Release of Endogenous Ascorbic Acid Preserves Extracellular Dopamine in the Mammalian Retina

Abstract

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.

The retina is a part of the central nervous system and uses most of the transmitters found in the brain. However, the only significant catecholamine in the retina is dopamine, which occurs in a subpopulation of amacrine cells, and in some species, in interplexiform cells. ^{1 2} Dopamine is released when the retina is stimulated with light ^{3 4 5 6 7 8} and is believed to have a mainly neuromodulatory role. ^{9 10} A wide variety of physiological effects have been ascribed to dopamine in the retina of various species. ⁹ These actions include decreasing electrical coupling between horizontal cells ¹¹ and enhancing their sensitivity to glutamate, the photoreceptor transmitter. ¹² Dopamine also has effects on ganglion cells in mammalian retinas and may be involved in light–dark adaptation processes. ⁹ A different subpopulation of amacrine cells possesses nitric oxide synthase (NOS), ^{13 14} and it has recently been shown that stimulation of the rabbit retina with light evokes a striking increase in nitric oxide (NO) release, ¹⁵ supporting previous suggestions that NO has a neurotransmitter–neuromodulator role in the retina. ¹⁶ The physiological roles of NO in the retina are unknown but because NO has effects on cone photoreceptors and horizontal cells that are similar to those normally seen during light adaptation, it has been suggested that NO may be another light-adaptive retinal signal. ^{17 18} Thus, both dopamine and NO may be involved in light adaptation. It seems that dopamine and NO interact in the retina because NO donors were found to inhibit the K⁺-evoked release of endogenous dopamine from the isolated bovine and rabbit retina. ^{19 20} Unfortunately, the conclusion that NO may inhibit dopamine release in the retina was cast into doubt by a report that an apparent decrease in dopamine release from rat pheochromocytoma (PC12) cells caused by NO was due to the destruction of dopamine by NO rather than to an effect on dopamine release. ²¹ Furthermore, an apparent inhibition by NO donors of K⁺-evoked dopamine release from rat striatal slices was converted to an increase in the presence of antioxidants, again suggesting the destruction of dopamine by NO. ²²  

In the present study, we report that although NO can rapidly destroy dopamine in pure physiological medium, in the presence of retinal tissue, sufficient endogenous antioxidants (mainly ascorbate) are released to prevent the oxidation of dopamine by NO. We therefore conclude that the inhibitory action of NO on the K⁺-evoked release of dopamine from the rabbit retina results from an action on the tissue and is not due simply to the chemical degradation of dopamine. 

Methods

All procedures involving animals were conducted in accordance with local animal care guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Dopamine Incubation

Krebs bicarbonate Ringer (0.5 ml) containing pargyline (50 μM) was placed in tubes in a shaking water bath at 25°C. Dopamine was added to achieve a final concentration of 53 nM. Samples (100 μl) were removed at t = 0 minutes and t = 10 minutes and acidified with 1 M perchloric acid (10 μl). The dopamine in the resultant samples was measured by HPLC. 

Effect of NO Donors on Dopamine Degradation

Dopamine was incubated as described but with the addition of sodium nitroprusside (SNP; 100–300 μM) or S-nitroso-N-acetyl-dl-penicillamine (SNAP; 100–300 μM) at t = 0 minutes. Samples were taken at t = 0 minutes and t = 10 minutes for dopamine analysis. Incubations with SNP were performed in room lighting (103 lux) and in the dark. 

Effect of Retinal Tissue and Antioxidants on NO–Dopamine Interaction

Male Wistar rats (250–300 g) were killed by stunning and cervical dislocation. The eyes were enucleated and the retinas dissected. Retinas (approximately 10 mg wet weight) were incubated for 10 minutes in Krebs bicarbonate Ringer (4 ml) containing pargyline (50 μM). The retinas were then removed and dopamine (53 nM) was added to samples of the medium (0.5 ml) at t = 0 minutes in the presence or absence of SNP (300 μM). Samples (100 μl) of the medium were removed at t = 0 minutes and t = 10 minutes and acidified for dopamine measurement. In other experiments, dopamine and SNP were added to the incubation medium in the presence or absence of ascorbate oxidase (5 U/ml) and presence or absence ofγ -glutamyltranspeptidase (0.1 U/ml). The dopamine content of samples taken at t = 0 minutes and t = 10 minutes was determined. 

