Hyposmotic shock stimulates insulin secretion by two distinct
mechanisms. Studies with the
HC9 cell
Susanne G.
Straub,
Samira
Daniel, and
Geoffrey W. G.
Sharp
Department of Molecular Medicine, College of Veterinary
Medicine, Cornell University, Ithaca, New York 14853
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ABSTRACT |
Exposure of
HC9 cells
to a Krebs-Ringer bicarbonate-HEPES buffer (KRBH) made hypotonic by a
reduction of 25 mM NaCl resulted in a prompt stimulation of insulin
release. The stimulation was transient, and release rates returned to
basal levels after 10 min. The response resembles that of the first
phase of glucose-stimulated insulin release. The response did not occur
if the reduction in NaCl was compensated for by the addition of an
equivalent osmolar amount of sorbitol, so the stimulation of release
was due to the osmolarity change and not the reduction in NaCl. The
hyposmotic shock released insulin in KRBH with or without
Ca2+. The L-type Ca2+ channel blocker
nitrendipine inhibited the response in normal KRBH but had no effect in
KRBH without Ca2+ despite the latter response being larger
than in the presence of extracellular Ca2+. Similar data
were obtained with calciseptine, which also blocks L-type channels. The
T-type Ca2+ channel blocker flunarizine was without effect,
as was the chloride channel blocker DIDS. In parallel studies, the
readily releasable pool of insulin-containing granules was monitored.
Immunoprecipitation of the target-SNARE protein syntaxin and
co-immunoprecipitation of the vesicle-SNARE VAMP-2 was used as an
indicator of the readily releasable granule pool. After
hypotonic shock in the presence of extracellular Ca2+, the
amount of VAMP-2 coimmunoprecipitated by antibodies against syntaxin
was much reduced compared with controls. Therefore, under these
conditions, hypotonic shock stimulates exocytosis of the readily
releasable pool of insulin-containing granules. No such reduction was
seen in the absence of extracellular Ca2+. In conclusion,
after reexamination of the effect of hyposmotic shock on insulin
secretion in the presence and absence of Ca2+ (with EGTA in
the medium), it is clear that two different mechanisms are operative
under these conditions. Moreover, these two mechanisms may be
associated with the release of two distinct pools of insulin-containing granules.
-cells; insulin secretion; hypotonicity; calcium
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INTRODUCTION |
In 1975, Blackard et al.
(5) reported that "an abrupt reduction of medium
osmolarity by as little as 20 mosM evoked a discrete short-lived
insulin secretory response from perifused chopped pancreas or isolated
islets." The response resembled that of the first phase of
glucose-stimulated insulin release and was due to the osmolarity change
per se and not to the absolute osmolarity, because a similar release of
insulin occurred when the osmolarity of a hypertonic buffer was
abruptly reduced to normal osmolarity. Increased osmolarity did not
stimulate insulin release in this study, although some stimulation of
release has been reported in response to large increases of 400 and 500 mosM (13, 33). One remarkable feature of the response to
hyposmolarity was that it was greater in "Ca2+-free
buffer" than in normal Ca2+-containing buffer. It should
be noted, however, that the Ca2+-free buffer did not
contain EGTA and was likely to contain a low concentration of
Ca2+ (28). Since this first report, the
phenomenon of insulin release induced by a decrease in osmolarity has
been confirmed by others (3, 8, 13, 16, 22, 23), but as
yet without any complete understanding of the mechanisms involved. It
is clear that the decrease in osmolarity depolarizes the
-cell and
that the depolarization is largely due to an outwardly rectifying
chloride channel (3, 4, 8, 16, 17), perhaps diminished
somewhat by a transient activation of the ATP-sensitive K+
channel (8). However, it is not at all clear how such a
depolarization would lead to a secretory response (in fact, a greater
secretory response) in the nominally Ca2+-free buffer than
in the presence of normal extracellular Ca2+, because
Ca2+ entry under such conditions would be minimal.
