Smith R G, Alexianu M E, Crawford G, Nyormoi O, Stefani E, Appel S H
Department of Neurology, Baylor College of Medicine, Houston, TX 77030.
Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3393-7. doi: 10.1073/pnas.91.8.3393.
Patients with amyotrophic lateral sclerosis possess antibodies (ALS IgGs) that bind to L-type skeletal muscle voltage-gated calcium channels (VGCCs) and inhibit L-type calcium current. To determine whether interaction of ALS IgGs with neuronal VGCCs might influence motoneuron survival, we used a motoneuron-neuroblastoma hybrid (VSC 4.1) cell line expressing binding sites for inhibitors of L-, N-, and P-type VGCCs. Using direct viable cell counts, quantitation of propidium iodide- and fluorescein diacetate-labeled cells, and lactate dehydrogenase release to assess cell survival, we document that ALS IgG kills 40-70% of cAMP-differentiated VSC 4.1 cells within 2 days. ALS IgG-mediated cytotoxicity is dependent on extracellular calcium and is prevented by peptide antagonists of N- or P-type VGCCs but not by dihydropyridine modulators of L-type VGCCs. Preincubating IgG with purified intact L-type VGCC or with isolated VGCC alpha 1 subunit also blocks ALS IgG-mediated cytotoxicity. These results suggest that ALS IgG may directly lead to motoneuron cell death by a mechanism requiring extracellular calcium and mediated by neuronal-type calcium channels.
肌萎缩侧索硬化症患者体内存在能与L型骨骼肌电压门控钙通道（VGCCs）结合并抑制L型钙电流的抗体（ALS IgGs）。为了确定ALS IgGs与神经元VGCCs的相互作用是否会影响运动神经元的存活，我们使用了一种运动神经元-神经母细胞瘤杂交（VSC 4.1）细胞系，该细胞系表达L型、N型和P型VGCCs抑制剂的结合位点。通过直接活细胞计数、碘化丙啶和荧光素二乙酸酯标记细胞的定量分析以及乳酸脱氢酶释放来评估细胞存活情况，我们发现ALS IgG在2天内可杀死40%-70%的经cAMP分化的VSC 4.1细胞。ALS IgG介导的细胞毒性依赖于细胞外钙，可被N型或P型VGCCs的肽拮抗剂阻断，但不能被L型VGCCs的二氢吡啶调节剂阻断。用纯化的完整L型VGCC或分离的VGCC α1亚基预孵育IgG也可阻断ALS IgG介导的细胞毒性。这些结果表明，ALS IgG可能通过一种需要细胞外钙且由神经元型钙通道介导的机制直接导致运动神经元细胞死亡。
