Bishara Nour B, Dunlop Marjorie E, Murphy Timothy V, Darby Ian A, Sharmini Rajanayagam M A, Hill Michael A
Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.
J Cell Physiol. 2002 Oct;193(1):80-92. doi: 10.1002/jcp.10153.
Studies have shown diabetes to be associated with alterations in composition of extracellular matrix and that such proteins modulate signal transduction. The present studies examined if non-enzymatic glycation of fibronectin or a mixed matrix preparation (EHS) alters endothelial cell Ca(2+) signaling following agonist stimulation. Endothelial cells were cultured from bovine aorta and rat heart. To glycate proteins, fibronectin (10 microg/ml), or EHS (2.5 mg/ml) were incubated (37 degrees C, 30 days) with 0.5 M glucose-6-phosphate. Matrix proteins were coated onto cover slips after which cells (10(5) cells/ml) were plated and allowed to adhere for 16 h. For measurement of intracellular Ca(2+), cells were loaded with fura 2 (2 microM) and fluorescence intensity monitored. Bovine cells on glycated EHS showed decreased ability for either ATP (10(-6) M) or bradykinin (10(-7) M) to increase Ca(2+) (i). In contrast, glycated fibronectin did not impair agonist-induced increases in Ca(2+) (i). In the absence of extracellular Ca(2+), ATP elicited a transient increase in Ca(2+) (i) consistent with intracellular release. Re-addition of Ca(2+) resulted in a secondary rise in Ca(2+) (i) indicative of store depletion-mediated Ca(2+) entry. Both phases of Ca(2+) mobilization were reduced in cells on glycated mixed matrix; however, as the ratio of the two components was similar in all cells, glycation appeared to selectively impair Ca(2+) release from intracellular stores. Thapsigargin treatment demonstrated an impaired ability of cells on glycated EHS to increase cytoplasmic Ca(2+) consistent with decreased endoplasmic reticulum Ca(2+) stores. Further support for Ca(2+) mobilization was provided by increased baseline IP(3) levels in cells plated on glycated EHS. Impaired ATP-induced Ca(2+) release could be induced by treating native EHS with laminin antibody or exposing cells to H(2)O(2) (20-200 microM). Glycated EHS impaired Ca(2+) signaling was attenuated by treatment with aminoguanidine or the antioxidant alpha-lipoic acid. The results demonstrate that matrix glycation impairs agonist-induced Ca(2+) (i) increases which may impact on regulatory functions of the endothelium and implicate possible involvement of oxidative stress.
研究表明，糖尿病与细胞外基质成分的改变有关，且这类蛋白质可调节信号转导。本研究检测了纤连蛋白或混合基质制剂（EHS）的非酶糖基化是否会改变激动剂刺激后内皮细胞的钙离子信号。内皮细胞取自牛主动脉和大鼠心脏。为使蛋白质糖基化，将纤连蛋白（10微克/毫升）或EHS（2.5毫克/毫升）与0.5 M葡萄糖-6-磷酸一起孵育（37摄氏度，30天）。将基质蛋白包被在盖玻片上，然后接种细胞（10⁵个细胞/毫升）并使其贴壁16小时。为测量细胞内钙离子浓度，用fura 2（2微摩尔）加载细胞并监测荧光强度。在糖基化EHS上的牛细胞对ATP（10⁻⁶ M）或缓激肽（10⁻⁷ M）增加钙离子浓度（i）的能力降低。相比之下，糖基化纤连蛋白并不损害激动剂诱导的钙离子浓度（i）增加。在无细胞外钙离子的情况下，ATP引起钙离子浓度（i）短暂增加，这与细胞内释放一致。重新添加钙离子导致钙离子浓度（i）二次升高，表明储存耗竭介导的钙离子内流。在糖基化混合基质上的细胞中，钙离子动员的两个阶段均降低；然而，由于所有细胞中两种成分的比例相似，糖基化似乎选择性地损害了细胞内储存钙离子的释放。毒胡萝卜素处理表明，在糖基化EHS上的细胞增加细胞质钙离子浓度的能力受损，这与内质网钙离子储存减少一致。接种在糖基化EHS上的细胞中基础肌醇三磷酸水平升高，进一步支持了钙离子动员。用层粘连蛋白抗体处理天然EHS或使细胞暴露于H₂O₂（20 - 200微摩尔）可诱导ATP诱导的钙离子释放受损。用氨基胍或抗氧化剂α-硫辛酸处理可减轻糖基化EHS对钙离子信号的损害。结果表明，基质糖基化损害激动剂诱导的钙离子浓度（i）增加，这可能影响内皮细胞的调节功能，并提示氧化应激可能参与其中。
