The purpose of these experiments was to determine the specific role of reactive oxygen species (ROS) in the blood-retinal barrier (BRB) breakdown that characterizes the early stages of vascular dysfunction in diabetes. Based on our data showing that high glucose increases nitric oxide, superoxide, and nitrotyrosine formation in retinal endothelial cells, we hypothesized that excess formation of ROS causes BRB breakdown in diabetes. Because ROS are known to induce increases in expression of the well-known endothelial mitogen and permeability factor vascular endothelial growth factor (VEGF) we also examined their influence on the expression of VEGF and its downstream target urokinase plasminogen activator receptor (uPAR). After 2 weeks of streptozotocin-induced diabetes, analysis of albumin leakage confirmed a prominent breakdown of the BRB. This permeability defect was correlated with significant increases in the formation of nitric oxide, lipid peroxides, and the peroxynitrite biomarker nitrotyrosine as well as with increases in the expression of VEGF and uPAR. Treatment with a nitric oxide synthase inhibitor (N--nitro-L-arginine methyl ester, 50 mg/kg/day) or peroxynitrite scavenger (uric acid, 160 mg/kg/day) blocked the breakdown in the BRB and prevented the increases in formation of lipid peroxides and tyrosine nitration as well as the increases in expression of VEGF and uPAR. Taken together, these data indicate that early diabetes causes breakdown of the BRB by a mechanism involving the action of reactive nitrogen species in promoting expression of VEGF and uPAR.

In glaucoma, the increased release of glutamate is the major cause of retinal ganglion cell death. Cannabinoids have been demonstrated to protect neuron cultures from glutamate-induced death. In this study, we test the hypothesis that glutamate causes apoptosis of retinal neurons via the excessive formation of peroxynitrite, and that the neuroprotective effect of the psychotropic Delta9-tetrahydroxycannabinol (THC) or nonpsychotropic cannabidiol (CBD) is via the attenuation of this formation. Excitotoxicity of the retina was induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats, which also received 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL,a superoxide dismutase-mimetic), N-omega-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), THC, or CBD. Retinal neuron loss was determined by TDT-mediated dUTP nick-end labeling assay, inner retinal thickness, and quantification of the mRNAs of ganglion cell markers. NMDA induced a dose- and time-dependent accumulation of nitrite/nitrate, lipid peroxidation, and nitrotyrosine (foot print of peroxynitrite), and a dose-dependent apoptosis and loss of inner retinal neurons. Treatment with L-NAME or TEMPOL protected retinal neurons and confirmed the involvement of peroxynitrite in retinal neurotoxicity. The neuroprotection by THC and CBD was because of attenuation of peroxynitrite. The effect of THC was in part mediated by the cannabinoid receptor CB1. These results suggest the potential use of CBD as a novel topical therapy for the treatment of glaucoma.

PURPOSE. Analyses in diabetic rats have shown that breakdown of the blood-retina barrier is associated with increased formation of peroxynitrite, nitric oxide, and lipid peroxidation. The permeability increase is blocked by treatments that also prevent the increases in reactive oxygen species, suggesting their causal role in vascular dysfunction. The purpose of this study was to determine the specific effects of high glucose and high osmolarity on the formation of nitrotyrosine, nitric oxide, and superoxide anion in retinal vascular endothelial cells and to evaluate the metabolic pathways involved. METHODS. Cultured retinal endothelial cells were maintained for 5 days in media with different concentrations of glucose or osmotic control reagents and tested for effects on protein tyrosine nitration and nitric oxide synthase (NOS) expression, using immunoblot techniques. NOS activity was determined by assays for nitrite formation and conversion of arginine to citrulline. Superoxide anion formation was assayed by hydroethidine staining. RESULTS. Increased concentrations of glucose or 3-methyL-oglucose stimulated formation of nitric oxide (NO) and superoxide induced protein nitration on tyrosine and increased expression and activity of endothelial nitric oxide synthase (eNOS). The effects of glucose were more potent: Inhibiting NOS or aldose reductase (AR), scavenging superoxide or peroxynitrite, or supplementing the NOS substrate L-arginine or cofactor tetrahydrobiopterin blocked the formation of reactive oxygen species and prevented protein tyrosine nitration. CONCLUSIONS. Increases in glucose levels and osmotic stress similar to those in diabetic patients increase the formation of nitrotyrosine in retinal endothelial cells because of their actions increasing NOS activity and causing superoxide formation due to eNOS uncoupling and AR activation. (Invest Ophthalmol Vis Sci. 2003;44:3135-3143) D iabetic retinopathy is characterized by an initial period of vascular injury and increased permeability that is followed by the active proliferation of new vessels. 1 The relation between diabetes-associated hyperglycemia and vascular disease is well established. However, the molecular mechanisms of hyperglycemia-induced endothelial dysfunction are not fully understood. 2 Oxidative stress has been strongly implicated in the macrovascular complications in diabetes. This hypothesis is supported by evidence that many biochemical pathways strictly associated with hyperglycemia (glucose auto-oxidation, polyol pathway, protein glycation) can increase the production of free radicals. 3 Recent studies in diabetic rats have supported the role of reactive oxygen and nitrogen species in retinal vascular disease associated with diabetic retinopathy. 4 -6 Furthermore, studies using cultured aortic endothelial cells have shown that treatment with high glucose increases endothelial nitric oxide synthase (eNOS) activity and promotes superoxide formation, reducing bioactivity of nitric oxide (NO). 7-9 Studies showing a reduction in NO bioavai...

