yo-pro-1 and Diabetic-Retinopathy

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.

Topics: Adenosine Diphosphate; Adenosine Diphosphate Ribose; ADP Ribose Transferases; Animals; Apoptosis; Benzoxazoles; Cell Membrane Permeability; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Fluorescent Dyes; Microscopy, Fluorescence; NAD; Pericytes; Purinergic P2 Receptor Antagonists; Quinolinium Compounds; Rats; Rats, Long-Evans; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Retinal Vessels

A sight-threatening complication of diabetes is cell death in retinal capillaries. Currently, the mechanisms responsible for this classic manifestation of diabetic retinopathy remain uncertain. The hypothesis for the current study is that diabetes increases the vulnerability of retinal microvessels to the potentially lethal consequences of having their P2X(7) purinoceptors activated. A pathophysiological role is suspected for these receptor-operated channels because, in addition to transducing retinovascular responses to extracellular adenosine triphosphate (ATP), the sustained opening of these channels can induce the formation of large transmembrane pores.. In pericyte-containing retinal microvessels that were freshly isolated from nondiabetic and streptozotocin-injected rats, cells with pores were identified by the uptake of YO-PRO-1. Cell viability was assayed by trypan blue dye exclusion, and cleaved caspase-3 immunoreactivity, TUNEL positivity, and nuclear morphology were used to detect apoptotic cells. Patch-clamp recordings assessed electrophysiological parameters.. Activation of P2X(7) receptors caused large pores to form and apoptosis to occur in retinal capillaries of nondiabetic and diabetic rats. Of importance to diabetes, the agonist concentration needed to open pores and trigger apoptosis decreased markedly soon after the onset of streptozotocin-induced hyperglycemia. However, despite this increased sensitivity, diabetes minimally affected the P2X(7)-induced ionic currents. Thus, rather than upregulate the number of functional P2X(7) receptor/channels, diabetes appears to facilitate the channel-to-pore transition that occurs during activation of these purinoceptors. In this way, normally nonlethal concentrations of P2X(7) ligands may trigger apoptosis in microvessels of the diabetic retina.. A diabetes-induced increase in the vulnerability of retinal microvessels to the lethal effect of P2X(7) receptor activation may be a previously unrecognized mechanism by which diabetic retinopathy progresses.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Benzoxazoles; Cell Membrane Permeability; Cell Survival; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Electrophysiology; Endothelium, Vascular; Fluorescent Dyes; Immunoenzyme Techniques; In Situ Nick-End Labeling; Ion Channels; Pericytes; Quinolinium Compounds; Rats; Rats, Long-Evans; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Retinal Vessels