Ranki Harri J, Budas Grant R, Crawford Russell M, Davies Anthony M, Jovanović Aleksandar
Tayside Institute of Child Health, Ninewells Hospital & Medical School, University of Dundee, Dundee DD1 9SY Scotland, UK.
J Am Coll Cardiol. 2002 Jul 17;40(2):367-74. doi: 10.1016/s0735-1097(02)01947-2.
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.
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.
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 Ca(2+) imaging were performed on living H9c2 cells. All experiments were done in cells incubated 24 h with or without 100 nM E2.
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 K(ATP) 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 microM), 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 Ca(2+) in H9c2 cells exposed to hypoxia-reoxygenation. Intracellular Ca(2+) loading induced by hypoxia-reoxygenation was significantly decreased by treatment with E2. This E2-mediated protection was inhibited by HMR 1098 (30 microM), but not by 5-hydroxydecanoate (50 microM).
In conclusion, this study has demonstrated that E2 increases levels of SUR2A subunit, stimulates K(ATP) channel formation and protects cardiac cells from hypoxiareoxygenation.
本研究的主要目的是确定17β-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。
基于我们之前证明肌膜K(ATP)通道存在性别特异性差异的研究，我们推测主要雌激素E2可能调节心脏K(ATP)通道的表达。
使用针对Kir6.2和磺脲类受体2A（SUR2A）亚基的引物，对大鼠胚胎心脏来源的H9c2细胞的总核糖核酸（RNA）进行逆转录-聚合酶链反应（RT-PCR）。使用抗Kir6.2和抗SUR2A抗体对H9c2细胞的膜组分进行免疫沉淀和蛋白质印迹分析。对活的H9c2细胞进行全细胞膜片钳电生理和数字落射荧光Ca(2+)成像。所有实验均在分别用或不用100 nM E2孵育24小时的细胞中进行。
RT-PCR显示，与未处理的细胞相比，E2处理的细胞中SUR2A的信使核糖核酸（mRNA）水平较高，但Kir6.2的mRNA水平未升高。只有当Kir6.2相对于SUR2A过量时，仅SUR2A亚基水平的增加才能改变肌膜K(ATP)通道的数量。实际上，RT-PCR分析表明SUR2A mRNA水平比Kir6.2 mRNA水平低得多。与未处理的细胞相比，在E2处理的细胞的膜组分中发现Kir6.2和SUR2A蛋白亚基的水平均显著更高，并且在E2处理的细胞中，K(ATP)通道开放剂吡那地尔（100 microM）诱发的电流密度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，我们在线测量了暴露于缺氧复氧的H9c2细胞中的细胞内Ca(2+)浓度。E2处理可显著降低缺氧复氧诱导的细胞内Ca(2+)负荷。HMR 1098（30 microM）可抑制这种E2介导的保护作用，但5-羟基癸酸（50 microM）则不能。
总之，本研究表明E2可增加SUR2A亚基水平，刺激K(ATP)通道形成，并保护心脏细胞免受缺氧复氧损伤。
