Enzymatic assay for perfluoro-tagged metabolites of l-DOPA using crude lysate from E. coli transformed with pKKAADCII

Isomers of l-DOPA and dopamine with a nine-atom ¹⁹F atom tag were exposed to aromatic acid decarboxylase (AADC) in the lysate of Escherichia coli JM109 that had been transformed with the plasmid pKKAADCII. The resulting samples were analyzed with HPLC. The first study investigated the conversion of the tagged l-DOPA into tagged dopamine, using the tagged dopamine as a standard. A second study was undertaken to identify the source of peaks seen in the enzymatic assays. l-DOPA with the tag bonded at position 5 served as the best substrate for AADC. Isomers that fit into the active site of AADC are likely to follow the biosynthetic path for dopamine in vivo and are potentially useful in magnetic resonance studies. The enzymatic assay described here provides an efficient and cost-effective tool for screening new compounds for use in the fluorine imaging of neural pathways.     

l-DOPA (l-3,4-dihydroxyphenylalanine) affects rooting potential and associated biochemical changes in hypocotyl of mung bean, and inhibits mitotic activity in onion root tips

A study was undertaken to explore the effect of l-DOPA (l-3,4-dihydroxyphenylalanine) on the rooting potential of hypocotyl cuttings of mung bean (Phaseolus aureus Roxb. var. SML-32) and related biochemical changes at the post-expression phase. At lower concentrations of (0.0001–0.1 mM) l-DOPA, there was no change in rooting potential, though the average number of roots per cutting and root length were significantly decreased (except at 0.0001 mM). However, at 1.0 mM concentration, a 50% inhibition in rooting potential was noticed and the root number and length were severely reduced. In contrast, at 2.5 mM l-DOPA, none of the hypocotyl cutting rooted. The decrease in rooting potential was associated with a significant effect on the biochemical changes measured in terms of protein and carbohydrate metabolism and activity of peroxidases. In the l-DOPA treated hypocotyl cuttings, there was a significant reduction in the protein and carbohydrate content, whereas activities of associated enzymes proteases and amylases decreased, particularly at higher treatment concentration (>1.0 mM). It indicated negative effect of l-DOPA on these two important metabolic processes. Likewise, activity of peroxidases also decreased in the l-DOPA treated hypocotyl mung bean cuttings thereby indicating its role in suppressing rhizogenesis as the enzyme is involved in lignification process during cell division. l-DOPA suppressed mitotic activity in the root tip cells of onion indicating thereby its interference with the cell division, which is required for the formation of new meristematic tissue during rhizogenesis. Based on the obtained results, it is concluded that l-DOPA interferes with the various biochemical processes in the mung bean hypocotyl cuttings thereby affecting their rooting potential.     

The Roles of Striatal Serotonin and <Emphasis Type="SmallCaps">l</Emphasis>-Amino-acid Decarboxylase on <Emphasis Type="SmallCaps">l</Emphasis>-DOPA-induced Dyskinesia in a Hemiparkinsonian Rat Model

The administration of l-DOPA is the standard treatment for Parkinson’s disease (PD). However, the symptomatic relief provided by long-term administration may be compromised by l-DOPA-induced dyskinesia (LID) that presents as adverse fluctuations in motor responsiveness and progressive loss of motor control. In the later stages of PD, raphestriatal serotonin neurons compensate for the loss of nigrostriatal dopamine (DA) neurons by converting and releasing DA derived from exogenous l-DOPA. Since the serotonin system does not have an autoregulatory mechanism for DA, raphe-mediated striatal DA release may fluctuate dramatically and precede the development of LID. The 6-hydroxydopamine lesioned rats were treated with l-DOPA (6 mg/kg) and benserazide (15 mg/kg) daily for 3 weeks to allow for the development of abnormal involuntary movement score (AIMs). In rats with LID, chronic treatment with l-DOPA increased striatal DA levels compared with control rats. We also observed a relative increase in the expression of striatal l-amino-acid decarboxylase (AADC) in LID rats, even though tyrosine hydroxylase (TH) expression did not increase. The administration of l-DOPA also increased striatal serotonin immunoreactivity in LID rats compared to control rats. Striatal DA and 5-hydroxytryptamine (5-HT) levels were negatively correlated in l-DOPA-treated rats. These results of this study reveal that 5-HT contributes to LID. Striatal DA positively influences LID, while 5-HT is negatively associated with LID. Finally, we suggest that by strategic modification of the serotonin system it may be possible to attenuate the adverse effects of chronic l-DOPA therapy in PD patients.     

