Ca2+ influx and cAMP elevation overcame botulinum toxin A but not tetanus toxin inhibition of insulin exocytosis

Xiaohang Huang1,2,*, You-Hou Kang1,2,*, Ewa A. Pasyk1,2,*, Laura Sheu1,2, Michael B. Wheeler1,2, William S. Trimble2,3,4, Annemarie Salapatek2, and Herbert Y. Gaisano1,2

Departments of 1 Medicine, 2 Physiology, and 3 Biochemistry, University of Toronto, Toronto M5S 1A8; and 4 Program in Cell Biology, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada M5G 1X8


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous reports showed that cleavage of vesicle-associated membrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting beta -cells inhibited Ca2+-evoked insulin secretion. In these reports, the soluble N-ethylmaleimide-sensitive factor attachment protein target receptor proteins might have formed complexes, which preclude full accessibility of the putative sites for neurotoxin cleavage. In this work, VAMP-2 and SNAP-25 were effectively cleaved before they formed toxin-insensitive complexes by transient transfection of insulinoma HIT or INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A (BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca2+ (glucose or KCl)-evoked insulin release proportionate to the transfection efficiency (40-50%) and an accumulation of insulin granules. With the use of patch-clamp capacitance measurements, Ca2+-evoked exocytosis by membrane depolarization to -10 mV was abolished by TeTx (6% of control) but only moderately inhibited by BoNT/A (30% of control). Depolarization to 0 mV to maximize Ca2+ influx partially overcame BoNT/A (50% of control) but not TeTx inhibition. Of note, cAMP activation potentiated Ca2+-evoked secretion by 129% in control cells but only 55% in BoNT/A-transfected cells and had negligible effects in TeTx-transfected cells. These results indicate that, whereas VAMP-2 is absolutely necessary for insulin exocytosis, the effects of SNAP-25 depletion on exocytosis, perhaps on insulin granule pool priming or mobilization steps, could be partially reversed by higher levels of Ca2+ or cAMP potentiation.

vesicle-associated membrane protein-2; synaptosomal-associated protein of 25 kilodaltons; clostridial neurotoxins; insulin exocytosis; capacitance measurements; adenosine 3',5'-cyclic monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN PANCREATIC ISLET beta -CELLS the molecular basis for insulin granule exocytosis is becoming clear (44). In regulated insulin secretion, insulin is synthesized and stored in secretory vesicles, which are then lodged beneath the plasma membrane of pancreatic islet beta -cells. A postprandial increase in plasma glucose causes membrane depolarization by activation of voltage-dependent Ca2+ channels. This results in a rise of cytosolic Ca2+, which causes the fusion of insulin granules with the plasma membrane to release insulin. Whereas glucose stimulates insulin secretion by this Ca2+-dependent pathway (36), this pathway becomes defective in diabetes (23). The cAMP-dependent pathway of insulin secretion is of importance, particularly in light of reports demonstrating that the glucagon-like peptide-1 agonists (6) acting on this pathway can bypass the defective Ca2+-activated pathways in diabetic islets (11, 14).

Much recent work has suggested that Ca2+-induced exocytosis of insulin granules is controlled by a set of proteins called soluble N-ethylmaleimide-sensitive factor attachment protein target receptors (SNAREs) (45). SNAREs were originally identified as the proteins required for synaptic vesicle fusion (32, 38). These proteins are specific substrates for proteolysis by the clostridial neurotoxins, enzymes known to inhibit neurotransmitter release (22). A current view of SNARE-mediated vesicle fusion is that the SNARE proteins on the donor vesicle v-SNARE (vesicle-associated membrane protein or VAMP) and the target membrane [t-SNAREs; synaptosomal-associated membrane protein of 25 kDa (SNAP-25) and syntaxin] interact to form a stable complex, which provides the energy to drive membrane fusion (41). In pancreatic islet beta -cells, the v-SNARES, VAMP-2 and VAMP-3, were found on insulin granules (16, 28, 29, 43), and the t-SNAREs, SNAP-25 and several syntaxin isoforms, were found on the plasma membrane (10, 33, 43). We and others reported (28, 29, 43) that addition of tetanus toxin light chain (TeTx-LC) into permeabilized islets and insulinoma cells inhibited Ca2+-evoked insulin secretion. Transient transfection of HIT cells with VAMP mutants resistant to proteolytic cleavage by TeTx-LC was able to rescue this toxin inhibition of Ca2+-evoked insulin release (28). Syntaxin 1 was also required for Ca2+-induced insulin release, shown by the fact that botulinum neurotoxin (BoNT)/C1 transfection of insulinoma HIT cells inhibited KCl-evoked insulin secretion (17). BoNT/C1, however, only minimally inhibited glucose-stimulated secretion (17). We recently showed that SNAP-25 is also required for Ca2+-evoked insulin secretion as well as for glucose-evoked insulin secretion, using the strategy of transfecting botulinum neurotoxin A light chain (BoNT/A-LC) into HIT cells to cleave and deplete endogenous SNAP-25 (15).

Whereas it is well established that Ca2+-evoked insulin secretion requires these SNARE proteins, the roles of these SNARE proteins are not clear within the distinct kinetic components (i.e., mobilization and release of insulin granule pools) of insulin exocytosis. It is even less clear whether cAMP-dependent insulin secretion also requires SNAREs. In support of a role for SNAREs in cAMP-mediated secretion is the report that BoNT/B cleavage of VAMP-2 inhibited cAMP-dependent enzyme secretion in parotid acinar cells (8). Exocrine acinar cells share similar properties with islet beta -cells in utilizing both Ca2+- and cAMP-dependent pathways to mediate secretion (44). In the present study, we have further explored the role of VAMP-2 and SNAP-25 in Ca2+- and cAMP-evoked insulin exocytosis from insulinoma cell populations (by radioimmunoassay) and from single beta -cells (by patch-clamp capacitance measurements). We used the strategy of transiently transfecting the TeTx-LC or BoNT/A-LC genes into insulinoma cells, which effectively cleaves VAMP-2 (9, 19, 22) or SNAP-25 (15). We demonstrated that both Ca2+ influx and elevated intracellular cAMP overcame BoNT/A but not TeTx-LC inhibition of insulin exocytosis. We conclude that VAMP-2 and SNAP-25 might be involved in the different kinetic components of insulin exocytosis. VAMP-2 is required for the insulin granule exocytotic fusion, whereas SNAP-25 may be involved in the priming and mobilization of insulin secretory granule pools in the exocytotic process, which is mediated by Ca2+ and cAMP.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. The HIT cell subclone T15 was a gift from Dr. Robert Santerre (Eli Lilly, Indianapolis, IN), and the INS-1 cell line was from American Type Culture Collection. A mouse monoclonal antibody generated against recombinant full-length TeTx-LC and TeTx-LC cDNA was a gift from H. Niemann (39) (Hanover, Germany), and anti-insulin antibody was from R. A. Pederson (University of British Columbia). A rabbit polyclonal antibody was generated against the NH2-terminal 16 amino acids of VAMP-2 (9). Forskolin and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma (St. Louis, MO).

