Diurnal patterns of dopamine release in chicken retina

The retinal dopaminergic system appears to play a major role in the regulation of global retinal processes related to light adaptation. Although most reports agree that dopamine release is stimulated by light, some retinal functions that are mediated by dopamine exhibit circadian patterns of activity, suggesting that dopamine release may be controlled by a circadian oscillator as well as by light. Using the accumulation of the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) in the vitreous as a measure of dopamine release rates, we have investigated the balance between circadian- and light control over dopamine release. In chickens held under diurnal light:dark conditions, vitreal levels of DOPAC showed daily oscillations with the steady-state levels increasing nine-fold during the light phase. Kinetic analysis of this data indicates that apparent dopamine release rates increased almost four-fold at the onset of light and then remained continuously elevated throughout the 12h light phase. In constant darkness, vitreal levels of DOPAC displayed circadian oscillations, with an almost two-fold increase in dopamine release rates coinciding with subjective dawn/early morning. This circadian rise in vitreal DOPAC could be blocked by intravitreal administration of melatonin (10 nmol), as predicted by the model of the dark-light switch where a circadian fall in melatonin would relieve dopamine release of inhibition and thus be responsible for the slight circadian increase in dopamine release. The increase in vitreal DOPAC in response to light, however, was only partially suppressed by melatonin. The activity of the dopaminergic amacrine cell in the chicken retina thus appears to be dominated by light-activated input.     

Investigation of Dopamine Content, Synthesis, and Release in the Rabbit Retina In Vitro: I. Effects of Dopamine Precursors, Reserpine, Amphetamine, and l-DOPA Decarboxylase and Monoamine Oxidase Inhibitors

The basal catecholamine content of rabbit retina was determined by liquid chromatography with electrochemical detection (LC-EC) and 3,4-dihydroxyphenylethylamine (dopamine, DA) found to be the major catecholamine. The immediate DA precursor, 3,4-dihydroxyphenylalanine (L-DOPA), and the metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were also detected at about 2.8% and 17% of DA levels, respectively. When added exogenously, L-tyrosine did not increase the rate of DA synthesis over the basal level. In contrast, exogenous L-DOPA led to a 3.5-fold increase in DA, and to a 20-fold increase in DOPAC content. The monoamine oxidase inhibitors pargyline and (-)-deprenyl differentially affected the degradation of DA, since 100 microM pargyline was apparently more effective than 100 microM (-)-deprenyl. Reserpine and (+/-)-amphetamine each induced a Ca2+-independent decrease of DA stores. The separate actions of reserpine and (+/-)-amphetamine in lowering tissue DA levels were additive, suggesting two separate pools of DA available for release from presynaptic stores. The present study demonstrates that the LC-EC technique may be used to investigate the modulation of the synthesis and release of retinal DA in vitro, without the prior uptake of radiolabelled transmitter.     

Argon laser irradiation of rabbits' eyes-changes in prostaglandin E2 levels

Laser irradiation of the eye is a widely used therapeutic measure in various ocular disorders. We investigated in laser-treated rabbits' eyes the changes in prostaglandin E2 (PGE2) levels of the tissue affected by the laser (the retina/choroid) and of its adjacent vitreous over a two-week period. The parameters studied were; PGE2 in vitro production by the retina/choroid, as well as PGE2 and protein levels in the vitreous, the latter indicative of a break in the blood retinal barrier (BRB). The effect of noncoherent light exposure used for illumination, and that of the mechanical manipulation involved (sham exposure) were also studied. Following laser exposure vitreal PGE2 levels were increased two-fold above baseline (days three and 14), whereas light exposure resulted in a single peak. PGE2 in vitro production by the retina/choroid in the laser-exposed group was elevated throughout the observation period, peaking twice (days 3 and 14), in the light-exposed group the enhanced production was evident during a shorter period, whereas in the sham group it remained unchanged from baseline. An elevation in vitreal protein levels to above baseline levels occurred in both the laser- and, to a lesser degree, in the noncoherent light-exposed groups, but not in the sham group. Our study demonstrated an enhanced PGE2 in vitro production by retina/choroid of laser-exposed eyes, which might be attributable to the additive effect of the laser induced trauma, and the noncoherent light photochemical changes; the clinical significance of the recurrent increase in vitreal PGE2 levels in laser-treated eyes might be related to its anti-inflammatory properties.     

