Abstract
P2X7 receptor/channels in the retinal microvasculature not only regulate vasomotor activity, but can also trigger cells in the capillaries to die. While it is known that this purinergic vasotoxicity is dependent on the transmembrane pores that form during P2X7 activation, events linking pore formation with cell death remain uncertain. To better understand this pathophysiological process, we used YO-PRO-1 uptake, dichlorofluorescein fluorescence, perforated-patch recordings, fura-2 imaging and trypan blue dye exclusion to assess the effects of the P2X7 agonist, benzoylbenzoyl-ATP (BzATP), on pore formation, oxidant production, ion channel activation, [Ca2+]i and cell viability. Experiments demonstrated that exposure of retinal microvessels to BzATP increases capillary cell oxidants via a mechanism dependent on pore formation and the enzyme, NADPH oxidase. Indicative that oxidation plays a key role in purinergic vasotoxicity, an inhibitor of this enzyme completely prevented BzATP-induced death. We further discovered that vasotoxicity was boosted 4-fold by a pathway involving the oxidation-driven activation of hyperpolarizing KATP channels and the resulting increase in calcium influx. Our findings revealed that the previously unappreciated pore/oxidant/KATP channel/Ca2+ pathway accounts for 75% of the capillary cell death triggered by sustained activation of P2X7 receptor/channels. Elucidation of this pathway is of potential therapeutic importance since purinergic vasotoxicity may play a role in sight-threatening disorders such as diabetic retinopathy.
Highlights
Functional P2X7 receptor/channels were discovered in retinal capillaries 15 years ago, much remains to be learned about their impact on retinovascular physiology and pathobiology
Based on our previous analysis of the electrophysiological effects of the oxidant H2 O2 [24], we postulated that the P2X7 -induced increase in oxidants may activate redox-sensitive KATP channels whose voltage-increasing effect would enhance the electro-gradient for the influx of calcium whose elevated intracellular concentration is known to increase the lethality of oxidative stress [24,25]
These considerations led us to posit that a pore/oxidant/KATP channel/Ca2+ pathway boosts the vulnerability of retinal capillaries to P2X7 vasotoxicity (Figure 1)
Summary
Functional P2X7 receptor/channels were discovered in retinal capillaries 15 years ago, much remains to be learned about their impact on retinovascular physiology and pathobiology. One possibility is that these purinergic receptor/channels transduce the putative glial-to-vascular signal, ATP [2], into an alteration in local perfusion They may play a role in mediating vascular responses to nicotinamide adenosine dinucleotide (NAD+ ), which is candidate signaling molecule [3]. Do P2X7 receptor/channels induce vasomotor activity, but we discovered that their sustained activation can trigger the death of capillary cells [4,5,6]. Noteworthy is that this toxic effect of vasoactive purinergic input is conceptually similar to the extensively studied phenomenon of the neurotransmitter glutamate being neurotoxic.
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