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) 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.