Acute regulation of norepinephrine transport: I. protein kinase C-linked muscarinic receptors influence transport capacity and transporter density in SK-N-SH cells.
Acute regulation of norepinephrine transport: I. protein kinase C-linked muscarinic receptors influence transport capacity and transporter density in SK-N-SH cells.
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发表时间：
1998-11
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通讯作者：
S. Apparsundaram;A. Galli;L. DeFelice;H. Hartzell;R. Blakely
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作者：
S. Apparsundaram;A. Galli;L. DeFelice;H. Hartzell;R. Blakely
使用 SK-N-SH 细胞，我们观察到醋甲胆碱 (MCh) 激活毒蕈碱乙酰胆碱受体会快速选择性地降低 l-NE 转运能力 (Vmax)，而去甲肾上腺素 (NE) Km 几乎没有变化或没有变化，并且对在电流钳下直接监测的膜电位没有明显影响。在同一时间范围内，MCh 暴露降低了完整细胞中 [3H]nisoxetine 结合位点 (Bmax) 的密度，但不降低总膜组分中的密度，这与转运蛋白隔离介导的转运能力损失一致，而不是内在转运活性或蛋白质降解的变化。佛波酯（β-PMA）处理后观察到 NE 转运和 [3H]nisoxetine 结合能力的类似变化。拮抗剂对 PKC 的抑制以及佛波酯长期治疗对 PKC 的下调消除了 β-PMA 介导的作用，但仅部分阻断了 MCh 诱导的作用。毒蕈碱乙酰胆碱受体和 PKC 激活都不需要细胞外 Ca++ 来降低 NET 活性。相比之下，用 Ca++/ATP 酶拮抗剂（无 Ca++ 培养基中的毒胡萝卜素）处理细胞，消除了 MCh 调节中星形孢菌素不敏感的成分。 [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）酯在无 Ca++ 培养基中应用以消除 MCh 的 NET 调节的能力进一步证实了这些发现。尽管它们可能有助于基础 NET 表达，但我们无法将 CaMKII、PKA 或一氧化氮相关途径与 MCh 调节联系起来。总之，这些发现 1) 提供了支持 G 蛋白偶联受体介导的儿茶酚胺转运调节的证据，2) 揭示了细胞内 Ca++ 敏感、PKC 依赖和独立的途径，用于调节 NET 表达，3) 表明 mAChR 和 PKC 激活后明显减少的 NE 转运能力涉及 NET 蛋白的重新分配。
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.