Ascorbic acid specifically enhances dopamine beta-monooxygenase activity in resting and stimulated chromaffin cells

Ascorbic acid enhancement of norepinephrine formation from tyrosine in cultured bovine chromaffin cells was characterized in detail as a model system for determining ascorbate requirements. In resting cells, ascorbic acid increased dopamine beta-monooxygenase activity without changing tyrosine 3-monooxygenase activity. [14C]Norepinephrine specific activity was increased by ascorbic acid, while [14C]dopamine specific activity was unchanged. Dopamine content, dopamine biosynthesis, tyrosine content, and tyrosine uptake were also unaffected by ascorbic acid. Furthermore, increased norepinephrine formation could not be attributed to changes in norepinephrine catabolism. Enhancement of dopamine beta-monooxygenase activity was specific for ascorbic acid, since other reducing agents with higher redox potentials were unable to increase norepinephrine formation. The specific effect of ascorbic acid on enhancement of norepinephrine formation was also observed in chromaffin cells stimulated to secrete with carbachol, acetylcholine, veratridine, and potassium chloride. In stimulated cells with and without ascorbate, there were no differences in dopamine content, tyrosine uptake, dopamine specific activity, and norepinephrine catabolism. These data indicate that, under a wide variety of conditions, only one catecholamine biosynthetic enzyme activity, dopamine beta-monooxygenase, is specifically stimulated by ascorbic acid alone in cultured chromaffin cells. This model system exemplifies a new approach for determining ascorbic acid requirements in cells and animals.     

Dopamine Metabolism in Hypoxanthine-Guanine Phosphoribosyltransferase-Deficient Variants of PC12 Cells

Lesch-Nyhan syndrome results from a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). It is manifest by behavioral abnormalities, including self-mutilation, and evidence of abnormal 3,4-dihydroxyphenylethylamine (dopamine) metabolism. To assess whether an HPRT deficiency in a dopaminergic cell can adversely affect dopamine metabolism in that cell, dopamine metabolism was examined in HPRT-deficient variants of PC12 pheochromocytoma cells and in cells that had regained HPRT activity by virtue of transformation with a recombinant retrovirus containing the human gene for HPRT. There was no correlation between HPRT activity and endogenous dopamine levels, dopamine uptake, dopamine release, or monoamine oxidase activity. Transformation with the HPRT retrovirus did not adversely affect dopamine metabolism.     

Ca2+ activates glycogenolysis in isolated mantle storage cells of Mytilus galloprovincialis Lmk.

Glycogenolytic activity (GA) in isolated mantle storage cells (MSC) from Mytilus galloprovincialis was studied, while glycogen and free-glucose content, as well as glucose released from cells were tested. In the period studied (November-December), the glucose releasing activity measured can be considered as an output of GA. In both, whole cells system (WCS) and crude cell-free system (CFS), a non-stimulated GA was detected. In WCS, dopamine and 5-hydroxytryptamine (5-HT) stimulated glycogenolysis, while epinephrine, norepinephrine and isoproterenol did not show any effect. Furthermore, mellitin and the Ca(2+)-ionophore, A23187, had a stimulating effect on the GA. In CFS, the absence of Ca2+ ions was a sufficient condition to depress GA. These and other findings suggest that: 1) GA in MSC may be stimulated by dopamine and 5-HT and not by adrenergic agonists; 2) cytosolyc Ca2+ signalling may have become an absolute requirement for activation of the glycogenolytic cascade in MSC; 3) a rapid high-affinity glucose transport may occur in these cells.     

