Nitric Oxide Prevents ATP-Induced Cell Death in Retinal Microvessels

Abstract

Abstract: : Purpose: Extracellular ATP, which is released within vascular lumens by erythrocytes and platelets and abluminally by glia, is likely to serve as a vasoactive signal in the retinal microvasculature. However, as we reported (ARVO 2002), activation of P2X₇ receptors by ATP initiates apoptotic cell death in retinal microvessels. The question arises, how is ATP-induced cell death prevented in the retinal microvasculature. Here, we tested the hypothesis that nitric oxide (NO) plays this protective role. Methods: Pericyte-containing microvessels were isolated from the adult rat retina. Cell viability was quantified by trypan blue exclusion after a 24 hr exposure to solutions with or without ATP plus or minus various pharmacological agents. The intracellular concentration of calcium ([Ca²⁺]_i) was measured in fura-loaded pericytes located on freshly isolated microvessels. Results: ATP induced cell death in a dose-dependent manner. However, in the presence of the NO donor, sodium nitroprusside (SNP, 100 µM), the half-maximal ATP-induced cell death decreased from 28% to 2.5 %. More specifically, microvascular cell death induced by 1 mM ATP was reduced by SNP from 19 ± 1% (n = 21 expts) to 5 ± 2% (P < 0.001); in 3 mM ATP the induced death decreased from 56 ± 4% (n = 9 expts) to 5 ± 3% (P < 0.001). Because many effects of NO are mediated via guanylate cyclase, we tested an inhibitor of this enzyme, methylene blue (0.5 µM). However, this inhibitor did not alter SNP’s protective effect (n = 3). Consistent with this, 8-bromo-cyclic GMP (1 mM) failed to mimic the action of SNP (n = 5). Because calcium can kill, we asked whether SNP, which inhibits calcium channels in pericyte-containing retinal vessels (Sakagami, 2001), blocked the ATP-mediated increase in [Ca²⁺]_i; but, SNP had no effect (P = 1.0, n = 75 cells). Conclusions: By a mechanism that involves neither cGMP nor inhibition of the ATP-evoked rise in [Ca²⁺]_i, NO protects the retinal microvasculature from cell death caused by the vasoactive signal ATP. CR: None

Keywords: nitric oxide • retina • vascular cells