Effects of Ca~(2+)/ calmodulin dependent protein kinase Ⅱ signal pathway in DPDPE long term treatment NG108-15 cells
GUO Qing-Min, LIU Jing-Sheng (Dept of Pharmacology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005)
AIM To observe the change of Ca 2+ /calmodulin dependent protein kinaseⅡ (CaMKⅡ) signal pathway in opioids dependent NG108-15 cells and the relationships between Ca 2+ /calmodulin dependent protein kinaseⅡ (CaMKⅡ) signal pathway and cAMP accumulation. METHODS NG108-15 cells were used as an in vitro model system. Competitive protein binding assay and radioimmunoassay were used to examine the intracellular cAMP accumulation. Calmodulin activity was assayed by PDE method. CaMKⅡ activity was assayed by γ- 32 P incorporation of syntide-2. mRNA expression of mDOR was measured by RT-PCR. RESULTS DPDPE long-term treatment also increased cAMP accumulation, and resulted in opioids dependence in NG108-15 cells; DPDPE long-term treatment increased calmodulin activity and CaMKⅡ activity in NG108-15 cells. Specific calmodulin antagonist W-7 was found to inhibit significantly the elevation of calmodulin and CaMKⅡ activity which resulted from DPDPE long-term treatment, and CaMKⅡ inhibitor KN-62 also inhibited elevation of CaMKⅡ activity by DPDPE long-term treatment. DPDPE long-term treatment increased cAMP accumulation, when W-7 or KN-62 was added at the same time, cAMP accumulation decreased. When naloxone was added to NG108-15 cells which were long-term treated by DPDPE, calmodulin and CaMKⅡactivity increased more. mRNA level of mDOR did not change significantly in opioids dependent NG108-15 cells. CONCLUSION Ca 2+ /CaMKⅡ signal pathway was involved in the mechanisms of opioids dependence through regulating cAMP level when DPDPE is long-term administered to NG108-15 cells.