Regulation of P2X7-induced pore formation and cell death in pericyte-containing retinal microvessels.
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.
Sugiyama
Tetsuya
T
Department of Ophthalmology and Visual Sciences, University of Michigan, 1000 Wall St., Ann Arbor, MI 48105, USA.
Kawamura
Hajime
H
Yamanishi
Shigeki
S
Kobayashi
Masato
M
Katsumura
Kozo
K
Puro
Donald G
DG
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D002196:Capillaries / Q000166:cytology* / Q000378:metabolism; D016923:Cell Death / Q000502:physiology*; D002470:Cell Survival; D008833:Microcirculation; D009569:Nitric Oxide / Q000378:metabolism; D018408:Patch-Clamp Techniques; D020286:Pericytes / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D012160:Retina / Q000033:anatomy & histology* / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
D015220:Calcium Channels; C546470:P2rx7 protein, rat; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C104749:purinoceptor P2Y4; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D014544:Uridine Triphosphate