IV. Determination of aromatic L-amino acid decarboxylase in serum of various animals by high-performance liquid chromatography with electrochemical detection

This paper describes the distribution of aromatic L-amino acid decarboxylase (AADC), using both L-DOPA and L-5-hydroxytryptophan (L-5-HTP) as substrates, in serum of various animals. The ratio of the activities of the enzyme towards both substrates was also determined in the same serum. AADC activity was discovered in serum using our new and highly sensitive assay for AADC activity by high-performance liquid chromatography (HPLC) with electrochemical detection (ED) with L-DOPA and L-5-HTP as substrates. It was found that among the species used, guinea pig serum had the highest activity, and we made a systematic study on guinea pig serum AADC.     

Lack of regulation of aromatic L-amino acid decarboxylase in intact bovine chromaffin cells

Aromatic l-amino acid decarboxylase (AADC) is the second enzyme in the catecholamine biosynthetic pathway, and its activity is generally considered not to be limiting, and therefore not involved, in regulating flux through this pathway. Recent studies showing that its activity can be regulated in vivo and that the enzyme can be phosphorylated and activated in vitro have raised the possibility that AADC may play more than an obligatory role in catecholamine biosynthesis. In the present study, the phosphorylation and activity of AADC was evaluated relative to that of tyrosine hydroxylase (TH; the first and rate-limiting enzyme in the pathway) in intact bovine chromaffin cells. Treatment of chromaffin cells with elevated potassium, acetylcholine, phorbol dibutyrate, forskolin, or okadaic acid each increased 32P incorporation into TH (after metabolic labeling of ATP pools with 32P(i)) and TH activity. In contrast, as measured in matched samples, 32P incorporation into AADC was not detected and none of the treatments altered AADC activity. Thus, that AADC can be phosphorylated and activated in vitro has questionable physiological significance.     

Regulation of Aromatic l-Amino Acid Decarboxylase by Dopamine Receptors in the Rat Brain

Decarboxylation of phenylalanine by aromatic L-amino acid decarboxylase (AADC) is the rate-limiting step in the synthesis of 2-phenylethylamine (PE), a putative modulator of dopamine transmission. Because neuroleptics increase the rate of accumulation of striatal PE, these studies were performed to determine whether this effect may be mediated by a change in AADC activity. Administration of the D1 antagonist SCH 23390 at doses of 0.01-1 mg/kg significantly increased rat striatal AADC activity in an in vitro assay (by 16-33%). Pimozide, a D2-receptor antagonist, when given at doses of 0.01-3 mg/kg, also increased AADC activity in the rat striatum (by 25-41%). In addition, pimozide at doses of 0.3 and 1 mg/kg increased AADC activity in the nucleus accumbens (by 33% and 45%) and at doses of 0.1, 0.3, and 1 mg/kg increased AADC activity in the olfactory tubercles (by 23%, 30%, and 28%, respectively). Analysis of the enzyme kinetics indicated that the Vmax increased with little change in the Km with L-3,4-dihydroxyphenylalanine as substrate. The AADC activity in the striatum showed a time-dependent response after the administration of SCH 23390 and pimozide: the activity was increased within 30 min and the increases lasted 2-4 h. Inhibition of protein synthesis by cycloheximide (10 mg/kg, 0.5 h) had no effect on the striatal AADC activity or on the increases in striatal AADC activity produced by pimozide or SCH 23390. The results indicate that the increases in AADC activity induced by dopamine-receptor blockers are not due to de novo synthesis of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of endogenous inhibitor of aromatic L-amino acid decarboxylase in monkey serum

Summary In the course of our studies on the developmental changes of aromatic L-amino acid decarboxylase (AADC) in the serum of Japanese monkeys (Macaca fuscata fuscata), we found the presence of an endogenous inhibitor of AADC in all stages of monkey life. This inhibitor inhibited the serum enzyme activity completely with L-5-hydroxytryptophan (L-5-HTP) as substrate, while the activity was partially inhibited with L-DOPA as substrate. The inhibitor was non-dialyzable, but it could be removed from the monkey serum by DEAE-Sephacel chromatography. After this treatment AADC activities could be detected in the monkey serum by using both L-DOPA and L-5-HTP as substrates. Moreover, the total activity for L-DOPA was augmented by 3-fold in the serum after the removal of the inhibitor. Serum AADC was partially purified from monkey and compared with that of rat using both L-DOPA and L-5-HTP as substrates, but the ratio of the activities for the two substrates did not change significantly in each fraction during purification from either monkey or rat serum.On leave from the University of Rajshahi, Rajshahi, Bangladesh.     

