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Research ArticleNEUROPHARMACOLOGY

Trafficking-Dependent and -Independent Pathways of Neurotransmitter Transporter Regulation Differentially Involving p38 Mitogen-Activated Protein Kinase Revealed in Studies of Insulin Modulation of Norepinephrine Transport in SK-N-SH Cells

Subbu Apparsundaram, Uhna Sung, Raymond D. Price and Randy D. Blakely
Journal of Pharmacology and Experimental Therapeutics November 2001, 299 (2) 666-677;
Subbu Apparsundaram
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Uhna Sung
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Raymond D. Price
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Randy D. Blakely
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Abstract

Presynaptic, cocaine- and antidepressant-sensitive norepinephrine (NE) transporters (NETs) dictate levels of extracellular NE after vesicular release. Recent studies suggest that G protein-coupled receptors linked to protein kinase C (PKC) down-regulate cell surface NET protein levels and diminish NE uptake capacity. We identified distinct phosphatidylinositol 3-OH kinase (PI3K)-linked pathways supporting basal and insulin-triggered NE transport in the human noradrenergic neuroblastoma, SK-N-SH. Acute (0–60 min) insulin treatments produced a time- and concentration-dependent stimulation of NE transport, resolved in kinetic studies as an enhancement of NE transport capacity (Vmax) without an alteration in NEKm. Basal and insulin-modulated NET activities were reduced by the tyrosine kinase inhibitor genistein and the PI3K inhibitors wortmannin and LY-294002, but not by the PKC inhibitor staurosporine. PI3K activation was found to support phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). However, basal and insulin-stimulated NET activities were differentiated by their reliance on p38 MAPK activation. Thus, the p38 MAPK inhibitor SB203580 and SB202190 abolished insulin activation of NE transport yet failed to impact basal NET activity. Moreover, p38 MAPK activation and insulin activation of NETs were found to be sensitive to external Ca2+ depletion, blockade of voltage-sensitive Ca2+ channels, and inhibition of protein phosphatase 2A. Effects of tyrosine kinase and PI3K inhibitors on basal NET uptake appear to arise from a loss of cell surface NET protein, whereas the p38 MAPK-dependent enhancement of NE transport occurs without a detectable enhancement of surface NET. Our findings establish two distinct pathways for regulation of NE uptake involving PI3K, one linked to transporter trafficking and a second linked to Ca2+-dependent, p38 MAPK phosphorylation that promotes activation of cell surface NETs.

Footnotes

  • ↵1 Current address: Department of Anatomy and Neurobiology, University of Kentucky Chandler Medical Center, Lexington, KY. 40536-0098.

  • This work was supported by National Institute of Neurological Disorders and Stroke award MH58921 and Diabetes Research Training Center pilot Grant DK20593 to R.D.B. and training grant T32 HL07323 to S.A.

  • Abbreviations:
    NE
    norepinephrine
    NET
    norepinephrine transporter
    GABA
    γ-aminobutyric acid
    CNS
    central nervous system
    PKC
    protein kinase C
    IR
    insulin receptor
    PI3K
    phosphatidylinositol 3-OH kinase
    IGF-1
    insulin-like growth factor-1
    β-PMA
    β-phorbol-12-myristate-13-acetate
    BAPTA-AM
    1,2-bis(o-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester
    fura-2 AM
    1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid acetoxy methylester
    PKB/Akt
    protein kinase B
    p38 MAPK
    p38 mitogen-activated protein kinase
    KRH
    Krebs-Ringer-HEPES
    PBS
    phosphate-buffered saline
    RIPA
    radioimmunoprecipitation assay
    PAGE
    polyacrylamide gel electrophoresis
    HBSS
    Hanks' balanced salt solution
    ANOVA
    analysis of variance
    [Ca2+]i
    intracellular calcium concentration
    ERK
    extracellular signal receptor-activated kinase
    PP2Ac
    catalytic subunit of protein phosphatase 2A
    PP1/PP2A
    protein phosphatase 1/2A
    • Received April 24, 2001.
    • Accepted August 9, 2001.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 299 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 299, Issue 2
1 Nov 2001
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Research ArticleNEUROPHARMACOLOGY

Trafficking-Dependent and -Independent Pathways of Neurotransmitter Transporter Regulation Differentially Involving p38 Mitogen-Activated Protein Kinase Revealed in Studies of Insulin Modulation of Norepinephrine Transport in SK-N-SH Cells

Subbu Apparsundaram, Uhna Sung, Raymond D. Price and Randy D. Blakely
Journal of Pharmacology and Experimental Therapeutics November 1, 2001, 299 (2) 666-677;

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Research ArticleNEUROPHARMACOLOGY

Trafficking-Dependent and -Independent Pathways of Neurotransmitter Transporter Regulation Differentially Involving p38 Mitogen-Activated Protein Kinase Revealed in Studies of Insulin Modulation of Norepinephrine Transport in SK-N-SH Cells

Subbu Apparsundaram, Uhna Sung, Raymond D. Price and Randy D. Blakely
Journal of Pharmacology and Experimental Therapeutics November 1, 2001, 299 (2) 666-677;
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