Dopamine Assay

The dopamine content of the samples was determined using an electrochemical detector (Intro, Antec, Leyden, The Netherlands) with the electrode set at 0.7 V oxidation. The system comprised a column (Spherisorb S5 ODS2; Waters, Milford, MA; 15 cm × 4.6 mm) with a 20-μl injection loop. The mobile phase (0.07 M KH₂PO₄, 0.0035% EDTA, 0.023% octyl sodium sulfate, and 12.5% methanol, [pH 2.75]) was filtered and degassed with helium before use, then pumped at a flow rate of 1 ml/min and a temperature of 35°C. 

Release of [³H]Dopamine

Male New Zealand rabbits (2–3 kg) were killed with an intravenous injection of pentobarbital, and the eyes were enucleated and the retinas dissected. The isolated retinas were cut in half, placed in 5 ml Krebs bicarbonate Ringer containing 50 μM pargyline, 200 μM ascorbate, and 4 × 10⁻⁸ M[ ³H]dopamine and incubated in a shaking water bath for 30 minutes at 32°C. They were then washed in 5 ml Krebs bicarbonate Ringer, and individual hemiretinas were placed in a small (1 ml) perspex chamber with 300 μl Krebs bicarbonate Ringer containing 50 μM pargyline and 10 μM nomifensine at room temperature and were gently bubbled with 5% CO₂ in oxygen. At 10-minute intervals the medium in the bath was replaced, and the resultant samples were assayed for total radioactivity. Potassium-evoked release of [³H]dopamine was obtained by exposing the retina for 10 minutes to medium containing KCl (50 mM). Two periods of K⁺-depolarization (S1 and S2) were separated by 20 minutes. The effect of NO donors was studied by adding them to the medium with the second pulse of KCl (S2). In control samples, no drug was added to the second pulse of KCl. The ratio of the two K⁺-evoked releases (S2/S1) was calculated. Results were compared between control samples and experiments in which a drug was present during S2 by use of Student’s unpaired t-test. Significant differences were assumed at a level of P < 0.05. 

Ascorbate Release and Assay

Isolated rat retinas (10 mg) were incubated in 300 μl Krebs bicarbonate medium for 10 minutes at 32°C, and then samples (20 μl) were subjected to HPLC analysis to measure ascorbate as previously described. ²³ In five experiments, ascorbate release from the retina of anesthetized rabbits was measured using a rabbit eyecup preparation that has been described previously. ¹⁵  

Materials

Krebs bicarbonate Ringer of the following composition was used (mM): NaCl 118.4, KCl 4.84, CaCl 2.4, MgSO₄ 1.8, KH₂PO₄ 1.2, NaHCO₃ 25, and glucose 9.5. It was oxygenated with 95% O₂-5% CO₂.[ 7,8-³H]dopamine (specific activity 1.78 TBq millimoles⁻¹) was obtained from Amersham (Amersham, UK). All other chemicals were obtained from Sigma (Poole, UK). 

Results

Degradation of Dopamine in Krebs Bicarbonate Ringer

When dopamine (53 nM) was incubated in Krebs bicarbonate Ringer at 25°C and in room light, the concentration gradually decreased over 40 minutes in a linear manner (not illustrated). After 30 minutes, the concentration of dopamine (measured by HPLC) was 50% of the initial value. In subsequent experiments 10-minute incubations were used, at which time the concentration of dopamine had decreased to 84% ± 3% of the initial value (n = 10). 

Effect of NO Donors on Dopamine Degradation

Incubation of dopamine (53 nM) with the NO donors SNP (300 μM) or SNAP (300 μM) strikingly increased the rate of degradation of dopamine. Thus, after 10 minutes’ incubation, the dopamine concentration decreased to 20% ± 5.0% (n = 4; P < 0.001) and 12% ± 2.1% (n = 4; P < 0.001) of initial values, respectively. This effect of NO donors was light dependent, with neither SNP nor SNAP exerting any significant effect on the concentration of dopamine in the dark (92% ± 3.2% and 85% ± 4.9% of control samples respectively). Using an NO meter, we have found that neither SNP nor SNAP liberates NO in the dark, ²⁴ indicating that it is not the donors but the released NO (or its breakdown products) that accelerates the degradation of dopamine. 

