促肾上腺皮质激素刺激H295R细胞分泌醛固酮和皮质醇的信号通道

Signal channel research on aldosterone and cortisol secretion in H295R cells after adrenocortico- tropin stimulation

目的观察促肾上腺皮质激素（ACTH）刺激人肾上腺皮质癌H295R细胞后对醛固酮和皮质醇的影响，探讨相关信号通道在介导该反应中的作用。方法用慢病毒包装的促ACTH受体（ACTHR）高表达载体感染H295R细胞作为实验组，同时设立对照组。感染后48h应用WesternMot和实时定量聚合酶链反应（ReM—timePCR）验证ACTHR表达。100nmol/LACTH刺激实验组和对照组细胞，同时加入ACTH相关信号通道抑制剂：环磷酸腺苷（cAMP）抑制剂PKI（终质量浓度4μmol／L）、蛋白激酶A（PKA）抑制剂H89（终质量浓度10μmol／L）、蛋白激酶c（PKC）抑制剂BIMl（终质量浓度3μmol／L）及钙离子通道抑制剂KN．93（终质量浓度10μmol／L），在ACTH刺激后5、30min及24h分别测定醛固酮合成酶（CYP11B2）和皮质醇合成酶（CYP11B1）的mRNA表达水平；用酶联免疫吸附试验（EHSA）法测定上述各时间点醛固酮和皮质醇激素的含量，对比各时间点醛固酮和皮质醇的抑制效果，判定在介导各反应中的关键信号通道。结果实验组细胞ACTHR在蛋白水平和mRNA水平分别增加2．4倍和18倍（P〈0．05）。对醛固酮而言，ACTH刺激5min及24h后各组细胞均无法被抑制；但在刺激30min时cAMP抑制组CYPllB2mRNA表达明显受到抑制（抑制率为39％，P〈0．05），而在同时点醛固酮激素水平，PKA抑制组降幅最明显[（79．31±5．21）ng/L比（86．45±6．37）ng／L，P〈O．05]；对皮质醇而言，无论是CYP11B1mRNA还是皮质醇激素水平，各时间点的cAMP或PKA抑制组表达水平均受到明显抑制（P〈0．05）。结论ACTH主要通过cAMP／PKA信号通道对皮质醇分泌发挥重要作用，但对刺激醛固酮激素分泌具有一定时限性，可能还有其他因素或信号通道参与。

Objective To study the changes of aldosterone and cortisol secretion in human adreno- cortical carcinoma H295R cells after adrenocorticotropin hormone （ACTH） stimulation, and investigate the effect of related signal channels. Methods Lentiviral vector with high ACTHR expression was transfected into the H295R cells. Similarly, the H295R cells without lentiviral vector transfection were used as control group. ACTHR alteration was measured by Western blotting and real - time quantitative polymerase chain reaction（Real- time PCR）） 48 h after transfection. CYPllB2 and CYPllB1 mRNA expression levels were detected at 5 min, 30 rain and 24 h after 100 nmol/L ACTH stimulation with each inhibitor： cAMP inhibitor PKI （4 μmol/L）, PKA inhibitor H89 （10 μmol/L）, PKC inhibitor BIM1 （3μmol/L） and cal- eium ehannel inhibitor KN -93 （10 μmol/L）. Aldosterone and cortisol levels were determined by enzyme linked immunosorbent assay （ELISA） kit. The possible signal ehannels of ACTI-I were determined by com- paring the inhibition efficiency of each inhibitor. Results Compared with those in control cells, the protein and mRNA levels of ACTHR in experimental eells were increased 2.4 times and 18 times respectively （P 〈 0. 05 ）. The aldosterone expression in each group could not be inhibited after 5 rain and 24 h stimula- tion of ACTH. However, CYPllB2 mRNA expression in eAMP- inhibited group was inhibited obviously after 30 - rain stimulation of ACTH （ about 39% , P 〈 0. 05 ）. Meanwhile, aldosterone production in PKA - inhibited group was inhibited too [ （79. 31± 5.21 ） vs. （ 86. 45± 6. 37 ） ng/L, P 〈 0. 05 ]. The CYP11 B1 mRNA and cortisol hormone eortisol levels in cAMP- or PKA -inhibited group were decreased at each time point （P 〈 0. 05 ）. Conclusion ACTH plays an important role in eortisol secretion through cAMP/PKA signal channels. However, it has certain time limit to aldosterone secretion or there may be other signal channels involved in.