Contribution of Serotonergic Transmission to the Motor and Cognitive Effects of High-Frequency Stimulation of the Subthalamic Nucleus or Levodopa in Parkinson’s Disease

Although they are effective at treating the motor impairments that are the core symptoms of Parkinson’s disease, current treatments, namely l-3,4-dihydroxyphenylalanine (l-DOPA), the gold standard medication and high-frequency stimulation of the subthalamic nucleus (HFS-STN), can lead to cognitive and mood alterations. Many of these side effects, such as depression, anxiety and sleep disturbances, could be related to abnormal functioning of the serotonergic system, but much basic research remains to be done. Molecular studies in humans and animal models of the disease have reported diverse drastic changes to the serotonergic system. It has also been shown that the serotonergic system both plays a major role in the mechanism of action of the current therapies and is altered by the therapies. It has been reported that HFS-STN decreases serotonin release in several regions, mostly via inhibition of serotonergic neuron activity. The involvement of serotonergic neurons in l-DOPA treatment is even more significant. First, serotonergic neurons, able to convert exogenous l-DOPA to dopamine, are a major site to release dopamine throughout the brain. Second, the substitution of serotonin by newly synthesized dopamine in serotonin neurons leads to acute and chronic alteration of serotonin release and metabolism. Therefore, both therapeutic approaches, via distinct mechanisms, decrease serotonergic system activity and, rather than alleviating cognitive or mood disorders, tend to aggravate them. Molecular strategies targeting the serotonergic system are being developed and could be decisive in limiting l-DOPA-induced dyskinesia, as well as mood and cognitive symptoms produced by antiparkinsonian therapies.     

Concomitant depletion of dopamine and secretory granules from cells in the ultimobranchial gland of vitamin D2-treated chicken

Summary The ultimobranchial gland (UBG) is a rich source of the polypeptide hormone calcitonin, which is present in a cell system analogous to the mammalian parafollicular cells (C cells) of the thyroid gland. Both types of cells are argyrophilic and, ultrastructurally, they are furnished with numerous electron-dense granules considered to contain the hormone. In the chicken, the main cells of the UBG contain large amounts of dopamine. The possible functional relationship between this amine and the hormone has been studied by a combination of fluorescence and electron microscopy of the UBG from chickens treated with vitamin D₂. This stimulus produced a depletion of dopamine and a pronounced degranulation of the UBG cells, concomitant with a loss in their argyrophilia. Administration of l-3,4-dihydroxyphenylalanine (l-DOPA) to vitamin D₂-treated animals was followed by a reappearance of dopamine in the cytoplasm of the UBG cells, whereas electron-dense granules or argyrophilia were not restored. It is suggested that this concomitant depletion of dopamine and the secretory granules from the UBG cells reflects a participation of the amine in the secretion of the polypeptide hormone.     

Immunohistochemistry of endogenous L-DOPA in the rat posterior hypothalamus

The aim of this work was to study L-DOPA-containing neuronal structures of the rat posterior and dorsal hypothalamus by means of immunohistochemistry using antiserum against glutaraldehyde conjugated L-DOPA. Aspects and distribution of L-DOPA immunoreaction among cells of the supramammillary nucleus and the A11, A13c and A13 cell groups are described and compared to dopamine immunoreactivity, mainly through a double colored labelling procedure employing a color modification of the DAB reaction by metallic ions. Differences between L-DOPA and dopamine stainings within cell groups as the presence of cells with predominant or exclusive L-DOPA coloration are tentatively explained under the light of previous findings using immunohistochemistry of catecholamines synthesizing enzymes and catecholamines histofluorescence.     