Development of Transmitter-Releasing Capacity in Neuron-Enriched Tissue Cultures

Dissociated cell cultures derived from whole brains of foetal rats (17 days of gestation) were maintained for periods of up to 21 days in vitro for the purpose of studying the transmitter-releasing properties of the dopaminergic neuronal cells and glial cells. In the neuron-enriched cultures, after 3 days in vitro, [3H]dopamine was released in response to depolarizing stimuli. Both the potassium and veratrine-evoked release of dopamine was Ca2+ dependent. Veratrine-evoked release was reduced in the presence of the calcium channel blocker verapamil and was tetrodotoxin sensitive. Glial cultures, after 7 days in vitro, did not respond to any depolarizing stimuli, although they displayed a significant ability to take up [3H]dopamine. Comparison between static incubations and perfused cultures showed no difference in the patterns of release resulting from veratrine stimulation. Tyrosine hydroxylase activity increased progressively in neuron-enriched cultures but was not detectable in glial cultures. These results show that neuron-enriched cultures respond to depolarizing stimuli in a manner similar to excised adult basal ganglia tissue, with the appearance of functional ionic channels after 3 days in vitro.     

Genetic deletion of vesicular monoamine transporter-2 (VMAT2) reduces dopamine transporter activity in mesencephalic neurons in primary culture

Our aim was to investigate whether a defect in vesicular monoamine transporter-2 (VMAT2) activities would affect dopaminergic cell functions or not. We examined mesencephalon dopaminergic cultures prepared from VMAT2 wild-type, heterozygous or homozygous knockout (KO) 14-day-old mouse fetuses to determine the number of tyrosine hydroxylase (TH)-positive cells and dopamine transporter activity. The number of TH-positive cells remained unchanged in the VMAT2-KO cultures. Of interest, the dopamine transporter activity in the homozygous cells was significantly decreased, but not in the heterozygous cells, suggesting that complete deletion of VMAT2 inhibited dopamine transporter function. Furthermore, dopamine transporter activity was prominently decreased in the synaptosomal fraction of neonatal homozygous VMAT2-KO mice compared with that of wild-type/heterozygous VMAT2-KO ones, indicating that VMAT2 activity might be one of the factors regulating dopamine transporter activities. To test this possibility, we used reserpine, a VMAT2 inhibitor. Reserpine (1muM) decreased dopamine transporter activity (approx. 50%) in wild-type and heterozygous VMAT2-KO cultures but not in homozygous ones, indicating that blockade of VMAT2 activity reduced dopamine transporter activity. To investigate possible mechanisms underlying the decreased dopamine transporter activity in VMAT2-KO mice, we measured dopamine transporter activities after 24-48h exposure of primary cultures of mesencephalic neurons to dopamine receptor antagonists, PKC inhibitor, PI(3)K inhibitor, and l-DOPA. Among these drugs, l-DOPA slightly reduced the dopamine transporter activities of all genotypes, but the other drugs could not. Since the ratios of reduction in dopamine transporter activity of each genotype treated with l-DOPA were similar, substrate inhibition of dopamine transporters was not the main mechanism underlying the reduced dopamine transporter activity due to genetic deletion of VMAT2. Our results demonstrate that genetic deletion of VMAT2 did not induce immediate cell death but did markedly inhibit dopamine transporter activity.     

Intrastriatal Grafting of Cos Cells Stably Expressing Human Aromatic l-Amino Acid Decarboxylase: Neurochemical Effects

Abstract: To study the possibility that increasing striatal activity of aromatic l -amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to l -3,4-dihydroxyphenylalanine ( l -DOPA) administration, we grafted Cos cells stably expressing the human AADC gene (Cos- haadc cells) into 6-hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of ¹⁴CO₂ from l -[¹⁴C]DOPA. The K _m value for l -DOPA in intact and disrupted cells was 0.60 and 0.56 m M , respectively. The cofactor, pyridoxal 5-phosphate, enhanced enzymatic activity with maximal effect at 0.1 m M . The pH optimum for enzyme activity was 6.8. Grafting Cos- haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of l -DOPA. When measured 2 h after l -DOPA administration, the mean dopamine level in the striata of Cos- haadc -grafted animals was 2 µg/g of tissue, representing 31% of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos-ut cells) was 1 µg/g. At 6–8 h after l -DOPA administration, striatal dopamine content in the Cos- haadc -grafted animals was 0.67 µg/g of tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos-ut-grafted animals was undetectable. These findings demonstrate that enhancing striatal AADC activity can improve dopamine bioformation in response to systemically administered l -DOPA.     

