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

Juan Carlos Gil-Redondo,Andreas Weber,María Teresa Miras-Portugal,José Luis Toca-Herrera,Jagoba Iturri,Raquel Pérez-Sen,Esmerilda G Delicado,Felipe Ortega

doi:10.3390/ijms22020624

Abstract

Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y1 and P2Y2 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 P2Y2 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 P2Y1 and P2Y13 receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y13 receptor, although they were negatively modulated by P2Y1 receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells.

Highlights

Nucleotides are universal extracellular messengers that regulate most bodily functions
Eight different P2Y receptors (P2YRs) have been identified in mammals and are subdivided into two subgroups, based on phylogenetic and structural similarities: the “P2Y1 R-like” group, that includes P2Y1R, P2Y2R, P2Y4R, P2Y6R, and P2Y11R, which are coupled to Gq, and the “P2Y12R-like” subgroup, coupled to Gi, that includes the new ones P2Y12R, P2Y13R, and P2Y14R [3]
The main goal of this work was to investigate the responses evoked after nucleotide receptor stimulation with a new methodological approach, Atomic Force Microscopy, which allows for the analysis of changes in the mechanical properties of the membrane in living cells at the nanometric level

Summary

Introduction

Nucleotides are universal extracellular messengers that regulate most bodily functions Their actions are mediated by membrane receptors called P2 receptors, which are key pieces of the complex puzzle of purinergic signaling. Nucleotide receptors have been classified into two groups: ionotropic P2X receptors (P2XRs), which are ATP-gated cationic channels, and metabotropic P2Y receptors (P2YRs), belonging to the G-protein coupled receptor (GPCR) superfamily. These P2YRs consist of 7-membrane-spanning proteins coupled to G proteins and, in contrast to ionotropic receptors, P2YRs are selectively activated by adenine and uracil nucleotides, and even discriminate between tri- and diphosphate nucleotides [1,2]. P2YRs are widely expressed in cells of all tissues and organs from neural and non-neural origin [2]

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