3-异丁基-1-甲基黄嘌呤对低钾诱导的大鼠小脑颗粒神经元凋亡的Ca^(2+)/cAMP不依赖性保护作用

cAMP-and Ca^( 2+) -independent protective effect of 3-isobuty-1-methylxanthine against apoptosis in cultured rat cerebellar granule neurons

目的 观察 3 异丁基 1 甲基黄嘌呤 ( 3 Isobutyl 1 methylxanthine ,IBMX)对 5mmol·L-1KCl诱导的大鼠小脑颗粒神经元凋亡的拮抗作用并探讨其与 [cAMP]i和Ca2 +的关系。方法 建立KCl 5mmol·L-1诱导的大鼠小脑颗粒神经元凋亡模型 ,用荧光二酯素法分析神经元存活 ,Hoechest332 5 8核染色和DNA琼脂糖凝胶电泳分析凋亡 ,放射免疫法测定cAMP浓度。结果 IBMX呈浓度依赖性拮抗 5mmol·L-1KCl诱导的神经元死亡 ,并明显减少核形态异常的神经元 ,使DNA电泳梯带变浅或完全消失 ;IBMX( 1.0mmol·L-1)可持续升高神经元 [cAMP]i,福司可林 2 .0 μmol·L-1升高 [cAMP]i作用与IBMX类似 ,但不能模拟IBMX的作用 ;此外 ,IBMX的拮抗作用不被cAMP竞争性抑制剂Rp cAMP和PKA的特异阻断药H 89阻断 ;单用或联用钙调素依赖的蛋白激酶Ⅱ的特异抑制剂KN 93和磷酯酰肌醇 3位羟基激酶的特异性抑制剂LY2 940 0 2也不能阻断。结论IBMX抗低钾诱导的大鼠小脑颗粒神经元凋亡作用不依赖cAMP和Ca2 +。

OBJECTIVE To investigate the protective effect of 3 isobutyl 1 methylxanthine(IBMX)against apoptosis induced by 5 mmol·L -1 KCl in cultured rat cerebellar granule neurons and the roles of calcium/cAMP in the protective effect of IBMX.METHODS 5 mmol·L -1 KCl induced apoptosis model was established in cultured rat cerebellar granule neuron.The neuronal viability was assayed by Fluorescemi Diacetate (FDA) staining, apoptosis was assayed by Hoechst33258 staining and DNA agarose gel electrophoresis.The [cAMP]i was measured by radio immunology.RESULTS IBMX (0.06～1.0 mmol·L -1 ) protected neurons in a concentration dependent manner with IC 50 0.5 mmol·L -1 . IBMX significantly decreased the number of abnormal nuclei and attenuated the DNA fragmentation, the typical biochemical character of apoptosis. These results indicated that IBMX protected neurons against apoptosis induced by 5 mmol·L -1 KCl in cultured cerebellar granule neurons.[cAMP]i was increased sustainedly by IBMX (1.0 mmol·L -1 ).The pattern of 2.0 μmol·L -1 forskolin increasing [cAMP]i was similar to that of IBMX, but apoptosis induced by 5 mmol·L -1 KCl was not blocked by 2.0 μmol·L -1 forskolin. Rp cAMP, a competent antagonist of cAMP, and H 89, a specific inhibitor of PKA, both did not block the protective effect of IBMX; same results were also observed with using KN 93, a specific calcium/calmodulin dependent protein kinase Ⅱ, or/and LY294002, a specific inhibitor of phosphatidylinositol 3 kinase.CONCLUSION IBMX protected neurons against apoptosis induced by 5 mmol·L -1 KCl in cultured rat cerebellar granule neurons independent of cAMP and calcium.