1 The goal of this work was to test the role of nitric oxide synthase (NOS) and its substrate Larginine in development of tolerance to nitroglycerin's (GTN) vasodilator actions. 2 GTN's eects on NOS activity and NO formation were tested in cultured bovine aortic endothelial cells (BAECs). The arginine to citrulline conversion assay showed that GTN stimulated NOS basal activity in BAECs by *40%, comparable with acetylcholine (ACh)-treated controls. Both eects were blocked by L-NMMA. Photometric assays showed that both GTN and AChstimulated NO formation. Both eects were potentiated by L-arginine and inhibited by L-NAME. L-NAME inhibited ACh responses *80% compared with *40% for GTN responses. 3 The aortic ring assay showed that 2 h pretreatment with GTN caused substantial tolerance to GTN's vasodilating eects as evidenced by a 38 fold rightward shift of the concentration-relaxation curve. In contrast to D-arginine, addition of L-arginine substantially inhibited this eect, reducing the rightward shift to 4.4 fold of control values. GTN tolerance was associated with a 40% reduction in L-arginine tissue levels. GTN had a biphasic eect on BAEC uptake of L-arginine, stimulating uptake at 5 and 15 min, and suppressing uptake after 1 and 4 h 4 In summary, acute GTN treatment stimulates endothelial NOS activity in producing NO and increases cellular uptake of L-arginine. Prolonged GTN exposure reduces GTN's vasodilator actions, decreases L-arginine tissue levels and depresses BAECs uptake of L-arginine. Supplementation of Larginine reduces development of GTN tolerance. These data indicate that GTN tolerance depends in part on activation of the NOS pathway.

Induction of nitric oxide synthase (iNOS) results in overproduction of nitric oxide (NO), which may be a principal cause of the massive vasodilatation and hypotension observed in septic shock. Since NO‐induced vasorelaxation is mediated via the soluble isoform of guanylate cyclase (sGC), the regulation of sGC activity during shock is of obvious importance, but yet poorly understood. The aim of the present study was to investigate the activation of sGC by sodium nitroprusside (SNP) before and after exposure of rat aortic smooth muscle cells to endotoxin (LPS) or interleukin‐1β (IL‐1β). Exposure of rat aortic smooth muscle cells to SNP (10 μm) elicited up to 200 fold increases in cyclic GMP. This effect was attenuated by 30–70% in IL‐1β‐ or LPS‐pretreated cells, in a pretreatment time‐and IL‐1β‐ or LPS‐concentration‐dependent manner. When, however, cells were exposed to IL‐1β or LPS and then stimulated with the particulate guanylate cyclase activator, atriopeptin II, no reduction in cyclic GMP accumulation was observed. Pretreatment of rats with LPS (5 mg kg−1, i.v.) for 6 h led to a decrease in aortic ring SNP‐induced cyclic GMP accumulation. The IL‐1β‐induced reduction in SNP‐stimulated cyclic GMP accumulation in cultured cells was dependent on NO production, as arginine depletion abolished the downregulation of cyclic GMP accumulation in response to SNP. Reverse‐transcriptase‐polymerase chain reaction analysis revealed that the ratio of steady state mRNA for the α1 subunit of sGC to glyceraldehyde phosphate dehydrogenase was decreased in LPS‐ or IL‐1β‐treated cells, as compared to vehicle‐treated cells. Protein levels of the α1 sGC subunit remained unaltered upon exposure to LPS or IL‐1β, suggesting that the early decreased cyclic GMP accumulation in IL‐1β‐ or LPS‐pretreated cells was probably due to reduced sGC activation. Thus, the observed decreased responsiveness of sGC to NO stimulation following cytokine or LPS challenge may represent an important homeostatic mechanism to offset the extensive vasodilatation seen in sepsis.