| 1. |
- Barg, Sebastian, et al.
(författare)
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Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells
- 2001
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Ingår i: Biophysical Journal. - 1542-0086 .- 0006-3495. ; 81:6, s. 3308-3323
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Tidskriftsartikel (refereegranskat)abstract
- The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.
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| 2. |
- 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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| 3. |
- Gromada, J, et al.
(författare)
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ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical actiflty by glucose in wild-type and SUR1(-/-) mouse alpha-cells
- 2004
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Ingår i: Diabetes. - 0012-1797. ; 53, s. 181-189
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Konferensbidrag (refereegranskat)abstract
- Patch-clamp recordings and glucagon release measurements were combined to determine the role of plasma membrane ATP-sensitive K+ channels (K-ATP channels) in the control of glucagon secretion from mouse pancreatic alpha-cells. In wild-type mouse islets, glucose produced a concentration-dependent (half-maximal inhibitory concentration [IC50] = 2.5 mmol/l) reduction of glucagon release. Maximum inhibition (similar to50%) was attained at glucose concentrations >5 mmol/l. The sulfonylureas tolbutamide (100 mumol/l) and glibenclamide (100 nmol/l) inhibited glucagon secretion to the same extent as a maximally inhibitory concentration of glucose. In mice lacking functional KATP channels (SUR1(-/-)), glucagon secretion in the absence of glucose was lower than that observed in wild-type islets and both glucose (0-20 mmol/l) and the sulfonylureas failed to inhibit glucagon secretion. Membrane potential recordings revealed that a-cells generate action potentials in the absence of glucose. Addition of glucose depolarized the alpha-cell by similar to7 mV and reduced spike height by 30% Application of tolbutamide likewise depolarized the alpha-cell (similar to17 mV) and reduced action potential amplitude (43%). Whereas insulin secretion increased monotonically with increasing external K+ concentrations (threshold 25 mmol/l), glucagon secretion was paradoxically suppressed at intermediate concentrations (5.6-15 mmol/l), and stimulation was first detectable at > 25 mmol/l K+. In alpha-cells isolated from SUR1(-/-) mice, both tolbutamide and glucose failed to produce membrane depolarization. These effects correlated with the presence of a small (0.13 nS) sulfonylurea-sensitive conductance in wild-type but not in SUR1(-/-) a-cells. Recordings of the free cytoplasmic Ca2+ concentration ([Ca2+](i)) revealed that, whereas glucose lowered [Ca2+](i) to the same extent as application of tolbutamide, the Na+ channel blocker tetrodotoxin, or the Ca2+ channel blocker Co2+ in wild-type alpha-cells, the sugar was far less effective on [Ca2+](i) in SUR1(-/-) alpha-cells. We conclude that the K-ATP channel is involved in the control of glucagon secretion by regulating the membrane potential in the alpha-cell in a way reminiscent of that previously documented in insulin-releasing beta-cells. However, because alpha-cells possess a different complement of voltage-gated ion channels involved in action potential generation than the beta-cell, moderate membrane depolarization in alpha-cells is associated with reduced rather than increased electrical activity and secretion.
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| 4. |
- Göpel, Sven, et al.
(författare)
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Capacitance measurements of exocytosis in mouse pancreatic {alpha}-, {beta}- and {delta}-cells studied in intact islets of Langerhans.
- 2004
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Ingår i: Journal of Physiology. - : Wiley. - 1469-7793 .- 0022-3751. ; 556:3, s. 711-726
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Tidskriftsartikel (refereegranskat)abstract
- To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.
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| 5. |
- Hansson, Karin M, et al.
(författare)
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The first gamma-carboxyglutamic acid-containing contryphan - A selective L-type calcium ion channel blocker isolated from the venom of conus marmoreus
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:31, s. 32453-32463
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Tidskriftsartikel (refereegranskat)abstract
- Contryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine, and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to {gamma}-carboxyglutamyl (Gla) residues. The amino acid sequence of glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-D-Trp-His-Pro-Trp-Cys. As with most other contryphans, glacontryphan-M is amidated at the C terminus and maintains the five-residue intercysteine loop. The occurrence of a D-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a KD of 0.63 mM. Cloning of the full-length cDNA encoding glacontryphan-M revealed that the primary translation product carries an N-terminal signal/propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calcium-dependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels.
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| 6. |
- Kanno, T, et al.
(författare)
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Large dense-core vesicle exocytosis in pancreatic beta-cells monitored by capacitance measurements
- 2004
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 33:4, s. 302-311
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Tidskriftsartikel (refereegranskat)abstract
- This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic P-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other. (C) 2004 Elsevier Inc. All rights reserved.
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| 7. |
- Kong, Xiangchen, et al.