Alpha-synuclein inhibits aromatic amino acid decarboxylase activity in dopaminergic cells

Alpha-synuclein is a presynaptic protein strongly implicated in Parkinson's disease (PD). Because dopamine neurons are invariably compromised during pathogenesis in PD, we have been exploring the functions of alpha-synuclein with particular relevance to dopaminergic neuronal cells. We previously discovered reduced tyrosine hydroxylase (TH) activity and minimal dopamine synthesis in stably-transfected MN9D cells overexpressing either wild-type or A53T mutant (alanine to threonine at amino acid 53) alpha-synuclein. TH, the rate-limiting enzyme in dopamine synthesis, converts tyrosine to l-dihydroxyphenylalanine (L-DOPA), which is then converted to dopamine by the enzyme, aromatic amino acid decarboxylase (AADC). We confirmed an interaction between alpha-synuclein and AADC in striatum. We then sought to determine whether wild-type or A53T mutant alpha-synuclein might have affected AADC activity in dopaminergic cells. Using HPLC with electrochemical detection, we measured dopamine and related catechols after L-DOPA treatments to bypass the TH step. We discovered that while alpha-synuclein did not reduce AADC protein levels, it significantly reduced AADC activity and phosphorylation in our cells. These novel findings further support a role for alpha-synuclein in dopamine homeostasis and may explain, at least in part, the selective vulnerability of dopamine neurons that occurs in PD.     

New and highly sensitive assay for l-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography—voltammetry

This paper describes a new, inexpensive and highly sensitive assay for aromatic l-amino acid decarboxylase (AADC) activity, using l-5-hydroxytryptophan (l-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. l-5-HTP was used as substrate and d-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from l-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using l-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.     

New and highly sensitive assay for l-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography—voltammetry

This paper describes a new, inexpensive and highly sensitive assay for aromatic l-amino acid decarboxylase (AADC) activity, using l-5-hydroxytryptophan (l-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. l-5-HTP was used as substrate and d-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from l-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using l-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.     

Parallel Up-Regulation of Catecholamine Biosynthetic Enzymes by Dexamethasone in PC12 Cells

Abstract: We sought to investigate whether dexamethasone produces a coordinated, time-dependent effect on all enzymes in the catecholamine biosynthetic pathway in PC12 cells. The levels of mRNAs of tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and dopamine γ-hydroxylase (DBH) were examined at 0, 6, 12, 24, and 48 h after dexamethasone (5 μ M ) treatment to PC12 cells. The levels of all enzyme mRNAs steadily increased for 24 h, although the increase of AADC mRNA content was slow. The increased mRNA levels of TH and AADC were maintained at 48 h, whereas the level of DBH mRNA was sharply decreased at 48 h. The maximally induced mRNA levels were ∼5.0-, 2.4-, and 7.0-fold higher than the control levels of TH, AADC, and DBH, respectively. The elevation of enzyme activities was detected later than the increase in levels of mRNAs. The maximal activities of TH, AADC, and DBH were reached between 48 and 72 h with 3.6-, 1.8-, and 8.0-fold increases, respectively. Low, but detectable, phenylethanolamine N -methyltransferase (PNMT) activity was observed in PC12 cells, and dexamethasone increased its activity 5.6-fold at 72 h. The PNMT mRNA was easily detected by northern blot analysis after exposure for 24 h to dexamethasone. The data suggest that, in PC12 cells, dexamethasone up-regulates all catecholamine biosynthetic enzyme genes in a parallel fashion.     