Changes in Sympathetic Nerve Terminals in the Heart of Cold-Exposed Rats

Abstract: Changes in sympathetic nerve terminals of the heart after varying periods of exposure of rats to 4°C were investigated. Two indices were used for changes in the number of noradrenaline storage vesicles, i.e., vesicular dopamine β-hydroxylase (DBH) activity and noradrenaline storage capacity. The latter was obtained after uptake of [³H]noradrenaline; endogenous content, uptake of exogenous noradrenaline, and degree of saturation of the vesicles were calculated using the specific activity of the [³H]noradrenaline. As a measure of tyrosine hydroxylase activity, whole ventricular noradrenaline, dopamine, and dihydroxyphenylacetic acid content were used. After 4 h of cold exposure there was an increase in vesicular endogenous noradrenaline content, uptake, storage capacity, and DBH activity as well as a large increase in whole ventricular dopamine. After 6 h in the cold, vesicular endogenous noradrenaline content, storage capacity, and DBH activity were decreased. The results suggest that during cold exposure there is an initial increase followed by a decrease in the number of functional vesicles in the nerve terminal, which could explain the fluctuations in the rate of noradrenaline release.     

Newly Synthesized Dopamine as the Precursor for Norepinephrine Synthesis in Bovine Adrenomedullary Chromaffin Cells

The precursor pool of dopamine for norepinephrine synthesis was investigated in cultured bovine adrenomedullary chromaffin cells incubated with [14C]tyrosine. Under conditions where the intracellular [14C]tyrosine specific activity was constant and [14C]dopamine synthesis was maximal, [14C]dopamine and [14C]norepinephrine accumulated over time, and the total intracellular dopamine content more than doubled within 120 min. When [14C]norepinephrine synthesis was calculated at different times based on the specific activity of [14C]dopamine, this rate was approximately equal to the rate of [14C]dopamine synthesis and was, thus, inconsistent with the observed dopamine accumulation. However, the rate of [14C]norepinephrine synthesis based on the [14C]tyrosine specific activity accounted for the dopamine accumulation, an observation suggesting that newly synthesized dopamine, i.e., dopamine with a specific activity equivalent to that of its precursor, [14C]tyrosine, is preferentially utilized for norepinephrine synthesis. Further studies showed that the subcellular distribution of [14C]dopamine was identical to that of norepinephrine and epinephrine and that the accumulated [14C]dopamine could be converted to norepinephrine within the chromaffin vesicle if dopamine uptake was blocked. Taken together, these results suggest that a small intravesicular dopamine pool, rapidly replenished by newly synthesized dopamine, serves as the substrate for dopamine beta-hydroxylase. Several mechanisms to account for this observation are discussed.     

Measurement of beta-galactosyltransferase activity in cell extracts with an ELISA-based assay

An enzyme-linked immunosorbent assay (ELISA)-based glycosyltransferase assay has been used to measure UDP-Gal:N-acetylglucosamine beta-1,4-galactosyl-transferase (EC 2.4.1.38) activity in detergent extracts of chinese hamster ovary (CHO) cells. LEC11 cells (a mutant of the CHO cell line, Pro -5), which are known to express a complex array of carbohydrate structures, were used to develop the assay for use with whole cell extracts. A detergent-solubilized preparation of the enzyme from whole cells was used to convert the substrate, lactotriglycosylceramide, to the product, neolactotetraglycosylceramide. The monoclonal antibody, 1B2, which specifically binds to the Gal beta 1-4GlcNAc epitope, was used in an ELISA to identify and quantify the product. The enzyme activity in the preparations was found to be similar to that obtained by conventional radioactive assay methods. The beta-galactosyltransferase found in LEC11 cell detergent extracts exhibited an absolute requirement for the nucleotide sugar and MnCl2. The activity of the enzyme was also strictly dependent on the presence of exogenous glycolipid acceptor. When Triton X-114 was used to solubilize the LEC11 beta-galactosyltransferase, activity was found in both the hydrophilic and the hydrophobic phases, suggesting the presence of two forms of the enzyme. The ELISA-based assay was used to compare beta-1,4-galactosyltransferase activity in detergent extracts of four CHO cell lines: Pro-5, Lec1, LEC11, and LEC12 and in detergent-solubilized microsomes from human leukemia cells. The results from this study demonstrate the utility of the ELISA-based assay for measuring glycosyltransferase activity in detergent-solubilized whole cells and microsome preparations.     

