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Maho Shibata, Atsuko Nakaizumi, Ting Zhang, Eisuke Ishizaki, Masanori Fukumoto, Alma Barajas-Espinosa, Donald Puro; Purinergic vasotoxicity in retinal capillaries. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2690.
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© ARVO (1962-2015); The Authors (2016-present)
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 KATP channels and influx of Ca2+.
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.
In retinal capillaries, BzATP resulted in (1) the formation of YO-PRO permeable pores, (2) a rise in intracellular oxidants, (3) the activation of KATP 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, FDCF fluorescence, KATP 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 KATP channels by glibenclamide prevented BzATP-induced cell death, but did not prevent increased YO-PRO uptake and FDCF fluorescence. How does KATP channel activation contribute to purinergic vasotoxicity? We hypothesized that P2X7-induced death is dependent on the influx of Ca2+ that occurs when a KATP-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.
In retinal capillaries, purinergic vasotoxicity is mediated by a pathway involving P2X7 receptors, transmembrane pores, oxidants, KATP 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.
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