Activation of P2X7 Receptors as a Death Signal in the Normal and Diabetic Retinal Microvasculature

Abstract

Abstract: : Purpose: Immunocytochemical and electrophysiological studies in our laboratory demonstrated that microvessels of the retina express functional P2X₇ receptors. Although not previously studied in capillaries, activation of this class of receptor initiates death in various cell types through the formation of membrane pores that are permeable to large molecules. Here, we tested the hypothesis that purinergic P2X₇ receptors induce cell death in the retinal microvasculature of normal and diabetic rats. Methods: We studied pericyte-containing microvessels that were freshly isolated from the adult rat retina. Microvascular cell viability was quantified by trypan blue exclusion. Apoptosis was assessed by the TUNEL method, Hoechst 33258 staining and immunoreactivity to cleaved caspase-3. Diabetes was induced by streptozotocin (STZ, 75 mg/kg) injection. Results: In microvessels isolated from the normal retina, ATP induced cell death (EC₅₀ = 1.6 mM). The P2X₇ agonist, Bz-ATP, mimicked this lethal effect with an EC₅₀ of 40 µM. A greater sensitivity to Bz-ATP is characteristic of P2X₇ receptors. Also indicating P2X₇ involvement, the blocker of these receptors, oxidized ATP, markedly reduced cell death during exposure to Bz-ATP. Consistent with an apoptotic mechanism, exposure to Bz-ATP significantly increased the number of cells that stained positively with cleaved caspase-3 antibodies, Hoechst 33258 and the TUNEL method. We also assessed the effect of Bz-ATP on cell death in microvessels isolated from diabetic retinas. After 4 weeks of STZ-induced diabetes, the EC₅₀ for cell death induced by Bz-ATP was 0.8 µM, which was 50-fold lower than in the controls. This increased vulnerability to P2X₇ receptor activation developed between one and two weeks after the onset of hyperglycemia. Conclusion: Activation of P2X₇ receptors initiates apoptotic cell death in microvessels isolated from the rat retina. Soon after the onset of STZ-induced diabetes, the retinal microvasculature becomes more vulnerable to cell death initiated via P2X₇ receptors.

Keywords: 323 apoptosis/cell death • 614 vascular cells • 388 diabetic retinopathy