Hyposmotic shock stimulates insulin secretion by two distinct mechanisms. Studies with the beta 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Exposure of beta 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.

beta -cells; insulin secretion; hypotonicity; calcium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 beta 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. beta 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. beta HC9 cells were used between passages 23-35.

Insulin secretion under perifusion conditions. The perifusion system was as described previously (6). In brief, beta 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 beta HC9 cells for immunoprecipitation, SDS-PAGE, and immunoblot analysis. Flasks (75 cm2) with beta 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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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In Fig. 1 are shown the insulin-secretory responses of beta 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 beta 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. beta 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+. beta 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 beta 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: beta 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: beta 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: beta 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 beta 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.

Parallel studies were performed in which beta 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 beta 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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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), beta -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 beta -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 beta -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 beta -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 beta -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 beta 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 282(5):E1070-E1076
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society




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