Dahmani Souhayl, Rouelle Danielle, Gressens Pierre, Mantz Jean
Institut National de la Santé et de la Recherche Médicale U 676, Hôpital Robert Debré, Paris, France.
Anesthesiology. 2005 Nov;103(5):969-77. doi: 10.1097/00000542-200511000-00011.
Dexmedetomidine is a potent and selective alpha2-adrenoceptor agonist that exhibits a broad pattern of actions, including sedation, analgesia, and neuroprotection. Some of these actions (e.g., neuroprotection) may require targets involved in long-term cellular changes. The authors hypothesized that dexmedetomidine increases the expression of active (autophosphorylated) focal adhesion kinase (FAK), a nonreceptor tyrosine kinase playing a pivotal role in cellular plasticity and survival. Therefore, we examined the cellular mechanisms involved in this effect and its sensitivity to oxygen-glucose deprivation (OGD) in rat hippocampal slices.
The effects of dexmedetomidine on phospho-tyrosine FAK phosphorylation were studied first with or without various pharmacologic agents in normoxic conditions, and second in a model of pharmacologic preconditioning of slices subjected to 30 min of OGD followed by 1 h of reperfusion. FAK phosphorylation and caspase-3 activation were examined by immunoblotting. Neuronal death was assessed by propidium iodide fluorescence.
Dexmedetomidine produced a dose-related increase in FAK phosphorylation (187 +/- 4%, mean +/- SD, from basal level, EC50 = 0.2 microm; 95% confidence interval, 0.09-0.5 microm). This effect was stereoselective and was completely blocked by yohimbine and the combination of the cyclic monophosphate permeant analog 8 bromo cyclic monophosphate and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It was mimicked by the protein kinase A inhibitor H 89. In contrast, prazosin and the protein kinase C inhibitors chelerythrine and bisindolylmaleimide I were ineffective. OGD induced a significant increase in immunoreactivity of the cleaved caspase-3 17-kd fragment (417 +/- 22; P < 0.001), a decrease in FAK phosphorylation (78 +/- 12% of control; P < 0.05), and production of significant neuronal death. In OGD conditions, a preconditioning application of dexmedetomidine (0.2 microm, 20-min application, 3 h before anoxia) significantly reduced neuronal death and cleaved caspase-3 expression and significantly attenuated the decrease in phosphorylated FAK content. The dexmedetomidine-induced reduction in caspase-3 expression was significantly decreased by the Src tyrosine kinase inhibitor PP2.
Dexmedetomidine exhibits a preconditioning effect against ischemic injury in hippocampal slices subjected to OGD. Increase in phosphorylation of FAK via stimulation of alpha2 adrenoceptors and decrease in cleaved caspase-3 expression correlate with dexmedetomidine-induced cell survival.
右美托咪定是一种强效且选择性的α2肾上腺素能受体激动剂，具有广泛的作用模式，包括镇静、镇痛和神经保护。其中一些作用（如神经保护）可能需要参与长期细胞变化的靶点。作者推测右美托咪定可增加活性（自磷酸化）粘着斑激酶（FAK）的表达，FAK是一种非受体酪氨酸激酶，在细胞可塑性和存活中起关键作用。因此，我们研究了大鼠海马切片中参与这种效应的细胞机制及其对氧糖剥夺（OGD）的敏感性。
首先在常氧条件下，研究右美托咪定在有无各种药理剂存在时对磷酸化酪氨酸FAK磷酸化的影响；其次在一个对经受30分钟OGD随后1小时再灌注的切片进行药理预处理的模型中进行研究。通过免疫印迹检测FAK磷酸化和半胱天冬酶-3激活。通过碘化丙啶荧光评估神经元死亡。
右美托咪定使FAK磷酸化呈剂量相关增加（相对于基础水平为(187\pm4%)，均值(\pm)标准差，(EC50 = 0.2)微摩尔；95%置信区间，(0.09 - 0.5)微摩尔）。这种效应具有立体选择性，并且被育亨宾以及环一磷酸通透类似物8-溴环一磷酸与磷酸二酯酶抑制剂3-异丁基-1-甲基黄嘌呤的组合完全阻断。它被蛋白激酶A抑制剂H 89模拟。相比之下，哌唑嗪以及蛋白激酶C抑制剂白屈菜红碱和双吲哚马来酰亚胺I无效。OGD导致裂解的半胱天冬酶-3 17-kD片段的免疫反应性显著增加（(417\pm22)；P < 0.001），FAK磷酸化降低（为对照值的(78\pm12%)；P < 0.05），并产生显著的神经元死亡。在OGD条件下，右美托咪定预处理应用（(0.2)微摩尔，应用20分钟，缺氧前3小时）显著减少神经元死亡和裂解的半胱天冬酶-3表达，并显著减轻磷酸化FAK含量的降低。Src酪氨酸激酶抑制剂PP2使右美托咪定诱导的半胱天冬酶-3表达降低显著减少。
右美托咪定对经受OGD的海马切片缺血损伤具有预处理作用。通过刺激α2肾上腺素能受体使FAK磷酸化增加以及裂解的半胱天冬酶-3表达降低与右美托咪定诱导的细胞存活相关。
