Purinergic Vasotoxicity: Role of the Pore/Oxidant/K_ATP Channel/Ca²⁺ Pathway in P2X₇-Induced Cell Death in Retinal...

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 K_ATP channels and the resulting increase in calcium influx. Our findings revealed that the previously unappreciated pore/oxidant/K_ATP 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.