Effect of Retinal Tissue and Antioxidants on Dopamine Degradation in the Presence of SNP

Medium was conditioned by incubating retinas for 10 minutes and then removing the tissue. When dopamine was added to this conditioned medium, the rate of degradation was identical with that observed in unconditioned, control medium. Thus, after 10 minutes’ incubation with dopamine, the concentration of the amine in conditioned and unconditioned medium was 90% ± 5.4% and 85% ± 6.2%, respectively, of the initial concentrations. However, the effect of SNP (300 μM) in increasing the rate of dopamine degradation was abolished by prior conditioning of the medium (Fig. 1) . Thus, it seems that substances released from the retina into the medium during the 10-minute incubation are able to protect exogenous dopamine from the degradation caused by SNP. 

The addition of ascorbic acid (10 μM) to the unconditioned medium inhibited the effect of SNP (300 μM) on dopamine degradation (Fig. 1) . This protective effect of ascorbic acid was abolished when ascorbate oxidase was included in the incubation medium (Fig. 1) . To test the possibility that the retina may release ascorbic acid, we conditioned medium as before by incubating retinas for 10 minutes but included ascorbate oxidase in the incubation medium. The presence of ascorbate oxidase in the medium greatly reduced the protective effect of conditioning the medium, the concentration of dopamine decreasing to 50% of the control values after 10 minutes incubation (cf. 92%, no ascorbate oxidase; P < 0.02; Fig. 1 ). In unconditioned medium, glutathione (10 μM) also protected dopamine from the effects of SNP, but in conditioned medium the presence ofγ -glutamyltranspeptidase had no effect, suggesting that significant amounts of glutathione are not released from the retina into the medium (not illustrated). 

Release of Ascorbic Acid from the Retina

To measure ascorbic acid release from the retina, samples of incubation medium (20 μl) were subjected to HPLC. These measurements revealed a spontaneous resting release of ascorbic acid from the isolated rat retina of 30 ± 3.9 nanomoles/g in 10 minutes (n = 6). Using an eyecup preparation in anesthetized rabbits, we found that ascorbic acid was released at a rate of 133 ± 17.0 nanomoles/g in 5 minutes (n = 5). 

Effect of SNP on [3H]Dopamine Release from the Rabbit Retina

The release of [³H]dopamine from the isolated rabbit retina was increased by exposure of the tissue to high-K⁺ (KCl 50 mM). In control experiments, the S2/S1 ratio was 0.85 ± 0.07 (n = 13; Fig. 2 ). Exposure of the retina to SNP (100 or 300 μM) during the second pulse of KCl (S2) reduced the S2/S1 ratio to 0.51 ± 0.05 (P < 0.01) and 0.56 ± 0.03 (P < 0.04), respectively. This inhibitory effect of SNP on the K⁺-evoked release of[ ³H]dopamine was completely abolished by the coapplication of the NO scavenger hemoglobin (Hb; 25 μM), which, by itself, had no effect on [³H]dopamine release (Fig. 2) . 

Discussion

The present study confirms that NO donors dramatically accelerate the oxidation of dopamine in physiological medium, an action that is completely prevented by the antioxidants, ascorbic acid and glutathione. We have reported previously ²⁰ that the K⁺-evoked release of dopamine from the rabbit retina is inhibited by NO. In these experiments dopamine was measured by HPLC using a physiological medium that did not contain antioxidants. Because NO rapidly oxidizes dopamine, the question arises whether the reduction in K⁺-evoked dopamine from the retina is due to an action on the tissue or merely to chemical oxidation of the amine. The latter effect has been reported in studies on the effect of NO on dopamine release from pheochromocytoma cells and rat striatal slices. ^{21 22} However, in the present study we found that NO inhibited the K⁺-evoked release of[ ³H]dopamine from the rabbit retina, and because, once released, the oxidation of radiolabeled dopamine is irrelevant to its measurement by liquid scintillation counting, it is clear that NO inhibits dopamine release from retinal neurons and does not simply oxidize it in the extracellular fluid. Although this conclusion is reassuring, it is clear that we were lucky in our original experiment on endogenous dopamine release, in that sufficient antioxidants (mainly ascorbic acid) were probably released from the retina to protect the dopamine from oxidation by exogenous NO. This was suggested by our finding that preincubation of the medium with retina protected dopamine from oxidation by NO, an action that was largely lost in the presence of ascorbate oxidase. It is clear from the present experiments using HPLC analysis that rat and rabbit retinas can release ascorbic acid at an impressive rate. It has previously been reported that the presence of neostriatal membranes inhibit considerably the decomposition of [³H]dopamine in Tris buffer. The mechanism of this protection was not determined, although a role for superoxide dismutase was ruled out. ²⁵ In the present experiments, it seems unlikely that glutathione played a significant role in protecting exogenous dopamine, but we cannot rule out the possibility that other antioxidants were released. For example, urate is released in brain, possibly from astrocytes. ²⁶  

