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Research ArticleArticle

Acute Regulation of Norepinephrine Transport: I. Protein Kinase C-Linked Muscarinic Receptors Influence Transport Capacity and Transporter Density in SK-N-SH Cells

Subramaniam Apparsundaram, Aurelio Galli, Louis J. DeFelice, H. Criss Hartzell and Randy D. Blakely
Journal of Pharmacology and Experimental Therapeutics November 1998, 287 (2) 733-743;
Subramaniam Apparsundaram
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Aurelio Galli
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Louis J. DeFelice
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H. Criss Hartzell
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Randy D. Blakely
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Abstract

Using SK-N-SH cells, we observe that muscarinic acetylcholine receptor activation by methacholine (MCh) rapidly and selectively diminishes l-NE transport capacity (Vmax) with little or no change in norepinephrine (NE) Km and without apparent effects on membrane potential monitored directly under current clamp. Over the same time frame, MCh exposure reduces the density of [3H]nisoxetine binding sites (Bmax) in intact cells but not in total membrane fractions, consistent with a loss of transport capacity mediated by sequestration of transporters rather than changes in intrinsic transport activity or protein degradation. Similar changes in NE transport and [3H]nisoxetine binding capacity are observed after phorbol ester (β-PMA) treatment. Inhibition of PKC by antagonists and downregulation of PKC by chronic treatment with phorbol esters abolishes β-PMA-mediated effects but produce only a partial blockade of MCh-induced effects. Neither muscarinic acetylcholine receptor nor PKC activation require extracellular Ca++ to diminish NET activity. In contrast, treatment of cells with the Ca++/ATPase antagonist, thapsigargin in Ca++-free medium, eliminates the staurosporine-insensitive component of MCh regulation. These findings were further corroborated by the ability of [1,2-bis(o-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester application in Ca++-free medium to abolish NET regulation by MCh. Although they may contribute to basal NET expression, we could not implicate CaMKII-, PKA- or nitric oxide-linked pathways in MCh regulation. Together, these findings 1) provide evidence in support of G-protein coupled receptor-mediated regulation of catecholamine transport, 2) reveal intracellular Ca++-sensitive, PKC-dependent and -independent pathways that serve to regulate NET expression and 3) indicate that the diminished capacity for NE transport evident after mAChR and PKC activation involves a redistribution of NET protein.

Footnotes

  • Send reprint requests to: Dr. Randy D. Blakely, Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232-6600.

  • ↵1 This work was supported by NINDS Award NS33373 and MH58921 to R.D.B. and NS34075 to L.J.D. and a NARSAD Young Investigator Award to A.G.

  • Abbreviations:
    BAPTA
    [1,2-bis(o-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic acid, BAPTA-AM, [1,2-bis(o-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester
    CaMK
    Ca++/calmodulin-dependent kinase
    CTX
    cholera toxin
    DA
    dopamine
    4-DAMP
    4-diphenylacetoxy-N-methylpiperidine methiodide
    KN-93
    [2-[N-(2-hydroxyethyl)-N-(4-methoxybezenesulfonyl]amino-N-(4-chlorocinnamul)-N-methylbenzylamine
    GPCR
    G-protein coupled receptor
    mAChR
    muscarinic acetylcholine receptor
    MCh
    methacholine
    NE
    norepinephrine
    NET
    norepinephrine transporters
    NOS
    nitric oxide synthase
    PMA
    phorbol 12-myristate 13-acetate
    PKC
    protein kinase C
    PTX
    pertussis toxin
    KRH
    Krebs-Ringers HEPES buffer
    SDS
    sodium dioctyl sulfate
    U-0521
    3,4-dihydroxymethylpropiophenone
    • Received January 13, 1998.
    • Accepted June 8, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics
Vol. 287, Issue 2
1 Nov 1998
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Research ArticleArticle

Acute Regulation of Norepinephrine Transport: I. Protein Kinase C-Linked Muscarinic Receptors Influence Transport Capacity and Transporter Density in SK-N-SH Cells

Subramaniam Apparsundaram, Aurelio Galli, Louis J. DeFelice, H. Criss Hartzell and Randy D. Blakely
Journal of Pharmacology and Experimental Therapeutics November 1, 1998, 287 (2) 733-743;

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Research ArticleArticle

Acute Regulation of Norepinephrine Transport: I. Protein Kinase C-Linked Muscarinic Receptors Influence Transport Capacity and Transporter Density in SK-N-SH Cells

Subramaniam Apparsundaram, Aurelio Galli, Louis J. DeFelice, H. Criss Hartzell and Randy D. Blakely
Journal of Pharmacology and Experimental Therapeutics November 1, 1998, 287 (2) 733-743;
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