Clarification of molecular mechanisms of endothelium-derived hyperpolarizing factor

本研究的目的是阐明内皮源性超极化因子（EDHF）介导的大鼠肠系膜动脉舒张和膜超极化的分子机制。我们试图确定离子通道，缝隙连接通道和trp通道参与EDHF的行动。RT-PCR结果显示，大鼠肠系膜动脉中存在小电导Ca^2+激活的K^+（SK）3、中电导Ca^2+激活的K^+（IK）、大电导Ca^2+激活的K^+（BK）通道和连接蛋白-37、-40、-43、-45的mRNA，而不存在SK 1和SK 2的mRNA。通过RT-PCR和cDNA文库筛选，克隆了这些离子通道和连接蛋白的cDNA。克隆了与SK 3通道相似的新基因（SK 3-B）。在SK 3-B中，SK 3的多聚谷氨酰胺位置被多聚丝氨酸取代。采用膜片钳技术的功能分析表明，SK 3-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，该通道的Ca^<2+>敏感性不如SK 3。免疫组化结果显示SK 3通道在内皮细胞中表达。CX 37和CX43在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天然动脉中的功能作用，构建了表达这些基因的腺病毒。腺病毒介导的SK 3或CX 43在培养的大鼠肠系膜动脉中的表达轻微地增强了EDHF的作用。卵巢切除降低了EDHF的作用，与CX40和CX43表达的降低一致

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