In vertebrates, ascorbic acid from the blood is concentrated in the brain ²⁷ and then transported into neurons and glial cells by sodium-dependent active uptake processes. ²⁸ The extracellular concentration of ascorbic acid in the brain is maintained between 200 and 400 μM, and the intracellular concentration is several times higher (1–2 mM). ^{29 30} Ascorbic acid may be released into the extracellular fluid of the brain by heteroexchange with glutamate ^{31 32} or by depolarization. ^{33 34} Much less is known about ascorbic acid in the retina but ocular tissues (including the retina) in several species have been found to have high concentrations of ascorbic acid. ^{35 36 37 38 39} Many studies have shown that ascorbic acid is released from the brain ⁴⁰ but as far as we are aware, ours is the first to demonstrate that ascorbic acid is released from both rat and rabbit retina. The cellular origin of ascorbic acid released in the retina is unknown. In the adrenal medulla, there is evidence that ascorbic acid is coreleased from vesicles together with catecholamines, ⁴¹ but in the brain the distribution of ascorbic acid and catecholamines is not correlated. ^{42 43} Furthermore, destruction of catecholamine terminals in the cerebellum and striatum with 6-hydroxydopamine did not reduce ascorbic acid release. ^{31 44} We do not know the origin of ascorbic acid released by the retina and cannot rule out the possibility that it originates from the dopaminergic amacrine cells. However, in the brain, most ascorbic acid release occurs by exchange with glutamate, ⁴⁰ and by analogy this suggests that in the mammalian retina ascorbic acid is more likely to be released from bipolar cells, photoreceptors, and glial Müller cells, which possess glutamate transporters. ^{45 46 47 48} In view of the high rate of ascorbic acid release in the retina, the Müller cells seem the most likely source, because these glial cells dominate total glutamate uptake. ⁴⁸ Furthermore, bipolar cells in rodent retina contain large amounts of glutaminase but not glutamine synthase, suggesting that heteroexchange of glutamate and ascorbate is unlikely in bipolar cells. ⁴⁹  

Ascorbic acid may act as a neuromodulator or neuroprotective agent in the brain. ⁴⁰ For example, ascorbic acid decreases dopamine binding to receptors in the brain, ⁵⁰ and it may have similar roles in the retina. Thus, ascorbic acid at physiological concentrations acts at D₁-receptors and inhibits voltage-dependent K⁺ currents in isolated goldfish bipolar cells through a G_s-protein–PKA system. ⁵¹ This is consistent with a report that ascorbate (0.5 mM), enhances the direct excitability of rat neocortical neurons in vitro. ⁵² Ascorbic acid has also been studied as a protector against light-induced damage. ^{53 54 55} Our experiments suggest that ascorbic acid in the retina may also have an important role in preserving dopamine released into the extracellular space from amacrine cells. Such an action would increase the efficiency of dopaminergic transmission in the retina, where the amine sometimes has to diffuse over considerable distances to reach its postsynaptic targets. ⁵⁶ It is possible that ascorbic acid also facilitates the transmitter role of NO, because it has been shown to protect NO against scavenger attack. ²³  

 Submitted for publication April 16, 1999; revised June 28, 1999; accepted July 6, 1999.

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Figure 1.

 

Release of ascorbic acid from tissue reduces degradation of dopamine in medium. Retinas were incubated in medium for 10 minutes (10 mg/4 ml) and then removed. In this conditioned medium, SNP had no effect on dopamine degradation. The protective effect of incubating dopamine in conditioned medium was greatly reduced in the presence of ascorbic acid oxidase. In unconditioned medium, ascorbic acid (AA) protected dopamine from oxidation, an effect that was abolished by ascorbic acid oxidase. Each result is the mean ± SEM of six to eight determinations.∗ P < 0.02.

Figure 2.

 

Effect of SNP on the K⁺-evoked release of[ ³H]dopamine from isolated rabbit retina. The ordinate S2/S1 shows the release as a ratio of the release evoked by exposing the retina to KCl (50 mM) on two occasions (S1 and S2). In control samples, no drugs were applied to the tissue. The effect of drugs was examined by exposing the retina to drug during the second period of potassium depolarization (S2). The K⁺-evoked release of[ ³H]dopamine was inhibited by SNP, an action that was abolished by Hb, which itself had no effect on release. Each result is the mean ± SEM of five to seven experiments.∗ P < 0.01.