells
but also from PC12 (14) and chromaffin
cells(15, 16) . Synaptobrevin, cellubrevin, SNAP-25,
and syntaxin are found in PC12 and chromaffin
cells(16, 17, 18) , and inhibition of
noradrenaline release from chromaffin cells has been associated with
cleavage of SNAP-25 by BoNT/A (17) and cleavage of
synaptobrevin and cellubrevin by BoNT/B(16) . Pancreatic
endocrine cells also contain SNAP-25, synaptobrevin, cellubrevin, and
syntaxin 1A, 4, and 5(19) . If these proteins are involved in
Ca We have confirmed the presence of SNAP-25,
syntaxin, and synaptobrevin immunoreactivities in the
The proteins were resolved
on a 4-20% Tris/glycine polyacrylamide gel and then transferred
to nitrocellulose membranes. The blots were probed with antibodies and
the immunoreactivities visualized using chemiluminescent detection
essentially as described previously(26) . Data was
quantified from Hyperfilm ECL (Amersham International plc) using a
Molecular Dynamics personal densitometer, and the effect of any
treatment was expressed as a percentage of the control value for each
experiment. The results were within the linear range of the technique. The efficiency of electroporation was determined by the use
of a fluorescein-dextran molecule of comparable molecular weight to the
toxin molecule. Electroporation was found to permeabilize >95% of
both the HIT-15 and RINm5F cells with only limited effects on cell
viability and no significant difference between the basal levels of
insulin release and protein in control and electroporated cells. Both HIT-15 and RINm5F cells were found to contain a
SNAP-25-immunoreactive protein of M
Figure 1:
Western blot analysis of cells
electroporated in the presence of botulinum neurotoxins. Duplicate
Triton X-114 extracts of HIT-15 (HIT) and RINm5F (RIN) cells that had
been electroporated in the absence of neurotoxin (C) or in the
presence of BoNT/A (A) or BoNT/B (B) were subjected
to Western blot analysis as detailed under ``Experimental
Procedures.'' The same blots were consecutively probed with
anti-synaptobrevin (SYB), anti-SNAP-25 (SNAP), and
10H5 (SYNT). The relative electrophoretic mobilities
(determined by comparison to prestained protein standards) were
16,000-17,000, 30,000, and 36,000,
respectively.
Electroporation of the HIT-15 and RINm5F cells with
BoNT/A (500 nM) resulted in a reduction of SNAP-25
immunoreactivity after 3 days of culture as assessed using the
anti-peptide antibody (Fig. 1). In HIT-15 cells 6.4 ±
5.3% of the protein remained compared with control (n =
3), and in RINm5F cells 14.5 ± 13.5% of the protein remained
compared with control (n = 3). Use of the monoclonal
antibody, SMI 81, revealed a small shift in electrophoretic mobility to M In
Ca
Figure 2:
The effect of botulinum toxins A and B on
calcium-dependent insulin release from HIT-15 cells. HIT-15 cells were
untreated (Con), electroporated (EP-Con),
electroporated in the presence of 500 nM BoNT/A (EP-A), or electroporated in the presence of 500 nM BoNT/B (EP-B). The cells were subsequently cultured for 3
days. The release of insulin from the cells during a 30-min incubation
period with 4.8 mM KCl-KRB (LK-KRB) or 30 mM KCl-KRB (HK-KRB) was determined by specific
radioimmunoassay. The secretion data presented are the means ±
S.E. of three experiments, each performed in duplicate; one star indicates p < 0.05 and two stars indicate p < 0.02 for differences from control, determined by a
two-tailed, unpaired t test.
Figure 3:
The effect of botulinum toxins A and B on
calcium-dependent release of insulin from RINm5F cells. RINm5F cells
were untreated (Con), electroporated (Con-EP),
electroporated in the presence of 500 nM BoNT/A (EP-A), electroporated in the presence of 500 nM BoNT/B (EP-B), or electroporated in the presence of 250
nM of BoNT/F (EP-F). The cells were subsequently
cultured for 3 days. The release of insulin from the cells during a
30-min incubation period with 4.8 mM KCl-KRB (LK-KRB) or 30
mM KCl-KRB (HK-KRB) was then determined by specific
radioimmunoassay. The secretion data presented are the means ±
S.E. of n separate experiments, each performed in duplicate; three stars indicate p < 0.001 for differences
from control, determined by a two-tailed, unpaired t test.
