Department of Pharmacology & Toxicology, University of Mississippi Medical Center, Jackson, MS 39216, USA.
Neurochem Int. 2010 May-Jun;56(6-7):760-7. doi: 10.1016/j.neuint.2010.02.011. Epub 2010 Feb 20.
Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [(3)H]NE uptake capacity, an effect characterized by a robust decrease in the V(max) of the transport of [(3)H]NE. As expected, reserpine did not displace the binding of [(3)H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [(3)H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [(3)H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca(2+)/Ca(2+)-calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [(3)H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, alpha-methyl-p-tyrosine, increased [(3)H]NE uptake and eliminated the inhibitory effects of reserpine on [(3)H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca(2+)-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.
用囊泡单胺转运体（VMAT）抑制剂利血平处理大鼠会耗尽去甲肾上腺素（NE）并调节去甲肾上腺素转运体（NET）的表达。本研究使用培养的细胞研究了利血平调节 NET 的分子机制。暴露于利血平的大鼠 PC12 细胞仅 5 分钟即可减少 [(3)H]NE 的摄取能力，这种作用的特征是 [(3)H]NE 转运的 V(max) 显著降低。正如预期的那样，利血平不会置换膜匀浆中 [(3)H]nisoxetine 与 NET 的结合。在具有降低儿茶酚胺储存能力的 SK-N-SH 细胞中，利血平降低 [(3)H]NE 摄取的效力显著降低。利血平对转染大鼠 NET（293-hNET）的 HEK-293 细胞或缺乏儿茶酚胺储存囊泡的细胞中 [(3)H]NE 的摄取没有影响。利血平对 NET 的调节与 NET 从细胞表面的运输无关。预先暴露于几种已知调节 NET 的细胞内信号转导途径的抑制剂（包括 Ca(2+)/Ca(2+)-钙调蛋白依赖性激酶和蛋白激酶 A、C 和 G）不会影响利血平降低 [(3)H]NE 摄取的能力。用儿茶酚胺耗竭剂 α-甲基-对酪氨酸处理 PC12 细胞会增加 [(3)H]NE 的摄取并消除利血平对 [(3)H]NE 摄取的抑制作用。利血平通过非竞争性抑制 NET 活性，这一过程需要儿茶酚胺储存囊泡，揭示了一种修饰 NET 功能的新药理学方法。对利血平作用的分子性质的进一步表征可能导致开发用于治疗已知受益于传统竞争性 NET 抑制剂治疗的疾病的替代治疗策略。