Dopamine but not l-dopa stimulates neural glutathione metabolism. Potential implications for Parkinson’s and other dopamine deficiency states

Dopamine is produced first by hydroxylalation of l-tyrosine to l-dihydroxyphenylalanine (l-dopa) and subsequently by the decarboxylation of l-dopa to dopamine catalysed by the enzymes tyrosine hydroxylase and aromatic l-amino acid decarboxylase (AADC) respectively. Reduced glutathione (GSH) acts as a major cellular antioxidant. We have investigated the role of dopamine in the control of GSH homeostasis in brain cells. The SH-SY5Y human neuroblastoma cell line was found to increase intracellular GSH levels in response to 50 μM dopamine treatment. Similarly the 1321N1 human astrocytoma cell line was found to increase GSH release in response to 50 μM dopamine. The same concentration of l-dopa was also found to increase intracellular GSH in SH-SY5Y cells, however when AADC was inhibited this affect was abolished. Furthermore 1321N1 cells which were found to have almost undetectable levels of AADC activity did not increase GSH release in response to 50 μM l-dopa. These results suggest that at these concentrations dopamine has the potential to act as a signal for the upregulation of GSH synthesis within neuronal-like cells and for the increased trafficking of GSH from astrocytes to neurons. This effect could potentially relate to the activation of antioxidant response elements leading to the induction of phase II detoxifying enzymes including those involved in GSH synthesis and release. The inability of l-dopa to produce a similar effect when AADC was inhibited or when AADC activity was absent indicates that these effects are relatively specific to dopamine. Additionally dopamine but not l-dopa treatment led in an increase in complex I activity of the respiratory chain in SH-SY5Y cells which may be related to the effect of dopamine on GSH levels.     

Dopamine inhibits calcium flux in the 7315a prolactin-secreting pituitary tumour

Cells of the 7315a prolactin-secreting tumour express biochemically normal cell-surface receptors for dopamine. However, dopamine inhibits prolactin release from these cells only when the basal rate of prolactin release is augmented by increasing the intracellular and/or extracellular calcium concentration of the tumour cells. This suggests that dopaminergic modulation of calcium ion flux could have a central physiological role in these neoplastic cells. In 7315a cells we examined the ability of dopamine to regulate 45Ca2+ influx and fractional 45Ca2+ efflux under conditions of enhanced calcium flux using the calcium channel activator, maitotoxin. It was observed that unidirectional calcium influx stimulated by maitotoxin was significantly inhibited by dopamine. Maitotoxin stimulated fractional efflux and prolactin release from the tumour cells and dopamine simultaneously inhibited both processes by a haloperidol-reversible mechanism. Therefore, in 7315a cells dopamine receptor activation is coupled to inhibition of calcium flux as at least one component in the regulation of prolactin release. These cells may provide further opportunity to study intracellular signalling mechanisms that are modulated by dopamine receptor activity.     

