Apparsundaram S, Galli A, DeFelice L J, Hartzell H C, Blakely R D
Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
J Pharmacol Exp Ther. 1998 Nov;287(2):733-43.
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 (beta-PMA) treatment. Inhibition of PKC by antagonists and downregulation of PKC by chronic treatment with phorbol esters abolishes beta-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.
利用SK-N-SH细胞，我们观察到，毒蕈碱型乙酰胆碱受体被乙酰甲胆碱（MCh）激活后，会迅速且选择性地降低左旋去甲肾上腺素（l-NE）的转运能力（Vmax），而去甲肾上腺素（NE）的米氏常数（Km）几乎没有变化，并且在电流钳直接监测下对膜电位没有明显影响。在相同的时间范围内，MCh处理会降低完整细胞中[3H]尼索西汀结合位点的密度（Bmax），但在总膜组分中则不会，这与通过转运体隔离介导的转运能力丧失一致，而不是内在转运活性或蛋白质降解的变化。佛波酯（β-PMA）处理后，NE转运和[3H]尼索西汀结合能力也出现类似变化。拮抗剂对蛋白激酶C（PKC）的抑制以及长期用佛波酯处理使PKC下调，可消除β-PMA介导的效应，但只能部分阻断MCh诱导的效应。毒蕈碱型乙酰胆碱受体和PKC的激活均不需要细胞外钙离子（Ca++）来降低去甲肾上腺素转运体（NET）的活性。相反，在无钙培养基中用Ca++/ATP酶拮抗剂毒胡萝卜素处理细胞，可消除MCh调节中对星形孢菌素不敏感的成分。在无钙培养基中应用[1, 2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸四（乙酰氧基甲基）酯]能够消除MCh对NET的调节，这进一步证实了这些发现。尽管它们可能有助于NET的基础表达，但我们无法证明CaMKII、PKA或一氧化氮相关途径参与了MCh的调节。总之，这些发现1）为G蛋白偶联受体介导的儿茶酚胺转运调节提供了证据，2）揭示了细胞内对Ca++敏感、PKC依赖和非依赖的途径，这些途径用于调节NET的表达，3）表明MChR和PKC激活后NE转运能力的降低涉及NET蛋白的重新分布。
