NAD+-Induced Vasotoxicity in the Pericyte-Containing Microvasculature of the Rat Retina: Effect of Diabetes

It was recently proposed that activation of P2X(7) purinoceptors may play a role in causing cell death in the pericyte-containing microvasculature of the diabetic retina. This hypothesis is supported by the observation that diabetes enhances lethal pore formation in retinal microvessels exposed to synthetic P2X(7) agonists. The goal of this study was to determine whether purinergic vasotoxicity can be triggered by the endogenous molecule nicotinamide adenosine dinucleotide (NAD(+)), which is a substrate for ecto-ribosylation reactions known to activate P2X(7) receptor/channels in other cell types. Pericyte-containing retinal microvessels were isolated from normal and streptozotocin-injected rats. Trypan blue dye exclusion was used to assess cell viability, YO-PRO-1 uptake was used to identify cells with P2X(7)-induced pores, and ethenoadenosine antibodies were used to detect ecto-adenosine diphosphate (ADP)-ribosyltransferase (ART) activity. In freshly isolated retinal microvessels, it was found that extracellular NAD(+), but not its catabolites, caused cell death (half-maximal effective concentration [EC(50)] = 2 nM) by a mechanism involving the activation of P2X(7) purinoceptors and the formation of transmembrane pores. A series of experiments provided evidence that NAD(+), which is not a direct purinergic agonist, serves as a substrate for ecto-ribosylation reactions that subsequently trigger P2X(7)-dependent cell death in the retinal microvasculature. Soon after the onset of diabetes, the sensitivity of retinal microvessels to the vasotoxic effect of extracellular NAD(+) increased by approximately 100-fold. Purinergic vasotoxicity triggered by extracellular NAD(+) is a newly recognized mechanism that may contribute to the cell death observed in the pericyte-containing microvascular of the diabetic retina.