Expression of dopamine receptors and transporter in neuroendocrine gastrointestinal tumor cells

C-11- or F-18-DOPA positron emission tomography (DOPA PET) is a new sensitive imaging technique for small neuroendocrine gastrointestinal tumors which evaluates the decarboxylase activity. To further characterize the dopaminergic system in neuroendocrine gastrointestinal tumor cells, we investigated the expression of both dopamine receptors and the transmembrane dopamine transporter (DAT) in the human neuroendocrine pancreatic cell line BON and in the neuroendocrine gut cell line STC-1. Both BON and STC-1 cells expressed mRNA of the dopamine receptors D2-D5 and DAT. mRNA of the dopamine receptor D1 was detected in BON cells only. Both in BON and STC-1 cells, expression of D2 and D5 receptors and DAT was also demonstrated immunocytochemically. For functional receptor characterization intracellular cAMP levels ([cAMP]i) were determined. Whereas in STC-1 cells dopamine and the D1-like (D1/D5) receptor agonist SKF 38393 increased [cAMP]i, [cAMP]i was decreased by dopamine or the D2-like (D2-D4) receptor agonist quinpirole in BON cells. Functional DAT activity was, however, not detected in either cell line. The presence of both dopamine receptors and of the DAT suggests an autocrine and/or paracrine function of dopamine in neuroendocrine gastrointestinal tumor cells. Yet neither the transmembrane dopamine transporter nor dopamine receptors are likely to contribute to positive DOPA PET imaging of neuroendocrine gastrointestinal tumors. However, these molecules may be of diagnostic importance when applying other dopaminergic system tracers.     

THE EFFECT OF HYPOXIA ON MONOAMINE SYNTHESIS, LEVELS AND METABOLISM IN RAT BRAIN

Abstract— Rats were exposed to 5.6% oxygen environments for up to 2 h. The accumulation of brain DOPA and 5-hydroxytryptophan at 30 min after decarboxylase inhibition was used to estimate cerebral tryosine and tryptophan hydroxylase activity, respectively, in vivo. There was a continuing decrease in tryosine hydroxylase activity during the 2 h in whole brain as well as five brain regions. Tryptophan hydroxylase activity declined during the 1st h, but then increased towards control levels during the 2nd h. There was an increase in brain tryptophan during the 2nd h as well. In whole brain and the five brain regions, there was no significant change in the levels of noradrenaline, dopamine or 5-hydroxytrypamine. During a 1 h exposure to 5.6% oxygen, there was decreased accumulation of noradrenaline, dopamine and 5-hydroxytryptamine after MAO inhibition and decreased accumulation of homovanillic acid and 5-hydroxyindoleacetic acid after probenecid administration. The dercreased synthesis and metabolism of the monoamines is most likely attributable to insufficient brain tissue oxygen as a substrate for the two hydroxylase enzymes.     

Enhancement of tyrosine hydroxylase phosphorylation and activity by glial cell line-derived neurotrophic factor

Although glial cell-line derived neurotrophic factor (GDNF) acts as a potent survival factor for dopaminergic neurons, it is not known whether GDNF can directly alter dopamine synthesis. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for dopamine biosynthesis, and its activity is regulated by phosphorylation on three seryl residues: Ser-19, Ser-31, and Ser-40. Using a TH-expressing human neuroblastoma cell line and rat primary mesencephalic neuron cultures, the present study examined whether GDNF alters the phosphorylation of TH and whether these changes are accompanied by increased enzymatic activity. Exposure to GDNF did not alter the TH protein level in either neuroblastoma cells or in primary neurons. However, significant increases in the phosphorylation of Ser-31 and Ser-40 were detected within minutes of GDNF application in both cell types. Enhanced Ser-31 and Ser-40 phosphorylation was associated with increased TH activity but not dopamine synthesis in neuroblastoma cells, possibly because of the absence of l-aromatic amino acid decarboxylase activity in these cells. In contrast, increased phosphorylation of Ser-31 and Ser-40 was found to enhance dopamine synthesis in primary neurons. Pharmacological experiments show that Erk and protein kinase A phosphorylate Ser-31 and Ser-40, respectively, and that their inhibition blocked both TH phosphorylation and activity. Our results indicate that, in addition to its role as a survival factor for dopaminergic neurons, GDNF can directly increase dopamine synthesis.     

