Clarification of molecular mechanisms of endothelium-derived hyperpolarizing factor

本研究旨在阐明内皮衍生超极化因子(EDHF)介导的大鼠肠系膜动脉松弛和膜超极化的分子机制。我们试图确定参与EDHF作用的离子通道、缝隙连接通道和色氨酸通道。RT-PCR结果显示，大鼠肠系膜动脉存在小电导钙激活钾通道、中导钙激活钾通道、大电导钙激活钾通道和连接蛋白-37、-40、-43、-45通道，而不存在连接蛋白-1和连接蛋白-2。通过RT-PCR和文库筛选的方法，克隆了这些离子通道和连接蛋白的基因。克隆了与SK3通道相似的新基因(SK3-b)。SK3-b中SK3的聚谷氨酰胺位被取代为聚丝氨酸。利用膜片钳技术进行的功能分析表明，SK3-b通道具有几乎相同的离子通道功能，如电压依赖性和毒素敏感性。然而，与SK3相比，该通道对钙离子的敏感性较低。免疫组织化学分析显示，SK3通道在内皮细胞中有表达。CX37和CX43在血管内皮细胞和平滑肌细胞中均有表达，而CX40仅在内皮细胞中表达。为了阐明这些基因在动脉中的功能作用，构建了表达这些基因的腺病毒。腺病毒介导的SK3或Cx43对培养的大鼠肠系膜动脉的EDHF作用略有增强。卵巢切除与CX40和Cx43表达减少有关的EDHF作用

The purpose of this study was to clarify the molecular mechanisms of endothelium-derived hyperpolarizing factor (EDHF)-mediated arterial relaxation and membrane hyperpolarization in the rat mesenteric arteries. We tried to identify the ion channels, gap junctional channels and trp channels involved in the EDHF action. RT-PCR experiment showed that mRNA of small-conductance Ca^<2+>-activated K^+ (SK)3, intermediate-conductance Ca^<2+>-activated K^+(IK), large-conductance Ca^<2+>-activated K^+ (BK) channels and connexin-37, -40, -43, -45 but not SK1 and SK2 were exist in the rat mesenteric artery. cDNA of these ion channels and connexins were cloned by RT-PCR and cDNA library screening. New gene (SK3-b) similar to SK3 channel was cloned. The poly glutamine position of SK3 was substituted to poly serine in SK3-b. Functional analysis using patch clamp techniques showed that the SK3-b channel has almost the same ion channel functions such as voltage dependency and toxin sensitivity. However the Ca^<2+> sensitivity of the channel was less sensitive compared with that of the SK3. Immunohistochemical analysis showed that the SK3 channel was expressed in the endothelium. The CX37 and 43 were expressed in both the endothelial and smooth muscle cells, however CX40 was expressed in only the endothelial cells. To clarify the functional role of these genes in the native artery, adenoviruses expressing these genes were constructed. Adenovirus-mediated expression of SK3 or CX43 to the cultured rat mesenteric artery enhanced EDHF action slightly. Ovariectomy reduced the EDHF action in accordance with the reduced expression of CX40 and CX43