Rhythmic changes in metabolism of dopamine in the chick retina: the importance of light versus biological clock

Rhythmic changes in dopamine (DA) content and metabolism were studied in retinas of chicks that were adapted to three different lighting conditions: 12-h light : 12-h dark (LD), constant darkness (DD) and continuous light (LL). Retinas of chicks kept under LD conditions exhibited light-dark-dependent variations in the steady-state level of DA and the two metabolites of DA, i.e. 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA). Concentrations of DA, DOPAC and HVA were high in light hours and low in dark hours of the LD illumination cycle. In retinas of chicks kept under DD, the content of DA, DOPAC and HVA oscillated in a rhythmic manner for 2 days, with higher values during the subjective light phase than during the subjective dark phase. The amplitudes of the observed oscillations markedly and progressively declined compared with the amplitudes recorded under the LD cycle. In retinas of chicks kept under LL conditions, levels of DA, DOPAC and HVA were similar to those found during the light phase of the LD cycle. Changes in the retinal contents of DA and HVA did not exhibit pronounced daily oscillations, while on the first day of LL the retinal concentrations of DOPAC were significantly higher during the subjective light phase than during the subjective dark phase. Acute exposure of chicks to light during the dark phase of the LD cycle markedly increased DA and DOPAC content in the retina. In contrast, light deprivation during the day decreased the retinal concentrations of DA and DOPAC. It is suggested that of the two regulatory factors controlling the level and metabolism of DA in the retina of chick, i.e. light and biological clock, environmental lighting conditions seem to be of major importance, with light conveying a stimulatory signal for the retinal dopaminergic cells.     

Antidepressants and the monoamine masquerade

Neurotransmitter transporters have long been known to recognize related compounds as substrates, resulting in the accumulation and release of so-called "false transmitters." In this issue of Neuron, Zhou et al. show that when serotonin levels are elevated by inhibition of either serotonin reuptake or of monoamine oxidase, dopamine neurons accumulate serotonin. The results suggest that release of serotonin by dopamine neurons may contribute to the effects of multiple major classes of antidepressants.     

INHIBITION BY APOMORPHINE OF DOPAMINE DEAMINATION IN THE RAT BRAIN

Abstract— Apomorphine (A) inhibited dopamine deamination by rat brain mitochondria, but did not influence catechol- O -methyltransferase (COMT) activity by brain homogenates. The administration of apomorphine (10mg/kg i.p.) to normal rats increased brain dopamine (DA) by 34 per cent and decreased homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) by 60 per cent. In rats treated with reserpine 15 min prior to A, the latter prevented the rise of cerebral HVA and DOPAC and the depletion of DA produced by the former. Finally, A decreased the L-DOPA-induced accumulation of HVA and DOPAC in the rat basal ganglia. These results indicate that A inhibits DA deamination by monoamine oxidase.
This inhibition seems to be specific since apomorphine did not influence 5-HIAA levels in normal rats and prevented neither central 5-HT depletion nor 5-HIAA rise induced by reserpine.     

