From the Center for Neuroscience and Pain Research, Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030.
From the Center for Neuroscience and Pain Research, Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030
J Biol Chem. 2014 Nov 7;289(45):31111-20. doi: 10.1074/jbc.M114.600320. Epub 2014 Sep 24.
Microtubule-stabilizing agents, such as paclitaxel (Taxol), are effective chemotherapy drugs for treating many cancers, and painful neuropathy is a major dose-limiting adverse effect. Cation-chloride cotransporters, such as Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) and K(+)-Cl(-) cotransporter-2 (KCC2), critically influence spinal synaptic inhibition by regulating intracellular chloride concentrations. Here we show that paclitaxel treatment in rats significantly reduced GABA-induced membrane hyperpolarization and caused a depolarizing shift in GABA reversal potential of dorsal horn neurons. However, paclitaxel had no significant effect on AMPA or NMDA receptor-mediated glutamatergic input from primary afferents to dorsal horn neurons. Paclitaxel treatment significantly increased protein levels, but not mRNA levels, of NKCC1 in spinal cords. Inhibition of NKCC1 with bumetanide reversed the paclitaxel effect on GABA-mediated hyperpolarization and GABA reversal potentials. Also, intrathecal bumetanide significantly attenuated hyperalgesia and allodynia induced by paclitaxel. Co-immunoprecipitation revealed that NKCC1 interacted with β-tubulin and β-actin in spinal cords. Remarkably, paclitaxel increased NKCC1 protein levels at the plasma membrane and reduced NKCC1 levels in the cytosol of spinal cords. In contrast, treatment with an actin-stabilizing agent had no significant effect on NKCC1 protein levels in the plasma membrane or cytosolic fractions of spinal cords. In addition, inhibition of the motor protein dynein blocked paclitaxel-induced subcellular redistribution of NKCC1, whereas inhibition of kinesin-5 mimicked the paclitaxel effect. Our findings suggest that increased NKCC1 activity contributes to diminished spinal synaptic inhibition and neuropathic pain caused by paclitaxel. Paclitaxel disrupts intracellular NKCC1 trafficking by interfering with microtubule dynamics and associated motor proteins.
微管稳定剂，如紫杉醇（Taxol），是治疗多种癌症的有效化疗药物，但会引起痛性周围神经病变，这是主要的剂量限制不良反应。阳离子-氯离子共转运体，如 Na(+)-K(+)-2Cl(-) 共转运蛋白-1（NKCC1）和 K(+)-Cl(-) 共转运蛋白-2（KCC2），通过调节细胞内氯离子浓度，对脊髓突触抑制有重要影响。我们的研究表明，紫杉醇处理可显著降低大鼠背角神经元 GABA 诱导的膜超极化，并导致 GABA 反转电位去极化。然而，紫杉醇对初级传入纤维到背角神经元的 AMPA 或 NMDA 受体介导的谷氨酸能输入没有显著影响。紫杉醇处理显著增加了脊髓中 NKCC1 的蛋白水平，但对 NKCC1 的 mRNA 水平没有影响。用布美他尼抑制 NKCC1 可逆转紫杉醇对 GABA 介导的超极化和 GABA 反转电位的作用。鞘内给予布美他尼也可显著减轻紫杉醇引起的痛觉过敏和痛觉异常。共免疫沉淀显示 NKCC1 与脊髓中的β-微管蛋白和β-肌动蛋白相互作用。值得注意的是，紫杉醇增加了 NKCC1 蛋白在质膜上的水平，减少了 NKCC1 在脊髓胞浆中的水平。相比之下，用肌动蛋白稳定剂处理对 NKCC1 在质膜或胞浆部分的蛋白水平没有显著影响。此外，抑制动力蛋白 dynein 可阻止紫杉醇诱导的 NKCC1 亚细胞重新分布，而抑制 kinesin-5 则模拟了紫杉醇的作用。我们的研究结果表明，NKCC1 活性的增加导致紫杉醇引起的脊髓突触抑制和神经病理性疼痛。紫杉醇通过干扰微管动力学和相关的运动蛋白，破坏细胞内 NKCC1 的运输。
