Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic β-cell ATP-sensitive K+ currents

Zünkler, Bernd J.; Wos-Maganga, Maria; Panten, U.

doi:10.1016/j.bcp.2003.12.011

Cited by 34 publications

(33 citation statements)

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“…Insulin secretion and calcium levels were each measured in intact islet ␤-cells by using distinct and direct methods. Together with the identification of K ATP channels at insulin granules (21,47,50), the results provide evidence for a second, granule-localized Fig. 5.…”

Section: Discussionsupporting

confidence: 63%

“…Glibenclamide is distinguished from other antidiabetic sulfonylureas, not only by its superior secretagogue potency but also by its exceptional ability to be internalized within ␤-cells (23,25,26). Furthermore, glibenclamide has been shown to localize to high-affinity sites of the insulin secretory granule membrane (7,34), which recently have been identified as K ATP channel subunits (21,47,50). Of functional relevance, SUR1 knockout mice, which have no K ATP channels, exhibit chronically elevated ␤-cell calcium level yet no detectable secondphase insulin release by high glucose, unless cholinergic modulatory pathways are stimulated (15,44).…”

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confidence: 99%

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Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane K_ATPchannels

Geng¹,

Li²,

Bottino³

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

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Drain P. Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATP channels. Am J Physiol Endocrinol Metab 293: E293-E301, 2007. First published April 3, 2007; doi:10.1152/ajpendo.00016.2007.-Understanding mechanisms by which glibenclamide stimulates insulin release is important, particularly given recent promising treatment by glibenclamide of permanent neonatal diabetic subjects. Antidiabetic sulfonylureas are thought to stimulate insulin secretion solely by inhibiting their high-affinity ATP-sensitive potassium (KATP) channel receptors at the plasma membrane of ␤-cells. This normally occurs during glucose stimulation, where ATP inhibition of plasmalemmal KATP channels leads to voltage activation of L-type calcium channels for rapidly switching on and off calcium influx, governing the duration of insulin secretion. However, growing evidence indicates that sulfonylureas, including glibenclamide, have additional KATP channel receptors within ␤-cells at insulin granules. We tested nonpermeabilized ␤-cells in mouse islets for glibenclamide-stimulated insulin secretion mediated by granule-localized KATP channels by using conditions that bypass glibenclamide action on plasmalemmal KATP channels. High-potassium stimulation evoked a sustained rise in ␤-cell calcium level but a transient rise in insulin secretion. With continued high-potassium depolarization, addition of glibenclamide dramatically enhanced insulin secretion without affecting calcium. These findings support the hypothesis that glibenclamide, or an increased ATP/ADP ratio, stimulates insulin secretion in part by binding at granule-localized KATP channels that functionally contribute to sustained second-phase insulin secretion.␤-cells; glibenclamide; permanent neonatal diabetes; exocytosis; endocytosis; adenosine 5Ј-triphosphate-sensitive potassium channels TO UNDERSTAND THE MECHANISMS underlying insulin secretion by glibenclamide, its functional sites of action in the ␤-cell of the endocrine pancreas must be identified. The plasmalemmal ATP-sensitive potassium (K ATP ) channel site for glibenclamide-stimulated insulin release is well studied (3). In glucose-stimulated insulin secretion (GSIS), the plasmalemmal K ATP channel transduces the signal of elevated glucose metabolism into calcium influx across the plasmalemma. The calcium then completes the final exocytic step by fusing previously primed insulin granules to the plasmalemma, experimentally observed as a transient first-phase insulin release (36).Generally, K ATP channels couple glucose metabolism and membrane electrical signaling (1, 2, 10, 16, 27). K ATP channels are ideal receptors for ligands signaling changes in glucose metabolism, because they are designed as sensors of adenine nucleotide levels. ATP binding to the K ir 6.2 subunit of the K ATP channel inhibits the potassium efflux that otherwise maintains the electrically negative resting state of the cell. ADP binding to the sulfonylurea receptor 1 (SUR1) subunit of the K ATP channel can antagonize the in...