Melatonin and its generating system in vertebrate retina: circadian rhythm, effect of environmental lighting and interaction with dopamine

Retinas of rats, rabbits, chicks and carp possess enzymes, i.e. serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), which convert serotonin (5-HT) to melatonin, NAT activity and melatonin levels, but not HIOMT activity, show distinct circadian rhythms, with peak values occurring during the dark (night) phase of the 12 h light-dark cycle. Exposure of the animals to light at night inhibited the night-stimulated NAT activity. Treatment of rats and rabbits with the dopaminergic agonist, apomorphine, inhibited the retinal NAT activity. Dopamine levels in the rabbit retina showed diurnal variations, with higher contents seen during the light phase of both the 12 h light-dark cycle with lights on between 06:00–18:00, and that with reversed periods of illumination (lights on between 18:00–06:00). Melatonin potently inhibited the electrically-evoked calcium-dependent release of [³H]dopamine from pieces of retina from both albino and pigmented rabbits. Our results indicate that the light-regulated melatonin-generating system does operate in the vertebrate retina. The present data, together with other findings, suggest that in the retina there is an antagonistic interplay between melatonin and dopamine. Thus, melatonin inhibits dopamine synthesis in, and release from, the retinal dopaminergic cells, whilst dopamine inhibits the night (dark)-stimulated melatonin formation by decreasing NAT activity. Since light increases metabolic activity of the retinal dopaminergic cells (it enhances the amine synthesis, levels and release), it seems likely that the retinal dopamine plays a role of a “light” messenger in the inhibition of melatonin synthesis. It is suggested that an interplay between melatonin and dopamine in the retina is responsible for regulation of those retinal events which follow circadian rhythmicity, and/or are dependent on light-dark conditions.     

Lack of collagen XVIII/endostatin results in eye abnormalities

Mice lacking collagen XVIII and its proteolytically derived product endostatin show delayed regression of blood vessels in the vitreous along the surface of the retina after birth and lack of or abnormal outgrowth of retinal vessels. This suggests that collagen XVIII/endostatin is critical for normal blood vessel formation in the eye. All basement membranes in wild-type eyes, except Descemet's membrane, showed immunogold labeling with antibodies against collagen XVIII. Labeling at sites where collagen fibrils in the vitreous are connected with the inner limiting membrane and separation of the vitreal matrix from the inner limiting membrane in mutant mice indicate that collagen XVIII is important for anchoring vitreal collagen fibrils to the inner limiting membrane. The findings provide an explanation for high myopia, vitreoretinal degeneration and retinal detachment seen in patients with Knobloch syndrome caused by loss-of-function mutations in collagen XVIII.     

An In Vivo Study of Dopamine Release and Metabolism in Rat Brain Regions Using Intracerebral Dialysis

Intracerebral dialysis was used with a specifically designed HPLC with electrochemical detection assay to monitor extracellular levels of endogenous 3,4-dihydroxyphenylethylamine (dopamine, DA) and its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in brain regions of the halothane-anesthetized rat. Significant amounts of DA, DOPAC, and HVA were detected in control perfusates collected from striatum and n. accumbens whereas the medial prefrontal cortex showed lower monoamine levels. The ratio of DA in perfusate to DA in whole tissue suggests that in f. cortex, compared to n. accumbens and striatum, there is a greater amount of DA in the extracellular space relative to the intraneuronal DA content. The DOPAC/HVA ratio in control perfusates varied between regions in accordance with whole tissue measurements. This ratio was highest in n. accumbens and lowest in f. cortex. The monoamine oxidase inhibitor pargyline (100 mg/kg i.p.) caused an exponential decline in DOPAC, but not of HVA, in regional perfusates, an effect that was associated with an increase in DA. The data indicated a higher turnover of extracellular DOPAC in n. accumbens than in striatum and the lowest DOPAC turnover in f. cortex. The rate of decline in extracellular DA metabolite levels was slow compared to whole tissue measurements. In the perfusates there was no statistical correlation between basal amounts of DA in the perfusates and DOPAC and HVA levels or DOPAC turnover for any of the areas, indicating that measurement of DA metabolism in the brain under basal conditions does not provide a good index of DA release. In summary, this study shows clear regional differences in basal DA release and metabolite levels, metabolite patterns, and DOPAC turnover rates in rat brain in vivo.     

