吗啡长时程作用对新生鼠TM神经细胞cAMP和cGMP的影响以及青藤碱的干预作用

目的：观察体外原代培养的新生鼠下丘脑结节乳头核（TM）神经细胞在吗啡长时程作用下胞内cAMP及cGMP 水平的变化以及青藤碱对它的影响。方法：采用原代培养7d的TM神经细胞，加入含100µmol/L吗啡的培养液培养48 h，形成类吗啡依赖神经细胞，经100 µmol/L纳洛酮戒断后，用EIA法测定细胞内cAMP和cGMP含量。不同剂量的组胺或青藤碱在纳洛酮戒断前30 min作用于细胞，同时测定药物对正常TM细胞内cAMP和cGMP水平的影响。结果：以100 µmol/L吗啡作用于新生鼠TM细胞48 h后，胞内cAMP及cGMP含量明显升高，经100 µmol/L纳洛酮戒断后，胞内cAMP含量出现反弹性超高现象，而cGMP含量则明显下降，cAMP与cGMP比值明显增大。30、100 µmol/L青藤碱 及40 µmol/L组胺对正常TM神经细胞的cAMP及cGMP含量无明显影响，300 µmol/L青藤碱及80 µmol/L组胺可使正常细胞内cAMP含量明显升高。三个剂量的青藤碱及40 µmol/L组胺预先作用可明显降低吗啡依赖的TM细胞经纳洛酮戒断时的cAMP超高水平，同时明显升高cGMP水平，使细胞内增大的cAMP与cGMP比值减小。结论：在吗啡（100 µmol/L）长时程（48 h）作用下，TM神经细胞内cAMP及cGMP水平发生了明显的变化，中枢组胺能神经系统可能参与了吗啡依赖的形成与戒断过程。青藤碱可显著降低吗啡依赖细胞戒断时的cAMP超高水平，同时升高cGMP水平，使两者比值趋于正常。

Objective: To explore the effects of long-term morphine exposure on cAMP and cGMP levels in primary cultured tuberomammillary nucleus (TM) neurons of neonatal rats and the effects of sinomenine on morphine-dependent TM cells. Methods: TM neurons after a 7-day primary culture were further cultured in the medium containing 100 µmol/L morphine for 48 h to prepare the cell model of morphine dependence. Serial doses of histamine or sinomenine wereadministered 30 min naloxone treatment, the cAMP and cGMP levels of the TM cells were determined by enzyme immunoassay. cAMP and cGMP levels were also determined in normal TM cells treated by histamine or sinomenine. Results: After treatment with 100 µmol/L morphine for 48 h, cAMP and cGMP levels in the TM neurons were increased markedly. Treatment with 100 µmol/L nalox­one added in the culture media caused an overshoot of cellular cAMP and a marked declination of cGMP, resulting in significantly increased cAMP/cGMP ratio. Sinomenine at 30 and 100 µmol/L and histamine at 40 µmol/L failed to obviously affect cAMP and cGMP levels in normal TM neurons, but sinomenine at 300 µmol/L and histamine at 80 µmol/L significantly in­creased the intracellular cAMP level. After pre-treatment with sinomenine at the above 3 doses or histamine at 40 µmol/L, the TM neurons with morphine dependence exhibited significant reduction in intracellular cAMP level but increment in cGMP level after naloxone treatment, with significantly reduced cAMP/cGMP ratio. Conclusion: Long-term morphine (100 µmol/L) exposure for 48 h can induce marked changes of cAMP and cGMP levels in the TM neurons. The central histaminergic nervous system may be responsible for the development of morphine dependence and withdrawal. Sinomenine can significantly reduce the cAMP level and enhance cGMP level of morphine-dependent TM neurons precipitated by naloxone, which results in a near-normal ratio of cAMP and cGMP.