Furthermore, we have found that chloride channel blockade by DIDS
(4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) fails to block
hyposmotic stimulation of insulin release. It seems likely, therefore,
that the chloride channel activation is responsible for the regulatory
volume decrease that follows the obligatory cell swelling under
hyposmotic conditions and not for the stimulation of secretion, even in
the presence of extracellular Ca2+.
We have reexamined the effect of hyposmotic shock on insulin secretion
in the presence and absence of Ca2+ (with EGTA present) and
determined that two different mechanisms are operative under these
conditions. Also, because the response to hyposmotic shock resembles
that of the first phase of glucose-stimulated insulin release and
because we have identified a readily releasable pool of granules in the
-cell as responsible for the first phase of release
(6), we looked to see whether the same pool was responsible for the insulin release following hyposmotic shock. The
experiments, which were performed on
HC9 cells, demonstrated that
the first-phase response to glucose and the response to hypotonic shock
in the presence of extracellular Ca2+ are due to the
discharge of the same rapidly releasable granule pool. However,
hypotonic shock in the absence of extracellular Ca2+ may
discharge a different pool of granules.
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METHODS |
Cell culture.
HC9 cells were cultured in Dulbecco's modified Eagle's medium
containing 25 mM glucose, 1 mM pyruvate, 15% horse serum, 2.5% fetal
bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin at
37°C in a 95% air-5% CO2 atmosphere.
HC9 cells were
used between passages 23-35.
Insulin secretion under perifusion conditions.
The perifusion system was as described previously (6). In
brief,
HC9 cells were grown on glass coverslips, which were then
placed in each of four 0.7-ml perifusion chambers with the cells facing
the inside of the chambers. Krebs-Ringer bicarconate-HEPES (KRBH)
composed of 129 mM NaCl, 5 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO2, 1.0 mM CaCl2, 1.2 mM
MgSO4, 0.1% bovine serum albumin, and 10 mM HEPES at pH
7.4, with 0.1 mM glucose at 37°C, flowed through the chambers at a
rate of 1 ml/min. The experiments were carried out after a 30-min
equilibration period in normal or Ca2+-free KRBH as
appropriate. Samples of 1 ml of the perifusate were collected every
minute for the measurement of insulin secretion from 5 or 10 min before
the first hyposmotic shock to the end of the experiment. During the
experimental period, the cells were perifused with hyposmotic KRBH
(minus 25 mM NaCl) for 5 min and returned to normal KRBH for 15 min,
and the hypotonic shock was repeated for a further period of 5 min
followed by a return to normal KRBH, as described in the figure
legends. When nitrendipine, calciseptine, and DIDS were applied, they
were present in the perifusate for 5 min before the hypotonic
challenge. The perifusate samples were frozen at
20°C until the
insulin release rates were measured by RIA (12).
Treatment of
HC9 cells for immunoprecipitation, SDS-PAGE, and
immunoblot analysis.
Flasks (75 cm2) with
HC9 cells were washed twice in KRBH
containing 0.1 mM glucose. Preincubation was carried out at 37°C in
KRBH buffer for 40 min, similar to the conditions for the perifusion experiments. After the medium was aspirated out, 10 ml of fresh hyposmolar KRBH buffer containing 0.1 mM glucose were used for the test
condition. The experiments were stopped after the first or second 5-min
period of hypotonic shock by removing the medium and washing the cells
twice with cold PBS solution. After any leftover solution was
completely aspirated out, the cells were incubated in 700 µl of lysis
buffer (150 mM NaCl, 15.7 mM NaH2PO4, 1.47 mM
KH2PO4, 2.68 mM KCl, 1% NP-40, 1 mM
dithiothreitol, protease inhibitors, pH 7.4) and rocked at 4°C for 30 min. The samples were collected in 1.5-ml Eppendorf tubes and
centrifuged at 15,000 rpm for 5 min, and the resulting supernatants
were collected as the cell lysates.
