Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Instituto Universitario de Investigación en Neuroquímica (IUIN), Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdiSSC), Universidad Complutense Madrid, 28040 Madrid, Spain.
Department of Nanobiotechnology (DNBT), Institute for Biophysics, BOKU University for Natural Resources and Life Sciences, Muthgasse 11 (Simon Zeisel Haus), A-1190 Vienna, Austria.
Int J Mol Sci. 2021 Jan 10;22(2):624. doi: 10.3390/ijms22020624.
Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y and P2Y receptors appear as main actors, since their stimulation induces intracellular calcium mobilization and activates signaling cascades linked to cytoskeletal reorganization. In the present work, we have analyzed, by means of atomic force microscopy (AFM) in force spectroscopy mode, the mechanical response of human umbilical vein endothelial cells (HUVEC) and astrocytes upon 2MeSADP and UTP stimulation. This approach allows for simultaneous measurement of variations in factors such as Young's modulus, maximum adhesion force and rupture event formation, which reflect the potential changes in both the stiffness and adhesiveness of the plasma membrane. The largest effect was observed in both endothelial cells and astrocytes after P2Y receptor stimulation with UTP. Such exposure to UTP doubled the Young's modulus and reduced both the adhesion force and the number of rupture events. In astrocytes, 2MeSADP stimulation also had a remarkable effect on AFM parameters. Additional studies performed with the selective P2Y and P2Y receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y receptor, although they were negatively modulated by P2Y receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells.
内皮细胞和星形胶质细胞优先表达代谢型 P2Y 核苷酸受体，这些受体参与维持血管和神经功能。在这些受体中，P2Y 和 P2Y 受体似乎是主要的作用者，因为它们的刺激会引起细胞内钙动员，并激活与细胞骨架重排相关的信号级联。在本工作中，我们通过原子力显微镜（AFM）在力谱模式下分析了 2MeSADP 和 UTP 刺激对人脐静脉内皮细胞（HUVEC）和星形胶质细胞的机械响应。这种方法允许同时测量杨氏模量、最大粘附力和破裂事件形成等因素的变化，这些变化反映了质膜的刚度和粘附性的潜在变化。在用 UTP 刺激 P2Y 受体后，内皮细胞和星形胶质细胞均观察到最大的效应。这种暴露于 UTP 会使杨氏模量增加一倍，并降低粘附力和破裂事件的数量。在星形胶质细胞中，2MeSADP 刺激对 AFM 参数也有显著影响。用选择性 P2Y 和 P2Y 受体拮抗剂进行的额外研究表明，2MeSADP 诱导的机械变化是由 P2Y 受体介导的，尽管它们受到 P2Y 受体刺激的负调节。因此，我们的结果表明，AFM 可以成为评估活细胞中功能性天然核苷酸受体的非常有用的工具。
