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

本研究旨在寻找药物依赖形成的共同生物标记物，并尝试开发药物基因组学疗法。以心理依赖小鼠脑内为对照，研究了药物短期(24小时)作用于小鼠大脑皮层神经元后L型高电压门控钙通道(CAV1)的功能变化。Kc1(30 MM)刺激的[^&lt；45&gt；Ca^&lt；2+&gt；]内流随着药物作用时间和浓度的增加而增加。这种增强作用可被Cav1抑制剂、兰尼定和IP_3受体拮抗剂完全消除。在滥用药物的神经元中，Cav1.2和Cavα2-δ蛋白表达增加。药物作用24 h后，[^&lt；3&gt；H]PN200-110的结合力增加是由于Kd值降低。在大脑皮质和伏隔核中，…的结合参数和蛋白表达也发生了类似的变化心理依赖小鼠较多。这些结果表明，Ryanodine受体刺激钙离子诱导的钙释放和随后的IP_3受体激活诱导Cav1上调。此外，我们还研究了乙醇(Etoh)诱导的Cav1上调中Cav3亚单位辅助蛋白的功能参与。短期乙醇暴露使Cavβ3亚基水平显著增加，而Cavβ1、β2和β4亚基水平无明显变化。乙醇可显著增加高钾刺激的[^&lt；45；gt；Ca^&lt；2+&gt；]内流，而用针对Cavβ3亚基的吗啡低聚物预处理的神经元则未见这种增加。β3亚基基因敲除后，乙醇引起的Kd值降低也被阻断。心理依赖小鼠大脑皮层的蛋白质表达也发生了类似的变化。这些结果表明，短期乙醇暴露通过增加神经细胞膜上Cavβ3亚单位蛋白的表达来上调CaV1的功能。较少

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