Immunoprecipitation reactions were carried out by first incubating the
antibody against syntaxin with protein G-agarose beads for 2 h at
4°C with shaking followed by three washes with the lysis buffer. The
lysate samples were incubated with the antibody-bound protein G-agarose
beads for 2 h at 4°C with shaking. After several washes with the
lysis buffer, the beads were resuspended in Laemmli sample buffer [65
mM Tris, 3% SDS, 10% glycerol, bromphenol blue (0.025 mg/ml) and 5%
2-mercaptoethanol] and boiled for 5 min. The supernatant samples were
separated by SDS-polyacrylamide gel electrophoresis (PAGE) and then
transferred electrophoretically to a polyvinylidene difluoride
membrane. The transfer solution contained 20 mM Tris, 192 mM glycine,
and 20% methanol. The membrane was blocked in a buffer containing
13.24 mM Tris-HCl, 1.94 mM Tris base, 32.72 mM NaCl, 1.79 mM
EDTA, 0.05% Tween 20, and 1% BSA (TBST) and 5% nonfat dried milk for
at least 1 h at 4°C. Primary antibody incubations were carried
out for 1 h at 4°C. After being washed with TBST buffer at least
three times for 5 min each, the membrane was incubated for 1 h at
4°C with horseradish peroxidase-conjugated donkey antibodies to mouse
IgG diluted 1:5,000. The membrane was washed as described, and the
immune complexes were visualized by enhanced chemiluminescence (ECL kit).
Materials.
DIDS and calciseptine were purchased from Calbiochem (La Jolla, CA).
Human monoclonal antibody to syntaxin (SP6) and mouse monoclonal
antibody to vesicle-associated membrane protein-2 (VAMP-2; SP10), were
purchased from Upstate Biotechnology (Lake Placid, NY). Mouse IgG and
the ECL reagents were obtained from Amersham Pharmacia Biotech
(Piscataway, NJ). Flunarizine, nitrendipine, the protein G-agarose
beads (from GpC streptococcus), and the prestained SDS-PAGE standards
were purchased from Sigma (St. Louis, MO).
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RESULTS |
In Fig. 1 are shown the
insulin-secretory responses of
HC9 cells subjected to two
consecutive 50 mosM reductions in the osmolarity of the perifusing KRBH
buffer, each lasting 5 min. For comparison, the responses to two
consecutive 5-min challenges with 30 mM glucose are shown also.
Stimulation of secretion in response to the first hyposmolar challenge
occurred promptly and resembled the first phase of glucose-stimulated
insulin secretion, although the response was of smaller magnitude.
After the first response and after a 15-min period of reperifusion in
normal KRBH buffer, the insulin secretory response to a second
hypotonic shock was markedly reduced in size to less than one-fourth of
the first one. The response to hypotonicity was transient and did not
differ when the hypotonicity was applied for 5 min or for longer
periods (data not shown). The magnitude of the secretory response to
hypotonicity varied considerably according to the passage number of the
cells used. The response to the second glucose challenge was also
reduced but to a lesser extent. In control experiments, when the change in osmolarity due to the reduction in NaCl was compensated for by the
addition of sorbitol, there were no insulin secretory responses (data
not shown), confirming previous findings (5). Thus it is
the hyposmotic shock that elicits the rapid secretory response and not
the reduction of NaCl per se. We next performed experiments with KRBH
made up with and without CaCl2 (without added EGTA). The
cells were exposed to both KRBH solutions, i.e., with and without
CaCl2, for the duration of the experiment and not just for
the period of the hypotonic shock. Under the "nominally"
Ca2+-free conditions, hypotonicity stimulated insulin
secretion to a greater extent than in the presence of normal
Ca2+ levels (Fig. 2),
confirming the result reported by Blackard et al. (5) on
pancreatic islets. This result was also seen when the experiments were
repeated using Ca2+-free KRBH plus 1 mM EGTA (again the
cells were exposed to the Ca2+-free KRBH for the duration
of the experiment). Importantly, the L-type voltage-dependent
Ca2+ channel blocker nitrendipine, which at a concentration
of 10 µM largely blocked the response to hypotonicity in the presence of extracellular Ca2+ (Fig.
