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

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.

Nucleotide-Induced Nanoscale Changes in the Mechanical Properties of Rat Cerebellar Astrocytes: Selective Stimulation and Blocking of the Purinergic Receptor P2X7

“…At first glimpse, and compared to untreated astrocytes, an increase in the stiffness (E app ) of the astrocytes after nucleotide stimulation of P2X7 receptors with BzATP can be observed (4.84 ± 4.69 kPa in control conditions vs. 5.93 ± 4.42 kPa after P2X7 stimulation), while initial treatment with the antagonist compound (A43) did not cause any significant effect in these same factors, and cells resembled those used as control (untreated). Interestingly, subsequent exposure of A43 treated cells to BzATP (A43 + BzATP) also caused a significant increase in the stiffness of the cells (8.60 ± 7.76 kPa), therefore indicating that the intended blocking by A43 was ineffective.. A similar behavior was described for pre-blocked P2YRs treated with 2MeSADP, which was finally connected to the intervention of two distinguishable contributors (P2Y 1 and P2Y 13 ) [ 26 ]. Then, and attending to our current observation, the intervention of a second element, which is not suppressed by action of the A43, can also be envisaged.…”

“…It was previously shown how the stimulation of metabotropic P2Y 13 receptor was sufficient to modify the cell motility of postnatal cerebellar astrocytes in primary cell cultures [ 26 ]. To explore whether the activation of the ionotropic P2X7 receptor may also induce changes in the cell size and migration of this cell population, cerebellar astrocytes were incubated in the presence or absence of the selective agonist of the P2X7R, BzATP (300 µM), for 1 h. Astrocytes were subsequently placed on a time-lapse video microscopy device and monitored for 3 h, acquiring bright field images every 5 min.…”

“…On the basis of these principles, previous collaborative work between our groups showed how nucleotide receptor regulation can influence the mechanical properties of cells at the nanoscale, as concluded from the studies performed on P2YR-expressing endothelial HUVEC cells and rat cerebellar astrocytes [ 26 ]. Stimulation of P2Y 2 Rs with UTP in both cell types caused an increase in cell stiffness and a decrease in adhesive properties.…”

The Interplay of Endothelial P2Y Receptors in Cardiovascular Health: From Vascular Physiology to Pathology

“…BAPTA-AM also prevented phosphorylation of FAK on tyrosine 397. In addition, P2Y 2 -R activation in endothelial cells caused change in both the stiffness and adhesiveness of the plasma membrane [ 67 ] and cytoskeletal rearrangements, as shown in endothelial cells by an increase in stress fiber formation [ 64 ]. Exposure to ATP or UTP also led to an increase in α v integrin expression at the protein level, which has previously been reported to play a role in mediating cell adhesion to the extracellular matrix, cell migration, and angiogenesis.…”

Dissecting the Purinergic Signaling Puzzle

“…In astrocytes, P2Y 1 /P2Y 13 receptor activation also modify AFM parameters. The results indicate that nucleotides alter mechanical properties of the plasma membrane in living cells modifying cytoskeleton rearrangements, focal adhesion kinase (FAK) being one of the targeted proteins [15].…”