吗啡长时程作用对SH-SY5Y细胞cAMP系统及c-Fos磷酸化的影响

Effects of Long Term Morphine Exposure on the cAMP System and c Fos Phosphorylation in Differentiated SH SY5Y Cells

目的 探讨吗啡长时作用下分化的人成神经瘤ＳＨＳＹ５Ｙ细胞中ｃＡＭＰ系统变化以及ＰＫＡ对转录因子ｃＦｏｓ的磷酸化调节。方法 采用蛋白竞争结合法、酶活性测定法及同位素掺入法分别观察ｃＡＭＰ、ＰＫＡ 及ｃＦｏｓ 的变化。结果 （１） １００μｍ ｏｌ／Ｌ吗啡作用２ ｍ ｉｎ～３６ ｈ 可使ＳＨＳＹ５Ｙ细胞ｃＡＭＰ水平呈双相变化：短时作用下ｃＡＭＰ水平降低，随着吗啡作用时间的延长，ｃＡＭＰ水平逐渐回升，至３６ ｈ 时明显高于对照，这时加入１００ μｍ ｏｌ／Ｌ纳洛酮可诱发ｃＡＭＰ水平的反弹性超高现象。表明吗啡作用３６ ｈ 可使分化的ＳＨＳＹ５Ｙ细胞产生类吗啡依赖样变化；（２） 吗啡长时作用下，胞质可溶相ＰＫＡ活性也呈双相变化，而膜相ＰＫＡ 活性未见明显变化；（３）吗啡作用３６ ｈ 时，细胞ｃＦｏｓ磷酸化水平显著降低，ＰＫＡ抑制剂可抑制此作用；（４）纳洛酮可抑制上述ＰＫＡ 活性及ｃＦｏｓ 磷酸化水平的改变。结论 ＳＨＳＹ５Ｙ 细胞ｃＡＭＰＰＫＡ 系统的上调可能与吗啡依赖的形成有关，而且在吗啡依赖样细胞中ＰＫＡ可能通过磷酸酶而使细胞ｃＦｏｓ 发生去磷酸化，从而激活某些基因的转录。

Objective To further understand the effects of long term morphine exposure on the cAMP system and c Fos phosphorylation in differentiated SH SY5Y human neuroblastoma cells. Methods Cellular changes of cAMP, PKA and c Fos were detected by protein competitive conjunction, enzyme activity and isotope incorporation methods respectively.Results (1) Long term exposure (2 min～36 h) to morphine (100 μmol/L) could induce the biphasic changes in cAMP contents. Treament for 2 min to 1 h, morphine caused rapid and siginificant decrease of the cAMP level and then gradully recovered and apparently increased at 36 h. At that time, naloxone added to the incubation media caused an overshoot of cellular cAMP; (2) Long term exposure to morphine could also induce the biphasic changes in cytosolic PKA activity. This is consistent with the changes of cAMP during the chronic treatment of cells with morphine. But no changes were observed in membrane PKA activity; (3) In morphine dependent like cells decreased c Fos phosphorylation level was observed. PKA inhibitor could significantly inhibit this change; (4) Concomitant administration of naloxone could block the changes in PKA activity and c Fos phosphorylation described above.Conclusions The up regulation of cAMP system in differentiated SH SY5Y cells may be involved in the development of morphine dependent and in morphine dependent like SH SY5Y cells and PKA was suggested to regulate c Fos dephosphorylation through activating phosphatase and then activate some genes transcription, which might be one of the important mechanism regardingas cellular adaptive responses underlying dependence to opioid drugs.# Corresponding author