3A), had no effect on the
response occurring in KRBH made up without CaCl2 (Fig.
3B). Similar results were obtained when 10 µM
calciseptine, another L-type channel Ca2+ blocker
(7), was used (data not shown). That T-type
Ca2+ channels were not involved was shown by the complete
lack of effect of the T-type Ca2+ channel blocker
flunarizine (data not shown). The release of Ca2+ from
intracellular calcium stores was also not involved, because similar
responses to hyposmotic shock were given by control cells and cells
that had their calcium stores depleted by pretreatment with
thapsigargin (data not shown). It has also been shown under Ca2+-free conditions that an osmotic shock due to a
reduction of 40 mM NaCl did not raise Ca2+ concentration
([Ca2+]i) (8). All of these
results suggest that the response to hypotonicity in the presence of
Ca2+ is due, in large part, to increased Ca2+
entry via L-type Ca2+ channels but that, in the absence of
extracellular Ca2+, the response is due to a totally
different mechanism that is Ca2+ independent. Because the
action of hyposmotic shock has been ascribed to a depolarization of the
cell by activation of chloride channels, we tested the response in the
presence of the chloride channel blocker DIDS. Exposure of the cells to
100 µM DIDS for 5 min before and during the hyposmotic shock was
without effect in either the presence or the absence of
Ca2+ (Fig. 4). This result
demonstrates that depolarization of the cell by chloride channel
activity in the presence of Ca2+ is not the cause of the
increased activity of the L-type Ca2+ channels; rather, the
increased activity of the L-type Ca2+ channels appears to
be caused by a direct effect of the hypotonic shock.

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Fig. 1.
The insulin-secretory responses of HC9 cells subjected
to 2 successive 50 mosM reductions in the osmolarity of the perifusing
Krebs-Ringer bicarbonate-HEPES (KRBH) buffer, each lasting for 5 min,
and to 2 successive 5-min challenges by 30 mM glucose. IRI,
immunoreactive insulin. HC9 cells were perifused with KRBH under
basal conditions for 30 min until equilibrated (0 time). From 0 to 10 min, the cells remained under basal conditions and were then subjected
to KRBH minus 25 mM (50 mosM) NaCl ( ) or 30 mM glucose
( ) for 5 min. The cells were then returned to normal
basal glucose conditions for 15 min. At 30 min, the challenges were
repeated. Data are expressed as means ± SE; n = 4-8.
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Fig. 2.
Effect of hypotonic shock in the presence and absence of
extracellular Ca2+. HC9 cells were perifused under basal
conditions for 35 min until equilibrated (at the 5-min point) in KRBH
made up with or without CaCl2. From 5 to 10 min and from 25 to 30 min, the cells were subjected to KRBH (with or without
CaCl2) minus 25 mM (50 mosM) NaCl. From 10 to 25 min, the
cells were returned to the isotonic KRBH solutions and basal glucose
conditions. The hyposmotic shock was applied to HC9 cells in KRBH
without Ca2+ ( ) and with Ca2+
( ). Data are expressed as means ± SE;
n = 4.
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Fig. 3.
Inhibitory effect of nitrendipine on hypotonic
shock-induced insulin release in the presence of extracellular
Ca2+ (A) and the lack of effect of nitrendipine
on hyposmotic shock-induced insulin secretion in the absence of
extracellular Ca2+ (B). A: HC9
cells were perifused under basal conditions for 30 min until
equilibrated (at 0 time) in KRBH made up with CaCl2. From 0 time to 35 min, the test islets were exposed also to 10 µM
nitrendipine ( ). At 5 and 25 min, the cells were
subjected to KRBH minus 25 mM (50 mosM) NaCl. The hyposmotic shock was
applied from 5 to 10 min and from 25 to 30 min in the absence
( ) and presence ( ) of 10 µM
nitrendipine. In the absence of nitrendipine, the hyposmotic shock
caused a "first phase-like" release of insulin, as anticipated. In
the presence of nitrendipine, the response to hypotonicity was largely
inhibited. Data are expressed as means ± SE; n = 4. B: HC9 cells were perifused under basal conditions for
30 min until equilibrated (at time 0) in KRBH made up
without CaCl2. From time 0 to 35 min, the test
islets were exposed also to 10 µM nitrendipine ( ).
