Acute regulation of norepinephrine transport: I. Protein kinase C- linked muscarinic receptors influence transport capacity and transporter density in SK-N-SH cells

Academic Article


  • Using SK-N-SH cells, we observe that muscarinic acetylcholine receptor activation by methacholine (MCh) rapidly and selectively diminishes I-NE transport capacity (V(max)) with little or no change in norepinephrine (NE) K(m) 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.
  • Authors

    Pubmed Id

  • 20655992
  • Author List

  • Apparsundaram S; Galli A; DeFelice LJ; Hartzell HC; Blakely RD
  • Start Page

  • 733
  • End Page

  • 743
  • Volume

  • 287
  • Issue

  • 2