凝血酶增高NMDA受体敏感性与组织型谷氨酰胺转移酶的关系

Enhancement of NMDA receptor sensitivity by thrombin and its relationship with tissue transglutaminase

目的探讨凝血酶增高大鼠的小脑颗粒细胞N-甲基-D-天冬氨酸受体(NMDA)敏感性是否与细胞内组织型谷氨酰胺转移酶(tTG)变化有关.方法以0.01 U/ml浓度凝血酶单独或与tTG抑制剂单丹磺酰尸胺(MDC) 0.5×10-4 mol/L处理原代培养的Wistar大鼠小脑颗粒细胞30 min后,以0.1×10-4 mol/L、0.25×10-4 mol/L和0.5×10-4 mol/L 3种浓度NMDA作用3 h诱导上述处理的细胞凋亡,比较流式细胞仪检测到的细胞凋亡率差异以判断NMDA受体敏感性的变化;RT-PCR和Gelpro图像分析软件检测各NMDA浓度有/无凝血酶预处理的细胞内tTG mRNA变化差异.结果凝血酶预处理细胞后上述3种浓度NMDA诱导的细胞凋亡率均升高[(8.3±0.5～15.1±0.8)%, (14.1±1.1～23.8±0.7)%, (26.8±1.9～33.7±2.1)%].预处理细胞时加入MDC可阻断凝血酶对NMDA受体的这种影响,细胞凋亡率降至(5.8±1.2)%、(11.5±1.5)%和(19.0±1.7)%;凝血酶预处理后各浓度NMDA处理细胞内tTG mRNA量均同一浓度单独NMDA处理的细胞高.结论凝血酶通过增高细胞内tTG使小脑颗粒细胞NMDA受体敏感性增高.

Objective To explore the possible relationship between the thrombin induced potentiation of rat cerebellum granular cell N-methyl-D-aspartate （NMDA） receptor function and the change of intracellular tissue transglutaminase （tTG）. Methods Primary cultured rat cerebellum granular cells were pretreated by using thrombin （0. 01 U/ml） with or without co-pretreatement of 0. 5 × 10^-4 mol/L MDC （monodansylcadaverine, a tTG inhibitor） 30 minutes. Induce cells mentioned above to undergo apoptosis with different concentration of NMDA （0. 1 × 10^-4 tool/L,0. 25 × 10^-4mol/L and 0. 5 × 10^-4 mol/L） . Flowcytometer was applied 3 hours later to detect the changed function of NMDA receptor by counting percentage of apoptotic cells. RT-PCR was used to compare the tTG mRNA level in the cerebellum granular cells treated with NMDA of same dosage with/without thrombin pretreatment. Results Thrombin pretreatment increased the percentage of apoptosis cells induced by NMDA [ （ 8. 3 ± 0.5 ） % to （ 15. 1 ± 0.8）% at0. 1 xl0-4 mol/L,（14. 1 ±1.1）% to （23.8±0.7）% at 0.25×10^-4 mol/L, （26.821.9）% to （33.7 22. 1）% at 0.5 × 10^-4 mol/L） ] ; and this effect was inhibited by co-pretreatment MDC with thrombin, cell apoptosis decreased to （ 5. 8±1.2 ） %, （ 11.5±1.5 ） % and （ 19. 0±1.7 ） % ; Thrombin increased intracellular tTG mRNA levels comparing with those without thrombin pretreatment. Conclusion Thrombin potentiates rat cerebellum granular cell NMDA receptor function by increasing intracellular tissue transglutaminase.