The hyposmotic shock was applied from 5 to 10 min and from 25 to 30 min
in the absence ( ) and presence ( ) of 10 µM nitrendipine and still in KRBH made up without Ca2+.
Under these Ca2+-free conditions, the response to the
hyposmotic shock was unaffected by the presence of nitrendipine. Data
are expressed as means ± SE; n = 4.
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Fig. 4.
Lack of effect of DIDS on hyposmotic shock-induced insulin
secretion in the presence (A) or absence (B) of
extracellular Ca2+. A: HC9 cells were
perifused under basal conditions for 30 min in KRBH with normal
extracellular Ca2+ until equilibrated (at time
0). From 0 to 5 min, the cells remained under basal conditions but
in the absence ( ) and presence ( ) of
DIDS. The hyposmotic shock was applied from 5 to 10 min in the absence
( ) and presence ( ) of DIDS and returned
to normal KRBH from 10 to 20 min. B: the HC9 cells were
treated in the same way as for those in A, with the
exception that the KRHB was made up without Ca2+. Data are
expressed as means ± SE; n = 4.
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Parallel studies were performed in which
HC9 cell lysates were
prepared under basal conditions just before each of the 5-min periods
of hypotonicity and at the end of the 5-min exposures to the hypotonic
conditions, thus mimicking the conditions used in the perifusion
studies. After immunoprecipitation of the lysates with antiserum
against syntaxin and subsequent SDS-PAGE of the immunoprecipitates, the
membranes were immunoblotted for VAMP-2. Under basal conditions, in the
presence of extracellular Ca2+, synaptobrevin was
coimmunoprecipitated with syntaxin (see time 0 in Fig.
5), a finding that identifies the readily
releasable granule pool (6). After the first 5 min
exposure to the hypotonic buffer, the band of VAMP-2 was much reduced,
indicating that the readily releasable granules had been largely
discharged. In four similar experiments, the density of the VAMP-2 band
was reduced by the hypotonic shock and in one case was undetectable.
Therefore, the hypotonic stimulation of insulin release in the presence
of extracellular Ca2+ is due to the readily releasable pool
of insulin-containing granules i.e., the same readily releasable pool
of granules that is responsible for the first phase of
glucose-stimulated insulin release. The readily releasable pool before
the second period of hypotonicity (at the 25 min point) was not fully
restored to its previous size under basal conditions (at the 0 min
point). No consistent change occurred in response to the second period
of hypotonicity, presumably because of the small size of the insulin
secretory response. When a second series of experiments was performed
in the absence of extracellular Ca2+ and in the presence of
1 mM EGTA, the results were different. In seven experiments, hypotonic
stimulation was not associated with any reduction in the amount of
coimmunoprecipitated VAMP-2 (data not shown). This suggests that the
different mechanisms of hyposmolar stimulation of insulin secretion, in
the presence and absence of extracellular Ca2+, are
associated with the release of granules from different cellular pools.

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Fig. 5.
Effect of hyposmotic shock on the coimmunoprecipitation
(IP) of vesicle-associated membrane protein-2 (VAMP-2) by syntaxin.
Antibodies against syntaxin were used to immunoprecipitate HC9 cell
lysates prepared under basal, nonstimulated conditions (equivalent to
the 10-min point in Fig. 1) and after 5 min of hypotonic shock
(equivalent to the 15-min point in Fig. 1). Similar
immunoprecipitations were performed at the beginning and end of the
second hypotonic shock (equivalent to the 30- and 35-min points in Fig.