Generation of constructs and transfections. Plasmids that contained VAMP-2 (40) and TeTx-LC cDNAs (7, 39) were subcloned into pcDNA3 as previously described (15). The HIT-T15 cells were grown at 37°C in 5% CO2-95% air in RPMI 1640 medium supplemented with 20 mM glutamine, 10% fetal calf serum (GIBCO, Gaithersburg, MD), penicillin (100 U/ml), and streptomycin (100 mg/ml). The cells were grown to a density of 3 × 106 cells/well in 12 (20-mm)-well plates. They were then transiently transfected with 2 µg of plasmid DNA (in Optimum media) or empty vector as control using Lipofectamine (GIBCO) and incubated for 4-6 h before normal RPMI was added. The medium was discarded the following day, and fresh RPMI 1640 medium was replaced. Transfection efficiency was determined by using pcDNA3/His/Lac Z (Invitrogen, La Jolla, CA) as a control plasmid for transfections, followed by determination of beta -galactosidase expression and also by visualization of cells by confocal microscopy.

Immunoblotting. SDS-PAGE and immunoblotting were performed as previously described (15, 43). Protein concentrations of each cell lysate sample were determined by the Bio-Rad (Hercules, CA) protein assay kit. The cell lysates were then dissolved in sample buffer, and proteins were separated on a 15% polyacrylamide gel, followed by transfer to a nitrocellulose membrane. The blots were then incubated with the primary antibody (anti-VAMP-2 antibody, 1:1,000; anti-TeTx-LC, 1:1,000; and anti-actin, 1:1,000 in 1% BSA and 0.05% Tween 20 in PBS) for 2 h at room temperature. Immunodetection was by enhanced chemiluminescence (Amersham ECL). Quantification of each band was determined by measuring the band area with NIH Image and Adobe Photoshop programs.

Insulin secretion. Forty-eight hours after transfection, the cells were cultured to ~80% confluence. As previously described (15), cells were washed and then incubated in 0.5 ml of low-K+ Krebs-Ringer bicarbonate (KRB) buffer (in mM: 4.8 KCl, 129 NaCl, 5 NaHCO3, 1.2 KH2PO4, 1 CaCl2, 1.2 MgSO4, 0.5 glucose, and 10 HEPES, pH 7.4) for 0.5 h. Each well was then exposed to basal conditions to obtain basal insulin secretion, followed by stimulatory conditions. The value of insulin released during the stimulatory conditions was subtracted from the value obtained from the basal conditions, which reflects the true stimulated secretion above basal levels (see Figs. 3, 4, and 6A). Specifically, for K+ stimulation studies, each well of cells was incubated with 0.5 ml of the same buffer containing low K+ (4.8 mM KCl and 0.5 mM glucose at 1 h) for basal secretion, and the media were collected after the incubation. This was followed by an exchange with 0.5 ml of a high-K+ buffer (30 mM KCl at 1 h), and the media were collected. For glucose stimulation, the wells were exposed to KRB buffer (with 4.8 mM KCl at 1 h) in which glucose was removed to determine basal secretion and then exchanged with a high-glucose buffer (11 mM with 4.8 mM KCl at 1 h). For cAMP stimulation, the cells were exposed to low-K+ buffer (0.5 mM glucose and 4.8 mM KCl) for basal level of insulin secretion, followed by an exchange with the same buffer containing 10 µM forskolin and 100 µM IBMX. We found that basal insulin secretion at 0.5 mM glucose was not different from that with no glucose, although for the cAMP-evoked secretion, the presence of low-glucose concentration (0.5 mM) was necessary. At the end of each incubation (basal and stimulated), the media collected were centrifuged at 3,000 g for 3 min to remove detached cells. The resulting top half of the supernatants was collected and determined for insulin release by radioimmunoassay (Linco, MO) according to the manufacturer's instructions. For the determination of total insulin content, the cell lysates remaining in the wells were scraped from the plates, and appropriate dilutions of the samples were carried out to allow the values to fall within the standard curve. As previously reported (15), we controlled the inherent variability of transfection efficiency and cell line passage by using the same plate of cells in each experiment, which included all the variables being tested. At least 3-5 independent experiments with triplicate or four replicate wells per experimental variable were performed. Values shown in the figures are the mean increases above basal levels, which were also determined in triplicate wells for each experiment. The data are expressed as means ± SE of the total number of wells and were analyzed by both Student's t-test and analysis of variance (ANOVA). A P value < 0.05 by both tests (shown in the text by ANOVA) indicates that the difference is considered significant.

Confocal immunofluorescence microscopy. Microscopy was performed as previously described (10, 15, 43). Briefly, the cells were plated on round polylysine-coated coverslips and placed in 12-well plates. Forty-eight hours after transfection, the cells were fixed in 2% formaldehyde for 0.5 h at room temperature, blocked with 5% normal goat serum with 0.1% saponin for 0.5 h at room temperature, and then immunolabeled with primary antibodies (1:100 anti-VAMP-2 and anti-TeTx-LC antibody) overnight at 4°C. After being rinsed with 0.1% saponin/PBS, the coverslips were then incubated with the appropriate rhodamine-labeled secondary antiserum for 1 h at room temperature, mounted on slides in a fading retarder 0.1% p-phenylenediamine in glycerol, and examined with a laser scanning confocal imaging system (Carl Zeiss). Transfected cells were identified by coexpression and visualization of the green fluorescence protein (GFP) (Clontech, Palo Alto, CA).

Patch-clamp capacitance measurements. Insulinoma beta -cell lines HIT-T15 and INS-1 were studied under standard whole cell patch-clamp conditions as previously described (34). To identify the TeTx-LC- and BoNT/A-LC-transfected HIT or INS-1 cells, we coexpressed the GFP, which we previously demonstrated to very reliably take up multiple plasmids (15). All intracellular solution contained (in mM) 140 KCl, 10 HEPES, 1 EGTA, 1 MgCl2, and 3 MgATP, pH 7.3 with KOH. Extracellular solution contained (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, and 10 HEPES, pH 7.3 with NaOH. In experiments with cAMP stimulation (INS-1 cells), 0.5 mM glucose was added to the above bath solution. Forskolin and IBMX were dissolved in DMSO (1,000× stock solutions). They were added directly to the bath solution to get a final concentration as indicated. Experiments were performed at room temperature. Capacitance measurements (Cm) were then performed similarly to that described by Ammala et al. (1) and Renstrom et al. (30). Specifically, the cells were held at -70 mV and depolarized to -10 or 0 mV (as indicated in the figures) for 250 ms. Immediately after depolarization, cells were exposed to a sine wave centered at -70 mV, and the changes in Cm stimulated by this depolarizing voltage pulse were observed. These experiments were performed with a quick-response lock-in patch-clamp amplifier (HEKA Electronics, Lambrecht, Germany) and a high-speed and high-memory computer (PC, Pentium III) running specialized patch-clamp Pulse software (HEKA). With the use of this equipment configuration, very small changes (fF range) in whole cell capacitance were monitored to indicate secretion granule (SG) fusion. This analysis is based on the fact that the specific capacitance of all biological membranes [including the plasma membrane (PM) and SG membranes] is ~10 fF/µm2. Because the fusion of an SG to the PM adds ~0.2 µm2 of membrane to the PM, a corresponding incremental increase in Cm by 2 fF would result. To minimize the possible influence of inherent variability of transfection and the passage of the cell lines on capacitance, data from three to four cells were averaged for each experiment (n = 1), and each group had the same chance of coming from the same passage. Data were presented as means ± SE. Data were compared by unpaired Student's t-test for single comparisons and by ANOVA for multiple comparisons. P < 0.05 was considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of TeTx-LC cleaves endogenous and overexpressed VAMP-2 in HIT-T15 cells. We and others reported that insulin secretion from permeabilized HIT-T15 is inhibited by TeTx-LC (29, 43). However, in those studies, only a modest inhibition was demonstrated (43). The lack of complete inhibition of insulin secretion could be due to the following: 1) VAMP-2 could be complexed with other proteins that render the cleavage site inaccessible to toxin cleavage (4, 12); 2) temporal limitations prevent the toxin from effectively acting on VAMP-2; and 3) inhibitory effects of cleavage products could be lost if these products leak out of the cell. To overcome these limitations, we transiently transfected HIT-T15 cells with the cDNA of the TeTx-LC (7, 39). This strategy should result in high levels of TeTx-LC within intact cells over a prolonged period of time, permitting VAMP-2 cleavage before it forms a complex with other SNAREs. The intact cell system would also permit the inhibitory effects on insulin secretion to be measured in response to a variety of secretagogues.