Cytofluorometric demonstration of a catecholamine in rat mast cells after the administration of DOPA

Synopsis A catecholamine, probably dopamine, was identified in rat peritoneal mast cells after subcutaneous injection of DOPA. Its identity was established by cytofluorometry of cells treated with hot formaldehyde vapour according to the Falck-Hillarp technique. Injections of 50–200 mg/kgl-DOPA were followed by a dose-dependent increase in fluorescence intensity, measured at the emission maximum for catecholamines. The increase in fluorescence intensity was accompanied by a change in the emission spectrum with displacement of the fluorescence maximum towards a shorter wavelength characteristic for a catecholamine. Recordings of rates of photodecomposition showed a rapid exponential fading of the fluorescence in mast cells of control rats comparable to that of 5-hydroxytryptamine-containing protein droplets, whereas the mast cells of DOPA-treated rats showed a slower fading rate, intermediate between that of dopamine- and 5-hydroxytryptamine-containing models.     

Tyrosine hydroxylase and Parkinson's disease

A consistent neurochemical abnormality in Parkinson's disease (PD) is degeneration of dopaminergic neurons in substantia nigra, leading to a reduction of striatal dopamine (DA) levels. As tyrosine hydroxylase (TH) catalyses the formation ofl-DOPA, the rate-limiting step in the biosynthesis of DA, the disease can be considered as a TH-deficiency syndrome of the striatum. Similarly, some patients with hereditaryl-DOPA-responsive dystonia, a neurological disorder with clinical similarities to PD, have mutations in the TH gene and decreased TH activity and/or stability. Thus, a logical and efficient treatment strategy for PD is based on correcting or bypassing the enzyme deficiency by treatment withl-DOPA, DA agonists, inhibitors of DA metabolism, or brain grafts with cells expressing TH. A direct pathogenetic role of TH has also been suggested, as the enzyme is a source of reactive oxygen species (ROS) in vitro and a target for radical-mediated oxidative injury. Recently, it has been demonstrated thatl-DOPA is effectively oxidized by mammalian TH in vitro, possibly contributing to the cytotoxic effects of DOPA. This enzyme may therefore be involved in the pathogenesis of PD at several different levels, in addition to being a promising candidate for developing new treatments of this disease.     

Multicore Vesicles: Hyperosmolarity and <Emphasis Type="SmallCaps">l</Emphasis>-DOPA Induce Homotypic Fusion of Dense Core Vesicles

Aim A fraction of vesicles in cells treated with hypertonic solution exhibit multiple dense cores and this is enhanced by treatment with l-3,4–dihydroxyphenylalanine (l-DOPA). These cells were examined to determine if the multicore vesicles are the product of endocytosis or homotypic fusion. Methods Electron microscopy was used to determine the number of multicore vesicles and amperometry was used to examine if the multicore vesicles are a competent fraction of the readily releasable pool. Results In this study, we observed that a substantial portion (15.3%) of large dense core vesicles in PC12 cells contained multiple cores in hypertonic saline loaded with l-DOPA, and amperometric measurements show a bimodal distribution of quantal sizes in treated cells. Conclusions The results suggest that the multicored vesicles are formed from homotypic fusion of LCDVs prior to exocytosis.     

Production of 3,4-dihydroxy phenyl-<Emphasis Type="SmallCaps">l</Emphasis>-alanine (<Emphasis Type="SmallCaps">l</Emphasis>-DOPA) by Egyptian halophilic black yeast

Production of 3,4-dihydroxy phenyl-l-alanine (l-DOPA) using an Egyptian isolate of halophilic black yeast was studied. Optimum aeration level and incubation period for high yield production of l-DOPA were 50 ml medium/250 ml flask with 200 rpm and 36 h, respectively. Two new techniques (addition of aniline or NaCl to the medium) have been investigated to enhance the monophenolase activity and inhibit or reduce diphenolase activity of tyrosinase to form high yield of l-DOPA without more oxidation to melanin. Addition of aniline to tyrosine medium at 3 μl/ml medium enhanced l-DOPA production 2.9 fold, however, addition of NaCl at 20% showed the same amount of l-DOPA as the control. Peptone and ram horn hydrolysate were studied as nitrogen sources instead of tyrosine in the medium and they showed good nitrogen sources for l-DOPA production as tyrosine. Finally, addition of aniline (3 μl/ml) to ram horn hydrolysate was economically feasible and cost effective for l-DOPA production by Egyptian halophilic black yeast.     

