Identification of common biological markers underlying establishment of drug dependence and an attempt to develop pharmacogenomic therapy

在这个研究项目中，我们已经开展了确定药物依赖的共同生物标志物，并尝试开发药物基因组疗法。研究了小鼠脑皮质神经元短期（24小时）暴露于药物滥用后l型高压门控钙通道（Cav1）与心理依赖小鼠脑的功能变化。KC1 （30 mM）刺激[^<45>Ca^<2+>]内流进入神经元随着药物暴露时间和浓度的增加而增加。这种增强作用被Cav1抑制剂、ryanodine和IP_3受体拮抗剂完全消除。滥用药物处理的神经元中Cav1.2和Cav α2-δ蛋白升高。滥用药物24 H后[^<3>H]PN200-110结合增加是由于Kd值降低所致。心理依赖小鼠的大脑皮层和伏隔核的结合参数和蛋白表达也发生了类似的变化。这些结果表明，ryanodine受体刺激钙诱导的钙释放和随后IP_3受体的激活可诱导Cav1上调。此外，我们还研究了Cav 3亚基的辅助蛋白在乙醇（EtOH）诱导的Cav1上调中的功能参与。短期暴露于EtOH后，Cav β3亚基水平显著升高，而Cav β1、β2和β4亚基水平无明显变化。高钾刺激[^<45>Ca^<2+>]流入神经元显著增加了EtOH暴露，而这种增加在Cav β3亚基特异性的morpholino低聚物预处理的神经元中没有观察到。[^3H] P: N200-110在EtOH暴露后的Kd值下降也通过Cav β3亚基敲除而消除。心理依赖小鼠的大脑皮层也出现了类似的蛋白表达变化。这些结果表明，短期暴露于EtOH下的神经元通过增加神经元膜中Cav β3亚基蛋白的表达来上调Cav1功能。少

In this research project, we have been carried out to identify the common biological markers underlying establishment of drug dependence and an attempt to develop pharmacogenomic therapy.Functional alterations in L-type high voltage-gated calcium channels (Cav1) after short-term (24h) exposure of mouse cerebrocortical neurons to drugs of abuse were examined as compared with those in psychological dependent mouse brains. KC1 (30 mM)-stimulated [^<45>Ca^<2+>] influx into the neurons increased with increasing the duration of the drugs exposure and its concentrations. This enhancement was completely abolished by Cav1 inhibitors, and antagonists for ryanodine and IP_3 receptors. Cav1.2 and Cav α2-δ proteins were increased in abused drugs-treated neurons. Increased binding of [^<3>H]PN200-110 after 24 h exposure to drugs of abuse was due to decreased Kd value. Similar changes in the binding parameters and protein expressions were obtained in cerebral cortices as well as nucleus accumbens fro … More m psychological dependent mice. These results indicate that stimulation of calcium-induced calcium release by ryanodine receptors and subsequent activations of IP_3 receptors induces Cav1 up-regulation.Furthermore, we examined the functional involvement of accessory proteins of Cav 3 subunits in ethanol (EtOH)-induced Cav1 up-regulation. Short-term exposure of the neurons to EtOH significantly increased in Cav β3 subunit levels, whereas Cav β1, β2 and β4 subunits showed no changes. High potassium-stimulated [^<45>Ca^<2+>] influx into the neurons significantly increased by EtOH exposure and this increase was not observed in the neurons pretreated with morpholino oligomers specific for Cav β3 subunits. Decreased Kd value of [^3H] P: N200-110 after EtOH exposure was also abolished by Cav β3 subunits knockdown. Similar changes in protein expressions were obtained in cerebral cortices from psychological dependent mice. These results indicate that short-term exposure of the neurons to EtOH up-regulates Cav1 functions via increased expression of Cav β3 subunit proteins in the neuronal membrane. Less