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

¹From the Department of Ophthalmology and Visual Sciences and the ²Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan.

PURPOSE. It was recently proposed that activation of P2X₇ 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 P2X₇ 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 P2X₇ receptor/channels in other cell types.

METHODS. 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 P2X₇-induced pores, and ethenoadenosine antibodies were used to detect ecto-adenosine diphosphate (ADP)-ribosyltransferase (ART) activity.

RESULTS. In freshly isolated retinal microvessels, it was found that extracellular NAD⁺, but not its catabolites, caused cell death (half-maximal effective concentration [EC₅₀] = 2 nM) by a mechanism involving the activation of P2X₇ 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 P2X₇-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.

CONCLUSIONS. 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.

This article has been cited by other articles:

				T. Kobayashi and D. G. Puro Loss of Insulin-Mediated Vasoprotection: Early Effect of Diabetes on Pericyte-Containing Microvessels of the Retina Invest. Ophthalmol. Vis. Sci., May 1, 2007; 48(5): 2350 - 2355. [Abstract] [Full Text] [PDF]