After 3 days in culture
following electroporation in the presence of BoNT/A (500 nM),
high K As with the
HIT-15 cells, electroporation of RINm5F cells did not significantly
affect subsequent basal or high K Electroporation of RINm5F cells with BoNT/F (250
nM) caused a reduction in the synaptobrevin immunoreactivities
equivalent to that caused by BoNT/B; 6.2 ± 4% of the protein
remained compared with control (n = 2, mean ±
range). As observed for BoNT/B, insulin secretion was unaffected by the
BoNT/F treatment (Fig. 3). A docking/fusion complex composed of SNAP-25, syntaxin, and
synaptobrevin has been proposed as an essential step in
exocytosis(3) . In this study we show that all these proteins
are present in the pancreatic cell lines studied. The detection of a
syntaxin-like immunoreactive band is consistent with the reported
expression of syntaxin 1A in cells of this type (19) and the
known specificity of this monoclonal antibody(30) . BoNT/A,
which cleaves SNAP-25 in both neurons and chromaffin cells, also
cleaves the SNAP-25 immunoreactive protein in the HIT-15 and RINm5F
insulinoma cells. None of the other proteins studied was found to be
cleaved by BoNT/A. In addition to cleavage of SNAP-25, electroporation
with BoNT/A was found to inhibit high K Two synaptobrevin-like
immunoreactivities were observed in this study. Both were equally
sensitive to proteolysis by BoNT/B or BoNT/F. Given that one of the
cell lines, RINm5F, is of rat origin, the similar extent of cleavage of
both immunoreactivities by BoNT/B or BoNT/F is consistent with this
cell line containing synaptobrevin 2 and cellubrevin but not
synaptobrevin 1, a conclusion in agreement with a recent
report(19) . The identification of the two synaptobrevin
immunoreactivities as synaptobrevin 2 and cellubrevin is consistent
with the electrophoretic mobilities observed. The degree of cleavage
of synaptobrevin in bovine chromaffin cells has been found to be
closely correlated with the degree of inhibition of calcium-stimulated
secretion(16) . In this study, however, despite the almost
total cleavage of synaptobrevin and cellubrevin seen, only in HIT-15
cells was a blockade of insulin release detected, and this was only
partial. As discussed above, insensitivity of
Ca Thus data for
SNAP-25 supports the involvement of a SNARE complex in insulin
secretion; however, for synaptobrevin or cellubrevin to be the v-SNARE
in RINm5F cells would require them to be present in at least a 10-fold
excess. Alternatively, there could be an unidentified v-SNARE in these
cells, which is not a substrate for either BoNT/B or BoNT/F and which
is not recognized by the HV62 antiserum. Whichever explanation proves
to be the case, the results presented show that there is no simple
relationship between the presence of synaptobrevin 2 and SNAP-25 in
cells and the mechanism of Ca
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-cell lines
HIT-15 and RINm5F with anti-SNAP-25 (synaptosomal associated protein of
25 kDa), anti-synaptobrevin, and anti-syntaxin 1 antibodies revealed
the presence of proteins with the same electrophoretic mobility as
found in neural tissue. Permeabilization of both of these insulinoma
cell lines to botulinum neurotoxin A by electroporation resulted, after
3 days of culture, in the loss of
90% of SNAP-25 immunoreactivity.
A similar permeabilization of these cells with botulinum neurotoxin B
resulted in the cleavage of
90% of the synaptobrevin-like
immunoreactivities. Botulinum neurotoxin F also cleaved
90% of the
synaptobrevin-like immunoreactivity in RINm5F cells. The
permeabilization of both insulinoma cells to neurotoxin A resulted in a
>90% inhibition of potassium-stimulated, calcium-dependent insulin
release. By contrast, permeabilization of the insulinoma cell lines to
neurotoxin B resulted in only a
60% inhibition of
potassium-stimulated insulin release in HIT-15 cells, and neither
neurotoxin B nor F caused inhibition in RINm5F cells. Thus HIT-15 and
RINm5F cells contain the components of the putative exocytotic docking
complex described in cells derived from the neural crest. In HIT-15
cells both SNAP-25 and synaptobrevin appear to be involved in
calcium-dependent insulin secretion, whereas in RINm5F cells SNAP-25
but not synaptobrevin is involved.
