Guse A H, Kiess W, Funk B, Kessler U, Berg I, Gercken G
University of Hamburg, Department of Biochemistry, Germany.
Endocrinology. 1992 Jan;130(1):145-51. doi: 10.1210/endo.130.1.1309323.
Cultured cardiac myocytes from adult Sprague-Dawley rats express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man6P) receptors and respond to IGF-I with a dose-dependent accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2]. Specific binding of [125I]IGF-I to isolated membranes from cultured cardiac myocytes amounted to 1-1.2%. Binding of [125I]IGF-I was inhibited by unlabeled IGF-I at nanomolar concentrations and insulin at much higher concentrations. These data suggest that IGF-I binds to its own receptor on rat cardiac myocytes. Competitive binding studies using isolated membranes from cardiac myocytes and [125I]IGF-II showed 2-4% specific binding. Binding of [125I]IGF-II was inhibited by IGF-II and much less potently by IGF-I and insulin. Immunoglobulin G (IgG) 3637 (an IgG directed against the IGF-II/Man6P receptor) partially inhibited binding of [125I]IGF-II whereas nonimmune IgG did not. Affinity cross-linking studies with [125I]IGF-II and cardiac myocyte membranes and subsequent analysis of the ligand-receptor complex using SDS-PAGE and autoradiography showed a radiolabeled band of approximately 250 kilodalton (kDa). The formation of the [125I]IGF-II-receptor complex was inhibited by incubation with IGF-II and IgG 3637 but not by insulin or nonimmune IgG. Western blotting of protein extracts from cultured cardiac myocytes was performed using IgG 3637 and an immunoperoxidase technique for the visualization of the IGF-II/Man6P receptor protein. A specific band at 220 kDa under nonreducing conditions was detected on the blots, providing further evidence for the expression of the IGF-II/Man6P receptor by cardiac myocytes. The effect of IGFs on the accumulation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[3H]inositol-labeled cultured cardiac myocytes. IGF-I (50 ng/ml) stimulated the accumulation both of Ins(1,4,5)P3 and Ins(1,4)P2 after 30 sec by 43% and 63%. IGF-II (up to 500 ng/ml) had no significant effect on inositol phosphate accumulation under the same conditions. However, in the presence of millimolar concentrations of Man6P, IGF-II (500 ng/ml) also increased Ins(1,4,5)P3 accumulation by 59%. We conclude that cardiac myocytes from adult rats express IGF receptors and respond to IGFs with the accumulation of Ins(1,4,5)P3 and Ins(1,4)P2. This effect seems to be mediated by an IGF-I receptor-specific pathway.
成年Sprague-Dawley大鼠的培养心肌细胞表达胰岛素样生长因子-I（IGF-I）受体和胰岛素样生长因子-II/甘露糖6-磷酸（IGF-II/Man6P）受体，并对IGF-I产生反应，使肌醇1,4,5-三磷酸[Ins(1,4,5)P3]和肌醇1,4-二磷酸[Ins(1,4)P2]呈剂量依赖性积累。[125I]IGF-I与培养心肌细胞分离膜的特异性结合率为1%-1.2%。[125I]IGF-I的结合在纳摩尔浓度的未标记IGF-I和高得多浓度的胰岛素作用下受到抑制。这些数据表明IGF-I与大鼠心肌细胞上自身的受体结合。使用心肌细胞分离膜和[125I]IGF-II进行的竞争性结合研究显示特异性结合率为2%-4%。[125I]IGF-II的结合受到IGF-II抑制，而IGF-I和胰岛素的抑制作用较弱。免疫球蛋白G（IgG）3637（一种针对IGF-II/Man6P受体的IgG）部分抑制[125I]IGF-II的结合，而非免疫IgG则无此作用。用[125I]IGF-II和心肌细胞膜进行亲和交联研究，随后使用SDS-PAGE和放射自显影分析配体-受体复合物，显示出一条约250千道尔顿（kDa）的放射性标记条带。[125I]IGF-II-受体复合物的形成在与IGF-II和IgG 3637孵育时受到抑制，但不受胰岛素或非免疫IgG抑制。使用IgG 3637和免疫过氧化物酶技术对培养心肌细胞的蛋白质提取物进行蛋白质印迹，以可视化IGF-II/Man6P受体蛋白。在非还原条件下，印迹上检测到一条220 kDa的特异性条带，为心肌细胞表达IGF-II/Man6P受体提供了进一步证据。通过对肌醇-[3H]肌醇标记的培养心肌细胞的高氯酸提取物进行HPLC分析，测定IGF对肌醇磷酸积累的影响。IGF-I（50 ng/ml）在30秒后使Ins(1,4,5)P3和Ins(1,4)P2的积累分别增加43%和63%。在相同条件下，IGF-II（高达500 ng/ml）对肌醇磷酸积累无显著影响。然而，在存在毫摩尔浓度的Man6P时，IGF-II（500 ng/ml）也使Ins(1,4,5)P3积累增加59%。我们得出结论，成年大鼠的心肌细胞表达IGF受体，并通过Ins(1,4,5)P3和Ins(1,4)P2的积累对IGF产生反应。这种效应似乎由IGF-I受体特异性途径介导。
