| 1. |
- Braun, Matthias, et al.
(författare)
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GABAB-receptor activation inhibits exocytosis in rat pancreatic {beta}-cells by G-protein-dependent activation of calcineurin.
- 2004
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Ingår i: Journal of Physiology. - : Wiley. - 1469-7793 .- 0022-3751. ; 559:2, s. 397-409
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the regulation of hormone secretion from rat pancreatic islets by the GABAB receptors (GABABRs). Inclusion of the specific GABABR antagonist CGP 55845 in the extracellular medium increased glucose-stimulated insulin secretion 1.6-fold but did not affect the release of glucagon and somatostatin. Conversely, addition of the GABABR agonist baclofen inhibited glucose-stimulated insulin secretion by ∼60%. Using RT-PCR, transcription of GABABR1a-c,f and GABABR2 subunits was detected in β-cells. Measurements of membrane currents and cell capacitance were applied to single β-cells to investigate the mechanisms by which GABABR activation inhibits insulin secretion. In perforated-patch measurements, baclofen inhibited exocytosis elicited by 500-ms voltage-clamp depolarizations to 0 mV by ≤ 80% and voltage-gated Ca2+ entry by only ∼30%. Both effects were concentration-dependent with IC50 values of ∼2 μm. The inhibitory action of baclofen was abolished in the presence of CGP 55845. The ability of baclofen to suppress exocytosis was prevented by pre-treatment with pertussis toxin and by inclusion of GDPβS in the intracellular medium, and became irreversible in the presence of GTPγS as expected for a process involving inhibitory G-proteins (Gi/o-proteins). The inhibitory effect of baclofen resulted from activation of the serine/threonine protein phosphatase calcineurin and pre-treatment with cyclosporin A or intracellular application of calcineurin autoinhibitory peptide abolished the effect. Addition of baclofen had no effect on [Ca2+]i and electrical activity in glucose-stimulated β-cells. These data indicate that GABA released from β-cells functions as an autocrine inhibitor of insulin secretion in pancreatic islets and that the effect is principally due to direct suppression of exocytosis.
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| 2. |
- Braun, Matthias, et al.
(författare)
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Regulated Exocytosis of GABA-containing Synaptic-like Microvesicles in Pancreatic {beta}-cells.
- 2004
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Ingår i: Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 123:3, s. 191-204
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Tidskriftsartikel (refereegranskat)abstract
- We have explored whether {gamma}-aminobutyric acid (GABA) is released by regulated exocytosis of GABA-containing synaptic-like microvesicles (SLMVs) in insulin-releasing rat pancreatic ß-cells. To this end, ß-cells were engineered to express GABAA-receptor Cl--channels at high density using adenoviral infection. Electron microscopy indicated that the average diameter of the SLMVs is 90 nm, that every ß-cell contains ~3,500 such vesicles, and that insulin-containing large dense core vesicles exclude GABA. Quantal release of GABA, seen as rapidly activating and deactivating Cl--currents, was observed during membrane depolarizations from -70 mV to voltages beyond -40 mV or when Ca2+ was dialysed into the cell interior. Depolarization-evoked GABA release was suppressed when Ca2+ entry was inhibited using Cd2+. Analysis of the kinetics of GABA release revealed that GABA-containing vesicles can be divided into a readily releasable pool and a reserve pool. Simultaneous measurements of GABA release and cell capacitance indicated that exocytosis of SLMVs contributes ~1% of the capacitance signal. Mathematical analysis of the release events suggests that every SLMV contains 0.36 amol of GABA. We conclude that there are two parallel pathways of exocytosis in pancreatic ß-cells and that release of GABA may accordingly be temporally and spatially separated from insulin secretion. This provides a basis for paracrine GABAergic signaling within the islet.
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| 3. |
- Eliasson, Lena, et al.
(författare)
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SUR1 Regulates PKA-independent cAMP-induced Granule Priming in Mouse Pancreatic B-cells.
- 2003
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Ingår i: Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 121:3, s. 181-197
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Tidskriftsartikel (refereegranskat)abstract
- Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS–insensitive) component correlated with a rapid increase in membrane capacitance of ~80 fF that plateaued within ~200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the Kd values were 6 and 29 µM for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1-/- mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1-/- mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl- into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane KATP-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca2+-induced exocytosis.
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| 4. |
- Rorsman, Patrik, et al.
(författare)
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The Cell Physiology of Biphasic Insulin Secretion
- 2000
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Ingår i: News in Physiological Sciences. - 1522-161X .- 0886-1714. ; 15:2, s. 72-77
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Tidskriftsartikel (refereegranskat)abstract
- Glucose-stimulated insulin secretion consists of a transient first phase followed by a sustained second phase. Diabetes (type II) is associated with abnormalities in this release pattern. Here we review the evidence that biphasic insulin secretion reflects exocytosis of two functional subsets of secretory granules and the implications for diabetes.
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| 5. |
- Wendt, Anna, et al.
(författare)
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Glucose Inhibition of Glucagon Secretion From Rat alpha-Cells Is Mediated by GABA Released From Neighboring beta-Cells.
- 2004
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Ingår i: Diabetes. - : American Diabetes Association. - 1939-327X .- 0012-1797. ; 53:4, s. 1038-1045
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Tidskriftsartikel (refereegranskat)abstract
- γ-Aminobutyric acid (GABA) has been proposed to function as a paracrine signaling molecule in islets of Langerhans. We have shown that rat β-cells release GABA by Ca2+-dependent exocytosis of synaptic-like microvesicles. Here we demonstrate that GABA thus released can diffuse over sufficient distances within the islet interstitium to activate GABAA receptors in neighboring cells. Confocal immunocytochemistry revealed the presence of GABAA receptors in glucagon-secreting α-cells but not in β- and δ-cells. RT-PCR analysis detected transcripts of α1 and α4 as well as β1–3 GABAA receptor subunits in purified α-cells but not in β-cells. In whole-cell voltage-clamp recordings, exogenous application of GABA activated Cl− currents in α-cells. The GABAA receptor antagonist SR95531 was used to investigate the effects of endogenous GABA (released from β-cells) on pancreatic islet hormone secretion. The antagonist increased glucagon secretion at 1 mmol/l glucose twofold and completely abolished the inhibitory action of 20 mmol/l glucose on glucagon release. Basal and glucose-stimulated secretion of insulin and somatostatin were unaffected by SR95531. The L-type Ca2+ channel blocker isradipine evoked a paradoxical stimulation of glucagon secretion. This effect was not observed in the presence of SR95531, and we therefore conclude that isradipine stimulates glucagon secretion by inhibition of GABA release.
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