Journal
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
Volume 288, Issue 3, Pages C568-C576Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00380.2004
Keywords
calcium channels; capillaries; purinoceptors; vasotoxicity
Categories
Funding
- NEI NIH HHS [EY-07003, EY-12505] Funding Source: Medline
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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 Ca2+ 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 Ca2+ 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 Ca2+ 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.
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