In Fig. 1A (top), HIT cells were transfected with TeTx-LC, and the anti-TeTx-LC antibody was used to detect expression of this protein. Recombinant TeTx-LC (9) used as a control (lane 3) was detected, and no TeTx-LC was detected in untransfected cells (lane 1). The lower part of the blot (bottom) was then probed with the anti-VAMP-2 antibody. This showed that endogenous VAMP-2 was cleaved by TeTx-LC (lane 2) compared with the intact VAMP-2 in untransfected cells (lane 1). The residual VAMP-2 (lane 1) comes from the untransfected cells, since transfection efficiency is ~40-50% based on cotransfection with the reporter beta -galactosidase gene or visualization of the coexpressed GFP (Fig. 2). The reduction of the endogenous VAMP-2 signal is therefore proportionate to the transfection efficiency.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 1.   Western analyses of tetanus toxin light chain (TeTx-LC) expression in HIT-T15 cells. A: effects of TeTx-LC expression in HIT cells on endogenous vesicle-associated membrane protein-2 (VAMP-2) proteins (lane 2). TeTx-LC and VAMP-2 proteins were detected by specific antibodies. Untransfected HIT cells (lane 1) and TeTx-LC fusion proteins (lane 3) were used as negative and positive controls, respectively. B: HIT cells were transfected with TeTx-LC and/or VAMP-2 and probed with VAMP-2, synaptosomal-associated protein of 25 kDa (SNAP-25), and anti-actin antibodies. Both endogenous (lane 1) and overexpressed VAMP-2 (lane 3) were cleaved by TeTx-LC (lanes 2 and 4) but had no effects on endogenous SNAP-25 and actin.



View larger version (101K):
[in this window]
[in a new window]
 
Fig. 2.   Confocal microscopy visualization of the effects of TeTx-LC expression on HIT-T15 cellular VAMP-2. Untransfected HIT cells were plated on the coverslips and probed with anti-VAMP-2 (A) and anti-insulin (B) antibodies to show the colocalization of endogenous VAMP-2 and insulin granules. C and D: HIT-T15 cells were transfected with TeTx-LC and then double labeled with anti-VAMP-2 (C) and anti-TeTx-LC (D) antibodies. Cells expressing TeTx-LC (arrows in D) demonstrate an absence or depletion of VAMP-2 (arrows in C).

Figure 1B demonstrates that expression of TeTx-LC (lane 2) caused a 74% cleavage of VAMP-2 compared with the control (lane 1), and the cleavage was specific since the toxin had no effect on endogenous SNAP-25 or actin. However, no truncated cleavage products were detectable in these experiments. To rule out the possibility that our VAMP-2 antibody might have a low affinity for VAMP-2 cleavage products, we coexpressed VAMP-2 along with TeTx-LC (lane 4). Overexpression of VAMP-2 alone (lane 3) caused a greater than threefold increase in VAMP-2 compared with its endogenous levels (lane 1). Coexpression of VAMP-2 with TeTx-LC resulted in >75% cleavage of the overexpressed VAMP-2 (lane 4). Again, we did not detect a smaller VAMP-2 cleavage product that would have been an NH2-terminal 77-amino acid protein migrating to ~12 kDa and would have been detected by our antibody. The absence of this cleavage product strongly suggests that these products might have undergone accelerated proteolysis, as was also noted in the neuron (19). In fact, we reported that BoNT/A-LC overexpression in HIT cells also resulted in accelerated proteolysis of SNAP-25 (15).

To determine the effects of TeTx-LC overexpression on cellular VAMP-2 and insulin, laser confocal immunofluorescence microscopy was performed using antibodies specific to VAMP-2, TeTx-LC, and insulin in Fig. 2. In untransfected HIT-T15 cells, punctate VAMP-2 staining (Fig. 2A) was found throughout the cytosol and was shown to colocalize with the insulin staining (Fig. 2B). In HIT cells transfected with TeTx-LC, the TeTx-LC could be detected with the anti-TeTx-LC antibody (Fig. 2D, arrows). VAMP-2 was present in cells that did not express TeTx-LC. Double labeling of these cells with anti-VAMP-2 antibody (Fig. 2C) shows the absence of VAMP-2 in these cells (arrows). Because the NH2-terminal 77-amino acid cleavage product of VAMP-2 would have been detected in the TeTx-LC-transfected cells, its absence is consistent with our results in Fig. 1B. The results shown in Figs. 1 and 2 indicate that transfection of TeTx-LC into HIT cells is capable of depleting VAMP-2 in these cells.

