17β-estradiol regulates expression of KATP channels in heart-derived H9c2 cells
17β-estradiol regulates expression of KATP channels in heart-derived H9c2 cells
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DOI：
10.1016/s0735-1097(02)01947-2
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发表时间：
2002-07-17
影响因子：
24
通讯作者：
Jovanovic, A
中科院分区：
文献类型：
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作者：
Ranki, HJ;Budas, GR;Jovanovic, A
目的本研究的主要目的是确定17 β-雌二醇（E2）是否调节心脏三磷酸腺苷敏感性钾（K-ATP）通道的表达。方法采用逆转录-聚合酶链反应（RT-PCR）技术，对Kir6.2和磺酰脲受体2A（SUR 2A）特异性引物进行扩增，并对K-ATP通道的表达进行定量分析。对来自大鼠胚胎心脏来源的H9 c2细胞的总核糖核酸（RNA）进行了亚基分析。使用抗Kir6.2和抗SUR 2A抗体对H9 c2细胞的膜部分进行免疫沉淀和Western印迹。对活的H9 c2细胞进行全细胞电生理学和数字荧光Ca 2+成像。结果RT-PCR检测结果显示，E2处理组细胞中SUR 2A mRNA表达水平高于对照组，Kir6.2 mRNA表达水平低于对照组。只有当Kir6.2超过SUR 2A时，仅SUR 2A亚基水平的增加才能改变肌膜KA（TP）通道的数量。事实上，RT-PCR分析表明，与Kir6.2 mRNA相比，SUR 2A mRNA的水平相当低。与未处理的细胞相比，在E2处理的细胞的膜部分中发现Kir6.2和SUR 2A蛋白亚基的水平显著更高，并且由K-ATP通道开放剂吡那地尔（100 μ M）诱发的电流密度在E2处理的细胞中显著高于未处理的细胞。为了测试E2对细胞缺氧-复氧反应的影响，我们在线测量了缺氧-复氧暴露的H9 c2细胞内Ca 2+浓度。缺氧-复氧诱导的细胞内钙负荷显着降低与E2的治疗。HMR 1098（30 μ M）可抑制E2介导的保护作用，而5-hydroxydecanoate（50 μ M）则不抑制E2介导的保护作用。结论E2可增加SUR 2A亚单位的表达，刺激K-ATP通道的形成，保护心肌细胞免受缺氧-复氧损伤。
Objectives The main objective of the present study was to establish whether 17beta-estradiol (E2) regulates expression of cardiac adenosine triphosphate-sensitive potassium (K-ATP) channel.Background Based on our previous studies that demonstrate gender-specific differences in sarcolemmal K-ATP channels, we have hypothesized that the main estrogen, E2, may regulate expression of cardiac K-ATP channels.Methods Reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for Kir6.2 and sulfonylurea receptor 2A (SUR2A) subunits was performed on total ribonucleic acid (RNA) from rat embryonic heart-derived H9c2 cells. Immunoprecipitation and Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was done on membrane fraction of H9c2 cells. Whole cell electrophysiology and digital epifluorescent Ca2+ imaging were performed on living H9c2 cells. All experiments were done in cells incubated 24 h with or without 100 nM E2.Results The RT-PCR revealed higher levels of SUR2A, but not Kir6.2, messenger RNA (mRNA) in E2-treated, relative to untreated, cells. Increase of the level of only the SUR2A subunit could change the number of sarcolemmal KA(TP) channels only if the Kir6.2 is in excess over SUR2A. Indeed, RT-PCR analysis demonstrated considerably lower levels of SUR2A mRNA compared with Kir6.2 mRNA. Significantly higher levels of both Kir6.2 and SUR2A protein subunits were found in the membrane fraction of E2-treated cells compared with untreated ones, and the density of current evoked by pinacidil (100 muM), a K-ATP channel opener, was significantly higher in E2-treated compared with untreated cells. To test the effect of E2 on cellular response to hypoxia-reoxygenation, we have measured on-line, intracellular concentration of Ca2+ in H9c2 cells exposed to hypoxia-reoxygenation. Intracellular Ca2+ loading induced by hypoxia-reoxygenation was significantly decreased by treatment with E2. This E2-mediated protection was inhibited by HMR 1098 (30 muM), but not by 5-hydroxydecanoate (50 muM).Conclusions In conclusion, this study has demonstrated that E2 increases levels of SUR2A subunit, stimulates K-ATP channel formation and protects cardiac cells from hypoxia-reoxygenation.