Leptin activation of ATP-sensitive K+ (KATP) channels in rat CRI-G1 insulinoma cells involves disruption of the actin cytoskeleton

J. Harvey, S. C. Hardy, A. J. Irving, M. L. J. Ashford

    Research output: Contribution to journalArticle

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    Abstract

    1. The role of the cytoskeleton in leptin-induced activation of ATP-sensitive K+ (KATP) channels was examined in rat CRI-G1 insulin-secreting cells using patch clamp and fluorescence imaging techniques. 2. In whole cell recordings, dialysis with the actin filament stabiliser phalloidin (10 µM) prevented KATP channel activation by leptin. 3. Application of the actin filament destabilising agents deoxyribonuclease type 1 (DNase 1; 50 µg ml-1) or cytochalasin B (10 µM) to intact cells or inside-out membrane patches also increased KATP channel activity in a phalloidin-dependent manner. 4. The anti-microtubule agents nocodazole (10 microM) and colchicine (100 µM) had no effect on KATP channel activity. 5. Fluorescence staining of the cells with rhodamine-conjugated phalloidin revealed rapid disassembly of actin filaments by cytochalasin B and leptin, the latter action being prevented by the phosphoinositide 3 (PI 3)-kinase inhibitor LY 294002. 6. Activation of KATP channels by the PI 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) was also prevented by phalloidin. This is consistent with the notion that leptin activates KATP channels in these cells by an increase in PtdIns(3,4,5)P3 or a similar 3-phosphorylated phosphoinositol lipid, resulting in actin filament disruption.

    Original languageEnglish
    Pages (from-to)95-107
    Number of pages13
    JournalJournal of Physiology
    Volume527
    Issue number1
    DOIs
    Publication statusPublished - 2000

    Fingerprint

    KATP Channels
    Insulinoma
    Leptin
    Actin Cytoskeleton
    Adenosine Triphosphate
    Phalloidine
    Cytochalasin B
    1-Phosphatidylinositol 4-Kinase
    Deoxyribonucleases
    Nocodazole
    2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
    Optical Imaging
    Colchicine
    Insulin-Secreting Cells
    Patch-Clamp Techniques
    Cytoskeleton
    Microtubules
    Dialysis
    Fluorescence
    Staining and Labeling

    Cite this

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    abstract = "1. The role of the cytoskeleton in leptin-induced activation of ATP-sensitive K+ (KATP) channels was examined in rat CRI-G1 insulin-secreting cells using patch clamp and fluorescence imaging techniques. 2. In whole cell recordings, dialysis with the actin filament stabiliser phalloidin (10 µM) prevented KATP channel activation by leptin. 3. Application of the actin filament destabilising agents deoxyribonuclease type 1 (DNase 1; 50 µg ml-1) or cytochalasin B (10 µM) to intact cells or inside-out membrane patches also increased KATP channel activity in a phalloidin-dependent manner. 4. The anti-microtubule agents nocodazole (10 microM) and colchicine (100 µM) had no effect on KATP channel activity. 5. Fluorescence staining of the cells with rhodamine-conjugated phalloidin revealed rapid disassembly of actin filaments by cytochalasin B and leptin, the latter action being prevented by the phosphoinositide 3 (PI 3)-kinase inhibitor LY 294002. 6. Activation of KATP channels by the PI 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) was also prevented by phalloidin. This is consistent with the notion that leptin activates KATP channels in these cells by an increase in PtdIns(3,4,5)P3 or a similar 3-phosphorylated phosphoinositol lipid, resulting in actin filament disruption.",
    author = "J. Harvey and Hardy, {S. C.} and Irving, {A. J.} and Ashford, {M. L. J.}",
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    Leptin activation of ATP-sensitive K+ (KATP) channels in rat CRI-G1 insulinoma cells involves disruption of the actin cytoskeleton. / Harvey, J.; Hardy, S. C.; Irving, A. J.; Ashford, M. L. J.

    In: Journal of Physiology, Vol. 527, No. 1, 2000, p. 95-107.

    Research output: Contribution to journalArticle

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    AU - Harvey, J.

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    N2 - 1. The role of the cytoskeleton in leptin-induced activation of ATP-sensitive K+ (KATP) channels was examined in rat CRI-G1 insulin-secreting cells using patch clamp and fluorescence imaging techniques. 2. In whole cell recordings, dialysis with the actin filament stabiliser phalloidin (10 µM) prevented KATP channel activation by leptin. 3. Application of the actin filament destabilising agents deoxyribonuclease type 1 (DNase 1; 50 µg ml-1) or cytochalasin B (10 µM) to intact cells or inside-out membrane patches also increased KATP channel activity in a phalloidin-dependent manner. 4. The anti-microtubule agents nocodazole (10 microM) and colchicine (100 µM) had no effect on KATP channel activity. 5. Fluorescence staining of the cells with rhodamine-conjugated phalloidin revealed rapid disassembly of actin filaments by cytochalasin B and leptin, the latter action being prevented by the phosphoinositide 3 (PI 3)-kinase inhibitor LY 294002. 6. Activation of KATP channels by the PI 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) was also prevented by phalloidin. This is consistent with the notion that leptin activates KATP channels in these cells by an increase in PtdIns(3,4,5)P3 or a similar 3-phosphorylated phosphoinositol lipid, resulting in actin filament disruption.

    AB - 1. The role of the cytoskeleton in leptin-induced activation of ATP-sensitive K+ (KATP) channels was examined in rat CRI-G1 insulin-secreting cells using patch clamp and fluorescence imaging techniques. 2. In whole cell recordings, dialysis with the actin filament stabiliser phalloidin (10 µM) prevented KATP channel activation by leptin. 3. Application of the actin filament destabilising agents deoxyribonuclease type 1 (DNase 1; 50 µg ml-1) or cytochalasin B (10 µM) to intact cells or inside-out membrane patches also increased KATP channel activity in a phalloidin-dependent manner. 4. The anti-microtubule agents nocodazole (10 microM) and colchicine (100 µM) had no effect on KATP channel activity. 5. Fluorescence staining of the cells with rhodamine-conjugated phalloidin revealed rapid disassembly of actin filaments by cytochalasin B and leptin, the latter action being prevented by the phosphoinositide 3 (PI 3)-kinase inhibitor LY 294002. 6. Activation of KATP channels by the PI 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) was also prevented by phalloidin. This is consistent with the notion that leptin activates KATP channels in these cells by an increase in PtdIns(3,4,5)P3 or a similar 3-phosphorylated phosphoinositol lipid, resulting in actin filament disruption.

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