NMDA受体和IP3受体介导NMDA诱导的小脑颗粒神经元内Ca^(2+)超载

NMDA-induced calcium overload via NMDA and IP3 receptors in cultured cerebellar granular neurons

目的探讨N-甲基-D-天门冬氨酸(NMDA)诱导的小脑颗粒神经元(CGNs)钙超载与NMDA受体(NMDAR)及细胞内钙库受体三磷酸肌醇受体(IP3R)和兰尼定受体(RyR)之间的关系。方法取出生后8 d SD大鼠小脑进行颗粒神经元体外培养。用NMDA(100μmol/L)急性损伤神经元,在激光扫描共聚焦显微镜(LSCM)扫描开始前30 min,或扫描开始后4,9,14 min分别加入NMDAR、IP3R、RyR拮抗剂MK-801、2-APB和DAN,检测神经元内Ca2+浓度的动态变化。结果 MK-801预孵育神经元经NMDA急性刺激后,神经元内Ca2+的荧光强度不再升高,NMDA刺激后加入MK-801,上升的Ca2+水平立即下降,最终下降至基线水平;NMDA急性刺激2-APB预孵育神经元,神经元内Ca2+的荧光强度升高,但升高幅度明显低于未经NMDA刺激组,NMDA刺激后加入2-APB,细胞内升高的Ca2+水平急剧下降,最终下降至接近基线水平;DAN预孵育的神经元经NMDA急性刺激后,胞内Ca2+的荧光强度急剧升高,达到NMDA刺激组水平,NMDA刺激后加入DAN,神经元内Ca2+水平无明显下降。结论 NMDA诱导的神经元Ca2+超载,主要由细胞膜钙通道NMDAR和细胞内钙释放通道IP3R介导,而细胞内钙释放通道RyR不起主导作用。

Objective To explore the relationship between NMDA-induced calcium overload and calcium receptors in the cerebellar granular neurons （CGNs）. Methods CGNs from postnatal 8-day rats were cultured for 8 days. On the 8th day, NMDA（ 100 μmol/L） was added to the onedinm to stimulato the neurons. MK-801 （ NMDAR antagonist）, 2-APB （ IP3 R antagonist）, and DAN （ RyR antagonist） were added before and after Laser scanning confocal microscope （LSCM） scanning. Dynamic changes of intra- cellular Ca2＋ fluorescence intensity in the neurons were measured during 15 min of the NMDA stimula- tion. Results The NMDA stimulation of the CGNs without any pretreatment caused a notable and sus- taining enhancement of Ca2＋ fluorescence intensity in the cultured neurons; MK-801 （NMDAR antago- nist）, 2-APB （IP3R antagonist）, and DAN （RyR antagonist） were added into the culture before NMDAfor 30 min or after the beginning of LSCM scanning at the 4th, 9th and 14th min respectively. MK-801 or 2- APB, but not DAN reduced Ca2＋ concentrations sharply induced by NMDA. When pretreatment with MK- 801, 2-APB, and DAN for 30 min before LSCM scanning, fluorescence intensity of Ca2＋ in neurons no longer increased in MK-801 group after the NMDA acute stimulation, while enhanced in 2-APB but still lower than NMDA group, and increased rapidly in DAN group. Conclusion NMDA-induced calcium o- verload via NMDA and IP3 receptors in cultured cerebellar granular neurons, and Ryanodine receptor is not playing a leading role.