Expression of TeTx-LC results in accumulation of insulin secretory granules. Because VAMP-2 is a v-SNARE protein involved in insulin granule exocytosis, its cleavage by TeTx-LC might result in accumulation of insulin granules. Indeed, in HIT cells lacking VAMP-2 (indicated by arrows in Fig. 3A) as a result of TeTx-LC expression, double labeling with anti-insulin antibodies demonstrated an accumulation of cytoplasmic insulin puncta in these cells (indicated by arrows in Fig. 3B) compared with the untransfected cells, wherein VAMP-2 remains intact. Because the immunofluorescence study in Fig. 3B suggested that TeTx-LC-transfected cells appeared to contain more insulin than nontransfected cells, we determined the total insulin content of these cells by radioimmunoassay in Fig. 3C. Total insulin content in cells expressing TeTx-LC was 322.3 ± 49 ng/well and in control cells was 252.9 ± 14 ng/well, which was an increase of 27.4% (14 wells, 5 experiments, P = 0.15). In cells cotransfected with VAMP-2 and TeTx-LC, total insulin content was 243.6 ± 20.9 ng/well compared with cells transfected with VAMP-2 alone (203.1 ± 54.5 ng/well), which was an increase of 20% (n = 14 wells, 5 experiments; data not shown). These increases in total insulin content were, however, not statistically significant from control, but they at least indicate that the inhibition of secretion shown in Figs. 4 to 6A was not due to the inhibition of insulin synthesis. This apparent discrepancy could in part be explained by the following inherent limitations of these assays. First, fluorescence microscopy is not sufficiently quantitative and may overemphasize differences. Second, the measurement of total insulin content in cell lysates includes insulin in both transfected and untransfected cells, as well as insulin in subcellular compartments other than secretory granules. These cells may tend to minimize differences due to averaging. We have, in fact, observed similar immunofluorescence and insulin content results with BoNT/A-LC transfection of HIT cells, which we previously reported (15). In that report, we performed electron microscopy and morphometric analysis and found that BoNT/A-LC transfection resulted in accumulation of insulin granules at the plasma membrane.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   Expression of TeTx-LC causes an accumulation of insulin secretory granules in HIT-T15 cells. A and B: HIT-T15 cells transfected with TeTx-LC were double labeled with anti-VAMP-2 (A) and anti-insulin (B) antibodies and then visualized by confocal microscopy (as in Fig. 2). Note that cells depleted of VAMP-2 (arrows in A) by TeTx-LC expression resulted in an excess accumulation of insulin (arrows in B). C: HIT-T15 cells transfected with TeTx-LC were harvested in 1 M acetic acid and lysed by two freeze-thaw cycles. Insulin content of the total lysate was then determined by RIA as described in MATERIALS AND METHODS. The number of wells for each value and their statistical significance are indicated in RESULTS.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4.   TeTx-LC expression inhibits Ca2+-evoked insulin release from HIT cells. A and C: HIT cells were transfected with TeTx-LC. B and D: HIT cells were transfected with VAMP-2 together with or without TeTx-LC. These were then stimulated with either a high-K+ buffer in A and B or high glucose in C and D. For K+ stimulation (A and B), HIT cells were first incubated with a low-K+ buffer to determine basal release and then stimulated with a high-K+ buffer as described in MATERIALS AND METHODS. For glucose stimulation (C and D), HIT cells were first incubated with a buffer with no glucose to determine basal secretion and then followed by stimulation with a high-glucose (10 mM) buffer. Insulin released into the media was detected by RIA, and each value represents the insulin release above basal levels (see MATERIALS AND METHODS) and is the means ± SE. Each experiment was compared with control cells transfected with the empty vector. The number of wells for each value and their statistical significance are indicated in RESULTS.

Effects of TeTx-LC transfections on Ca2+-stimulated insulin secretion. We next examined the effects of VAMP-2 depletion by TeTx-LC transfection on Ca2+-evoked insulin secretion. In neuroendocrine islet beta -cells, Ca2+ influx across the Ca2+ channel is brought about by membrane depolarization caused by glucose-mediated closure of the ATP-sensitive K+ channel, which leads to K+ influx (27). Membrane depolarization can therefore be directly affected by the addition of high concentrations of K+ (30 mM) into a Ca2+-containing buffer with low-K+ concentrations (4.8 mM) (15). We therefore examined the effects of TeTx-LC transfection on glucose- and KCl-stimulated insulin secretion (Fig. 4).

We determined secretagogue-stimulated insulin secretion above the basal levels first by determining the basal levels of every well (first hour) and then by stimulating the cells of each well with the secretagogue (second hour). We then subtracted the insulin secreted in the second hour from that secreted in the first hour from the cells in each well (see MATERIALS AND METHODS). To minimize inter- and intra-assay variations and the possible variation in transfection efficiency, the following controls were performed. For control of the effect of transfection on insulin release, cells were transfected with the empty vector. That had no effect on insulin secretion compared with the untransfected cells (data not shown). Cells in each set of experiments were from a single plate of cells of the same passage, plated in an identical manner into each well. Triplicate wells for each variable and the control were performed for each experiment, and at least three independent experiments were performed. The viability and confluence of the cells in each well were confirmed visually and found to be uniform. Transfection efficiencies in the range of 40-50% were confirmed by coexpression of the beta -galactosidase gene in a control well performed in every set of experiments. These conditions were uniformly used for all the secretory studies (Figs. 4 and 6A). Typically, in control untransfected cells, high-K+ buffer (30 mM KCl) evoked insulin release of two- to fourfold that of the basal levels.

Figure 4A demonstrates that transient expression of TeTx-LC caused a significant 45% reduction in high K+-evoked insulin release (0.89 ± 0.15 ng/well) compared with the control untransfected cells (1.63 ± 0.24 ng/well, n = 9 wells from 3 experiments, P = 0.019). We next examined the effects of overexpressing VAMP-2 on TeTx-LC inhibition of insulin secretion to see whether the overexpressed VAMP-2 would rescue this TeTx-LC inhibition (Fig. 4B). First, we did not observe a significant difference between control (2.14 ± 0.56 ng/well, P = 0.53) and VAMP-2-transfected cells (2.5 ± 0.42 ng/well) in a high K+-evoked insulin release. Second, cotransfection of VAMP-2 along with TeTx-LC resulted in a high K+-evoked insulin release of 1.0 ± 0.32 ng/well (n = 9 wells, 3 experiments), which was significantly reduced by ~60% compared with the insulin released from the VAMP-2-transfected cells (P = 0.0024).

The effects of TeTx-LC on glucose-evoked secretion in HIT cells (Fig. 4, C and D) were similar to the effects on KCl-evoked secretion, as would be anticipated, since both act primarily on the Ca2+-signaling pathway. In the TeTx-LC-transfected HIT cells (Fig. 4C), maximal stimulatory glucose concentrations (11 mM, 1 h, 37°C) stimulated insulin release (above basal levels) of 0.75 ± 0.08 ng/well, a significant reduction of 49% (n = 9 wells, 3 experiments, P = 0.0003) compared with the control untransfected cells (1.48 ± 0.06 ng/well). In cells coexpressing VAMP-2 and TeTx-LC (Fig. 4D), insulin release was also significantly reduced by 58% (0.35 ± 0.06 ng/well, 9 wells, 3 experiments, P = 0.008) compared with the cells transfected with VAMP-2 alone (0.83 ± 0.16 ng/well).