Effects of Light on Dopamine Metabolism in the Chick Retina

The effect of prolonged exposure to light on the activity of dopaminergic neurons and dopamine (DA) metabolism of chick retinae was investigated. alpha-Fluoromethyldopa, a potent and specific irreversible inactivator of aromatic amino acid decarboxylase, was used to assess DA turnover after inhibition of synthesis, and also to assess in vivo tyrosine hydroxylase activity by dihydroxyphenylalanine accumulation. After 48 h of light exposure, retinal DNA in 12-day-old chicks was about 30% higher (p less than 0.005) whereas dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were elevated two to three times (p less than 0.005) the level of controls kept in the dark for the same period. DA turnover was about twofold faster in the light (t 1/2 = 31 min) than in the dark (t 1/2 = 65 min). Tyrosine hydroxylase, assayed in vitro with saturating levels of cofactor and substrate, increased by about 50% after light exposure. The apparent tyrosine hydroxylase activity in vivo was approximately sixfold higher in the light than the dark. These results are interpreted and discussed in terms of the regulation of DA synthesis, and the use of DOPAC and HVA as indices of DA function in the retina.     

Formation and Clearance of Interstitial Metabolites of Dopamine and Serotonin in the Rat Striatum: An In Vivo Microdialysis Study

In vivo microdialysis was employed in order to characterize the steady-state kinetics of the turnover of specific dopamine and serotonin metabolites in the rat striatum 48 h after surgery. Inhibitors of monoamine oxidase (MAO; pargyline) and catechol-O-methyltransferase (COMT; Ro 40-7592) were administered, either separately or in conjunction, at doses sufficient to block these enzymes in the CNS. In some experiments, the acid metabolite carrier was blocked with probenecid. Temporal changes were then observed in the efflux of interstitial dopamine, 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA). The fractional rate constants for the accumulation or disappearance of the metabolites could be determined after pharmacological blockade of catabolic enzymes or the acid metabolite carrier. Interstitial 5-HIAA was found to be cleared with a half-life of approximately 2 h. After blockade of either MAO or COMT, HVA disappeared with a half-life of 17 min. Experiments employing probenecid suggested that some of the interstitial HVA was cleared by the acid metabolite carrier, the remainder being cleared by a probenecid-insensitive process, possibly conjugation. After MAO inhibition, DOPAC disappeared with an apparent half-life of 11.3 min. The rate of 3-MT accumulation after pargyline indicated that the majority of interstitial HVA (> 95%) is formed from DOPAC rather than 3-MT. The formation of 3-MT from interstitial dopamine, calculated from the accumulation rate of 3-MT after pargyline, appeared to follow first-order kinetics (k = 0.1 min-1).     

Dopamine metabolism and free-radical related mitochondrial injury during transient brain ischemia in gerbils

Regional extracellular release of dopamine (DA) and its metabolites, 3,4-dihydroxy-phenylacetic acid (DOPAC), homovanillic acid (HVA) and 3-methoxytyramine (3-MT) was measured in gerbils (with or without pargyline pretreatment) subjected to bilateral carotid artery occlusion (15 min) and various periods of recirculation (up to 6 hr), utilizing intracerebral microdialysis and high-performance liquid chromatography (HPLC) with electrochemical detection. Mitochondrial monoamine oxidase (MAO) and superoxide dismutase (SOD) activities andin vitro stimulated lipid peroxidation (TBARM) were determined in separate experimental groups of animals. The ischemically induced DA release, decrease of MAO-derived DA metabolites DOPAC and HVA, and accumulation of 3-MT were potentiated and prolonged by pargyline pretreatment. Mitochondrial MAO and SOD activities were significantly reduced during ischemia alone and up to 1 hr of reperfusion, whereas TBARM was enhanced during reflow only. The data suggest that reduced activity of mitochondrial antioxidative enzyme(s) but not DA metabolism by MAO may contribute to free radical-mediated injury of (mitochondrial) membranes.     

l-3,4-Dihydroxyphenylalanine-Induced Dopamine Release in the Striatum of Intact and 6-Hydroxydopamine-Treated Rats: Differential Effects of Monoamine Oxidase A and B Inhibitors

