Regulation of Retinal Glutamate Release by Peroxides
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Authors
Leday, Angela Marie
Issue Date
2002
Volume
Issue
Type
Dissertation
Language
en_US
Keywords
Alternative Title
Abstract
Hydrogen peroxide (H2O2), a reactive oxygen metabolite, has been localized in the neural retina and pigment epithelium of some vertebrate species. Although oxidative stress has been implicated in some retinal disorders such as age-related macular degeneration and diabetic retinopathy, no study has addressed the potential pharmacological/toxicological effect of H2O2 on the release of neurotransmitters in this tissue. The overall objective of the present study was to investigate the effect of oxidative stress induced by peroxides on basal and evoked release of glutamate from the bovine retina both in riho and ex two. Bovine neural retinae were incubated in Krebs solution containing PH]D- aspartate (as a marker for glutamate) and then set up for studies of neurotransmitter release using the Superfusion Method. Release of PH]D- aspartate was elicited by depolarization caused by an iso-osmotic concentration of potassium chloride (K+, 50 mM). Both naturally-occurring (hydrogen peroxide, H2O2; 1 pM - 1 mM) and synthetic (cumene hydroperoxide, cuOOH; 1 pM - 1 mM) peroxides caused a concentration-dependent inhibition of K+- evoked PH]D-aspartate release without affecting basal tritium efflux. cuOOH was more potent than H2O2 in eliciting an inhibitory effect on depolarization- evoked neurotransmitter release. The free radical scavenger, trolox (2 mM) prevented the inhibition of evoked PH]D-aspartate overflow elicited by both H2O2 (30 pM) and cuOOH (10 pM). Inhibition of catalase by 3-amino-triazole (3-AT, 100 mM) unmasked an inhibitory effect of a low concentration of H2O2 (1 pM) on K^-induced pH]D-aspartate release. Furthermore, 3-AT attenuated the inhibition of evoked PH]D-aspartate release caused by a higher concentration of H2O2 (30 pM). These results show that peroxides such as H2O2 can inhibit the release of [T l]D-aspartate from the bovine retina. The ThCb-induced inhibition of neurotransmitter release can be mimicked by the synthetic peroxide, cuOOH, and may involve the generation of reactive oxygen species. Metabolism of exogenous H2O2 by catalase alters the effect of this oxidant on excitatory amino acid neurotransmission in the bovine isolated retina.
In ex vivo experiments, exogenously applied H2O2 (1-100 pM) caused a concentration-related decrease in glutamate and glycine levels in the bovine retina. However, H2O2 (1-10 pM) had no significant effect on glutamate levels, but caused a concentration-dependent increase in glycine levels in the vitreous humor. At a high concentration (100 pM), H2O2 produced a significant increase in both glutamate and glycine levels in the vitreous humor. These observations support the view that exogenous H2O2 can alter endogenous pools of glutamate as well as glycine in the bovine retina. Inhibition of catalase activity with 3-AT (100 mM) caused a significant reduction in both glutamate and glycine levels in the bovine retina and vitreous humor, gx Hro. These results suggest that endogenous H2O2 also plays a role in the regulation of these amino acids in the retina.
Agents that release nitric oxide (NO) such as S-nitroso-N- acetylpenicillamine and 3-morpholinylsdnoneimine chloride (SIN-1) attenuated K+-depolarization evoked PH]D-aspartate overflow. Interestingly, both NO donors completely abolished the inhibition caused by H2O2 (30pM). Following treatment with different concentrations of peroxides, retinal NO levels were not significantly altered. These findings indicate that NO may be involved in the pathway leading to PFTjD-aspartate release in the bovine isolated retina. However, H2O2 does not directly stimulate NO production in this tissue.
Inhibition of prostaglandin synthase by flurbiprofen and blockade of isoprostane receptors by SQ 29548 had no significant effect on K+-evoked [HT]D- aspartate release from the bovine isolated retina. However, both flurbiprofen and SQ 29548 were able to reverse the inhibition of KP-evoked PH]D-aspartate release caused by H2O2. In Hho, both H2O2 and cuOOH were able to directly stimulate PGE2 and 8-isoprostane PGF2a production in this tissue. These results suggest that prostaglandins and isoprostanes are involved in H202-induced inhibition of glutaminergic neurotransmission.
I conclude that peroxides can inhibit neurotransmitter release from glutaminergic nerves in the bovine retina in niho. Nitric oxide and arachidonic acid metabolites account, at least in part, for the effect of peroxides on glutaminergic neurotransmission in the bovine retina. The inhibitory effects produced by H2O2 on glutamate levels in the retina ex Huo mimics the effects seen in in Him superfusion assays. Furthermore, H2O2 can alter glycine concentrations in the bovine retina and vitreous humor. The observed ability of peroxides to inhibit the production of glutamate in the retina may play a role in the regulation of the levels of this excitatory amino acid in diseases of the retina such as retrolental fibroplasia, retinal degeneration, retinopathies, and glaucoma.
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Creighton University
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Copyright is retained by the Author.
A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.
Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.
Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.