Institute of Pharmacology, Heidelberg University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany.
Department of Medicine I and Clinical Chemistry, Heidelberg University Hospital, Germany; German Center for Diabetes Research (DZD), Germany.
Cell Calcium. 2019 Mar;78:66-75. doi: 10.1016/j.ceca.2019.01.002. Epub 2019 Jan 9.
Methylglyoxal (MG) is a by-product of glucose metabolism and its accumulation has been linked to the development of diabetic complications such as retinopathy and nephropathy by affecting multiple signalling pathways. However, its influence on the intracellular Ca homeostasis and particularly Ca entry, which has been reported to be mediated via TRPA1 channels in DRG neurons, has not been studied in much detail in other cell types. In this study, we report the consequences of acute and long-term MG application on intracellular Ca levels in endothelial cells. We showed that acute MG application doesn't evoke any instantaneous changes in the intracellular Ca concentration in immortalized mouse cardiac endothelial cells (MCECs) and murine microvascular endothelial cells (muMECs). In contrast, an MG-induced rise in intracellular Ca level was observed in primary mouse mesangial cells within 30 s, indicating that the modulation of Ca homeostasis by MG is strictly cell type specific. The formation of the MG-derived advanced glycation end product (AGE) MG-H1 was found to be time and concentration-dependent in MCECs. Likewise, MG pre-incubation for 6 h increased the angiotensin II-evoked Ca entry in MCECs and muMECs which was abrogated by inhibition of Calcium release activated calcium (CRAC) channels with GSK-7975A, but unaffected by an inhibitor specific to TRPA1 channels. Quantitative PCR analysis revealed that MG pre-treatment did not affect expression of the genes encoding the angiotensin receptors AT1R (Agtr 1a & Agtr 1b), Trpa1 nor Orai1, Orai2, Orai3, Stim1, Stim2 and Saraf which operate as constituents or regulators of CRAC channels and store-operated Ca entry (SOCE) in other cell types. Together, our results show that long-term MG stimulation leads to the formation of glycation end products, which facilitates the agonist-evoked Ca entry in endothelial cells, and this could be a new pathway that might lead to MG-evoked vasoregression observed in diabetic vasculopathies.
甲基乙二醛 (MG) 是葡萄糖代谢的副产物，其积累通过影响多种信号通路与糖尿病并发症的发展有关，如视网膜病变和肾病。然而，其对细胞内钙稳态的影响，特别是钙内流，据报道在背根神经节神经元中是通过 TRPA1 通道介导的，但在其他细胞类型中尚未进行详细研究。在这项研究中，我们报告了急性和长期 MG 应用对内皮细胞细胞内 Ca 水平的影响。我们表明，急性 MG 应用不会引起永生的小鼠心脏内皮细胞 (MCEC) 和小鼠微血管内皮细胞 (muMEC) 细胞内 Ca 浓度的瞬时变化。相反，在原代小鼠系膜细胞中观察到 30s 内 MG 诱导的细胞内 Ca 水平升高，表明 MG 对钙稳态的调节是严格的细胞类型特异性的。在 MCEC 中发现 MG 衍生的晚期糖基化终产物 (AGE) MG-H1 的形成具有时间和浓度依赖性。同样，MG 预孵育 6 小时增加了 MCEC 和 muMEC 中血管紧张素 II 诱导的 Ca 内流，该作用被 GSK-7975A 抑制钙释放激活钙 (CRAC) 通道所阻断，但不受 TRPA1 通道抑制剂的影响。定量 PCR 分析显示，MG 预处理不会影响编码血管紧张素受体 AT1R (Agtr1a 和 Agtr1b)、Trpa1 以及 Orai1、Orai2、Orai3、Stim1、Stim2 和 Saraf 的基因的表达，这些基因在其他细胞类型中作为 CRAC 通道和储存操作钙内流 (SOCE) 的组成部分或调节剂起作用。总之，我们的结果表明，长期 MG 刺激导致糖基化终产物的形成，这促进了内皮细胞中激动剂诱导的 Ca 内流，这可能是导致糖尿病血管病变中观察到的 MG 诱导的血管舒张的新途径。
