Role of tyrosine phosphorylation in leptin activation of ATP-sensitive K+ channels in the rat insulinoma cell line CRI-G1

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    Abstract

    1. Using whole-cell and cell-attached recording configurations, the role of phosphorylation in leptin activation of ATP-sensitive K+ (KATP) channels was examined in the rat CRI-G1 insulinoma cell line. 2. Whole-cell current clamp recordings demonstrated that, following dialysis with the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP; 3-5 mM), the leptin-induced hyperpolarization and increase in K+ conductance were completely inhibited. 3. Under current clamp conditions, application of the broad-spectrum protein kinase inhibitor H-7 (10 µM) had no effect on the resting membrane potential or slope conductance of CRI-G1 insulinoma cells and did not occlude the actions of leptin. 4. Application of the tyrosine kinase inhibitors genistein (10 µM), tyrphostin B42 (10 µM) and herbimycin A (500 nM) all resulted in activation of KATP channels. In cell-attached recordings, the presence of tyrphostin B42 (10 microM) in the pipette solution activated tolbutamide-sensitive KATP channels in CRI-G1 cells. In contrast, the inactive analogues of genistein and tyrphostin B42 were without effect. 5. The serine/threonine-specific protein phosphatase inhibitors okadaic acid (50 nM) and cyclosporin A (1 µM) did not prevent or reverse leptin activation of KATP channels. In contrast, whole-cell dialysis with the tyrosine phosphatase inhibitor orthovanadate (500 µM) prevented the actions of both leptin and tyrphostin B42. 6. In conclusion, leptin activation of KATP channels appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. This process is likely to occur prior to activation of phosphoinositide 3-kinase (PI 3-kinase) as wortmannin prevented activation of KATP channels by tyrphostin B42.

    Original languageEnglish
    Pages (from-to)47-61
    Number of pages15
    JournalJournal of Physiology
    Volume510
    Issue number1
    DOIs
    Publication statusPublished - 1998

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    Insulinoma
    KATP Channels
    Leptin
    Tyrosine
    Adenosine Triphosphate
    Phosphorylation
    Cell Line
    Adenylyl Imidodiphosphate
    Genistein
    Protein-Tyrosine Kinases
    Dialysis
    1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
    Okadaic Acid
    Tolbutamide
    1-Phosphatidylinositol 4-Kinase
    Vanadates
    Phosphoprotein Phosphatases
    Protein Kinase Inhibitors
    Phosphoric Monoester Hydrolases
    Membrane Potentials

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    title = "Role of tyrosine phosphorylation in leptin activation of ATP-sensitive K+ channels in the rat insulinoma cell line CRI-G1",
    abstract = "1. Using whole-cell and cell-attached recording configurations, the role of phosphorylation in leptin activation of ATP-sensitive K+ (KATP) channels was examined in the rat CRI-G1 insulinoma cell line. 2. Whole-cell current clamp recordings demonstrated that, following dialysis with the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP; 3-5 mM), the leptin-induced hyperpolarization and increase in K+ conductance were completely inhibited. 3. Under current clamp conditions, application of the broad-spectrum protein kinase inhibitor H-7 (10 µM) had no effect on the resting membrane potential or slope conductance of CRI-G1 insulinoma cells and did not occlude the actions of leptin. 4. Application of the tyrosine kinase inhibitors genistein (10 µM), tyrphostin B42 (10 µM) and herbimycin A (500 nM) all resulted in activation of KATP channels. In cell-attached recordings, the presence of tyrphostin B42 (10 microM) in the pipette solution activated tolbutamide-sensitive KATP channels in CRI-G1 cells. In contrast, the inactive analogues of genistein and tyrphostin B42 were without effect. 5. The serine/threonine-specific protein phosphatase inhibitors okadaic acid (50 nM) and cyclosporin A (1 µM) did not prevent or reverse leptin activation of KATP channels. In contrast, whole-cell dialysis with the tyrosine phosphatase inhibitor orthovanadate (500 µM) prevented the actions of both leptin and tyrphostin B42. 6. In conclusion, leptin activation of KATP channels appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. This process is likely to occur prior to activation of phosphoinositide 3-kinase (PI 3-kinase) as wortmannin prevented activation of KATP channels by tyrphostin B42.",
    author = "J. Harvey and Ashford, {M. L. J.}",
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    TY - JOUR

    T1 - Role of tyrosine phosphorylation in leptin activation of ATP-sensitive K+ channels in the rat insulinoma cell line CRI-G1

    AU - Harvey, J.