-dependent proteolysis of specific synaptic
proteins(2) . The different botulinum neurotoxin serotypes
specifically cleave one of three proteins, synaptobrevin, syntaxin, or
SNAP-25. (
)A recent model of synaptic vesicle fusion
proposes that the vesicle protein synaptobrevin, termed a v-SNARE, and
the plasma membrane-associated proteins syntaxin and SNAP-25, t-SNARES,
interact to form a receptor for the assembly of a fusion complex
involving the N-ethylmaleimide-sensitive fusion
protein(3, 4, 5) . Botulinum neurotoxin type
A (BoNT/A) selectively cleaves SNAP-25(6) , whereas botulinum
neurotoxin type B (BoNT/B), in common with tetanus neurotoxin (7) and botulinum neurotoxin F (BoNT/F), cleave
synaptobrevin(8, 9) . Two forms of synaptobrevin,
synaptobrevin 1 and 2, have been described in mammals. In most species
both forms are substrates for BoNT/B; however, in the rat synaptobrevin
1 is not a substrate(10) . Synaptobrevin 1 and 2 in the rat are
substrates for BoNT/F(9) . A homologue of synaptobrevin,
cellubrevin, has been identified in non-neuronal cells and is a toxin
substrate(11) .
-dependent secretion from these cells, then their
cleavage by botulinum toxins should inhibit
Ca
-dependent release of hormones or enzymes from
pancreatic cells. Indeed cleavage of synaptobrevin by tetanus toxin
inhibits enzyme secretion from rat pancreatic acinar
cells(20) .
-pancreatic
cell lines, HIT-15 and RINm5F, and determined the effect of BoNT/A, -B,
and -F both on these proteins and on the release of insulin. To study
the effects of the toxins in these cells it was necessary to introduce
them into the cell cytoplasm, and electroporation was used for this
purpose(21) .
Materials
BoNT/A, BoNT/B, and BoNT/F were
purified as described
previously(22, 23, 24) . The rat insulin
radioimmunoassay kit and ECL reagents were obtained from Amersham
International plc. The rabbit polyclonal antiserum used to detect
SNAP-25 immunoreactivity was raised using a synthetic peptide of
sequence CANGRATKMLGSG following published
methods(25, 26) . A mouse monoclonal antibody against
SNAP-25, SMI 81, was obtained from Affiniti Research Products Ltd.
(Nottingham, UK). The polyclonal antibody used to detect synaptobrevin
immunoreactivity was raised in guinea pig against a synthetic peptide
(HV62) comprising amino acids 33-94 from the conserved
cytoplasmic domain of human synaptobrevin 2(24) . This antibody
has been shown to recognize sequences common to both synaptobrevin I
and II. The anti-syntaxin antibody, 10H5, was a gift from Prof.
Takahashi (Mitsubishi Kasei Institute, Tokyo, Japan). HIT-15 and RINm5F
cells were a gift from Dr. Irene Green (Sussex University, Brighton,
UK). Electroporation cuvettes were from Bio-Rad. The SDS-polyacrylamide
gel electrophoresis gels and nitrocellulose were from R& Systems
Europe Ltd. (Abingdon, UK). All other reagents and chemicals used in
this study were obtained from Sigma Chemical Co. Ltd. (Fancy Road,
Poole, UK).Cell Culture
HIT-15 cells and RINm5F cells were
cultured in 80-cm flasks in DMEM containing 2% (v/v) fetal
calf serum (HIT-15 cells) or 10% (v/v) fetal calf serum (RINm5F cells).
Cultures were maintained at 37 °C in a humidified atmosphere of 95%
air and 5% CO
.
Electroporation
Cells (HIT-15 or RINm5F) were
selected when they were subconfluent and harvested using cell
dissociation reagent (Sigma). After washing in phosphate-buffered
saline the cells were resuspended in DMEM and 0.1% (w/v) bovine serum
albumin at a density of 2-5 million cells/ml, and 0.8 ml was
added to each of a number of 0.4-cm electroporation cuvettes. Toxins
were added as required, and the cells were then immediately
electroporated at 960 microfarads and 0.28 kV using a Bio-Rad
electroporator(21) . After electroporation the cells were
washed three times in DMEM and bovine serum albumin by centrifugation
(1000 g for 5 min). The cells were then resuspended in
the appropriate medium containing antibiotics and plated out at a
density of 0.3-0.6 million cells/well in a 24-multiwell plate.
The cells were cultured for 3 days at 37 °C in a humidified
atmosphere of 95% air and 5% CO
.
Cell Secretion Experiments
After 3 days in culture
the cells were washed three times with 1 ml of DMEM. Secretion of
insulin was then assessed by incubation of the cells for 30 min at 37
°C in a humidified atmosphere of 95% air and 5% CO with
0.3 ml of Krebs-Ringer bicarbonate (KRB) buffer containing 129 mM NaCl, 5 mM NaHCO
, 4.8 mM KCl, 1.2
mM KH
PO
, 1.0 mM CaCl
, 1.2 mM MgSO
, 2.8 mM glucose, and 10 mM HEPES, pH 7.4 (27) or in
HK-KRB in which KCl was increased to 30 mM and NaCl was
reduced to 103.8 mM. In some experiments calcium was omitted
from the KRB buffer, and EGTA was added to a final concentration of 0.1
mM, Ca
-free KRB. At the end of the
experimental incubation the supernatants were removed and centrifuged
at 13,000
g for 4 min. The supernatants and cells were
frozen at -80 °C until analysis. Insulin was assayed using
the Amersham International plc rat insulin radioimmunoassay kit
according to the manufacturer's instructions.