1). The immunoprecipitated proteins were subjected to SDS-PAGE and
Western immunoblotted (IB) for synaptobrevin. Results are
representative of 5 such experiments.
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DISCUSSION |
The work described here confirms an observation made 27 years ago
that a decrease in osmolarity (hypotonic shock) stimulates the release
of insulin (5). Several other hormones and peptides are
also released in response to hypotonicity, as reviewed by Strbak and
Greer (26). Among these are glucagon (5),
luteinizing hormone (9-11), thyrotropin (9,
30), prolactin (9, 25, 30),
-endorphin
(1), gonadotropin-releasing hormone (14), thyrotropin-releasing hormone (TRH) (18, 24), and even TRH from rat pancreatic islets (2). It is clear from several
of these studies, and from the work we report here, that regulated secretion can be stimulated in a Ca2+-independent manner
(26). Our data also demonstrate that hypotonic shock
stimulates the release of insulin by two distinct mechanisms, depending
on the presence and absence of extracellular Ca2+. The
substantial inhibition of the response in the presence of extracellular
Ca2+ by the L-type Ca2+ channel blockers
nitrendipine and calciseptine shows that hypotonicity activates L-type
voltage-dependent Ca2+ channels and increases
Ca2+ influx. The resulting elevated
[Ca2+]i then stimulates insulin release. This
conclusion is in apparent agreement with the results of other studies
showing that hypotonicity depolarizes the
-cell by activation of
chloride channels (3, 8, 16). However, the chloride
channel blocker DIDS failed to inhibit the hypotonically induced
insulin release. This suggests, surprisingly, that the
Ca2+-mediated stimulation of insulin secretion in response
to hypotonicity is not a result of the chloride channel-mediated
depolarization. That a second and Ca2+-independent
mechanism for the hypotonic stimulation of insulin release exists is
clear from the following. First, increased Ca2+ influx and
[Ca2+]i cannot be responsible for the
stimulation of insulin release that is seen in the absence of
extracellular Ca2+ and presence of EGTA. Second, as shown
here and in the original article on this topic (5), the
stimulation of insulin release in the absence of extracellular
Ca2+ is greater than the stimulation that occurs in
response to the same hypotonic challenge in the presence of
extracellular Ca2+. The simplest explanation for these
phenomena is that two mechanisms are involved and that the mechanism
responsible for the stimulation of release in the absence of
extracellular Ca2+ is suppressed in the presence of
extracellular Ca2+. That it is extracellular
Ca2+ and not Ca2+ influx or elevated
[Ca2+]i that is responsible for the
suppression is shown by the experiments with nitrendipine. In the
presence of extracellular Ca2+, a supermaximal
concentration of nitrendipine reduced the amount of insulin released by
>80%. Because nitrendipine, which blocks Ca2+ influx and
prevents the increase in [Ca2+]i, had no
effect on the larger stimulation of insulin release by hypotonicity in
the absence of extracellular Ca2+, it follows that it is
the presence of extracellular Ca2+ that is suppressing the
mechanism responsible for the Ca2+-independent stimulation
of release.
Under normal conditions, with Ca2+ present, it has been
shown that the hypotonicity-induced depolarization of the
-cell is due to an inward current generated by activation of an outwardly rectifying chloride channel. However, the chloride channel blocker DIDS
failed to inhibit the stimulation of insulin release in either the
presence or the absence of extracellular Ca2+. This is not
compatible with the idea that activation of chloride channels is the
cause of the increased Ca2+ influx or the
nitrendipine-sensitive stimulation of insulin release seen in the
presence of extracellular Ca2+. Consequently, it seems
likely that hypotonic shock has a direct effect to activate L-type
Ca2+ channels and that the chloride channel response to
hyposmolarity is involved only in the regulatory volume decrease after
cell swelling. Evidence for such an action of hyposmolar cell swelling on voltage-dependent Ca2+ channels is already available for
rat and guinea pig myocytes (19, 31, 32). Additional
evidence for this comes from the work of Kinard et al.
