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

Abstract

As members of the family of nucleotide receptors, P2X7 receptors are of particular interest due to their unique structural and pharmacological characteristics. As ATP-gated ionic channels, P2X7 receptors in their activation elicit membrane depolarization; extracellular calcium influx; and activation of several downstream intracellular signaling pathways, some of them independent of the ionic channel activity. Further interactions of P2X7 receptors and cytoskeleton-related proteins have also been confirmed, and we previously described the effects of P2X7 receptor stimulation on the morphology of rat cerebellar astrocytes. In the present work, we used time-lapse video microscopy and atomic force microscopy (AFM) to elucidate the effects of P2X7 receptor stimulation on the morphology, migratory capabilities, and mechanical properties of rat cerebellar astrocytes in vitro. Stimulation of P2X7 receptors with the selective agonist BzATP specifically caused an increase in cell size, motility, and number of membrane protrusions of the astrocytes in culture. These effects were reverted when cells were previously treated with the competitive antagonist of P2X7R, A 438079. AFM analysis also showed an increase in cell stiffness and viscosity after P2X7 receptor stimulation. Surprisingly, these effects on the mechanical properties of the cell were not blocked by the treatment with the antagonist. Fluorescence microscopy analysis of the actin cytoskeleton showed an increase in actin stress fibers after BzATP treatment, an effect that again was not blocked by previous treatment with the antagonist, further confirming that the effects of P2X7 receptors on the cytoskeleton of astrocytes are, at least in part, independent of the ionic channel activity.

Role of the P2 × 7 receptor in neurodegenerative diseases and its pharmacological properties

“…Therefore, the unique structure of P2 × 7R is crucial for its function. The functional receptor is composed of a trimeric subunit and its structure is composed of an ionic pore with six transmembrane (TM) domains for each subunit, a longer intracellular domain, and three ATP-binding sites in a chalice-like extracellular domain [ 25 ]. The three-dimensional structure of the P2 × 7R subunit is dolphin-shaped, with the extracellular region serving as the body (including the head and fins) and two transmembrane helices (TM1 and TM2) serving as the tail [ 26 ].…”

DUSP1/MKP-1 represents another piece in the P2X7R intracellular signaling puzzle in cerebellar cells: our last journey with Mª Teresa along the purinergic pathways of Eden

“…Considering that BDNF exerts neuroprotective effects that combat glutamate neurotoxicity in cerebellar granule neurons, DUSP1 could underlie these actions, as well as those triggered by P2X7R [ 22 , 40 ]. In addition, DUSP1 may also be involved in the cytoskeletal rearrangements induced by P2X7R in cerebellar astrocytes [ 20 , 41 ].…”