ACUTE REGULATION OF NOREPINEPHRINE TRANSPORT - I - PROTEIN-KINASE-C-LINKED MUSCARINIC RECEPTORS INFLUENCE TRANSPORT CAPACITY AND TRANSPORTER DENSITY IN SK-N-SH CELLS

Using SK-N-SH cells, we observe that muscarinic acetylcholine receptor activation by methacholine (MCh) rapidly and selectively diminishes I -NE transport capacity (Vmax) with little or no change in norepinephri ne (NE) K-m and without apparent effects on membrane potential monitor ed directly under current clamp. Over the same time frame, MCh exposur e reduces the density of [H-3]nisoxetine binding sites (Bmax) in intac t cells but not in total membrane fractions, consistent with a loss of transport capacity mediated by sequestration of transporters rather t han changes in intrinsic transport activity or protein degradation. Si milar changes in NE transport and [H-3]nisoxetine binding capacity are observed after phorbol ester (beta-PMA) treatment. Inhibition of PKC by antagonists and downregulation of PKC by chronic treatment with pho rbol esters abolishes beta-PMA-mediated effects but produce only a par tial blockade of MCh-induced effects. Neither muscarinic acetylcholine receptor nor PKC activation require extracellular Ca++ to diminish NE T activity. In contrast, treatment of cells with the Ca++/ATPase antag onist, thapsigargin in Ca++-free medium, eliminates the staurosporine- insensitive component of MCh regulation. These findings were further c orroborated by the ability of 2-bis(o-amino-phenoxy)ethane-N,N,N',N'-t etraacetic acid tetra(acetoxymethyl)ester application in Ca++-free med ium to abolish NET regulation by MCh. Although they may contribute to basal NET expression, we could not implicate CaMKII-, PKA- or nitric o xide-linked pathways in MCh regulation. Together, these findings I) pr ovide evidence in support of G-protein coupled receptor-mediated regul ation of catecholamine transport, 2) reveal intracellular Ca++-sensiti ve, PKC-dependent and -independent pathways that serve to regulate NET expression and 3) indicate that the diminished capacity for NE transp ort evident after mAChR and PKC activation involves a redistribution o f NET protein.