(16), who also showed that insulin secretion stimulated by
hypotonic shock persisted in the presence of the chloride channel
blockers DIDS and niflumic acid, despite their demonstrated ability to
block the chloride currents. Thus we favor the idea that, in the
presence of extracellular Ca2+, the influx of water or
stretching of the plasma membrane activates L-type Ca2+
channels and that the depolarization via activated chloride channels is
redundant to the release of insulin. Under these conditions, most of
the insulin released is due to Ca2+ influx and increased
[Ca2+]i, as is shown by the experiments with
the Ca2+ channel blockers, and little or none of the
released insulin is due to the largely suppressed
Ca2+-independent mechanism.
In studying the source of the insulin released by the hyposmotic shock,
we have used a coimmunoprecipitation method to detect and study the
readily releasable pool of
-cell granules. An association of plasma
membrane and granule membrane soluble
N-ethylmaleimide-sensitive factor attachment protein
receptor (SNARE) proteins involved in exocytosis can be detected by
immunoprecipitation and used to study the size of a readily releasable
granule pool and changes in the composition of this pool under
physiological and pharmacological challenges (6).
Exocytosis involves carefully orchestrated interactions between granule
membrane proteins, plasma membrane proteins, and cytosolic factors as
described by the modified SNARE hypothesis (15, 20, 27).
This states that the complex formed by the granule/vesicle-associated
membrane protein VAMP-2, the plasma membrane protein syntaxin 1A, and
SNAP-25 (synaptosome-associated protein of molecular weight 25,000) was
the receptor for SNAP(s) and the soluble
N-ethylmaleimide-sensitive factor (NSF) attachment protein(s) and that these proteins were intimately involved in exocytosis. Although the original hypothesis has been markedly changed
by recent studies, it remains true that the core complex of VAMP-2,
syntaxin 1A, and SNAP-25 is critical for exocytosis. Consequently, we
used the coimmunoprecipitation of the granule membrane protein VAMP-2
with the plasma membrane protein syntaxin 1A as a marker for a readily
releasable pool of insulin-containing granules. Although the
coimmunoprecipitation technique could conceivably be expected to pull
down core complex proteins that associate randomly in the cell lysate
or could have remained complexed after exocytosis had occurred, the
assay was validated by appropriate changes in the coimmunoprecipitation
under physiological conditions. For example, stimulation of release by
glucose and other secretagogues that would be expected to discharge a
readily releasable pool (6) was associated with decreased
amounts of VAMP-2 coimmunoprecipitated by antibody against syntaxin.
Such decreases were blocked by norepinephrine along with inhibition of
secretion. As a result, it was possible to draw the following
conclusions. 1) Coimmunoprecipitation of the membrane
protein VAMP-2 with syntaxin can be used as a marker of a readily
releasable granule pool. 2) The readily releasable granules
defined in this way are responsible for the first phase of
glucose-stimulated insulin release. 3) The readily
releasable granules are rapidly released by other secretagogues such as
mastoparan and by depolarizing concentrations of KCl. These conclusions
allowed us to test the hypothesis that the insulin released in response to hypotonic shock is also derived from this pool of granules. The
results demonstrate that the readily releasable granules are indeed the
source of the insulin released by hyposmotic shock in the presence of
extracellular Ca2+. These data are similar in some respects
to those of Lonart and Sudhof (21), who demonstrated that
SNARE core complexes assembled before neurotransmitter release.
Furthermore, treatment of synaptic vesicles with
N-ethylmaleimide to block the action of NSF (and dissociation of core complexes) increased the amount of the core complex in the synaptosomes and the size of the readily releasable pool, as judged by glutamate release in response to hypertonic shock.
They suggested that the degree of core complex assembly could determine
the size of the readily releasable pool.
