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Ca2+/cAMP不依赖性保护作用

 目的观察3-异丁基-1-甲基黄嘌呤（3-Isobutyl-1-methylxanthine, IBMX）对5 mmol·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 5 mmol·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，Hoechest 33258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（1.0 mmol·L^-1）可持续升高神经元cAMP]i，福司可林2.0μmol·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LY294002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⁺。

cAMP-and Ca2+-independent protective effect of 3-isobuty-1-methylxanthine against apoptosis in cultured rat cerebellar granule neurons

OBJECTIVE To investigate the protective effect of 3-isobutyl-1-methylxanthine（IBMX）against apoptosis induced by 5 mmol·L^-1KCl in cultured rat cerebellar granule neurons and the roles of calcium/cAMP in the protective effect of IBMX.METHODS 5 mmol·L^-1KCl 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.ThecAMP]I was measured by radio immunology.RESULTS IBMX (0.06～1.0 mmol·L^-1) protected neurons in a concentration dependent manner with IC₅₀0.5mmol·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^-1KCl in cultured cerebellar granule neurons.cAMP]I was increased sustainedly by IBMX (1.0 mmol·L^-1).The pattern of 2.0μmol·L^-1forskolin increasingcAMP]I was similar to that of IBMX, but apoptosis induced by 5 mmol·L^-1KCl was not blocked by 2.0μmol·L^-1forskolin. 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^-1KCl in cultured rat cerebellar granule neurons independent of cAMP and calcium.