Localization of α-Galactosidase in an Alkalophilic Strain of Micrococcus†

Localization of α-galactosidase in an alkalophilic strain of Micrococcus was investigated in relation to the cell membrane as a permeability barrier. The most α-galactosidase appered to be intracellular; only about 4% of α-galactosidase was released by lysozyme or freeze-thaw treatments of the whole cells. The enzyme activity was not inhibited by treatment of the whole cells with diazo-7-amino-1,3-naphthalene disulfonic acid (NDS) which penetrated the cell wall but not the cytoplasmic membrane. The enzyme activity of the whole cells increased about four-fold by toluene-acetone treatment which caused an alteration in the membrane permeability. The enzyme in such cells became to be relatively sensitive to pH. These results showed that cell membrane played a protective role as a permeability barrier against alkaline environment.     

Assessment of the direct and indirect effects of MPP+ and dopamine on the human proteasome: implications for Parkinson's disease aetiology

Mitochondrial impairment, glutathione depletion and oxidative stress have been implicated in the pathogenesis of Parkinson's disease (PD), linked recently to proteasomal dysfunction. Our study analysed how these factors influence the various activities of the proteasome in human SH-SY5Y neuroblastoma cells treated with the PD mimetics MPP⁺ (a complex 1 inhibitor) or dopamine. Treatment with these toxins led to dose- and time-dependent reductions in ATP and glutathione and also chymotrypsin-like and post-acidic like activities; trypsin-like activity was unaffected. Antioxidants blocked the effects of dopamine, but not MPP⁺, suggesting that oxidative stress was more important in the dopamine-mediated effects. With MPP⁺, ATP depletion was a prerequisite for loss of proteasomal activity. Thus in a dopaminergic neuron with complex 1 dysfunction both oxidative stress and ATP depletion will contribute independently to loss of proteasomal function. We show for the first time that addition of MPP⁺ or dopamine to purified samples of the human 20S proteasome also reduced proteasomal activities; with dopamine being most damaging. As with toxin-treated cells, chymotrypsin-like activity was most sensitive and trypsin-like activity the least sensitive. The observed differential sensitivity of the various proteasomal activities to PD mimetics is novel and its significance needs further study in human cells.     

Dopamine: an old target in a new therapy

Dopamine, a molecule of joy and emotions, plays vital role in regulation cancer growth and tumor angiogenesis. Dopamine secrets from neural cells in brain and peripheral cells as well. Peripheral dopamine is associated with tumorigenic events. Recent publication [Sarkar et al. Int. J. Cancer: doi:10.1002/ijc.29414, 2014] suggests that dopamine can be an ideal substitute as an anti-vascular endothelial growth factor A (VEGF-A) agent for the treatment tumor angiogenesis as dopamine is less expensive, minimum side-effect and more sensitive than other drugs. The studies also found that dopamine prevent the 5FU-induced neutropenia in tumor-bearing mice. Collectively, these pre-clinical studies claim that dopamine could be a novel therapy for managing cancer growth and chemotherapy related disorder.     

Diabetes induces changes of catecholamines in primary mesangial cells

Diabetes mellitus is a frequent cause of kidney function damage with diabetic nephropathy being predominantly related to glomerular dysfunction. Diabetes is capable of interfering with distinct hormonal systems, as well as catecholamine metabolism. Since mesangial cells, the major constituent of renal glomerulus, constitute a potential site for catecholamine production, the present study was carried out to investigate alterations in catecholamine metabolism in cultured mesangial cells from the nonobese diabetic mouse, a well-established model for type I diabetes. We evaluated mesangial cells from normoglycemic and hyperglycemic nonobese diabetic mice, as well as cells from normoglycemic Swiss mice as control. Mesangial cells from normoglycemic mice presented similar profiles concerning all determinations. However, cells isolated from hyperglycemic animals presented increased dopamine and norepinephrine production/secretion. Among the studied mechanisms, we observed an upregulation of tyrosine hydroxylase expression accompanied by increased tetrahydrobiopterin consumption, the tyrosine hydroxylase enzymatic cofactor. However, this increase in synthetic pathways was followed by decreased monoamine oxidase activity, which corresponds to the major metabolic pathway of catecholamines in mesangial cells. In addition, whole kidney homogenates from diabetic animals also presented increased dopamine and norepinephrine levels when compared to normoglycemic animals. Thus, our results suggest that diabetes alters catecholamine production by interfering with both synthesizing and degrading enzymes, suggesting a possible role of catecholamine in the pathogenesis of acute and chronic renal complications of diabetes mellitus.     

