Expression of cloned aromatic L-amino acid decarboxylase in Xenopus laevis oocytes.

Sense mRNA coding for bovine adrenal medulla aromatic L-amino acid decarboxylase (AADC) was expressed following microinjection into Xenopus laevis oocytes. The expressed enzyme activity was stereoselective for L-5-hydroxytryptophan and L-DOPA and blocked by NSD-1015 an inhibitor of AADC. Heating the expressed enzyme at 55 degrees C resulted in a parallel loss of activity towards both substrates. Our findings are consistent with the prevailing notion that a single enzyme is able to decarboxylate both substrates in vivo.     

Induction of aromaticl-amino acid decarboxylase mRNA by interleukin-1β and prostaglandin E2 in PC12 cells

Aromatic 1-amino acid decarboxylase (AADC) is involved in the synthesis of the putative neurotransmitters dopamine (DA), norepinephrine (NA) and 5-hydroxytryptamine (5-HT). We report here that the gene expression of AADC can be regulated by interleukin (IL) 1- and prostaglandin (PG) E₂ in PC12 cells. The cells were treated with different doses of IL 1- and PGE₂ for 3 days. Slot blot hybridization was performed to detect AADC mRNA and Western immunoblot to detect AADC protein. The cDNA probe for rat AADC was generated by the PCR method. IL 1- and PGE₂ produced a dose- and time-dependent up-regulation in AADC mRNA levels (up to 200% of the control values) which was followed by a stable increase in AADC protein. The data further support the suggestion that AADC is a regulated enzyme and that the regulation occurs at the level of gene expression. Because IL-1 is synthesized, and acts locally, within the brain to influence neuronal and glial functions, it has been proposed to be a mediator with both beneficial and detrimental responses to inflammation and injury. The regulation of AADC by IL-1 may indicate a possible involvement for AADC in neuronal injury and recovery. Since IL-1 promotes PGE₂ formation, its effects may be occurring by increasing level of PGE₂.Abbreviations AADC aromatic 1-amino acid decarboxylase - IL-1 interleukin 1 - PGE₂ prostaglandin E₂ - GITC guanidinium isothiocyanate - DEPC diethyl pyrocarbonate - MOPS 3-(4-morpholino)propanesulfonic acid - SSPE 0.18M NaCl, 0.001M sodium phosphate, and 0.001M EDTA Special issue dedicated to Dr. Bernard W. Agranoff.     

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.     

Altered hypothalamic monoamine metabolism and pituitary prolactin regulation in female spontaneously hypertensive rats

Prolactin (PRL) secretion after aromatic amino acid decarboxylase inhibition with NSD-1015 was significantly elevated in female spontaneously hypertensive rats (SHR) as compared to normotensive (WKY) controls. Although basal PRL levels tended to be elevated in SHR rats, the differences were not significant. In vitro PRL secretion was also significantly elevated in the SHR rats as compared to the WKY rats, but the SHR rats were more responsive to the inhibitory effects of dopamine (DA). Despite changes in pituitary PRL secretion and DA response, there was no apparent difference in tubero-infundibular DA activity between the two rat strains. Hypothalamic serotonin levels were elevated in SHR rats, but metabolism did not appear to be significantly changed based on measurements of 5-hydroxytryptophan accumulation after NSD-1015 treatment.     

Transmitter-Like Basal and K+-Evoked Release of 3,4-Dihydroxyphenylalanine from the Striatum in Conscious Rats Studied by Microdialysis

Using microdialysis and HPLC, characteristics of the release of endogenous 3,4-dihydroxyphenylalanine (DOPA) from striatum in conscious rats were studied in comparison with those of 3,4-dihydroxyphenylethylamine (dopamine; DA). Purified L-aromatic amino acid decarboxylase (AADC) converted a putative peak of DOPA to DA. The retention time of DOPA differed from that of DA and major metabolites of DA and norepinephrine. The DOPA peak of dialysates comigrated with that of authentic DOPA when the pH of the HPLC buffer was modified. The ratio of the basal release of DOPA:DA was 1:2. 3-Hydroxybenzylhydrazine (NSD-1015; 100 mg/kg, i.p.), an AADC inhibitor, markedly increased the basal release of DOPA but produced no effect on DA. The basal release of DOPA was markedly decreased by alpha-methyl-p-tyrosine (200 mg/kg, i.p.), substantially tetrodotoxin (1 microM) sensitive, and Ca2+ (removal plus 12.5 mM Mg2+ addition) dependent. Fifty millimolar K+ released DOPA and this release was also Ca2+ dependent. These characteristics of the basal and evoked release of DOPA were similar to those of DA. The ratio of the evoked release of DOPA:DA was 1:3. These results indicate that DOPA is released under physiological conditions and by K(+)-induced depolarization in a manner similar to that for transmitter DA from striatum in freely moving rats.     