In the absence of extracellular Ca2+, the situation is
quite different. Surprisingly, as determined by the
coimmunoprecipitation method, we found no evidence that the readily
releasable pool of insulin-containing granules was discharged by
hypotonicity. This suggests, therefore, that the insulin released by
hypotonic shock under Ca2+-free conditions is not derived
from the readily releasable pool that is discharged by an increase in
[Ca2+]i. Rather, the data suggest that it is
a separate and distinct pool of granules, perhaps the morphologically
docked, unprimed granule pool, that is discharged. The question arises
as to the mechanism of the Ca2+-independent stimulation of
insulin secretion by hypotonic shock. It seems likely that the effect
is physical and could be due to the influx of water per se or the
mechanical effect of cell swelling to stretch the plasma membrane. The
idea of a physical input fits with current ideas concerning regulated
exocytosis in those cell types with a pool of readily releasable
granules. The granules at the plasma membrane can be defined as
morphologically docked and subdefined as 1) granules that
are unprimed and unable to be released by physiological mechanisms in
their current state, and 2) granules that are primed for
release and comprise the readily releasable pool. It seems likely that
the assembly of syntaxin 1A, SNAP-25, and VAMP-2 into the tightly bound
trans-SNARE complexes that are essential for exocytosis
(15, 20, 27) is a determinant of the readily releasable
granule pool (21, 29). As syntaxin and SNAP-25 at the
plasma membrane and VAMP-2 on the granule membrane begin to combine,
they form a loose trans-SNARE complex, which, as it forms
and tightens into a coiled-coil helical structure, pulls the granule
closer to the plasma membrane. This "zippering up" is thought to be
a key step in overcoming much of the energy barrier preventing fusion
of the two membranes (15, 27). Thus the distinction
between loose and tight states of the trans-SNARE complexes
could be one of the distinctions between unprimed and primed granules
(29). However, even the primed, readily releasable granules still require a final input of energy for exocytosis to take
place. This is normally due to a rise in
[Ca2+]i and a conformational change induced
by a Ca2+ sensor (29). However, this last
input of energy or reduction of the final energy barrier could be due
to a physical perturbation, as, for instance, by a reduction in
osmolarity, cell swelling, and/or membrane stretching. Such input would
be responsible for the hypotonic stimulation of insulin exocytosis in
the
-cell (3, 5, 8, 16, 22, 23) and other cell types
(26). Similarly, the physical input of hypertonicity and
cell shrinkage is responsible for the release of synaptic vesicles in
response to hypertonicity (21).
In summary, the stimulation of insulin secretion by hypotonic shock in
the presence of extracellular Ca2+ is ultimately due to the
physiological mechanism of Ca2+ influx via L-type
Ca2+ channels and increased
[Ca2+]i. It would be expected, therefore,
that this would stimulate exocytosis of the readily releasable
granules. In the absence of extracellular Ca2+, the
situation is different. Hypotonic release is not Ca2+
mediated; it is due to a different mechanism: insulin may be released
from a different granule pool from that released by increased [Ca2+]i and is likely due to physical changes
in the plasma and/or granule membranes rather than to any physiological
mechanism. This mechanism is largely or completely suppressed in the
presence of extracellular Ca2+.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Douglas Hanahan for the
HC9 cells and to
Dr. Troitza Bratanova-Tochkova for excellent tissue culture.
 |
FOOTNOTES |
The work was supported by grants to G. W. G. Sharp from the
National Institute of Diabetes and Digestive and Kidney Diseases (RO1-DK-42063 and RO1-DK-54243) and by a Mentor-Based Postdoctoral Fellowship from the American Diabetes Association.
Address for reprint requests and other correspondence: G. W. G. Sharp, Dept. of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca NY 14853-6401 (E-mail:
gws2{at}cornell.edu).
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.
First published January 15, 2002;10.1152/ajpendo.00176.2001
Received 23 April 2001; accepted in final form 20 December 2001.
 |
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