L-amino Acid Decarboxylase Research Articles

Unraveling the Molecular Determinants of Catalytic Efficiency and Substrate Specificity in l-Amino Acid Decarboxylases.

l-Tryptophan decarboxylase (TDC) and l-3,4-dihydroxyphenylalanine decarboxylase (DDC) catalyze the decarboxylation of l-tryptophan, 5-hydroxytryptophan, and l-3,4-dihydroxyphenylalanine. In this study, we analyzed the amino acid compositions of the substrate-binding pockets of TDC from Catharanthus roseus (CrTDC) and DDC from Sus scrofa (SsDDC), explored the specificity of key amino acids within these pockets, and elucidated mechanisms influencing substrate selectivity and catalytic activity in both enzymes, using whole-cell catalysis to screen mutants and determine enzymatic kinetic parameters. The results demonstrated that residues Ala-103 and Val-122 in CrTDC, along with their corresponding sites Thr-82 and Ile-101 in SsDDC, significantly influence substrate selectivity and catalytic efficiency. Molecular dynamics simulations revealed that substrate selectivity and catalytic efficiency depends on the nucleophilic attack distance between the substrate's amino group and the C4' of pyridoxal 5'-phosphate. This study elucidates the catalytic mechanisms and structural bases of TDC and DDC, guiding enhancements in the related aromatic monoamine biosynthesis.

Gene Therapy for Parkinson's Disease Using Midbrain Developmental Genes to Regulate Dopaminergic Neuronal Maintenance.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder. It is characterized by the progressive loss of dopaminergic (DAnergic) neurons in the substantia nigra and decreased dopamine (DA) levels, which lead to both motor and non-motor symptoms. Conventional PD treatments aim to alleviate symptoms, but do not delay disease progression. PD gene therapy offers a promising approach to improving current treatments, with the potential to alleviate significant PD symptoms and cause fewer adverse effects than conventional therapies. DA replacement approaches and DA enzyme expression do not slow disease progression. However, DA replacement gene therapies, such as adeno-associated virus (AAV)-glutamic acid decarboxylase (GAD) and L-amino acid decarboxylase (AADC) gene therapies, which increase DA transmitter levels, have been demonstrated to be safe and efficient in early-phase clinical trials. Disease-modifying strategies, which aim to slow disease progression, appear to be potent. These include therapies targeting downstream pathways, neurotrophic factors, and midbrain DAnergic neuronal factors, all of which have shown potential in preclinical and clinical trials. These approaches focus on maintaining the integrity of DAnergic neurons, not just targeting the DA transmitter level itself. In particular, critical midbrain developmental and maintenance factors, such as Nurr1 and Foxa2, can interact synergistically with neighboring glia, in a paracrine mode of action, to protect DAnergic neurons against various toxic factors. Similar outcomes could be achieved by targeting both DAnergic neurons and glial cells with other candidate gene therapies, but in-depth research is needed. Neurotrophic factors, such as neurturin, the glial-cell-line-derived neurotrophic factor (GDNF), the brain-derived neurotrophic factor (BDNF), and the vascular endothelial growth factor (VEGF), are also being investigated for their potential to support DAnergic neuron survival. Additionally, gene therapies targeting key downstream pathways, such as the autophagy-lysosome pathway, mitochondrial function, and endoplasmic reticulum (ER) stress, offer promising avenues. Gene editing and delivery techniques continue to evolve, presenting new opportunities to develop effective gene therapies for PD.

Expression Analysis of Serotonin Receptors, Serotonin Transporter and l-Amino Acid Decarboxylase in the Mouse Sphenopalatine Ganglion by RT-PCR, Northern Blot Analysis and In Situ Hybridization

The sphenopalatine ganglion (SPG) is a gathering of the cell bodies of parasympathetic fibers that dominate the nasal gland, lacrimal gland and cerebral blood vessels. The SPG controls nasal secretions, tears, and the dilation of cerebral blood vessels. However, it is unclear how serotonin regulates SPG functions. In this study, we investigated the expression of genes involved in the serotonergic system in the mouse SPG. We examined the mRNA expression levels of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3A, 5-HT3B, 5-HT4, 5-HT5A, 5-HT5B, 5-HT6 and 5-HT7 receptors, as well as serotonin transporter, tryptophan hydroxylases 1 and 2, and L-amino acid decarboxylase (AADC) by RT-PCR. It revealed that the 5-HT3A and 5-HT3B ionotropic receptors and AADC were likely to be highly expressed in the SPG, as measured by RT-PCR. We next performed in situ hybridization on the SPG to examine the expression of these three genes at the cellular level after validating the specificity of each cRNA probe by northern blotting. The 5-HT3A receptor, 5-HT3B receptor, and AADC were expressed in 96.5% ± 1.0%, 29.7% ± 10.7%, and 57.4% ± 2.9% of neuronal cell bodies in the SPG, respectively, indicating that the 5-HT3A receptor was virtually expressed in all SPG neurons. Our results on the expression of these critical serotonin system genes in the parasympathetic SPG provide insight into the pathogenetics of rhinitis, conjunctivitis and headache. Furthermore, our findings suggest that targeting the 5-HT3A receptor might have therapeutic potential in the treatment of these ailments.