酪氨酸羟化酶和左旋芳香族氨基酸脱羧酶基因的细胞表达及活性检测

由于在帕金森病中合成多巴胺所需的酪氨酸羟化酶(tyrosine hydroxylase,TH)和左旋芳香族氨基酸脱羧酶(aromatic L-amino acid decarboxylase,AADC)活性明显降低,所以补充多巴胺合成酶成为基因治疗帕金森病研究的主要手段。我们分别构建了重组逆转录病毒载体pLHCX／TH及pLNCX2／AADC,通过脂质体介导将带有目的基因的载体分别转到包装细胞PA317中,经筛选得到产病毒的细胞PA317／TH和PA317／AADC,采用免疫组化、原位杂交方法检测目的基因表达;细胞经裂解后进行的酶促反应产物多巴胺以高压液相电化学方法检测证明所克隆的T‘H及AADC基因具有功能活性;这两种基因工程改造细胞可以完成酶促动力学的功能,使L-dopa及多巴胺产生明显增加。本研究为用TH和AADC双基因对帕金森病进行基因治疗提供了一定的依据。     

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.     

Effects of glutamate antagonists on the activity of aromatic L-amino acid decarboxylase

Summary This study examines the hypothesis that glutamate tonically suppresses the activity of the enzyme aromatic L-amino acid decarboxylase (AADC), and hence the biosynthesis of dopamine, to explain how antagonists of glutamate receptors might potentiale the motor actions of L-DOPA in animal models of Parkinson's disease. A variety of glutamate antagonists were therefore administered acutely to normal rats, which were sacrificed 30–60 min later and AADC activity assayed in the substantia nigra pars reticulate (SNr) and corpus striatum (CS). The NMDA receptor-ion channel antagonists MK 801, budipine, amantadine, memantine and dextromethorphan all caused a pronounced in creased in AADC activity, more especially in the SNr than CS. The NMDA glycine site antagonist (R)-HA 966 produced a modest increase in AADC activity in the CS but not SNr, whilst the NMDA polyamine site antagonist eliprodil, the NMDA competitive antagonist CGP 40116 and the AMPA antagonist NBQX were without effect. The results suggest that an increase in dopamine synthesis might contribute to the L-DOPA-facilitating actions of some glutamate antagonists.     

Analysis of the Alternative Promoters that Regulate Tissue-Specific Expression of Human Aromatic l-Amino Acid Decarboxylase

Abstract: Previously we identified two alternative first exons (exon N1 and exon L1) coding for 5' untranslated regions of human aromatic l -amino acid decarboxylase (AADC) and found that their alternative usage produced two types of mRNAs in a tissue-specific manner. To determine the cis -acting element regulating the tissue-specific expression of human AADC, we produced three kinds of transgenic mice harboring 5' flanking regions of the human AADC gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The transgene termed ACA contained −7.0 kb to −30 bp in exon N1, including the entire exon L1; ACN contained −3.6 kb to −30 bp in exon N1; and ACL contained −2.8 kb to −42 bp in exon L1. The ACA transgenic mice expressed CAT at extremely high levels in peripheral nonneuronal tissues, such as pancreas, liver, kidney, small intestine, and colon, that contained endogenous high AADC activity, whereas CAT immunoreactivity was not detected in either catecholaminergic or serotonergic neurons in the CNS. Thus, it was suggested that the ACA transgene contained the major part of cis -regulatory elements for the expression of AADC in peripheral nonneuronal tissues. On the other hand, the ACN transgenic mice moderately expressed CAT in various tissues except for the lung and liver, and the ACL transgenic mice showed moderate CAT expression only in the kidney.     

Identification of the aromatic L-amino acid decarboxylase (AADC) gene and its expression in the attachment and metamorphosis of the barnacle, Balanus amphitrite

Serotonin and dopamine are involved in the attachment and metamorphosis of cypris larvae of barnacles. Aromatic L-amino acid decarboxylase (AADC) gene, the product of which catalyzes the synthesis of serotonin and dopamine from L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine, respectively, was characterized. A DNA clone containing part of an AADC sequence was obtained from the genomic DNA library of the barnacle, Balanus amphitrite. This clone had four putative exons consisting of 226 amino acids with an identity of 63.2% and a similarity of 92.1% with human AADC. Northern blot analysis showed that AADC mRNA was expressed at all stages of barnacles: naupliar larvae, cypris larvae and adult barnacles. Two inducers of larval attachment and metamorphosis; that is, serotonin and extract of adult barnacles, obviously increased the expression of AADC mRNA at an early cypris larval stage. These results suggest that intracellular biosynthesis of serotonin, or dopamine, or both is at least partly involved in the control of the attachment and metamorphosis of cypris larvae.     

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.     

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.