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S. Liao, D.G. Puro; Enhancement of NAD–Induced Vasotoxicity: An Early Effect of Diabetes on Retinal Microvessels . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1747.
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© ARVO (1962-2015); The Authors (2016-present)
A sight–threatening complication of diabetes is apoptotic cell death in retinal capillaries. In this study, we tested the hypothesis that extracellular NAD+ (nicotinamide adenine dinucleotide) can induce cell death in pericyte–containing retinal microvessels. NAD+ is of interest because it is a putative extracellular signaling molecule in the retina and, from studies of immune cells, is reported to interact with P2X7 purinoceptors whose activation in retinal microvessels can trigger apoptosis.
Complexes of pericyte–containing microvessels were freshly isolated from papain–treated adult rat retinas. Cell viability was quantified by trypan blue dye exclusion. Diabetes was induced by streptozotocin–injection.
In pericyte–containing retinal microvessels, extracellular NAD+ caused cell death in a dose–dependent manner (EC50 = 2 nM). Of importance to diabetes, the NAD+ concentration needed to cause microvascular cell death decreased by ∼40–fold soon after the onset of streptozotocin–induced hyperglycemia. Consistent with NAD’s vasotoxicity being mediated via P2X7 receptor activation, two blockers of these purinoceptors, oxidized–ATP and Brilliant Blue G, dramatically reduced (P = 0.001) the microvascular death caused by NAD+. In addition, cell death induced by NAD+ was prevented (P < 0.001) by pre–exposure to UTP, which in retinal microvessels activates P2Y receptors and blocks the creation of pores formed by P2X7 activation. Indicative of the importance of NAD+ rather than its catabolites, neither ADP–ribose nor nicotinamide were vasotoxic. Suggestive that ecto–ADP–ribosyltransferases (ecto–ARTs) play a key role, pre–incubation of microvessels with a relatively high concentration of etheno–NAD+ (100 µM), which is an ART substrate that does not cause microvascular death, prevented (P = 0.001) NAD+ from inducing vasotoxicity.
Our findings support the hypothesis that extracellular NAD+ can kill retinal microvascular cells by a mechanism involving the activation of P2X7 purinoceptors. Of further importance, the vulnerability of pericyte–containing retinal microvessels to NAD–induced vasotoxicity was dramatically increased soon after the onset of experimental diabetes.
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