Turnover of Dopamine and Serotonin and Their Metabolites in the Striatum of Aged Rats

Turnover of dopamine (DA), serotonin [5-hydroxytryptamine (5-HT)], and their metabolites has been measured in adult and aged rats. Turnover rates of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxy-3-indoleacetic acid (5-HIAA) have been assayed from the disappearance rates after blocking by pargyline inhibition of monoamine oxidase (MAO) and from the accumulation rates by probenecid inhibition of the probenecid-sensitive transport system. DA and 5-HT turnover rates have been measured as accumulation rates of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively, after central decarboxylase inhibition by 3-hydroxybenzylhydrazine (NSD-1015) and as accumulation rates of DA and 5-HT after pargyline inhibition of MAO. The DA turnover rate after NSD-1015 was 23.9% lower in aged rats than in adults, whereas after pargyline there was no significant difference between the two age groups. The HVA fractional rate constant and turnover after pargyline were lower in aged rats than in adults, and HVA turnover after probenecid was higher in aged rats than in adults. The DOPAC-HVA pathway seems to be reinforced at the expense of DOPAC conjugation. In aged and adult rats whose 5-HT steady-state levels were not statistically different, the 5-HT turnover rate after pargyline and NSD-1015 treatment was lower in aged rats than in adults. An increase of 5-HIAA levels after pargyline and probenecid treatment in aged rats could be due to the handling stress.     

Alternate Promoters in the Rat Aromatic l-Amino Acid Decarboxylase Gene for Neuronal and Nonneuronal Expression: An In Situ Hybridization Study

Abstract: Aromatic l -amino acid decarboxylase (AADC) is found in both neuronal cells and nonneuronal cells, and a single gene encodes rat AADC in both neuronal and nonneuronal tissues. However, two cDNAs for this enzyme have been identified: one from the liver and the other from pheochromocytoma. Exons 1a and 1b are found in the liver cDNA and the pheochromocytoma cDNA, respectively. In the third exon (exon 2), there are two alternatively utilized splicing acceptors specific to these exons, 1a and 1b. Structural analysis of the rat AADC gene showed that both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. To demonstrate whether alternative promoter usage and splicing are tissue specific and whether the exons 1a and 1b are differentially and specifically transcribed in nonneuronal and neuronal cells, respectively, in situ hybridization histochemistry for the rat brain, adrenal gland, liver, and kidney was carried out using these two exon probes. The exon 1a probe specifically identified AADC mRNA only in nonneuronal cells, including the liver and kidney, and the exon 1b probe localized AADC mRNA to monoaminergic neurons in the CNS and the adrenal medulla. Thus, both alternative promoter usage and differential splicing are in fact operative for the tissue-specific expression of the rat AADC gene.     

Determination of Amino Acids and Monoamine Neurotransmitters in Caudate Nucleus of Seizure-Resistant and Seizure-Prone BALB/c Mice

Abstract: Amino acid and monoamine concentrations were examined in tissue extracts of caudate nucleus of genetic substrains of BALB/c mice susceptible or resistant to audiogenic seizures. Amino acids [aspartate, glutamate, glycine, taurine, serine, γ-aminobutyric acid (GABA)], monoamines, and related metabolites were separated by isocratic reverse-phase chromatography and detected by a coulometric electrode array system. In situ activity of tyrosine hydroxylase and tryptophan hydroxylase were determined by measuring the accumulation of L-DOPA and 5-hydroxytryptophan after administration of the decarboxylase inhibitor NSD-1015. Highly significant decreases in concentrations of both excitatory (glutamate and aspartate) and inhibitory amino acids (GABA and taurine) were observed in extracts of caudate nucleus of seizure-prone mice. Substantial decreases in concentrations of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were also noted. Decreased accumulation of L-DOPA after NSD-1015 administration provided evidence for decreased tyrosine hydroxylase activity and decreased DA synthesis in striatum of seizure-prone mice compared with seizure-resistant mice. Decreased concentrations of the DA metabolite 3-methoxytyramine (after NSD-1015 administration) suggested that DA release was also compromised in seizure-prone mice. No significant difference in 5-hydroxytryptophan accumulation in striatum of seizure-prone and seizure-resistant mice suggested that tryptophan hydroxylase activity and serotonin synthesis were not affected. The data suggest that seizure-prone BALB/c mice have a deficiency in intracellular content of both excitatory and inhibitory amino acids. The data also raise the issue of whether GABAergic interactions with the nigrostriatal DA system are important in the regulation of audiogenic seizure susceptibility.     