Distinct effects of BoNT/A-LC and TeTx-LC on Ca2+-evoked insulin secretion. The results in Figs. 3 and 4 appear similar to those we have reported for BoNT/A-LC transfection (15). It is, therefore, intuitive to assume that cleavage of VAMP-2 and SNAP-25 by these toxins would uniformly abrogate exocytic fusion. However, a more recent report on chromaffin cells using the patch-clamp capacitance measurements demonstrated that these neurotoxins exhibit distinct effects on the multiple components of exocytosis (47). The acute addition of these toxins used in that report would not reliably effect complete cleavage of the SNARE proteins, particularly those that already exist in the distinct SNARE complexes (12). In contrast, overexpression of the neurotoxin over a 24-h period ensures both very high levels of the neurotoxin and full cycling of SNARE complex assembly and disassembly. This ensures that the neurotoxin gains full access to not only the endogenously but also to exogenously expressed SNARE proteins (Figs. 1 and 2). We, therefore, use this strategy of neurotoxin overexpression in HIT-T15 and INS-1 cell lines to dissect the distinct effects of VAMP-2 and SNAP-25 proteolytic depletion by these clostridial neurotoxins on Ca2+-evoked secretion at the single cell level using patch-clamp capacitance measurements (1, 30) (Figs. 5 and 6, B and C). To identify the neurotoxin-transfected cells, we coexpressed GFP in the cells in which we have shown to reliably take up multiple plasmids (15). The cells with the highest GFP fluorescence were studied, which contained the corresponding high concentration of the coexpressed neurotoxins. Figure 5 shows the effects of TeTx-LC or BoNT/A-LC expression on membrane depolarization-evoked exocytosis in HIT-T15 cells compared with the control cells. Figure 5A shows representative capacitance recordings, and Fig. 5B shows a summary of these recordings. These cells were evoked by a depolarization to -10 mV (Fig. 5A, top, and Fig. 5B, open bars) or 0 mV (Fig. 5A, bottom, and Fig. 5B, filled bars). These levels of depolarization were selected on the basis of our previous study of HIT-T15 cells on voltage-evoked Ca2+ currents in which we showed that a depolarization to 0 mV resulted in a 41% increase in Ca2+ current over depolarization to -10 mV (34). At -10 mV, TeTx-LC expression virtually abolished (1.7 ± 0.7 fF, or 5.6% of control cells) the capacitive response compared with the control cells (30.3 ± 1.7 fF). A depolarization to 0 mV, which further increases Ca2+ influx and the capacitive response in the control cells (40.7 ± 1.9 fF), did not reverse the inhibition by TeTx-LC at all (1.6 ± 0.6 fF). In contrast, at -10 mV, BoNT/A-LC expression inhibited the capacitive response to 30% of control (9.52 ± 0.9 fF), demonstrating that BoNT/A-LC caused a smaller inhibition than TeTx-LC. More interestingly, at 0 mV, this inhibition by BoNT/A-LC was greatly reversed to ~50% of control cells (20.4 ± 3.1 fF), with the actual capacitive change at 0 mV to double that at -10 mV (Fig. 5B).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5.   Distinct effects of TeTx-LC or botulinum neurotoxin A light chain (BoNT/A-LC) expression on Ca2+-evoked exocytosis as determined by patch-clamp capacitance measurements (Cm). HIT-T15 cells expressing TeTx-LC, BoNT/A-LC, or green fluorescence protein alone (control cells) were studied with standard whole cell voltage-clamp techniques as described in MATERIALS AND METHODS. A: representative capacitance traces are shown for 3 separate cells in each study group: control (ovals), BoNT/A-LC-expressing (triangles), and TeTx-LC-expressing (squares) cells. Cells were depolarized to -10 mV (top) or to 0 mV (bottom) from a holding potential of -70 mV for 250 ms. B: summary of cumulative data from all studies on control (n = 4 experiments, 15 cells) and BoNT/A-LC- and TeTx-LC-expressing cells (n = 3 experiments, 12 cells each) performed with depolarization to both -10 mV (open bars) and 0 mV (filled bars). Cm changes are means ± SE above basal Cm levels.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6.   Distinct inhibitory effects of TeTx-LC or BoNT/A-LC expression on cAMP-mediated insulin release and potentiation of Ca2+-evoked insulin exocytosis. A: HIT cells transfected with TeTx-LC or BoNT/A-LC were incubated at basal conditions to determine basal insulin release and then stimulated with 10 µM forskolin and 100 µM IBMX (1 h at 37°C). Insulin released into the media was measured by RIA and analyzed as in Fig. 4. The insulin release is means ± SE of the insulin released above basal levels. B and C: INS-1 cells transfected with TeTx-LC or BoNT/A-LC were evoked by a membrane depolarization from -70 to 0 mV in the absence and then in the presence of 10 µM forskolin and 50 µM IBMX, and Cm increase was determined as described in MATERIALS AND METHODS and RESULTS. B: representative traces pre- and postpulse Cm values; a: (no cAMP) Ca2+-dependent Cm increase (postpulse traces above a dashed line); b: additional potentiation of this Ca2+-mediated response by elevation of intracellular cAMP with 10 µM forskolin and 50 µM IBMX. C: summary bar graph (means ± SE) of cAMP-dependent potentiation of Ca2+-mediated Cm increase in control (n = 8 cells), BoNT/A-LC- (n = 9 cells), and TeTx-LC (n = 4 cells)-expressing cells, which are expressed as a percent increase over the respective values of the postpulse Cm increase without cAMP stimulation (i.e., the Cm values, a, over the Cm values, b, in B). The Cm increases without cAMP stimulation are 88 ± 37 fF for control cells (n = 8), 56 ± 21 fF for BoNT/A-LC-expressing cells (n = 9), and 12 ± 9 fF for TeTx-LC-expressing cells (n = 4).

Distinct effects of TeTx-LC and BoNT/A-LC on cAMP-stimulated potentiation of Ca2+-evoked insulin secretion. Glucose is the physiological agonist for the islet beta -cells and activates Ca2+-dependent pathways to stimulate insulin secretion, which may become defective in the diabetic states (23). cAMP pathways, including those evoked by the incretin hormone, glucagon-like peptide-1 (GLP-1), can potentially bypass or potentiate glucose-mediated pathways (6, 11, 14). We therefore examined whether transient transfection of TeTx-LC or BoNT/A-LC might also affect cAMP-mediated insulin release (Fig. 6A) and potentiation of Ca2+-evoked secretion (Fig. 6B). The cAMP pathway was stimulated by forskolin and IBMX at the concentrations indicated. Figure 6A shows that, in the TeTx-LC-transfected HIT cells in the presence of the permissive glucose concentrations (0.5 mM), insulin secretion (above the basal levels) was 2.27 ± 0.29 ng/well (P = 0.02, n = 12 wells, 4 experiments) and 3.05 ± 0.22 ng/well in the control cells. TeTx caused a significant inhibition of 25.4% (P = 0.03) compared with the control. In BoNT/A-LC toxin-transfected cells, insulin secretion from HIT cells was 1.91 ± 0.29 ng/well, an inhibition of 37% (P = 0.02, n = 12 wells, 4 experiments) compared with the control, which was not significantly different from that caused by TeTx-LC (P = 0.31).