Signal transmission in the photosensitive pineal organ of the rainbow trout: Modulation of ganglion cell activity by intrinsic dopamine

The photosensitive pineal organ of the rainbow trout (Oncorhynchus mykiss) transduces photic information into nycthemeral neuronal signals. To investigate origin, cellular localization, and functional significance of pineal catecholamines, we performed HPLC-analysis of catecholamines and tyrosine hydroxylase (TH) activity, as well as immunocytochemical and electrophysiological studies. In biochemical and immunocytochemical investigations, pineal cells were found to contain endogenous TH. Using HPLC-analysis, the presence of a catecholamine precursor ( -dopa), catecholamines (dopamine, norepinephrine, epinephrine), and a metabolite (DOPAC) was demonstrated. The release of -dopa, dopamine and DOPAC from isolated pineal organs was shown by superfusion experiments. Extracellular recordings were used to monitor the action of dopaminergic drugs on electrical activity of ganglion cells. Dopamine increased the discharge activity of action potentials, whereas dopamine receptor antagonists resulted in a reduction of ganglion cell activity. Our data provide evidence for establishing dopamine as an intrinsic neurotransmitter or neuromodulator in the photosensitive pineal organ of the rainbow trout.     

Role of ascorbic acid in dopamine beta-hydroxylation. The endogenous enzyme cofactor and putative electron donor for cofactor regeneration

The role(s) of ascorbic acid in dopamine beta-hydroxylation was studied in primary cultures of bovine adrenomedullary chromaffin cells and in isolated bovine adrenomedullary chromaffin vesicles. Dopamine beta-hydroxylase activity was assessed by measuring the rate of conversion of tyramine to octopamine. The ascorbic acid content of chromaffin cells declined with time in culture and the dopamine beta-hydroxylase activity of ascorbate-depleted cells was low. Ascorbate additions to ascorbate-depleted cells increased both the intracellular ascorbate concentrations and the rates of dopamine beta-hydroxylation. Ascorbate uptake into the cells was rapid; however, the onset of enhanced octopamine synthesis by added ascorbate was delayed by several hours and closely followed the time course for accumulation of the newly taken up ascorbate into the chromaffin vesicle. The amount of octopamine synthesized by the chromaffin cells exceeded the intracellular ascorbate content and ascorbate levels were maintained during dopamine beta-hydroxylation in the absence of external ascorbate. This suggests an efficient recycling of ascorbate. In contrast to intact cells, ascorbic acid was depleted during octopamine synthesis in isolated chromaffin vesicles. The molar ratio of octopamine formed to ascorbate depleted was close to unity. Thus, the recycling of intravesicular ascorbate depends on an extravesicular factor(s). The depletion of intravesicular ascorbate during dopamine beta-hydroxylation was prevented by the addition of nonpermeant extravesicular electron donors such as ascorbate or glucoascorbate. This suggests that intravesicular ascorbate is maintained in the reduced state by electron transport across the vesicle membrane. These results are compatible with the hypothesis that both intra- and extravesicular ascorbate participate in the regulation of dopamine beta-hydroxylase. Intravesicular ascorbate is the cofactor for the enzyme. Cytosolic ascorbate is most likely the electron donor for the vesicle-membrane electron transport system which maintains the intravesicular cofactor concentration.