Abstract: Administration of l -DOPA (50 mg/kg) elicits a significant increase in extracellular dopamine in striata of rats treated with the catecholaminergic neurotoxin 6-hydroxydopamine but not in striata of intact rats. To assess the role of dopaminergic nerve terminals in determining the effects of exogenous l -DOPA on extracellular dopamine levels in striatum, we examined the relative contributions of monoamine oxidase A and monoamine oxidase B to the catabolism of dopamine synthesized from exogenous l -DOPA. Extracellular concentrations of dopamine and its catabolite, 3,4-dihydroxyphenylacetic acid, were monitored with in vivo dialysis in striata of intact rats and of rats with unilateral 6-hydroxydopamine lesions of striatal dopamine. Clorgyline (2 mg/kg), an inhibitor of monoamine oxidase A, significantly increased dopamine and decreased 3,4-dihydroxyphenylacetic acid in intact but not in dopamine-depleted striata. Inhibition of monoamine oxidase B with either l -deprenyl (1 mg/kg) or Ro 19-6327 (1 mg/kg) did not significantly affect dopamine or 3,4-dihydroxyphenylacetic acid in striata of intact or dopamine-depleted rats. In intact rats, administration of clorgyline in conjunction with l -DOPA produced a >20-fold increase in dopamine and prevented the l -DOPA-induced increase in 3,4-dihydroxyphenylacetic acid. Although both l -deprenyl and Ro 19-6327 administered in combination with l -DOPA elicited a small but significant increase in dopamine, levels of 3,4-dihydroxyphenylacetic acid were not affected. In rats pretreated with 6-hydroxydopamine, clorgyline had no significant effect on the increases in dopamine and 3,4-dihydroxyphenylacetic acid elicited by l -DOPA. Furthermore, neither l -deprenyl nor Ro 19-6327 affected l -DOPA-induced increases in dopamine and 3,4-dihydroxyphenylacetic acid in dopamine-depleted striata. The present findings indicate that deamination by monoamine oxidase A is the primary mechanism for catabolism of striatal dopamine, both under basal conditions and after administration of exogenous l -DOPA. Loss of dopaminergic terminals eliminates this action of monoamine oxidase A but does not enhance deamination by monoamine oxidase B. These data support a model in which exogenous l -DOPA elicits enhanced extracellular accumulation of dopamine in the dopamine-depleted striatum because some transmitter synthesis occurs at nondopaminergic sites and the dopamine terminals that normally take up and catabolize this pool of transmitter are absent.     

Circadian Regulation of Melatonin in the Retina of Xenopus laevis: Limitation by Serotonin Availability

Treatments expected to increase retinal serotonin levels were found to stimulate melatonin production by cultured eyecups from Xenopus laevis. The monoamine oxidase inhibitor pargyline (100 microM) caused a sixfold increase in melatonin release, and the serotonin precursor 5-hydroxy-L-tryptophan (100 microM) caused a 70-fold increase. Both acted synergistically with eserine, an inhibitor of melatonin deacetylation in the retina. The effect of 5-hydroxytryptophan was dose dependent, with effects increasing from 1 to 100 microM. Increasing the tryptophan level in the culture medium had no effect on melatonin release. These results indicate that the rate-limiting step in retinal melatonin synthesis is 5-hydroxylation of tryptophan. Melatonin released from individual eyecups in superfusion culture in constant darkness with and without added 5-hydroxy-L-tryptophan was monitored over a 5-day period. Control eyecups released low levels of melatonin, with circadian rhythmicity persisting for 1-3 days. With 5-hydroxy-L-tryptophan added, melatonin levels were elevated 10-20-fold at all times, and rhythmicity was apparent for as long as five cycles. This provides a model system for studies of the circadian clock in the eye.     