A primary role of the cAMP/protein kinase A (PKA) pathway is the potentiation of Ca2+-evoked insulin secretion, as demonstrated by capacitance measurements (1, 30). It was postulated that the underlying mechanism of cAMP/PKA potentiation of Ca2+-evoked secretion is a combination of an increase in the release probability of the readily releasable pool of insulin granules and an acceleration of the refilling of this pool from the reserve pool (30). We therefore examined the effects of BoNT/A-LC and TeTx-LC on PKA potentiation of Ca2+-evoked insulin exocytosis by capacitance measurements (Fig. 6, B and C). Here, we used INS-1 cells, which reliably have more insulin granules than the HIT cell line. Again, the TeTx-LC- and BoNT/A-LC-expressing cells were identified by the coexpressed GFP. The cells were depolarized only to 0 mV, the voltage at which a maximal Ca2+ influx current occurs (34) and at which the reversal of the BoNT/A inhibitory effect on Cm was more pronounced than at -10 mV (Fig. 5). After the initial Cm recording was completed, the cells were not stimulated for 3 min, allowing the cells to recover and replenish the readily releasable pool. Cells were then treated with forskolin (10 µM) and IBMX (50 µM). Cm responses were then elicited again with the same depolarization pulse to 0 mV. In control cells, depolarization to 0 mV increased Cm to 88 ± 37 fF (n = 8 cells), and the subsequent cAMP activation resulted in an additional Cm increase of 129% (Fig. 6C). In cells expressing TeTx-LC, the Cm increase by the depolarizing pulse was 12 ± 9 fF, and after cAMP activation, it was increased only by 15% (n = 4 cells, P > 0.05). In contrast, in BoNT/A-LC-transfected cells, the depolarizing pulse caused a Cm increase of 56 ± 21 fF (n = 9 cells), which was 63% that of the control cells. This 63% value was in a similar range as the percentage increase (~50%) observed in the HIT cell study (Fig. 5). Remarkably, subsequent cAMP activation was able to further increase the Cm by a very substantial 55% (P < 0.05), which approximates the level of Cm increase of control cells before the cAMP activation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SNARE proteins VAMP-2, SNAP-25, and syntaxin constitute the "minimal" machinery for membrane fusion (41), and, therefore, it would be intuitive to assume that cleavage of these proteins by BoNT should have identical effects on exocytosis. However, TeTx-LC (this study) and BoNT/A-LC (15), which cleave VAMPs and SNAP-25, respectively, possess distinct (18) and broader effects on the signaling pathways (Ca2+, cAMP, glucose) mediating insulin secretion than BoNT/C1 (17), which cleaves the syntaxins. This suggests that each of these SNARE proteins has additional distinct actions within the insulin secretory process that are differentially coupled to these signaling pathways. In fact, these SNARE proteins are potential substrates for phosphorylation by Ca2+/calmodulin-dependent protein kinase II, PKA, and protein kinase C (PKC) (13, 31, 37). Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of insulin exocytosis (1). However, mutations of Ca2+/calmodulin-dependent protein kinase II phosphorylation sites in VAMP-2 had no effect on Ca2+-stimulated insulin secretion (28). If these kinases act directly on the SNARE proteins to induce exocytosis, then SNAREs other than VAMP-2 must be the substrates (13, 31, 37). In support of this possibility, SNAP-25 in the insulinoma RIN cells was phosphorylated via an undefined tyrosine kinase mechanism after glucose or GLP-1 stimulation (49). SNAP-25 also contains consensus sequences for PKC and PKA phosphorylation (13, 31, 37). However, mutations of putative phosphorylation sites within SNAP-25 and syntaxins were recently shown to have little effect on SNARE complex assembly in vitro (31). Nonetheless, these researchers indicated that phosphorylation of SNARE proteins may still affect SNARE complex function through their actions on other proteins, such as synaptotagmins (31), which are also present in beta -cells (43). Functional assays are, therefore, necessary to demonstrate how these SNARE proteins are actually coupled to these signaling pathways.

We have indeed demonstrated that cleavage of VAMP-2 and SNAP-25 (15) by TeTx-LC and BoNT/A-LC, respectively, inhibited insulin secretion evoked by glucose, Ca2+ (by KCl-evoked membrane depolarization), and cAMP. The residual insulin secretion observed with the wells transfected with TeTx-LC or BoNT/A-LC were likely due to cells that were either not transfected or that expressed lower levels of TeTx-LC. The levels of reduction in evoked insulin secretion of 45-60% in these population cell studies are remarkably comparable to the transfection efficiency of 40-50%, based on the reported beta -galactosidase gene assays and the confocal microscopy studies (Fig. 2). An inhibition of secretion could be due to blockade of the secretion step or an alteration in the biosynthesis of secretory vesicles. It has been shown previously that SNARE proteins participate in vesicle formation at the stage of vesicle coating (20, 35). However, we do not believe that the reduction of secretion is related to an inhibition of granule synthesis since, as shown in Fig. 3, TeTx-expressing cells possess more insulin-containing granules, consistent with a block in release. To more precisely determine the extent and distinct kinetic components of insulin exocytosis affected, we have employed the method of patch-clamp capacitance measurements on cells expressing the highest levels of neurotoxins. With the use of single cell capacitance measurements, we showed that VAMP-2 and SNAP-25 might act at distinct sites within the Ca2+-evoked insulin exocytic machinery.

Differences between the inhibitory effects of BoNT/A and TeTx on neurotransmitter release have been observed many times over the past two decades in a variety of systems (2, 5, 18, 21, 24, 25 47). With the use of permeabilized chromaffin cells, Lawrence et al. (18) showed that BoNT/A partially inhibited MgATP-primed neurotransmitter release, whereas TeTx completely abolished it. Moreover, the inhibition by BoNT/A seen in autaptic hippocampal cultures resembled that obtained by reduced extracellular Ca2+ (24). Interestingly, reduced Ca2+ levels reduced the probability of release in these systems. More recently, by combining flash photolysis with capacitance measurements in chromaffin cells, Xu and colleagues (47) showed that BoNT/A affected the rate of both the exocytic burst phase and slow phase of catecholamine release. Together, these results suggest that the COOH-terminal nine amino acids of SNAP-25 appear to couple to the Ca2+ sensor to facilitate rapid exocytosis. Here we show that in the insulinoma HIT-T15 cells, BoNT/A and TeTx also inhibit insulin secretion differently. As in neurons and chromaffin cells, BoNT/A intoxication can be overcome by a stronger depolarization that results in more Ca2+ influx across the Ca2+ channels. In contrast, TeTx-LC inhibition of secretion was complete and could not be overcome by the stronger depolarization. BoNT/A-LC and TeTx-LC expression, rather than injection into cells (25, 47), ensures complete cleavage of SNAP-25 and VAMP-2, and, therefore, any lack of inhibition cannot be simply attributed to the uncleaved SNAREs in toxin-insensitive complexes.

cAMP and downstream PKA activation are known to increase the release probability of insulin secretory granules in the readily releasable pool and also increase the rate of refilling of this pool by accelerated mobilization of insulin secretory granules from the reserve pool (30). This is the postulated mechanism by which cAMP/PKA activation amplifies both the exocytic burst and sustained phase of insulin secretion (30), the latter of which involves the refilling of the readily releasable pool. These enhancements of the insulin exocytotic machinery by cAMP/PKA activation, even in the presence of maximal depolarization-induced Ca2+ influx to increase priming and mobilization of insulin granules, could not reverse the inhibitory effects of TeTx-LC expression in beta -cells. SNAP-25 and its ability to form SNARE complexes also plays a critical role in the sustained phase of secretion in chromaffin cells, which involves priming and granule pool mobilization steps (42, 47, 48). Here we show that cAMP/PKA activation is able to overcome the BoNT/A-LC inhibition, suggesting that a PKA-mediated mechanism may be able to rescue the unstable SNARE complex that is perhaps formed by the truncated SNAP-25 and enable it to still mediate exocytic membrane fusion (albeit a less efficient process). SNAP-25 indeed possesses PKA phosphorylation sites within this truncated segment (13, 31, 37), which might positively alter its binding to the cognate SNAREs to form the putative complex(es) that specifically affect these exocytic steps. Alternatively, another protein substrate for PKA, such as alpha -SNAP (13), could modulate the kinetics of priming and mobilization of insulin granule pools (46). Together, our results demonstrate that increased Ca2+ influx and/or cAMP/PKA activation can overcome the inhibitory effects of BoNT/A-LC cleavage of SNAP-25 but not TeTx-LC cleavage of VAMP-2.