Direct and decarboxylation-dependent effects of neurotransmitter precursors on firing of isolated monoaminergic neurons

To elucidate mechanisms that underlie the profound physiological effects of the monoamine precursors 5-hydroxy-l-tryptophan (5-HTP) and l-3,4-dihydroxyphenylalanine (l-DOPA), we examined their action on single monoaminergic neurons isolated from the ganglia of the gastropod snail Lymnaea stagnalis. In isolated serotonergic PeA motoneurons, 5-HTP produced excitation. The effect was mimicked by serotonin at 0.5–1 μM, masked by pretreatment with serotonin at higher concentrations, and abolished by the inhibitor of aromatic amino acid decarboxylase (AAAD) m-hydroxybenzylhydrazine (NSD-1015), the inhibitor of the vesicular monoamine transporter reserpine or the serotonin receptor antagonist mianserin. Exposure of the dopaminergic interneurons RPeD1 to l-DOPA caused a biphasic effect composed of a depolarization followed by a hyperpolarization. AAAD inactivation with NSD-1015, as well as the blockade of dopamine receptors with sulpiride, resulted in the enhancement of the excitatory effect, and the abolition of the inhibitory effect. Dopamine caused hyperpolarization and masked the inhibitory phase of l-DOPA action. The results show that precursors affect the rate of firing of isolated monoaminergic neurons and that their effect is completely or partially mediated by the enhanced synthesis of the respective neurotransmitter, followed by extrasynaptic release of the latter and activation of extrasynaptic autoreceptors.     

Bioconversion of L-tyrosine to L-DOPA by a novel bacterium Bacillus sp. JPJ

l-DOPA is an amino acid derivative and most potent drug used against Parkinson’s disease, generally obtained from Mucuna pruriens seeds. In present communication, we have studied the in vitro production of l-DOPA from l-tyrosine by novel bacterium Bacillus sp. JPJ. This bacterium produced 99.4% of l-DOPA from l-tyrosine in buffer (pH 8) containing 1 mg ml⁻¹ cell mass incubated at 40°C for 60 min. The combination of CuSO₄ and l-ascorbic acid showed the inducing effect at concentrations of 0.06 and 0.04 mg ml⁻¹, respectively. The activated charcoal 2 mg ml⁻¹ was essential for maximum bioconversion of l-tyrosine to l-DOPA and the crude tyrosinase activity was 2.7 U mg⁻¹ of tyrosinase. Kinetic studies showed significant values of Y _p/s (0.994), Q _s (0.500) and q _s (0.994) after optimization of the process. The production of l-DOPA was confirmed by analytical techniques such as HPTLC, HPLC and GC–MS. This is the first report on rapid and efficient production of l-DOPA from l-tyrosine by bacterial source which is more effective than the plant, fungal and yeast systems.     

Microarray analysis of <Emphasis Type="Italic">Arabidopsis</Emphasis> plants in response to allelochemical <Emphasis Type="SmallCaps">l</Emphasis>-DOPA

Velvetbean (Mucuna pruriens) plants impede the growth of neighboring plants. One compound, 3-(3′,4′-dihydroxyphenyl)-l-alanine (l-DOPA), is responsible for the allelopathic capacity of velvetbean. This compound is an active allelochemical that decreases root growth of several plant species. In mammals, l-DOPA is a well-known therapeutic agent for the symptomatic relief of Parkinson’s disease. However, its mode of action in plants is still not well understood. To address such issues, gene expression in Arabidopsis thaliana plants, which had been exposed to l-DOPA, was analyzed using DNA microarrays. After 6 h of l-DOPA exposure, the expression of 110 genes was significantly upregulated, and the expression of 69 genes was significantly downregulated. These induced genes can be divided into different functional categories, mainly on the basis of subcellular localization, metabolism, and proteins with a binding function or cofactor requirement. Based on these results, we suggest that l-DOPA acts by two mechanisms: it influences amino acid metabolism and deregulates metal homeostasis, especially that of iron, which is required for the fundamental biological processes of all organisms.     

