González Marcelo, Flores Carlos, Pearson Jeremy D, Casanello Paola, Sobrevia Luis
Cellular and Molecular Physiology Laboratory (CMPL), Department of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile.
Pflugers Arch. 2004 Jul;448(4):383-94. doi: 10.1007/s00424-004-1261-x. Epub 2004 Apr 3.
Insulin induces vasodilatation in human subjects and increases L-arginine transport and NO synthesis in human umbilical vein endothelial cells (HUVEC). Cell signalling events associated with insulin effects on activity and mRNA expression of the human cationic amino acid transporters 1 (hCAT-1) and 2B (hCAT-2B) are unknown. L-arginine transport and eNOS activity were determined in HUVEC exposed to insulin. mRNA levels for hCAT-1, hCAT-2B and eNOS were quantitated by real time RT-PCR and endothelial NO synthase (eNOS) protein was identified by Western blot analysis. Intracellular Ca2+, L-arginine and L-citrulline levels, L-[3H]citrulline formation from L-[(3)H]arginine, cGMP formation, nitrite level, ATP release and membrane potential were determined. Insulin increased L-arginine transport and the mRNA levels for hCAT-1 and hCAT-2B and eNOS expression and activity. Insulin also induced membrane hyperpolarization and increased intracellular Ca2+, L-[3H]citrulline, cGMP and nitrite formation. Insulin-mediated stimulation of the L-arginine/NO pathway is thus associated with increased hCAT-1 and hCAT-2B mRNA, and eNOS expression, via mechanisms involving membrane hyperpolarization, mitogen-activated protein kinases p42 and p44, phosphatidylinositol 3-kinase, NO and protein kinase C. We have characterized a cell signalling pathway by which hyperinsulinaemia could lead to vasodilatation in human subjects, and which could have implications in patients in whom plasma insulin levels are altered, such as in diabetes mellitus.
胰岛素可诱导人体受试者血管舒张，并增加人脐静脉内皮细胞（HUVEC）中L-精氨酸转运及一氧化氮（NO）合成。胰岛素对人阳离子氨基酸转运体1（hCAT-1）和2B（hCAT-2B）活性及mRNA表达影响相关的细胞信号转导事件尚不清楚。我们测定了暴露于胰岛素的HUVEC中的L-精氨酸转运及内皮型一氧化氮合酶（eNOS）活性。通过实时逆转录聚合酶链反应（RT-PCR）定量检测hCAT-1、hCAT-2B和eNOS的mRNA水平，并用蛋白质免疫印迹分析鉴定内皮型一氧化氮合酶（eNOS）蛋白。测定细胞内钙离子、L-精氨酸和L-瓜氨酸水平、L-[(3)H]精氨酸生成L-[3H]瓜氨酸、环磷酸鸟苷（cGMP）生成、亚硝酸盐水平、三磷酸腺苷（ATP）释放及膜电位。胰岛素增加了L-精氨酸转运、hCAT-1和hCAT-2B的mRNA水平以及eNOS的表达和活性。胰岛素还诱导膜超极化，并增加细胞内钙离子、L-[3H]瓜氨酸、cGMP和亚硝酸盐生成。因此，胰岛素介导的L-精氨酸/NO途径刺激与hCAT-1和hCAT-2B mRNA增加以及eNOS表达增加相关，其机制涉及膜超极化、丝裂原活化蛋白激酶p42和p44、磷脂酰肌醇3-激酶、NO和蛋白激酶C。我们已确定了一种细胞信号转导途径，高胰岛素血症可通过该途径导致人体受试者血管舒张，这可能对血浆胰岛素水平改变的患者（如糖尿病患者）具有重要意义。