Our recent report that levels of SNARE proteins, including VAMP-2, SNAP-25, and syntaxin 1 were diminished in islets of obese diabetic fa/fa rats, a model of type 2 diabetes, led us to postulate that this could contribute to the basal hypersecretion as well as the dampened insulin secretion in response to the high-glucose concentration (3). Patients with type 2 diabetes exhibit a similar presentation of basal hyperinsulinemia and low-insulin secretory responses to the postprandial high-glucose concentrations. GLP-1, likely by PKA-dependent mechanisms, was able to bypass these secretory defects in these patients and normalized insulin secretion (11, 14). It therefore behooves us and others to determine which steps within the dysregulated insulin exocytotic machinery (i.e., exocytotic fusion, priming, and mobilization of insulin granule pools) in these diabetic islet beta -cells, which are coupled to the Ca2+- and cAMP-signaling pathways, are specifically affected by the depletion of SNARE proteins. Such novel insights may lead to the identification of new therapeutic targets.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the Juvenile Diabetes Research Foundation, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-55160, and by grants from the Canadian Diabetes Association (to H. Gaisano) and the Canadian Institutes for Health Research (to H. Gaisano and A. M. Salapatek).


    FOOTNOTES

* X. Huang, Y. Kang, and E. A. Pasyk contributed equally to this work.

H. Gaisano is a Research Scholar of the American Gastroenterology Association/Pharmacia and Upjohn. A. M. Salapatek is a recipient of a scholarship jointly funded by Eli Lilly, Banting and Best Diabetes Centre of the University of Toronto, and the Canadian Institutes of Health Research. W. S. Trimble is a Canadian Institutes of Health Research Scientist.

Address for reprint requests and other correspondence: H. Y. Gaisano, Rm. 7226, Medical Sciences Bldg., Univ. of Toronto, Toronto, Ontario, Canada M5S 1A8 (E-mail: herbert.gaisano{at}utoronto.ca).

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.

Received 14 March 2000; accepted in final form 8 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ammala, C, Eliasson L, Bokvist K, Berggren PO, Honkanen RE, Sjopholm A, and Rorsman P. Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of exocytosis in mouse pancreatic beta -cells. Proc Natl Acad Sci USA 91: 4343-4347, 1994[Abstract].

2.   Ashton, AC, and Dolly JO. Characterization of the inhibitory action of botulinum neurotoxin type A on the release of several transmitters from rat cerebrocortical synaptosomes. J Neurochem 50: 1808-1816, 1988[ISI][Medline].

3.   Chan, CB, MacPhail RM, Sheu L, Wheeler M, and Gaisano HY. beta Cell hypertrophy in fa/fa rats is associated with basal glucose hypersensitivity and reduced SNARE protein expression. Diabetes 48: 997-1005, 1999[Abstract].

4.   Chapman, ER, An S, Barton N, and Jahn R. SNAP-25, a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils. J Biol Chem 269: 27427-27432, 1994[Abstract/Free Full Text].

5.   Dreyer, F, and Schmitt A. Transmitter release in tetanus and botulinum A toxin-poisoned mammalian motor endplates and its dependence on nerve stimulation and temperature. Pflügers Arch 399: 228-234, 1983[ISI][Medline].

6.   Drucker, DJ. Glucagon-like peptides. Diabetes 47: 159-169, 1998[Abstract].

7.   Eisel, U, Reynolds K, Riddick M, Zimmer A, Niemann H, and Zimmer A. Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis. EMBO J 12: 3365-3372, 1993[Abstract].

8.   Fujita-Yoshigaki, J, Dohke Y, Hara-Yokoyama M, Kamata Y, Kocaki S, Furuyama S, and Sugiya H. Vesicle-associated membrane protein 2 is essential for cAMP-regulated exocytosis in rat parotid acinar cells. J Biol Chem 271: 13130-13134, 1996[Abstract/Free Full Text].

9.   Gaisano, HY, Sheu L, Foskett JK, and Trimble WS. Tetanus toxin-light chain cleaves a vesicle-associated membrane protein (VAMP) isoform 2 in rat pancreatic zymogen granules and inhibits enzyme secretion. J Biol Chem 269: 17062-17066, 1994[Abstract/Free Full Text].

10.   Gaisano, HY, Sheu L, Wong PC, Klip A, and Trimble WS. SNAP23 is located in the basolateral plasma membrane of rat pancreatic acinar cells. FEBS Lett 414: 298-302, 1997[ISI][Medline].

11.   Gutniak, M, Orskov C, Holst JJ, Ahren B, and Efendic S. Antidiabetogenic effects of GLP-1 (7-36) amide in normal subjects and patients with diabetes mellitus. N Engl J Med 326: 1316-1322, 1992[Abstract].

12.   Hayashi, T, McMahon H, Yamasaki S, Binz T, Hata Y, and Sudhof TC. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J 13: 5051-5061, 1994[Abstract].

13.   Hirling, H, and Scheller RH. Phosphorylation of synaptic vesicle proteins: modulation of the alpha SNAP interaction with the core complex. Proc Natl Acad Sci USA 93: 11945-11949, 1996[Abstract/Free Full Text].

14.   Holz, GG, Kuhtreiber WM, and Habener JF. Pancreatic beta cells are rendered glucose-competent by insulinotropic hormone glucagon-like peptide 1 (7-37). Nature 361: 363-365, 1993.

15.   Huang, XH, Wheeler MB, Kang YH, Lukacs G, Sheu L, Trimble WS, and Gaisano HY. Truncated SNAP-25 (1-197), like botulinum neurotoxin A inhibits insulin secretion from HIT-T15 insulinoma cells. Mol Endocrinol 12: 1060-1070, 1998[Abstract/Free Full Text].

16.   Jacobson, G, Bean AJ, Scheller RH, Juntti-Berggren L, Deeney JT, Berggren P-O, and Meister B. Identification of synaptic proteins and their isoform RNAs in compartments of pancreatic endocrine cells. Proc Natl Acad Sci USA 91: 12487-12491, 1994[Abstract/Free Full Text].

17.   Lang, J, Zhang H, Vaidyanathan VV, Sadoul K, Niemann H, and Wollheim CB. Transient expression of botulinum neurotoxin C1 light chain differentially inhibits calcium and glucose induced insulin secretion in clonal beta cells. FEBS Lett 419: 13-17, 1997[ISI][Medline].

18.   Lawrence, GW, Weller U, and Dolly JO. Botulinum A and the light chain of tetanus toxins inhibit distinct stages of MgATP-dependent catecholamine exocytosis from permeabilized chromaffin cells. Eur J Biochem 222: 325-333, 1994[Abstract].