Striatal Dopaminergic Fiber Recovery After Acute <Emphasis Type="SmallCaps">l</Emphasis>-DOPA Treatment in 6-Hydroxydopamine (6-OHDA) Lesioned Rats

In order to examine the acute effects of l-DOPA treatment following 6-hydroxydopamine (6-OHDA) injection into rat medial forebrain bundle (MFB). Sprague–Dawley rats (n = 48) received either 6-OHDA, via intracranial unilateral injection, into the MFB (experimental group) or saline 0.9% (control group). Administration of l-DOPA or saline 0.9% began 1 month after the 6-OHDA injection for 10 consecutive days. Within 3 days, an increase in the density of striatal tyrosine hydroxylase (TH) immunoreactive fibers within the striatum, when compared to the control group was observed. There was no difference in the loss of substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons between. The greater density of TH fibers in the striatum following l-DOPA may be related to recovery of the DA phenotype and/or sprouting of TH axon terminals. Only animals with severe cell loss in the SNpc experienced abnormal involuntary movements (AIMs) or “dyskinesias” in response to l-DOPA, which did not correlate with striatal TH fiber density, suggesting that induction of TH-positive fibers does not contribute to the occurrence of dyskinesia. The relationship between cell loss, fiber density and AIM to the abundance of markers of microglial activation were also examined. Iba-1, a microglial marker, immunoreactivity was not affected by l-DOPA treatment, was not correlated with the severity of AIM indicating that microglial activation does not contribute to dyskinetic phenomena.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for improving <Emphasis Type="SmallCaps">l</Emphasis>-3,4-dihydroxyphenylalanine (<Emphasis Type="SmallCaps">l</Emphasis>-DOPA) synthesis from glucose

l-3,4-dihydroxyphenylalanine (l-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of l-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of l-tyrosine (l-Tyr), an l-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on l-Tyr production of PTS inactivation (PTS⁻ gluc⁺ phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of l-Tyr production (q _l-Tyr), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q _l-Tyr in the PTS⁺ and the PTS⁻ gluc⁺ strains, respectively. An 8.6-fold increase in l-Tyr yield from glucose was observed in the PTS⁻ gluc⁺ tyrR ⁻ strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for l-Tyr production caused the synthesis of l-DOPA. One of such strains, having the PTS⁻ gluc⁺ tyrR ⁻ phenotype, displayed the best production parameters in minimal medium, with a specific rate of l-DOPA production of 13.6 mg/g/h, l-DOPA yield from glucose of 51.7 mg/g and a final l-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of l-DOPA in 50 h.     

Effects of Hydrogen Sulfide-releasing l-DOPA Derivatives on Glial Activation: POTENTIAL FOR TREATING PARKINSON DISEASE*

The main lesion in Parkinson disease (PD) is loss of substantia nigra dopaminergic neurons. Levodopa (l-DOPA) is the most widely used therapy, but it does not arrest disease progression. Some possible contributing factors to the continuing neuronal loss are oxidative stress, including oxidation of l-DOPA, and neurotoxins generated by locally activated microglia and astrocytes. A possible method of reducing these factors is to produce l-DOPA hybrid compounds that have antioxidant and antiinflammatory properties. Here we demonstrate the properties of four such l-DOPA hybrids based on coupling l-DOPA to four different hydrogen sulfide-donating compounds. The donors themselves were shown to be capable of conversion by isolated mitochondria to H₂S or equivalent SH⁻ ions. This capability was confirmed by in vivo results, showing a large increase in intracerebral dopamine and glutathione after iv administration in rats. When human microglia, astrocytes, and SH-SY5Y neuroblastoma cells were treated with these donating agents, they all accumulated H₂S intracellularly as did their derivatives coupled to l-DOPA. The donating agents and the l-DOPA hybrids reduced the release of tumor necrosis factor-α, interleukin-6, and nitric oxide from stimulated microglia, astrocytes as well as the THP-1 and U373 cell lines. They also demonstrated a neuroprotective effect by reducing the toxicity of supernatants from these stimulated cells to SH-SY5Y cells. l-DOPA itself was without effect in any of these assays. The H₂S-releasing l-DOPA hybrid molecules also inhibited MAO B activity. They may be useful for the treatment of PD because of their significant antiinflammatory, antioxidant, and neuroprotective properties.     