Western Blot Analysis
Hydrophobic proteins were
extracted from the cells remaining after the secretion experiment by a
modification of the method of Bordier(28) . The cells in each
well were treated with 0.5 ml of 0.2 M NaOH for 10 min at room
temperature before being neutralized with 0.5 ml of 0.2 M HCl.
The mixture was then cooled to 4 °C, and 100 µl of Triton X-114
(10%, v/v) was added, mixed three times, and left for 1 h at 4 °C.
The resulting solution was then centrifuged at 13,000 g for 10 min at 4 °C, and the supernatant was incubated in a
water bath at 37 °C for 30 min until two distinct phases formed.
After separation by centrifugation the upper phase was removed and the
lower phase containing hydrophobic proteins was retained. The samples
were prepared for SDS-polyacrylamide gel electrophoresis by
precipitation with chloroform/methanol, and the precipitate was
dissolved in 20 µl of sample buffer.
30,000 as
determined by two separate antibodies. Both insulinoma cells contained
two synaptobrevin-immunoreactive proteins of M
16,000-17,000. The lower synaptobrevin immunoreactivity was
less intense in both cell lines, and in the HIT-15 cells it was not
always clearly visible. A protein of M
36,000 with
syntaxin immunoreactivity was also detected in both cell lines (Fig. 1).
29,000; this is consistent with the known site
of proteolysis of SNAP-25 by BoNT/A, which results in the loss of a
9-residue peptide from the C terminus of the protein(6) .
Electroporation of the HIT-15 and RINm5F cells with BoNT/B (500
nM) resulted in a reduction of both synaptobrevin-like
immunoreactivities after 3 days of culture (Fig. 1). In HIT-15
cells 7.8 ± 2.9% of the protein remained compared with control (n = 3), and in RINm5F cells 12.3 ± 4.8% of the
protein remained compared with control (n = 4).
-containing KRB, high K
treatment
significantly stimulated insulin release from both the HIT-15 and
RINm5F cells ( Fig. 2and Fig. 3). High K
did not stimulate insulin release from RINm5F cells in
Ca
-free KRB (94.8 ± 7% of basal release; means
± range of two determinations).
-stimulated insulin release from HIT-15 cells
was reduced by 95.0% (p < 0.02; n = 4; Fig. 2). Basal or high K
-stimulated insulin
release from HIT-15 cells was not significantly affected by
electroporation perse. Electroporation of HIT-15
cells with heat-treated BoNT/A did not significantly affect high
K
-stimulated insulin release (data not shown). After 3
days of culture following electroporation of BoNT/B (500 nM)
into HIT-15 cells, high K
-stimulated insulin release
was reduced by 63% (p < 0.05; n = 3). This
partial inhibition of high K
-stimulated insulin
release was not increased by electroporation of cells with higher
levels of BoNT/B (2.5 µM; data not shown).
-stimulated insulin
release (Fig. 3). Electroporation of RINm5F cells with BoNT/A
(500 nM) resulted, after 3 days in culture, in the inhibition
of insulin release by 91.3% (p < 0.001; n =
5). However, after 3 days in culture following electroporation of the
RINm5F cells with BoNT/B (500 nM), there was no significant
block of high K
-stimulated insulin release nor did
electroporation of the cells in the presence of a much higher
concentration of BoNT/B (2.5 µM) have any effect (data not
shown).
-stimulated
insulin secretion in both cell lines, which is in agreement with the
recent findings in pancreatic cells (29) . The inhibition of
calcium-dependent insulin release was almost complete in both cell
lines, consistent with the observation that the majority of the SNAP-25
immunoreactivity had disappeared from the cells. These results are
consistent with the involvement of a SNARE-based mechanism involving
SNAP-25 in insulin secretion.
-dependent insulin secretion from RINm5F cells to
BoNT/B is not due to the presence of synaptobrevin 1 in these cells nor
would this explain the lack of inhibition by BoNT/F.
-dependent secretion.
We are grateful to Prof. Masami Takahashi for the
anti-syntaxin antibody, 10H5, to Dr. I. Green for providing the
insulinoma cell lines, and to A. Jen for culturing them. We thank Dr.
J. R. North for critically reading the manuscript and Prof. J. O. Dolly
and Prof. B. Gomperts for helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.