19.   Link, E, Edelman L, Chou JH, Binz T, Yamasaki S, Eisel U, Baumert M, Sudhof TC, Niemann H, and Jahn R. Tetanus toxin action: inhibition of neurotransmitter release linked to synaptobrevin proteolysis. Biochem Biophys Res Commun 189: 1017-1023, 1992[ISI][Medline].

20.   Matsuoka, K, Morimitsu Y, Uchida K, and Schekman R. Coat assembly directs v-SNARE concentration into synthetic COPII vesicles. Mol Cell 2: 703-708, 1998[ISI][Medline].

21.   Molgo, J, Comella JX, Angaut-Petit D, Pecot-Dechavassine M, Tabti N, Faille L, Mallart A, and Thesleff S. Presynaptic actions of botulinal neurotoxins at vertebrate neuromuscular junctions. J Physiol (Paris) 84: 152-166, 1990[Medline].

22.   Niemann, H, Blasi J, and Jahn R. Clostridial neurotoxins: new tools for dissecting exocytosis. Trends Cell Biol 4: 179-185, 1994.

23.   Okamoto, Y, Ishida H, Tsuura Y, Yasuda K, Kato S, Matsubara H, Nishimura M, Mizuno H, Ikeda H, and Seino Y. Hyperresponse in Ca2+-induced insulin release from electrically permeabilized pancreatic islets of diabetic GK rats and its defective augmentation by glucose. Diabetologia 38: 772-778, 1995[ISI][Medline].

24.   Owe-Larsson, B, Kristensson K, Hill RH, and Brodin L. Distinct effects of clostridial toxins on activity-dependent modulation of autaptic responses in cultured hippocampal neurons. Eur J Neurosci 9: 1773-1777, 1997[ISI][Medline].

25.   Penner, R, Neher E, and Dreyer F. Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells. Nature 324: 719-721, 1986.

27.   Rajan, AS, Aguilar-Bryan L, Nelson DA, Yaney GC, Ysu WH, Kunze DL, and Boyd E. Ion channels and insulin secretion. Diabetes Care 13: 340-363, 1990[Abstract].

28.   Regazzi, R, Sadoul K, Meda P, Kelly RB, Halban PA, and Wollheim CB. Mutational analysis of VAMP domains implicated in Ca2+-induced insulin exocytosis. EMBO J 15: 6951-6959, 1996[Abstract].

29.   Regazzi, R, Wollheim CB, Lang J, Theler JM, Rossetto O, Montecucco C, Sadoul K, Weller U, Plamer M, and Thorens B. VAMP-2 and cellubrevin are expressed in pancreatic beta cells and are essential for Ca2+ but not for GTPgamma S-induced insulin secretion. EMBO J 12: 2723-2730, 1995[Abstract].

30.   Renstrom, E, Eliasson L, and Rorsman P. Protein kinase A-dependent and -independent stimulation of exocytosis by cAMP in mouse pancreatic beta cells. J Physiol 502: 105-118, 1997[Abstract].

31.   Risinger, C, and Bennett MB. Differential phosphorylation of syntaxin and SNAP-25 isoforms. J Neurosci 72: 614-624, 1999.

32.   Rothman, JE, and Wieland FT. Protein sorting by transport vesicles. Science 272: 227-234, 1996[Abstract].

33.   Sadoul, S, Lang J, Montecucco C, Weller U, Regazzi R, Catsicas S, Wollheim CB, and Halban PA. SNAP-25 is expressed in islets of Langerhans and is involved in insulin release. J Cell Biol 128: 1019-1028, 1995[Abstract].

34.   Salapatek, AM, MacDonald P, Gaisano HY, and Wheeler MB. Mutations to the third cytoplasmic domain of the glucagon-like peptide 1 (GLP-1) receptor can functionally uncouple from GLP-1-stimulated insulin secretion in HIT-T15 cells. Mol Endocrinol 13: 1305-1317, 1999[Abstract/Free Full Text].

35.   Salem, N, Aundez V, Horng JT, and Kelly RB. A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex. Nat Neurosci 1: 551-556, 1998[ISI][Medline].

36.   Sato, Y, Henquin M, and Henquin JC. Relative contribution of Ca2+-dependent and Ca2+-independent mechanisms to the regulation of insulin secretion by glucose. FEBS Lett 421: 115-119, 1998[ISI][Medline].

37.   Shimazaki, Y, Nishik T, Omori A, Sekiguchi M, Kamata Y, Kozaki S, and Takahashi M. Phosphorylation of SNAP-25. J Biol Chem 271: 14548-14553, 1996[Abstract/Free Full Text].

38.   Sudhof, TC. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375: 645-653, 1995[ISI][Medline].

39.   Sweeney, ST, Broadie K, Jeane J, Niemann H, and O'Kane CJ. Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioural defects. Neuron 14: 341-351, 1995[ISI][Medline].

40.   Trimble, WS, Gray TS, Elferink LA, Wilson MC, and Scheller RH. Distinct patterns of expression of two VAMP genes within the rat brain. J Neurosci 10: 1380-1387, 1990[Abstract].

41.   Weber, T, Zemelman BV, McNew JA, Westermann B, Gmachi M, Parlati F, Sollner TH, and Rothman JE. SNAREpins: minimal machinery for membrane fusion. Cell 92: 759-772, 1998[ISI][Medline].

42.   Wei, S, Xu T, Ashery U, Kollewe A, Matti U, Antonin W, Rettig J, and Neher E. Exocytotic mechanism studied by truncated and zero layer mutants of the C-terminus of SNAP-25. EMBO J 19: 1279-1289, 2000[Abstract/Free Full Text].

43.   Wheeler, MB, Sheu L, Ghai M, Bouquillon A, Grondin G, Weller U, Beaudoin AR, Bennett MK, Trimble WS, and Gaisano HY. Characterization of SNARE protein expression in beta cell lines and pancreatic islets. Endocrinology 137: 1340-1348, 1996[Abstract].

44.   Williams, JA, and Yule DI. Stimulus-secretion coupling in pancreatic acinar cells. In: The Pancreas: Biology, Pathobiology and Disease, edited by Go VLW, DiMagno E, Gardner JP, Lebenthal E, Reber HA, and Scheele G.. New York: Raven, 1993, p. 167-170.

45.   Wollheim, CB, Lang J, and Regazzi R. The exocytotic process of insulin secretion and its regulation by Ca2+ and G-proteins. Diabetes Metab Rev 4: 276-297, 1996.

46.   Xu, T, Ashery U, Burgoyne RD, and Neher E. Early requirement for alpha SNAP and NSF in the secretory cascade in chromaffin cells. EMBO J 18: 3293-3304, 1999[Abstract/Free Full Text].

47.   Xu, T, Binz T, Niemann H, and Neher E. Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nat Neurosci 1: 192-200, 1998[ISI][Medline].

48.   Xu, T, Rammner B, Margittai M, Artalejo AR, Neher E, and Jahn R. Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis. Cell 99: 713-722, 1999[ISI][Medline].

49.   Zhou, J, and Egan J. SNAP-25 is phosphorylated by glucose and GLP-1 in RIN 1046-38 cells. Biochem Biophys Res Commun 238: 297-300, 1997[ISI][Medline].


Am J Physiol Cell Physiol 281(3):C740-C750
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society