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Updated: Aug 13, 2026
A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
Release of endogenous ascorbic acid preserves extracellular dopamine in the mammalian retina
M J Neal1, J R Cunningham, K L Matthews
1Department of Pharmacology, King's College London, St Thomas' Hospital, United Kingdom. m.neal@umds.ac.uk
Purpose:
To investigate whether the inhibitory effect of nitric oxide (NO) on dopamine release from the retina is due to chemical oxidation of dopamine in the extracellular medium rather than to an inhibitory effect on dopamine release from retinal neurons.
Methods:
Dopamine was incubated in Krebs bicarbonate medium and its rate of chemical degradation measured by high-performance liquid chromatography (HPLC). The effects of NO donors and antioxidants on dopamine were assessed by comparing dopamine degradation in the presence and absence of drug. The effects of NO donors on the K-evoked release of [3H]dopamine were measured from isolated superfused rabbit retinas. The release of ascorbic acid from the isolated rat retina and from an eyecup preparation in anesthetized rabbits was measured by HPLC.
Results:
After 10 minutes' incubation in Krebs bicarbonate medium, the dopamine concentration decreased by 20%. This decline increased to 80% in the presence of S-nitroso-N-acetyl-DL-penicillamine (SNAP) or sodium nitroprusside (SNP). The increased rate of dopamine degradation was abolished if retina was incubated in the medium and then removed before the incubation of dopamine. The protective effect of preincubation with tissue was lost in the presence of ascorbate oxidase suggesting the release of ascorbic acid. HPLC analysis confirmed a substantial release of ascorbic acid from both rabbit and rat retinas. The K-evoked release of [3H]dopamine from the rabbit retina was inhibited by SNP.
Conclusions:
NO can rapidly, oxidize dopamine in physiological medium, but in the presence of retina, sufficient endogenous antioxidants (mainly ascorbate) are released to prevent this chemical reaction. Thus, the inhibitory action of NO on dopamine release results from an action on retinal neurons. Ascorbate release in the retina may have an important physiological role in prolonging the life of dopamine, which often has to diffuse long distances from axons in the inner plexiform layer to receptors in other retinal layers.
