Purinergic vasotoxicity in retinal capillaries

Abstract

Purpose: Purinergic vasotoxicity is a newly discovered pathophysiological mechanism (Puro, Prog Ret Eye Res, 2012). First identified in retinal microvessels, purinergic vasotoxicity is mediated by P2X₇ receptors, although it is unclear how P2X₇ activation triggers microvascular cell death. We now report that purinergic vasotoxicity in retinal capillaries is dependent on the formation of transmembrane pores, the elevation of intracellular oxidants, the activation of K_ATP channels and influx of Ca²⁺.

Methods: In microvascular complexes freshly isolated from the adult rat retina, we used trypan blue dye exclusion, YO-PRO uptake, DCF fluorescence, perforated-patch recordings and fura-2 to assess the effect of the P2X₇ agonist, BzATP, on cell viability, pore formation, oxidant level, ionic currents and [Ca²⁺]_i.

Results: In retinal capillaries, BzATP resulted in (1) the formation of YO-PRO permeable pores, (2) a rise in intracellular oxidants, (3) the activation of K_ATP channels and (4) an influx of Ca²⁺. Indicative of the importance of pores, prevention of pore formation by UTP treatment (Sugiyama et al., 2005) markedly lessened BzATP-induced increases in cell death, F_DCF fluorescence, K_ATP current and [Ca²⁺]_i. We found that pore formation alone is not sufficient to trigger vasotoxicity as the antioxidant, n-acetyl-cysteine, prevented BzATP-induced cell death without altering YO-PRO uptake. Furthermore, the combined formation of pores and oxidants is also insufficient because the inhibition of oxidant-sensitive K_ATP channels by glibenclamide prevented BzATP-induced cell death, but did not prevent increased YO-PRO uptake and F_DCF fluorescence. How does K_ATP channel activation contribute to purinergic vasotoxicity? We hypothesized that P2X₇-induced death is dependent on the influx of Ca²⁺ that occurs when a K_ATP-driven hyperpolarization increases calcium’s electrogradient. Consistent with this, BzATP-induced death was dependent on an influx-mediated [Ca²⁺]_i rise that was sensitive to glibenclamide.

Conclusions: In retinal capillaries, purinergic vasotoxicity is mediated by a pathway involving P2X₇ receptors, transmembrane pores, oxidants, K_ATP channels and Ca²⁺ influx. Elucidation of this pathway has potential therapeutic importance because Sugiyama et al. (2004) showed that the vulnerability of the capillaries to purinergic vasotoxicity is boosted in the diabetic retina.

Keywords: 688 retina • 436 blood supply • 569 ion channels