Astrocyte cultures exhibit P2X7 receptor channel opening in the absence of exogenous ligands

Abstract

P2X7 receptors (P2X7Rs) gate the opening of large channels when activated by ATP or other ligands. P2X7Rs are expressed by astrocytes in culture and by reactive astrocytes in vivo, and astrocytes in culture have been shown to release glutamate and ATP in response to P2X7R activation. However, P2X7Rs are activated by ATP only at concentrations greater than 1 mM. The conditions under which astrocyte P2X7Rs would be activated in vivo are, thus, unclear. Here we show that astrocytes in culture exhibit basal P2X7R activity. Primary mouse astrocytes were found to take up the P2X7R permeant dyes YO-PRO-1 (YP) and propidium iodide in absence of any added ligands. By contrast, cultured rat astrocytes took up very little YP, consistent with their much lower level of P2X7R expression. The uptake by mouse astrocytes was inhibited by oxATP, suramin, KN-62 and brilliant blue G, and by siRNA knock-down of P2X7R. Astrocyte uptake of YP was also inhibited by phenol red at concentrations above 50 muM, suggesting that phenol red present in standard cell culture media may influence P2X7R channel activity. Treatment with apyrase, an enzyme that degrades extracellular ATP, partially decreased YP uptake in astrocytes. Conversely, exposure to the ectonucleotidase inhibitor ARL67156 enhanced YP uptake and astrocytes plated without contiguous neighboring astrocytes showed reduced basal YP uptake. These results suggest that the basal uptake of YP may be due to activation of P2X7R by release of ATP by astrocytes themselves into intercellular spaces.

Impact of Manganese on and Transfer across Blood-Brain and Blood-Cerebrospinal Fluid Barrier in Vitro

“…How Mn crosses the BBB has been a subject of many studies, and it appears that several pathways are operative, including facilitated diffusion (12) and active transport. Mn is discussed to be transported among others via the divalent metal transporter 1 (DMT1), the transferrin receptor (TfR), the divalent metal/bicarbonate ion symporters ZIP8 and ZIP14, various calcium channels, the solute carrier-39 (SLC39) family of zinc transporters, park9/ATP13A2, the magnesium transporter hip14, the transient receptor potential melastatin 7 (TRPM7) channels/transporters, homomeric purinreceptors (P2X and P2Y), and the citrate transporter (3,(13)(14)(15)(16).…”

Astrocytes, but Not Neurons, Exhibit Constitutive Activation of P2X7 Receptors in Mouse Acute Cortical Slices under Non-stimulated Resting Conditions

“…12,24) After slices had been pre-incubated with or without the designated concentration of a P2X7R antagonist, brilliant blue G (BBG), 25) KN62, 26) or A438079, 27) for 10 min in aCSF equilibrated with 95% O 2 and 5% CO 2 at 30°C, they were incubated with 5 µM PI for 30 min, this time point being determined based on time-dependent PI uptake profiles, in which PI uptake increased linearly from 15 to 60 min (data not shown). The uptake was terminated by washing three times with warmed aCSF and then the slices were subjected to the aforementioned immunostaining procedure.…”

“…Previously, we demonstrated that in primary cultured mouse astrocytes, P2X7Rs were activated without any exogenous ligands, and that this constitutive activation was due, at least in part, to autocrine/paracrine stimulation by ATP released from astrocytes themselves into restricted intercellular space, 12) regulated by P2X7R splice variants, 13) and involved in regulation of astrocytic engulfing activity. 14) These findings suggest that under non-stimulated resting conditions, P2X7Rs expressed by astrocytes play roles in the maintenance of brain neuronal homeostasis.…”

Intracellular Signaling Pathways Integrating the Pore Associated with P2X7R Receptor with Other Large Pores

“…P2X7R receptor large conductance channel was inhibited by RNAi to pannexin-1, inhibitory peptide and pannexin antagonists . In contrast, other groups did not observe inhibition of the P2X7R large conductance channel for pannexin-1 inhibitors or RNAi (Faria et al, 2005(Faria et al, , 2010Nagasawa et al, 2009b;Reyes et al, 2008;Schchater et al, 2008;Yan et al, 2008Yan et al, , 2011. Up to now, for some groups the pannexin-1 seems to be an important player in this phenomenon, but apparently this protein is working in conjunction with other protein(s).…”