Gene therapy using AAV

AAV (adeno-associated virus) vectors are considered to be promising gene-delivery vehicles for gene therapy, because they are derived from non-pathogenic virus, efficiently transduce non-dividing cells, and cause long-term gene expression. Appropriate AAV serotypes are utilized depending on the type of target cells. Among various neurological disorders, Parkinson's disease (PD) is one of the most promising candidates of gene therapy. PD is a progressive neurodegenerative disorder that predominantly affects dopaminergic neurons in the substantia nigra. One of the major approaches to gene therapy of PD is the intrastriatal expression of dopamine (DA)-synthesizing enzyme genes. As for the initial step of clinical application, AAV vector-mediated AADC (aromatic L-amino acid decarboxylase; the enzyme converting L-DOPA to DA) gene transfer in combination with oral administration of L-DOPA would be appropriate, since DA production can be regulated by adjusting the dose of L-DOPA. Second, intramuscular injection of AAV vectors is appropriate to protein-supplement gene therapy. Monogenic diseases such as hemophilia and Fabry disease are suitable candidates. Regarding cancer gene therapy, AAV vectors may be utilized to inhibit tumor angiogenesis, metastasis, and invasion. When long-term transgene expression in stem cells is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS1 locus on the chromosome 19 (19q13.4) by using the integration machinery of AAV would be particularly valuable.     

Aromatic L-Amino Acid Decarboxylase (AADC) Is Crucial for Brain Development and Motor Functions

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric neuro-metabolic disease in children. Due to the lack of an animal model, its pathogenetic mechanism is poorly understood. To study the role of AADC in brain development, a zebrafish model of AADC deficiency was generated. We identified an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome. Whole-mount in situ hybridization analysis showed that the ddc gene is expressed in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO) reduced brain volume and body length. We observed increased brain cell apoptosis and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants (ddc MO-injected embryos). Seizure-like activity was also detected in ddc morphants in a dose-dependent manner. ddc morphants had less sensitive touch response and impaired swimming activity that could be rescued by injection of ddc plasmids. In addition, eye movement was also significantly impaired in ddc morphants. Collectively, loss of Ddc appears to result in similar phenotypes as that of ADCC deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of AADC deficiency in children.     

A Biochemical and Functional Protein Complex Involving Dopamine Synthesis and Transport into Synaptic Vesicles

Synaptic transmission depends on neurotransmitter pools stored within vesicles that undergo regulated exocytosis. In the brain, the vesicular monoamine transporter-2 (VMAT₂) is responsible for the loading of dopamine (DA) and other monoamines into synaptic vesicles. Prior to storage within vesicles, DA synthesis occurs at the synaptic terminal in a two-step enzymatic process. First, the rate-limiting enzyme tyrosine hydroxylase (TH) converts tyrosine to di-OH-phenylalanine. Aromatic amino acid decarboxylase (AADC) then converts di-OH-phenylalanine into DA. Here, we provide evidence that VMAT₂ physically and functionally interacts with the enzymes responsible for DA synthesis. In rat striata, TH and AADC co-immunoprecipitate with VMAT₂, whereas in PC 12 cells, TH co-immunoprecipitates with the closely related VMAT₁ and with overexpressed VMAT₂. GST pull-down assays further identified three cytosolic domains of VMAT₂ involved in the interaction with TH and AADC. Furthermore, in vitro binding assays demonstrated that TH directly interacts with VMAT₂. Additionally, using fractionation and immunoisolation approaches, we demonstrate that TH and AADC associate with VMAT₂-containing synaptic vesicles from rat brain. These vesicles exhibited specific TH activity. Finally, the coupling between synthesis and transport of DA into vesicles was impaired in the presence of fragments involved in the VMAT₂/TH/AADC interaction. Taken together, our results indicate that DA synthesis can occur at the synaptic vesicle membrane, where it is physically and functionally coupled to VMAT₂-mediated transport into vesicles.     

Conversion of p-tyrosine to p-tyramine in the isolated perfused rat kidney: modulation by perfusate concentrations of p-tyrosine

We used the isolated perfused rat kidney to evaluate the role of renal decarboxylation of p-tyrosine as the source of urinary p-tyramine. Kidneys were perfused with concentrations of p-tyrosine ranging from 0.02 mM to 2.0 mM. p-Tyramine was measured by a sensitive and specific radioenzymatic assay. An increase in the perfusate concentration of p-tyrosine resulted in a significant increase in p-tyramine production that was blocked by the addition of NSD-1015, an inhibitor of aromatic-1-amino decarboxylase (AADC). We conclude p-tyrosine is the precursor for the renal production of p-tyramine, renal AADC catalyzes the formation of urinary p-tyramine, synthesized p-tyramine is predominantly excreted in the urine, and p-tyramine synthesis is modulated by the arterial delivery of p-tyrosine to the kidney.     