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Section: Discussionsupporting

confidence: 63%

mentioning

confidence: 99%

Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane K_ATPchannels

Geng¹,

Li²,

Bottino³

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

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“…This localisation could be demonstrated in cells whose cytoplasmic staining was low, either spontaneously (variations between beta cells) or after imposed degranulation with glibenclamide, because of an increased contrast between the plasma membrane and the cytoplasm. Like others [16,19,20], we found the glibenclamide-BODIPY technique inadequate to localise SUR1 in the plasma membrane. In this respect our ultrastructural studies were also disappointing because of the relatively poor preservation of membranes by the technical conditions imposed by SUR1 immunodetection at the electron microscopic level.…”

Section: Discussionsupporting

confidence: 63%

“…This non-specific labelling in control islets was far less than the 80% of total labelling measured in RINm5F cells [20]. We attribute this difference to the higher density of insulin granules in normal beta cells compared with the cell line [42], hence to a greater proportion of specific binding sites.…”

Section: Discussionmentioning

confidence: 89%

“…Moreover, studies using 3 H-labelled glibenclamide (known as glyburide in the USA and Canada) have shown that, unlike other sulfonylureas, glibenclamide is taken up by beta cells [17] and binds to an intracellular protein that is much more abundant (more than tenfold) than in the plasma membrane [18]. These conclusions were extended recently by monitoring the binding of glibenclamide labelled with fluorescent dipyrromethane boron difluoride (BODIPY-FL) to islet cells [16,19], a technique that is, however, complicated by a significant degree of non-specific binding due to the lipophilicity of the probe [20].…”

Section: Introductionmentioning

confidence: 99%

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Morphological localisation of sulfonylurea receptor 1 in endocrine cells of human, mouse and rat pancreas

et al. 2007

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Aims/hypothesis Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of ATP-sensitive K channels in beta cells. Morphological methods (immunohistochemistry and sulfonylurea binding) were used to establish the cellular and subcellular location of SUR1 in human and rodent islets. Results In the human, mouse and rat pancreas, all endocrine cells of the islets were immunolabelled with an anti-SUR1 antibody, whereas tissues containing SUR2 were consistently negative, as were those from Sur1 (also known as Abcc8) −/− mice. In beta cells of the three species, the plasma membrane was distinctly stained, but SUR1 was mainly present over the cytoplasm, with an intensity that varied between cells. Electron microscopy showed that SUR1 was immunolocalised in insulin, glucagon and somatostatin granules. In rat beta cells degranulated by in vivo treatment with glibenclamide (known as glyburide in the USA and Canada), the insulin and SUR1 staining intensity was similarly decreased by ∼45%, whereas SUR1 staining was not changed in non-beta cells. In all islet cells, binding of glibenclamide labelled with fluorescent dipyrromethane boron difluoride (BODIPY-FL) was punctate over the cytoplasm, compatible with the labelling of endocrine granules. A faint labelling persisted in Sur1 −/− mice, but it was not different from that obtained with BODIPY-FL alone used as negative control. Conclusions/interpretation Our study immunolocalised SUR1 in alpha, beta and delta cells of human, mouse and rat islets, and for the first time visualised it in the plasma membrane. We also show that SUR1 is abundant in endocrine granules, where its function remains to be established. No specific sulfonylurea-binding sites other than SUR1 are identified in islet cells by the glibenclamide-BODIPY-FL technique.

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A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part II: Reagents for Non‐Vesicular Organelles

2013

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A wide range of fluorescent dyes and reagents exist for labeling organelles in live and fixed cells. Choosing between them can sometimes be confusing, and optimization for many of them can be challenging. Presented here is a discussion on the commercially‐available reagents that have shown the most promise for each organelle of interest, including endoplasmic reticulum/nuclear membrane, Golgi apparatus, mitochondria, nucleoli, and nuclei, with an emphasis on localization of these structures for microscopy. Included is a featured reagent for each structure with a recommended protocol, troubleshooting guide, and example image. Curr. Protoc. Cytom. 66:12.31.1‐12.34.24. © 2013 by John Wiley & Sons, Inc.

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Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic β-cell ATP-sensitive K+ currents

Cited by 34 publications

References 18 publications

Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane K_ATPchannels

Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane K_ATPchannels

Morphological localisation of sulfonylurea receptor 1 in endocrine cells of human, mouse and rat pancreas

A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part II: Reagents for Non‐Vesicular Organelles

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Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic β-cell ATP-sensitive K+ currents

Cited by 34 publications

References 18 publications

Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATPchannels

Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATPchannels

Morphological localisation of sulfonylurea receptor 1 in endocrine cells of human, mouse and rat pancreas

A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part II: Reagents for Non‐Vesicular Organelles

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Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane K_ATPchannels

Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane K_ATPchannels