Fluorescence microscopy of the 5-HTP turnover in the exocrine pancreas of mice and rats

Summary The turnover ofl-5-HTP,d-5-HTP and 5-HT in the exocrine pancreas have been studied by means of the fluorescence method ofFalck andHillarp. l- andd-5-HTP are easily taken up by the acinar cells, whereas 5-HT seems to pass into the cells only to a minor extent. After the administration ofl-5-HTP (and in some cases after 5-HT administration), specific fluorescence is seen in the form of apically located granules (probably identical with the zymogen granules) for a short period, which is prolonged, if the animals are pretreated with a MAO inhibitor. Decarboxylase inhibition prevents the appearance of these fluorescent granules. Administration ofd-5-HTP does not give rise to this granular fluorescence but to a diffuse fluorescence throughout the cells. Thus, there are reasons to assume that the granular fluorescence derives from 5-HT. The results obtained in this work correspond well with those from a similar study withl-DOPA and some of its analogues.abbreviations DOPA 3,4-dihydroxyphenylalanine - DA dopamine - NA noradrenaline - A adrenaline - 5-HTP 5-hydroxytryptophan - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase This work was supported by grants from the Swedish Medical Research Council (B68-12X-712-03B and B68-14X-56-04B), the United States Public Health Service (06701-02) and the Faculty of Medicine, University of Lund, Lund, Sweden.     

Cloning and expression of a tyrosinase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>: application in <Emphasis Type="SmallCaps">l</Emphasis>-DOPA biotransformation

Amplification of the tyrosinase gene (melO) from the genomic DNA of Aspergillus oryzae NCIM 1212 yielded a 1.6-kb product. This gene was cloned into pYLEX1, and the resulting pTyro-YLEX1 vector was transformed in Yarrowia lipolytica strain Po1g. A clone displaying the highest specific activity for tyrosinase (10.94 U/mg) was used for obtaining the complementary DNA (cDNA) and for protein expression studies. cDNA sequence analysis indicated the splicing of an intron present in the melO gene by Po1g. Native polyacrylamide gel electrophoresis, acidification at pH 3.0 followed by activity staining with l-DOPA indicated the expression of an active tyrosinase. The clone over-expressing the tyrosinase transformed l-tyrosine to l-DOPA. On optimization of conditions for the biotransformation (pH 4.0, temperature 60°C and with 3.5 mg of biomass), 0.4 mg/ml of l-DOPA was obtained.     

Histochemical studies on catecholaminergic cells in the carp retina

Histochemical studies on catecholaminergic cells were conducted with the carp (Cyprinus carpio) retina. Catecholamine (CA)-containing cell bodies appear sparsely distributed among amacrine cells in the innermost cellular row of the inner nuclear layer (INL) and occasionally in the outer half part of the inner plexiform layer (IPL); only exceptionally are they found among ganglion cells. The fluorescent cells interspersed with the amacrine cells and in the IPL send their fiber processes toward both the outer plexiform layer (OPL) and the IPL; the fine fibers form dense networks in the INL and IPL. Pretreatment of the fish with intramuscular injection of reserpine (20 hr prior to enucleation) completely depleted CA from the retina. The fluorescence of catecholaminergic cells was enhanced, and the number of fluorescent cells visible was increased, by intravitreous injection ofl-DOPA, DA, and NA (3 hr prior to enucleation). A combination of pretreatment with intramuscular reserpine and intravitreous NA was particularly effective. These results indicate that catecholamines may play an important role in the modulation of the membrane potential of horizontal cells.