Catecholamine Disorders and Hereditary Sensory and Autonomic Neuropathy

Hereditary disorders affecting catecholamine metabolism or development of sensory and autonomic neurons manifest with a wide range of autonomic disturbance. Genetic catecholamine disorders include deficiencies of tyrosine hydroxylase (TH), tetrahydrobiopterin (BH4), L-amino acid decarboxylase (L-ADCC), dopamine-beta-hydroxylase (DBH), vesicula monoamine transporter (VMAT) 2, and norepinephrine transporter (NET). BH4, L-ADCC and VMAT2 deficiency have similar manifestations; autonomic symptoms include hypersalivation, temperature instability, and pupil abnormalities, associated with various motor (eg, dystonia) and behavioral abnormalities. DBH deficiency manifests neonatally with hypotension, hypothermia, hypoglycemia, vomiting and dehydration; orthostatic hypotension is the major manifestation in childhood and later. NET deficiency produces orthostatic intolerance with postural tachycardia. Hereditary sensory and autonomic neuropathies (HSANs) are a heterogenous group of disorders that present with sensory loss and variable degrees of motor and autonomic involvement. Autosomal dominant HSANs include HSAN-1 (due SPTLC1 mutations), Charcot-Marie-Tooth (CMT) disease 2B (due to Rab7 mutations), and HSAN-1 with dementia and hearing loss (linked to DNTM1 mutations). HSAN-1 has a juvenile-to-adult onset sensory neuropathy with minor autonomic involvement; CMT2B has prominent motor manifestations. Autosomal recesssive forms include HSAN-II (linked to mutations in the WNK1, FAM134B, or KIF1A genes), HSAN-III (linked to IKBKAP mutation), HSAN-IV (linked to NTRK1 mutations), HSAN-V (linked to NGFB mutations), and HSAN with spastic paraplegia (linked to CCT5 mutations). Autosomal recessive HSANs have onset at birth or in early childhood and have prominent autonomic symptoms. In particular, HSAN-III (familial dysautonomia or Riley-Day syndrome) is characterized by severe small fiber sensory loss and life-threatening autonomic dysfunction. Prominent manifestations include absence of cold and pain sensation, temperature instability (hypothermia and excessive perspiration), blood pressure lability (due to impaired baroreflex afferent function), abnormal swallowing, vomiting crises, and recurrent bouts of aspiration pneumonia. HSAN-IV and HSN-V are characterized by congenital insensitivity to pain and reduced or absent sweating.

The role of the serotonergic system in suicidal behavior

Serotonin is a widely investigated neurotransmitter in several psychopathologies, including suicidal behavior (SB); however, its role extends to several physiological functions involving the nervous system, as well as the gastrointestinal and cardiovascular systems. This review summarizes recent research into ten serotonergic genes related to SB. These genes – TPH1, TPH2, SLC6A4, SLC18A2, HTR1A, HTR1B, HTR2A, DDC, MAOA, and MAOB – encode proteins that are vital to serotonergic function: tryptophan hydroxylase; the serotonin transporter 5-HTT; the vesicular transporter VMAT2; the HTR1A, HTR1B, and HTR2A receptors; the L-amino acid decarboxylase; and the monoamine oxidases. This review employed a systematic search strategy and a narrative research methodology to disseminate the current literature investigating the link between SB and serotonin.

Monoamine Oxidase A Down-Regulation Contributes to High Metanephrine Concentration in Pheochromocytoma