Formation and Metabolism of Dopamine in Nine Areas of the Rat Brain: Modifications by Haloperidol

The rate of removal of 3,4-dihydroxyphenylacetic acid (DOPAC) in nine rat brain areas (striatum, nucleus accumbens, tuberculum olfactorium, hypothalamus, lateral hippocampus, occipital cortex, brain stem, cerebellum, and retina) was calculated from its exponential decline after monoamine oxidase inhibition by pargyline. The experiments were carried out with rats pretreated with either saline or haloperidol. It appeared that the efficiency with which DOPAC was removed from the brain (expressed by the fractional rate constant k) varied considerably throughout the brain. Haloperidol dramatically decreased the k values, and in addition these effects differed widely in the various brain areas. Similarly to DOPAC, haloperidol had a pronounced retarding effect on the efflux of homovanillic acid (HVA) from the brain. These findings strongly suggest that great care should be taken when drug-induced alterations in DOPAC and HVA concentrations are interpreted as changes in dopaminergic activity. The dopamine (DA) concentrations were measured in the same experiments, but it appeared that the pargyline-induced rise in DA was of limited use for the estimation of the synthesis rate of the amine. We calculated the rate of catecholamine synthesis in the nine brain areas from the rise of 3,4-dihydroxyphenylalanine (DOPA) during decarboxylase inhibition. In saline- as well as in haloperidol-pretreated rats it was found that the total catecholamine synthesis rate in the typical dopaminergic areas (striatum, nucleus accumbens, and tuberculum olfactorium) was of the same order of magnitude as the DOPAC rate of removal. This confirms that DOPAC formation is quantitatively the main route of degradation in these brain areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative analysis of indices of central dopaminergic functions in man

Various postulated indices of central dopaminergic activity - cerebrospinal fluid (CSF) dopamine (DA), dihydroxy-phenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), plasma NA, serum prolactin, serum dopamine-β-hydroxylase (DBH), and platelet monoamine oxidase (MAO) activity - were measured in 30 drug-free inpatients. The mean values and the ranges were similar to those described in the literature. Plasma NA showed significant positive correlation with age. Significant positive correlation was found between CSF DA and its metabolites DOPAC and HVA. Serum DBH activity showed a slight but significant inverse correlation with CSF DA and its two metabolites. CSF NA showed a significant positive correlation with CSF DOPAC, but only in females. Serum DBH activity had no significant correlation either with CSF or with plasma NA levels. These findings suggest that either CSF HVA or DOPAC and DA may be useful indicators of DA metabolism in humans. Serum DBH activity may be in relationship with the central dopaminergic functions.     

Melatonin Increases Serotonin N-Acetyltransferase Activity and Decreases Dopamine Synthesis in Light-Exposed Chick Retina: In Vivo Evidence Supporting Melatonin-Dopamine Interaction in Retina

The administration of melatonin, either peripherally (0.01-10 mg/kg) or intraocularly (0.001-10 mumol/eye), to light-exposed chicks dose-dependently increased serotonin N-acetyltransferase (NAT) activity in retina but not in pineal gland. The effect of melatonin was slightly but significantly reduced by luzindole (2-benzyl-N-acetyltryptamine), and not affected by two other purported melatonin antagonists, N-acetyltryptamine and N-(2,4-dinitrophenyl)-5-methoxytryptamine (ML-23). The elevation of the enzyme activity induced by melatonin was substantially stronger than that evoked by 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, or 5-methoxytryptamine. The melatonin-evoked rise in the retinal NAT activity was counteracted by two dopamine D2 receptor agonists, quinpirole and apomorphine, and prevented by the dopamine D2 receptor blocker spiroperidol, and by an inhibitor of dopamine synthesis, alpha-methyl-p-tyrosine. Melatonin (0.1-10 mg/kg i.p.) dose-dependently decreased the levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), as well as the DOPAC/dopamine ratio, in chick retina but not in forebrain. The results obtained (1) indicate that melatonin in vivo potently inhibits dopamine synthesis selectively in retina, and (2) suggest that the increase in retinal NAT activity evoked by melatonin in light-exposed chicks is an indirect action of the compound, and results from the disinhibition of the NAT induction process from the dopaminergic (inhibitory) signal. The results provide in vivo evidence supporting the idea (derived on the basis of in vitro findings) that a mutually antagonistic interaction between melatonin and dopamine operates in retinas of living animals.     

