Regulation of P2X₇-induced pore formation and cell death in pericyte-containing retinal microvessels

Abstract

The purpose if this study was to elucidate how extracellular ATP causes cell death in the retinal microvasculature. Although ATP appears to serve as a vasoactive signal acting via P2X(7) and P2Y(4) purinoceptors, this nucleotide can kill microvascular cells of the retina. Because P2X(7) receptor activation causes transmembrane pores to form and microvascular cells to die, we initially surmised that pore formation accounted for ATP's lethality. To test this hypothesis, we isolated pericyte-containing microvessels from rat retinas, assessed cell viability using Trypan blue dye exclusion, detected pores by determining the uptake of the fluorescent dye YO-PRO-1, measured intracellular Ca(2+) with the use of fura-2, and monitored ionic currents via perforated patch pipettes. As predicted, ATP-induced cell death required P2X(7) receptor activation. However, we found that pore formation was minimal because ATP's activation of P2Y(4) receptors prevented P2X(7) pores from forming. Rather than opening lethal pores, ATP kills via a mechanism involving voltage-dependent Ca(2+) channels (VDCC). Our experiments suggest that when high concentrations of ATP caused nearly all microvascular P2X(7) receptor channels to open, the resulting profound depolarization opened VDCC. Consistent with lethal Ca(2+) influx via VDCC, ATP-induced cell death was markedly diminished by the VDCC blocker nifedipine or a nitric oxide (NO) donor that inhibited microvascular VDCC. We propose that purinergic vasotoxicity is normally prevented in the retina by NO-mediated inhibition of VDCC and P2Y(4)-mediated inhibition of P2X(7) pore formation. Conversely, dysfunction of these protective mechanisms may be a previously unrecognized cause of cell death within the retinal microvasculature.

Nucleotides in the Eye: Focus on Functional Aspects and Therapeutic Perspectives

“…Thus, diabetes appears to facilitate the channel-to-pore transition that occurs during activation of P2X7 receptors, although the mechanism by which diabetes induces this effect remains unclear. Interestingly, activation of P2Y 4 receptors inhibited P2X7 pore formation and microvascular cell death (Sugiyama et al, 2005). These findings suggest that ATP vasotoxicity may be improved by P2Y 4 -mediated inhibition of the lethal effects of P2X7 on the diabetic retina.…”

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

“…It has been reported recently that P2X 7 receptors are expressed in central and spinal cord neurons [19][20][21][22]. Brain glial cells (microglia, astrocytes, and Muller cells), bone cells (osteoblasts, osteoclasts, and osteocytes), and epithelial and endothelial cells are also rich in P2X 7 receptors [23][24][25][26][27][28][29]. Expression of P2X 7 receptors has also been demonstrated in the enteric nervous system of the small intestine, kidney, urinary tract, uterus, and liver [30][31][32][33].…”

Endothelial P2X7 receptors’ expression is reduced by schistosomiasis

“…The P2X7 receptor is a multifunctional protein related to multiple signalling pathways, including vasodilation, endothelial cell apoptosis, inflammasome activation and the production of pro-inflammatory cytokines, such as interleukin-1β (IL-1β). Thus, the P2X7 receptor causes a wide variety of biological effects associated with infection and inflammation [24,[54][55][56], eventually leading to killing of intracellular parasite [57,58]. IL-1β is a multifunctional cytokine upregulated in the initial phase of schistosomiasis [59].…”