    AU - Ashford, M. L. J.

    PY - 1998

    Y1 - 1998

    N2 - 1. Using whole-cell and cell-attached recording configurations, the role of phosphorylation in leptin activation of ATP-sensitive K+ (KATP) channels was examined in the rat CRI-G1 insulinoma cell line. 2. Whole-cell current clamp recordings demonstrated that, following dialysis with the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP; 3-5 mM), the leptin-induced hyperpolarization and increase in K+ conductance were completely inhibited. 3. Under current clamp conditions, application of the broad-spectrum protein kinase inhibitor H-7 (10 µM) had no effect on the resting membrane potential or slope conductance of CRI-G1 insulinoma cells and did not occlude the actions of leptin. 4. Application of the tyrosine kinase inhibitors genistein (10 µM), tyrphostin B42 (10 µM) and herbimycin A (500 nM) all resulted in activation of KATP channels. In cell-attached recordings, the presence of tyrphostin B42 (10 microM) in the pipette solution activated tolbutamide-sensitive KATP channels in CRI-G1 cells. In contrast, the inactive analogues of genistein and tyrphostin B42 were without effect. 5. The serine/threonine-specific protein phosphatase inhibitors okadaic acid (50 nM) and cyclosporin A (1 µM) did not prevent or reverse leptin activation of KATP channels. In contrast, whole-cell dialysis with the tyrosine phosphatase inhibitor orthovanadate (500 µM) prevented the actions of both leptin and tyrphostin B42. 6. In conclusion, leptin activation of KATP channels appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. This process is likely to occur prior to activation of phosphoinositide 3-kinase (PI 3-kinase) as wortmannin prevented activation of KATP channels by tyrphostin B42.

    AB - 1. Using whole-cell and cell-attached recording configurations, the role of phosphorylation in leptin activation of ATP-sensitive K+ (KATP) channels was examined in the rat CRI-G1 insulinoma cell line. 2. Whole-cell current clamp recordings demonstrated that, following dialysis with the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP; 3-5 mM), the leptin-induced hyperpolarization and increase in K+ conductance were completely inhibited. 3. Under current clamp conditions, application of the broad-spectrum protein kinase inhibitor H-7 (10 µM) had no effect on the resting membrane potential or slope conductance of CRI-G1 insulinoma cells and did not occlude the actions of leptin. 4. Application of the tyrosine kinase inhibitors genistein (10 µM), tyrphostin B42 (10 µM) and herbimycin A (500 nM) all resulted in activation of KATP channels. In cell-attached recordings, the presence of tyrphostin B42 (10 microM) in the pipette solution activated tolbutamide-sensitive KATP channels in CRI-G1 cells. In contrast, the inactive analogues of genistein and tyrphostin B42 were without effect. 5. The serine/threonine-specific protein phosphatase inhibitors okadaic acid (50 nM) and cyclosporin A (1 µM) did not prevent or reverse leptin activation of KATP channels. In contrast, whole-cell dialysis with the tyrosine phosphatase inhibitor orthovanadate (500 µM) prevented the actions of both leptin and tyrphostin B42. 6. In conclusion, leptin activation of KATP channels appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. This process is likely to occur prior to activation of phosphoinositide 3-kinase (PI 3-kinase) as wortmannin prevented activation of KATP channels by tyrphostin B42.

    U2 - 10.1111/j.1469-7793.1998.047bz.x

    DO - 10.1111/j.1469-7793.1998.047bz.x

    M3 - Article

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    SP - 47

    EP - 61

    JO - Journal of Physiology

    JF - Journal of Physiology

    SN - 0022-3751

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    ER -