Extracellular cAMP inhibits D1 dopamine receptor expression in CAD catecholaminergic cells via A2a adenosine receptors

The expression of D1 dopamine (DA) receptor gene is regulated during development, aging, and pathophysiology. The extracellular factors and signaling mechanisms that modulate the expression of D1 DA receptor have not been well characterized. Here, we present novel evidence that endogenous D1 DA receptor expression is inhibited by extracellular cAMP in the Cath.A Derived (CAD) catecholaminergic neuronal cell line. CAD cells express the multi-drug resistance protein 5 transporters and secrete cAMP. Addition of exogenous cAMP decreases D1 receptor mRNA and protein greater than fourfold in 24 h. The cAMP-induced decrease of D1 receptor mRNA levels is blocked by cGMP and by 1,3-dipropyl-8-(p-sulfo-phenyl)xanthine, an inhibitor of ecto-phosphodiestrase. Extracellular AMP, a metabolite of cAMP, also independently decreased D1 receptor mRNA levels. Inhibitors of ecto-nucleotidases, alpha,beta-methyleneadenosine 5'-di-phosphate and GMP, completely blocked the decrease of D1 receptor mRNA by extracellular cAMP, but only partially blocked the decrease induced by extracellular AMP. Levamisole, an inhibitor of tissue non-specific alkaline phosphatase, completely blocked the AMP-induced decrease of D1 receptor mRNA. The extracellular cAMP, AMP, and adenosine (ADO)-induced decrease in D1 receptor mRNA expression are mediated by A2a ADO receptor subtype. The results suggest a novel molecular mechanism linking activation of A2a ADO receptors with inhibition of D1 DA receptor expression.     

Role of endothelial AADC in cardiac synthesis of serotonin and nitrates accumulation

Serotonin (5-HT) regulates different cardiac functions by acting directly on cardiomyocytes, fibroblasts and endothelial cells. Today, it is widely accepted that activated platelets represent a major source of 5-HT. In contrast, a supposed production of 5-HT in the heart is still controversial. To address this issue, we investigated the expression and localization of 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC) in the heart. We also evaluated their involvement in cardiac production of 5-HT. TPH1 was weakly expressed in mouse and rat heart and appeared restricted to mast cells. Degranulation of mast cells by compound 48/80 did not modify 5-HT cardiac content in mice. Western blots and immunolabelling experiments showed an abundant expression of AADC in the mouse and rat heart and its co-localization with endothelial cells. Incubation of cardiac homogenate with the AADC substrate (5-hydroxy-L-tryptophan) 5-HTP or intraperitoneal injection of 5-HTP in mice significantly increased cardiac 5-HT. These effects were prevented by the AADC inhibitor benserazide. Finally, 5-HTP administration in mice increased phosphorylation of aortic nitric oxide synthase 3 at Ser (1177) as well as accumulation of nitrates in cardiac tissue. This suggests that the increase in 5-HT production by AADC leads to activation of endothelial and cardiac nitric oxide pathway. These data show that endothelial AADC plays an important role in cardiac synthesis of 5-HT and possibly in 5-HT-dependent regulation of nitric oxide generation.     

Effect of 1-methyl-4-phenylpyridinium ion (MPP+) on catecholamine levels and activity of related enzymes in clonal rat pheochromocytoma PC12h cells

1-Methyl-4-phenylpyridinium ion (MPP+), a metabolite of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, was found to reduce dopamine (DA) level and the activity of enzymes related to its metabolism in clonal rat pheochromocytoma PC12h cells. After 6 days' culture in the presence of 1 mM and 100 microM MPP+, DA content in PC12h cells was reduced markedly, but with MPP+ at concentrations lower than 10 microM, DA levels in the cells did not change. The amounts of 3,4-dihydrophenylacetic acid (DOPAC), a metabolite of DA were reduced markedly in culture medium and in PC12h cells cultured with MPP+ at concentrations higher than 1 microM. MPP+ was found to reduce the enzyme activity of tyrosine hydroxylase (TH), monoamine oxidase (MAO) and aromatic L-aminoacid decarboxylase (AADC). In the presence of MPP+ at concentrations higher than 10 microM, reduction of TH activity in the cells was more pronounced than reduction of cell protein or of the activity of a non-specific enzyme, beta-galactosidase. With 1 mM and 100 microM MPP+, MAO activity was reduced to about 30% of that in control cells. Reduction was observed with MPP+ at concentrations higher than 1 microM. AADC was the most sensitive to MPP+ and its activity was reduced markedly in the cells cultured with 100 nM MPP+. These results indicate that MPP+ inhibits not only the biosynthesis of catecholamines, but also the enzyme participating in their catabolism in cells, and may thus perturb catecholamine levels in the brain.