Biotransformation of dopamine to norlaudanosoline by Aspergillus niger

Norlaundanosoline is a key intermediate in the synthesis of the benzylisoquinoline alkaloids providing the upper isoquinoline portion of the morphinan skeleton. This study evaluates the feasibility of using Aspergillus niger as an in situ biotransformation system to produce norlaudanosoline from dopamine. A. niger was chosen because monoamine oxidase can be readily induced in this organism. Monoamine oxidase catalyzes the conversion of dopamine to 3,4-dihydroxyphenylacetaldehyde. In the presence of dopamine, this aldehyde will then undergo a spontaneous Picket-Spengler condensation to form norlaudanosoline. Fermentation condition to form norlaudanosoline. Fermentation conditions were optimized for the production monoamine oxidase by using a two-stage process consisting of a growth stage and an induction stage. pH control was found to be important, and at pH 4.5 dopamine accumulation in the cells was high as was the level of monoamine oxidase. With pH control at 4.5, up to 21% of the cellular dopamine was converted to norlaudanosoline. It is proposed that with further protein engineering improvements, this system may prove suitable for the in situ bio-transformation of dopamine to norlaudanosoline.     

Changes in extracellular levels of dopamine metabolites in somatosensory cortex after peripheral denervation

This study examined the effects of a nerve transection on monoamine release from primary somatosensory cortex. The technique of microdialysis was employed to sample extracellular levels of norepinephrine (NE), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HIAA) and homovanillic acid (HVA) in the barrel field of freely moving rats following the surgical transection of the contralateral infraorbital nerve. Microdialysates obtained 3, 4, and 5 days after deafferentation were analyzed using high-performance liquid chromatography with electrochemical detection. We found a significant increase in the release of the dopamine metabolites, DOPAC and HVA from the deafferented cortex. Three days after deafferentation the release of DOPAC was three-fold higher in the deafferented than in the control animals, and remained about 100% higher in the next two days in this group of animals. The release of HVA showed a gradual increase following the deafferentation procedure, since a 92% larger value on day 3 increased to a 338% difference on day 5. On the other hand, the release rate of NE and the levels of the serotonin metabolite 5-HIAA were not significantly affected by the deafferentation procedure. These results are discussed in the context of the possible participation of dopamine in the reorganization of the deafferented somatosensory cortex.     

Inhibition of vesicular monoamine transporter enhances vulnerability of dopaminergic cells: relevance to Parkinson's disease

Parkinson's disease is a neurodegenerative disorder associated with progressive loss of dopaminergic cells in the substantia nigra. Oxidative stress has been implicated in the pathogenesis of the disease, and dopamine has been suggested as a contributing factor that generates reactive oxygen species due to its unstable catechol moiety. We have previously shown that tetrahydrobiopterin (BH4), an obligatory cofactor for dopamine synthesis, also contributes to the vulnerability of dopamine-producing cells by generating oxidative stress. This study shows that the presence of dopamine in the cytosol enhances the cell's vulnerability to BH4. Upon exposure to ketanserin, a vesicular monoamine transporter inhibitor, BH4-induced dopaminergic cell death is exacerbated, accompanied by increased lipid peroxidation and protein bound quinone. While intracellular amount of DOPAC is elevated by ketanserin, the monoamine oxidase inhibitor pargyline showed no significant protection. Instead, the thiol agent N-acetylcysteine and quinone reductase inducer dimethyl fumarate abolish BH4/ketanserin-induced cell death, suggesting that quinone production plays an important role. Therefore, it can be concluded that the presence of dopamine in the cytosol seems to contribute to the cells' vulnerability to BH4 and that vesicular monoamine transporter plays a protective role in dopaminergic cells by sequestering dopamine not only from monoamine oxidase but also from BH4-induced oxidative stress.