| 1. |
- Jeans, Alexander F., et al.
(författare)
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A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse
- 2007
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 104:7, s. 2431-2436
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Tidskriftsartikel (refereegranskat)abstract
- The neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25) The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic beta-cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease.
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| 2. |
- Ma, Xiaosong, et al.
(författare)
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Glucagon stimulates exocytosis in mouse and rat pancreatic {alpha} cells by binding to glucagon receptors.
- 2005
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Ingår i: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 19:1, s. 198-212
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Tidskriftsartikel (refereegranskat)abstract
- Glucagon, secreted by the pancreatic alpha-cells, stimulates insulin secretion from neighboring beta-cells by cAMP- and protein kinase A (PKA)-dependent mechanisms, but it is not known whether glucagon also modulates its own secretion. We have addressed this issue by combining recordings of membrane capacitance (to monitor exocytosis) in individual alpha-cells with biochemical assays of glucagon secretion and cAMP content in intact pancreatic islets, as well as analyses of glucagon receptor expression in pure alpha-cell fractions by RT-PCR. Glucagon stimulated cAMP generation and exocytosis dose dependently with an EC50 of 1.6-1.7 nm. The stimulation of both parameters plateaued at concentrations beyond 10 nm of glucagon where a more than 3-fold enhancement was observed. The actions of glucagon were unaffected by the GLP-1 receptor antagonist exendin-(9-39) but abolished by des-His1-[Glu9]-glucagon-amide, a specific blocker of the glucagon receptor. The effects of glucagon on alpha-cell exocytosis were mimicked by forskolin and the stimulatory actions of glucagon and forskolin on exocytosis were both reproduced by intracellular application of 0.1 mm cAMP. cAMP-potentiated exocytosis involved both PKA-dependent and -independent (resistant to Rp-cAMPS, an Rp-isomer of cAMP) mechanisms. The presence of the cAMP-binding protein cAMP-guanidine nucleotide exchange factor II in alpha-cells was documented by a combination of immunocytochemistry and RT-PCR and 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP, a cAMP-guanidine nucleotide exchange factor II-selective agonist, mimicked the effect of cAMP and augmented rapid exocytosis in a PKA-independent manner. We conclude that glucagon released from the alpha-cells, in addition to its well-documented systemic effects and paracrine actions within the islet, also represents an autocrine regulator of alpha-cell function.
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| 3. |
- MacDonald, Patrick, et al.
(författare)
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Regulated Exocytosis and Kiss-and-Run of Synaptic-Like Microvesicles in INS-1 and Primary Rat {beta}-Cells.
- 2005
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Ingår i: Diabetes. - : American Diabetes Association. - 1939-327X .- 0012-1797. ; 54:3, s. 736-743
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Tidskriftsartikel (refereegranskat)abstract
- We have applied cell-attached capacitance measurements to investigate whether synaptic-like microvesicles (SLMVs) undergo regulated exocytosis in insulinoma and primary pancreatic beta-cells. SLMV and large dense-core vesicle (LDCV) exocytosis was increased 1.6- and 2.4-fold upon stimulation with 10 mmol/l glucose in INS-1 cells. Exocytosis of both types of vesicles was coupled to Ca(2+) entry through l-type channels. Thirty percent of SLMV exocytosis in INS-1 and rat beta-cells was associated with transient capacitance increases consistent with kiss-and-run. Elevation of intracellular cAMP (5 micromol/l forskolin) increased SLMV exocytosis 1.6-fold and lengthened the duration of kiss-and-run events in rat beta-cells. Experiments using isolated inside-out patches of INS-1 cells revealed that the readily releasable pool (RRP) of SLMVs preferentially undergoes kiss-and-run exocytosis (67%), is proportionally larger than the LDCV RRP, and is depleted more quickly upon Ca(2+) stimulation. We conclude that SLMVs undergo glucose-regulated exocytosis and are capable of high turnover. Following kiss-and-run exocytosis, the SLMV RRP may be reloaded with gamma-aminobutyric acid and undergo several cycles of exo- and endocytosis. Our observations support a role for beta-cell SLMVs in a synaptic-like function of rapid intra-islet signaling.
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| 4. |
- Vikman, Jenny, et al.
(författare)
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Antibody inhibition of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 reduces rapid exocytosis in insulin-secreting cells.
- 2006
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Ingår i: Journal of Molecular Endocrinology. - : Bioscientifica. - 1479-6813 .- 0952-5041. ; 36:3, s. 503-515
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Tidskriftsartikel (refereegranskat)abstract
- SNARE-proteins (soluble NSF-attachment protein receptor) are important for Ca2+-dependent exocytosis. We have used capacitance measurements and confocal imaging to dissect the role of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 in rapid exocytosis in insulin-secreting pancreatic ß-cells. Following immunoneutralization of syntaxin 1 and SNAP-25, exocytosis was strongly reduced and associated with a marked reduction in the size of the readily releasable pool (RRP) by 65% and 86% in the presence of the anti-SNAP-25 and anti-syntaxin 1 antibodies respectively. The size of the immediately releasable pool (IRP), a subset of RRP in close association with the voltage-dependent Ca2+-channels, was reduced to an equal extent. The reduction in IRP correlated with slowed release kinetics and the time constant ({tau}) increased from a control value of 16 to 36 ms and 51 ms after inclusion of anti-SNAP-25 and anti-syntaxin 1 antibodies respectively in the pipette solution. We further show that SNAP-25 and syntaxin 1 aggregate in clusters along the plasma membrane. The size of these clusters was estimated to be ~300 nm and every ß-cell contained ~400 SNAP-25/syntaxin 1 clusters. Whereas the inhibitory action of the anti-syntaxin 1 antibody on exocytosis could be attributed almost entirely to suppression of the voltage-dependent Ca2+-current (–40%), the effect of the anti-SNAP-25 antibody was not mediated by decreased Ca2+-entry and is more likely due to a direct interference with the exocytotic machinery. Our data are consistent with the concept that both syntaxin 1 and SNAP-25 are required for rapid exocytosis in ß-cells.
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