Lenox R H, McNamara R K, Watterson J M, Watson D G
Department of Psychiatry, University of Florida, University of Florida College of Medicine, Gainesville, USA.
J Clin Psychiatry. 1996;57 Suppl 13:23-31; discussion 32-3.
Lithium remains a first-line treatment for the acute and prophylactic management of bipolar illness. Previous studies in our laboratory have demonstrated that chronic, but not acute, exposure to therapeutic concentrations of lithium significantly reduces the expression of the protein kinase C (PKC) substrate MARCKS (myristoylated alanine-rich C kinase substrate) in the rat hippocampus and an immortalized hippocampal cell line (HN33). The anticonvulsant drugs valproate and carbamazepine are emerging as efficacious alternative and adjunctive treatments for bipolar disorder. In the present study, we sought to determine the effects of valproate and carbamazepine on MARCKS protein levels by using our hippocampal cell model.
HN33 immortalized hippocampal cells were exposed acutely or chronically to sodium valproate 1 mM, carbamazepine 100 microM, lithium chloride 5 mM, or lithium chloride 5 mM + sodium valproate 1 mM. Additionally, cells were exposed to lithium chloride 5 mM in the absence or presence of inositol 5 microM, or sodium valproate 1 mM in the absence or presence of inositol 40 microM. After drug exposure, cells were collected, separated into soluble and membrane fractions, and MARCKS protein assayed by Western blot analysis using polyclonal rabbit antibody. Immunoreactive bands were quantitated by densitometric analysis.
We report that chronic exposure of HN33 cells to either lithium or valproate produced a time-dependent down-regulation of MARCKS protein. Maximal reduction in MARCKS levels were observed after 3 days of exposure to valproate and after 7 days of exposure to lithium. The reduction of MARCKS produced by lithium and valproate alone were additive when the two drugs were combined. The reduction in MARCKS produced by lithium was reversed by the addition of inositol to the media, whereas the reduction produced by valproate was unaffected by the addition of inositol. Carbamazepine failed to affect MARCKS protein levels at each dose and time tested.
These data provide evidence that, like lithium, chronic exposure to valproate produces a significant time-dependent down-regulation of the PKC substrate MARCKS, whereas carbamazepine is without effect. The MARCKS reduction produced by valproate appears to occur independently of inositol concentrations yet is additive with the reduction produced by lithium, which is inositol-reversible. Valproate- and lithium-induced regulation of MARCKS expression appears to be mediated by different mechanisms that may utilize PKC, and may be associated with the clinical profile of these mood stabilizers. Regulation of MARCKS expression may be associated with the prophylactic efficacy of lithium in the long-term stabilization of the recurrent affective episodes in bipolar disorder, and valproate may share this property.
锂盐仍是双相情感障碍急性发作期及预防治疗的一线用药。我们实验室之前的研究表明，长期而非短期暴露于治疗浓度的锂盐会显著降低大鼠海马体及永生化海马细胞系（HN33）中蛋白激酶C（PKC）底物MARCKS（肉豆蔻酰化富含丙氨酸的C激酶底物）的表达。抗惊厥药物丙戊酸盐和卡马西平正逐渐成为双相情感障碍有效的替代治疗及辅助治疗药物。在本研究中，我们试图利用我们的海马细胞模型来确定丙戊酸盐和卡马西平对MARCKS蛋白水平的影响。
将HN33永生化海马细胞急性或长期暴露于1 mM丙戊酸钠、100 μM卡马西平、5 mM氯化锂或5 mM氯化锂 + 1 mM丙戊酸钠中。此外，细胞在不存在或存在5 μM肌醇的情况下暴露于5 mM氯化锂，或在不存在或存在40 μM肌醇的情况下暴露于1 mM丙戊酸钠。药物暴露后，收集细胞，分离为可溶性和膜部分，并用兔多克隆抗体通过蛋白质印迹分析测定MARCKS蛋白。通过光密度分析对免疫反应条带进行定量。
我们报告，HN33细胞长期暴露于锂盐或丙戊酸盐会导致MARCKS蛋白随时间依赖性下调。暴露于丙戊酸盐3天后和暴露于锂盐7天后观察到MARCKS水平的最大降低。当两种药物联合使用时，锂盐和丙戊酸盐单独产生的MARCKS降低是相加的。向培养基中添加肌醇可逆转锂盐引起的MARCKS降低，而添加肌醇对丙戊酸盐引起的降低没有影响。在测试的每个剂量和时间，卡马西平均未能影响MARCKS蛋白水平。
这些数据表明，与锂盐一样，长期暴露于丙戊酸盐会导致PKC底物MARCKS随时间显著下调，而卡马西平则无此作用。丙戊酸盐引起的MARCKS降低似乎与肌醇浓度无关，但与锂盐引起的降低是相加的，而锂盐引起的降低是可被肌醇逆转的。丙戊酸盐和锂盐诱导的MARCKS表达调节似乎是由可能利用PKC的不同机制介导的，并且可能与这些心境稳定剂的临床特征有关。MARCKS表达的调节可能与锂盐在双相情感障碍复发性情感发作的长期稳定中的预防效果有关，丙戊酸盐可能具有相同的特性。
