Nucleotides-Induced Changes in the Mechanical Properties of Living Endothelial Cells and Astrocytes, Analyzed by Atomic Force Microscopy

• Published in: International journal of molecular sciences Vol. 22; no. 2; p. 624
• Main Authors: Gil-Redondo, Juan Carlos, Iturri, Jagoba, Ortega, Felipe, Pérez-Sen, Raquel, Weber, Andreas, Miras-Portugal, María Teresa, Toca-Herrera, José Luis, Delicado, Esmerilda G
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 10.01.2021; MDPI
• Subjects: Adenosine Diphosphate - analogs & derivatives; Adenosine Diphosphate - metabolism; Adenosine Diphosphate - pharmacology; Adhesion; Astrocytes; Astrocytes - drug effects; Astrocytes - metabolism; Atomic force microscopy; Calcium (intracellular); Calcium signalling; Cell adhesion & migration; Cytoskeleton; Endothelial cells; Human Umbilical Vein Endothelial Cells - drug effects; Human Umbilical Vein Endothelial Cells - metabolism; Humans; Intracellular signalling; Mechanical properties; Metabotropic receptors; Microscopy; Microscopy, Atomic Force; Modulus of elasticity; nucleotide receptor; Nucleotides; P2Y nucleotide receptor; Purine P2Y receptors; Receptor mechanisms; Receptors, Purinergic P2 - metabolism; Receptors, Purinergic P2Y1 - metabolism; Rupture; Rupturing; Signal Transduction; Spectroscopy; Stiffness; Stimulation; Thionucleotides - metabolism; Thionucleotides - pharmacology; Umbilical vein; Uridine Triphosphate - metabolism; Uridine Triphosphate - pharmacology; endothelial cells; astrocytes; atomic force microscopy; nucleotide receptor; P2Y nucleotide receptor
• ISSN: 1422-0067; 1661-6596; 1422-0067
• 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.