(författare)
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Glucagon-Like Peptide 1 Stimulates Insulin Secretion via Inhibiting RhoA/ROCK Signaling and Disassembling Glucotoxicity-Induced Stress Fibers
- 2014
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Ingår i: Endocrinology. - : The Endocrine Society. - 0013-7227 .- 1945-7170. ; 155:12, s. 4676-4685
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Tidskriftsartikel (refereegranskat)abstract
- Chronic hyperglycemia leads to pancreatic beta-cell dysfunction characterized by diminished glucose-stimulated insulin secretion (GSIS), but the precise cellular processes involved are largely unknown. Here we show that pancreatic beta-cells chronically exposed to a high glucose level displayed substantially increased amounts of stress fibers compared with beta-cells cultured at a low glucose level. beta-Cells at high glucose were refractory to glucose-induced actin cytoskeleton remodeling and insulin secretion. Importantly, F-actin depolymerization by either cytochalasin B or latrunculin B restored glucotoxicity-diminished GSIS. The effects of glucotoxicity on increasing stress fibers and reducing GSIS were reversed by Y-27632, a Rho-associated kinase (ROCK)-specific inhibitor, which caused actin depolymerization and enhanced GSIS. Notably, glucagon-like peptide-1-(7-36) amide (GLP-1), a peptide hormone that stimulates GSIS at both normal and hyperglycemic conditions, also reversed glucotoxicity-induced increase of stress fibers and reduction of GSIS. In addition, GLP-1 inhibited glucotoxicity-induced activation of RhoA/ROCK and thereby resulted in actin depolymerization and potentiation of GSIS. Furthermore, this effect of GLP-1 was mimicked by cAMP-increasing agents forskolin and 3-isobutyl-1-methylxanthine as well as the protein kinase A agonist 6-Bnz-cAMP-AM whereas it was abolished by the protein kinase A inhibitor Rp-Adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt. To establish a clinical relevance of our findings, we examined the association of genetic variants of RhoA/ROCK with metabolic traits in homeostasis model assessment index of insulin resistance. Several single-nucleotide polymorphisms in and around RHOA were associated with elevated fasting insulin and homeostasis model assessment index of insulin resistance, suggesting a possible role in metabolic dysregulation. Collectively these findings unravel a novel mechanism whereby GLP-1 potentiates glucotoxicity-diminished GSIS by depolymerizing F-actin cytoskeleton via protein kinase A-mediated inhibition of the RhoA-ROCK signaling pathway.
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| 8. |
- 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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| 9. |
- Ma, Xiaosong
(författare)
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Regulation of exocytosis by Ca2+ and cAMP - A study on pancreatic beta- and alpha-cells
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Type-2 diabetes is characterized by impaired insulin secretion associated with excess glucagon release. Exocytosis of insulin- or glucagon-containing granules is initiated by Ca2+-influx through voltage-dependent Ca2+-channels and is modulated by the second messengers such as cAMP. Membrane capacitance measurements were used in this study to investigate the cellular mechanisms behind cAMP-regulated Ca2+-dependent exocytosis in pancreatic beta-and alpha-cells. In pancreatic beta-cells, the secretory granules have been demonstrated to co-localize with L-type Ca2+-channels. Experiments using photorelease of caged Ca2+ revealed that exocytosis of insulin-containing granules requires an increase in [Ca2+]i with a Kd of 17 µM, which is most likely achieved in the vicinity of the Ca2+-channels. It was further proved that a close association between the alpha1c L-type Ca2+-channels and the secretory granules is a prerequisite for rapid exocytosis and ensures maximum usage of Ca2+ influx in a cell with few Ca2+-channels (~500) as is the case in the beta-cell. Pharmacologically, L-type Ca2+-channels are suppressed by organic Ca2+-channel blockers such as dihydropyridines. Glacontryphan- M was isolated from the venom of Conus Marmoreus and was detected to reduce L-type Ca2+ currents and associated exocytosis. It was discovered that this novel antagonist needs the binding of Ca2+ to the Gla-residues for its function. Ca2+-dependent exocytosis in the beta-cell is enhanced by cAMP through both PKA-independent granular priming and PKA-dependent recruitment of granules from a reserve pool. This study revealed a concentration-dependent activation of the two cAMP-signalling pathways and that 5-fold higher [cAMP]i was needed for the PKA-independent mechanism. Interestingly, the sulfonylurea receptor SUR1 was required in the regulation of cAMP-stimulated PKA-independent exocytosis. This is an additional function of SUR1 apart from being a subunit of the KATP channel. Enhancement of exocytosis by cAMP in alpha-cells was discovered to involve similar mechanisms as in the beta-cell. In the present study glucagon was detected to elevate cAMP resulting in an enhanced Ca2+-dependent exocytotic response by binding to the glucagon-receptors in the plasma membrane of the alpha-cell. The stimulatory effect of glucagon was mainly mediated by PKA-dependent mechanisms, possibly due to the inability of glucagon to elevate [cAMP]i to the sufficient level needed for activation of the PKA-independent pathway.
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| 10. |
- MacDonald, Patrick E., et al.
(författare)
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A K-ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of langerhans
- 2007
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Ingår i: PLoS Biology. - : Public Library of Science (PLoS). - 1545-7885. ; 5:6, s. 1236-1247
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Tidskriftsartikel (refereegranskat)abstract
- Glucagon, secreted from pancreatic islet a cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the a cells themselves. We examined hormone secretion and Ca2+ responses of a and b cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn (2+) signalling was blocked, but was reversed by low concentrations (1-20 mu M) of the ATP-sensitive K+ (K-ATP) channel opener diazoxide, which had no effect on insulin release or b cell responses. This effect was prevented by the K-ATP channel blocker tolbutamide (100 mu M). Higher diazoxide concentrations (>= 30 mu M) decreased glucagon and insulin secretion, and alpha-and beta-cell Ca2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (< 1 mu M) stimulated glucagon secretion, whereas high concentrations (> 10 mu M) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the K-ATP channel, inhibition of voltage-gated Na+ (TTX) and N-type Ca2+ channels (omega-conotoxin), but not L-type Ca2+ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca2+ channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an a-cell K-ATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.
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