Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
Neuroscience. 2013 Feb 12;231:206-15. doi: 10.1016/j.neuroscience.2012.11.044. Epub 2012 Dec 10.
Neuronal electrical activity has been known to affect the viability of neurons in the central nervous system. Here we show that long-lasting membrane depolarization induced by elevated extracellular K(+) recruits nitric oxide (NO)/soluble guanylyl cyclase/protein kinase G signaling pathway, induces 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP)-mediated protein S-guanylation, and confers dopaminergic neuroprotection. Treatment of primary mesencephalic cell cultures with 1-methyl-4-phenylpyridinium (MPP(+)) for 72 h decreased the number of dopaminergic neurons, whereas the cell loss was markedly inhibited by elevated extracellular concentration of K(+) (+40 mM). The neuroprotective effect of elevated extracellular K(+) was significantly attenuated by tetrodotoxin (a Na(+) channel blocker), amlodipine (a voltage-dependent Ca(2+) channel blocker), N(ω)-nitro-l-arginine methyl ester (l-NAME) (a nitric oxide synthase inhibitor), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (a soluble guanylyl cyclase inhibitor), and KT5823 or Rp-8-bromo-β-phenyl-1,N(2)-ethenoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPS) (protein kinase G inhibitors). Elevated extracellular K(+) increased 8-nitro-cGMP production resulting in the induction of protein S-guanylation in cells in mesencephalic cultures including dopaminergic neurons. In addition, exogenous application of 8-nitro-cGMP protected dopaminergic neurons from MPP(+) cytotoxicity, which was prevented by zinc protoporphyrin IX, an inhibitor of heme oxygenase-1 (HO-1). Zinc protoporphyrin IX also inhibited the neuroprotective effect of elevated extracellular K(+). On the other hand, KT5823 or Rp-8-Br-PET-cGMPS did not inhibit the induction of HO-1 protein expression by 8-nitro-cGMP, although these protein kinase G inhibitors abrogated the neuroprotective effect of 8-nitro-cGMP. These results suggest that protein S-guanylation (leading to HO-1 induction) as well as canonical protein kinase G signaling pathway plays an important role in NO-mediated, activity-dependent dopaminergic neuroprotection.
神经元的电活动已被证实会影响中枢神经系统神经元的存活。在这里，我们表明，由细胞外液 K(+)升高引起的持续的膜去极化会招募一氧化氮（NO）/可溶性鸟苷酸环化酶/蛋白激酶 G 信号通路，诱导 8-硝基鸟苷 3'，5'-环单磷酸（8-硝基-cGMP）介导的蛋白质 S-鸟苷酸化，并赋予多巴胺能神经保护作用。用 1-甲基-4-苯基吡啶（MPP(+））处理原代中脑神经细胞培养物 72 小时会减少多巴胺能神经元的数量，而升高细胞外液 K(+)浓度（+40 mM）则明显抑制细胞丢失。升高细胞外液 K(+)的神经保护作用被河豚毒素（一种钠离子通道阻断剂）、氨氯地平（一种电压依赖性钙通道阻断剂）、N(ω)-硝基-L-精氨酸甲酯（l-NAME）（一种一氧化氮合酶抑制剂）、1H-[1,2,4]恶二唑[4,3-a]喹喔啉-1-酮（ODQ）（一种可溶性鸟苷酸环化酶抑制剂）和 KT5823 或 Rp-8-溴-β-苯基-1,N(2)-亚乙基鸟苷 3'，5'-环单磷酸硫代酯（Rp-8-Br-PET-cGMPS）（蛋白激酶 G 抑制剂）显著减弱。升高细胞外液 K(+)增加 8-硝基-cGMP 的产生，导致包括多巴胺能神经元在内的中脑神经细胞中的蛋白质 S-鸟苷酸化诱导。此外，外源性应用 8-硝基-cGMP 可保护多巴胺能神经元免受 MPP(+)细胞毒性，该作用被血红素加氧酶-1（HO-1）抑制剂锌原卟啉 IX 所阻止。锌原卟啉 IX 还抑制了升高细胞外液 K(+)的神经保护作用。另一方面，KT5823 或 Rp-8-Br-PET-cGMPS 并未抑制 8-硝基-cGMP 诱导的 HO-1 蛋白表达，尽管这些蛋白激酶 G 抑制剂消除了 8-硝基-cGMP 的神经保护作用。这些结果表明，蛋白质 S-鸟苷酸化（导致 HO-1 诱导）以及经典蛋白激酶 G 信号通路在 NO 介导的、活性依赖性多巴胺能神经保护中发挥重要作用。