The high diagnostic performance of plasma-free metanephrines (metanephrine and normetanephrine) (MN) for pheochromocytoma (PHEO) results from the tumoral expression of catechol-O-methyltransferase (COMT), the enzyme involved in O-methylation of catecholamines (CAT). Intriguingly, metanephrine, in contrast to epinephrine, is not remarkably secreted during a stress in hypertensive or normotensive subjects, whereas in PHEO patients CAT and MN are both raised to high levels. Because epinephrine and metanephrine are almost exclusively produced by the adrenal medulla, this suggests distinct CAT metabolism in chromaffin cells and pheochromocytes. The objective of the study was to compare CAT metabolism between adrenal medulla and PHEO tissue regarding related enzyme expression including monoamine oxidases (MAO) and COMT. A multicenter comparative study was conducted. The study included 21 patients with a histologically confirmed PHEO and eight adrenal glands as control. CAT, dihydroxyphenol-glycol, 3,4-dihydroxyphenylacetic acid, and MN were measured in adrenal medulla and PHEO tissue. Western blot, quantitative RT-PCR and immunofluorescence studies for MAOA, MAOB, tyrosine hydroxylase, dopamine β-hydroxylase, L-amino acid decarboxylase, and COMT were applied on tissue homogenates and cell preparations. At both the protein and mRNA levels, MAOA and COMT are detected less often in PHEO compared with adrenal medulla, conversely to tyrosine hydroxylase, L-amino acid decarboxylase, and dopamine β-hydroxylase, much more expressed in tumor tissue. MAOB protein is detected less often in tumor but not differently expressed at the mRNA level. Dihydroxyphenol-glycol is virtually absent from tumor, whereas MN, produced by COMT, rises to 4.6-fold compared with adrenal medulla tissue. MAOA down-regulation was observed in 100% of tumors studied, irrespectively of genetic alteration identified; on the other hand, MAOA was strongly expressed in all adrenal medulla collected independently of age, gender, or late sympathetic activation of the deceased donor. High concentrations of MN in tumor do not only arise from CAT overproduction but also from low MAOA expression, resulting in higher substrate availability for COMT.

L-Amino acid decarboxylase- and tyrosine hydroxylase-immunoreactive cells in the extended olfactory amygdala and elsewhere in the adult prairie vole brain

Neurons synthesizing dopamine (DA) are widely distributed in the brain and implicated in a tremendous number of physiological and behavioral functions, including socioreproductive behaviors in rodents. We have recently been investigating the possible involvement of sex- and species-specific TH-immunoreactive (TH-ir) cells in the male prairie vole (Microtus ochrogaster) principal bed nucleus of the stria terminalis (pBST) and posterodorsal medial amygdala (MeApd) in the chemosensory control of their monogamous pairbonding and parenting behaviors. These TH-ir cells are not immunoreactive for dopamine-beta-hydroxylase (DBH), suggesting they are not noradrenergic but possibly DAergic. A DAergic phenotype would require them to contain aromatic l-amino acid decarboxylase (AADC) and here we examined the existence of cells immunoreactive for both TH and AADC in the pBST and MeApd of adult virgin male and female prairie voles. We also investigated the presence of TH/AADC cells in the anteroventral periventricular nucleus (AVPV), medial preoptic area (MPO), arcuate nucleus (ARH), zona incerta (ZI), substantia nigra (SN) and ventral tegmental area (VTA). Among our findings were: (1) the pBST and MeApd each contained completely non-overlapping distributions of TH-ir and AADC-ir cells, (2) the AVPV contained surprisingly few AADC-ir cells and almost no TH-ir cells contained AADC-ir, (3) approximately 60% of the TH-ir cells in the MPO, ARH, and ZI also contained AADC-ir, (4) unexpectedly, only about half of TH-ir cells in the SN and VTA contained AADC-ir, and (5) notable populations of AADC-ir cells were found outside traditional monoamine-synthesizing regions, including some sites that do not contain AADC-ir cells in adult laboratory rats or cats (medial septum and cerebral cortex). In the absence of the chemical requirements to produce DA, monoenzymatic TH-ir cells in the virgin adult prairie vole pBST, MeApd, and elsewhere in their brain may instead produce l-DOPA as an end product and use it as a neurotransmitter or neuromodulator, similar to what has been observed for monoenzymatic TH-synthesizing cells in the laboratory rat brain.

Opposite sexual dimorphism of 3,4-dihydroxyphenylalanine decarboxylase in the kidney and small intestine of mice

3,4-Dihydroxyphenylalanine decarboxylase (DDC; also known as l-amino acid decarboxylase) is involved in the synthesis of dopamine, norepinephrine, and serotonin, and also acts as an androgen receptor co-regulator protein. In contrast to other amino acid decarboxylases that are modulated by sex hormones, little is known about the influence of these hormones on DDC regulation. In the present work, we studied the influence of gender in the expression of DDC in different mouse tissues. Among the different organs studied, including brain, liver, kidney, intestine, heart, adrenal gland, and skeletal muscle, only kidney and small intestine showed a sex-dependent dimorphism in DDC expression. In the kidney, levels of DDC activity, DDC mRNA, and protein were remarkably higher in females than in males. On the contrary, in the small intestine, male mice displayed higher levels of DDC activity than females but they did not correlate precisely with mRNA levels. This dimorphism was dependent on androgens, since male castration and treatment of female mice with testosterone propionate, oppositely affected DDC levels in kidney and small intestine. However, estrogen ablation or treatment with estradiol did not significantly affect DDC activity in these tissues. Immunocytochemical analysis revealed that DDC was mainly located in the proximal straight tubular cells of the kidney and in the cytoplasm of enterocytes. These data and the fact that renal DDC inversely correlated with renal sodium reabsorption suggest that renal and intestinal gender dimorphism in DDC could be related to sex-related differences in sodium balance observed between males and females.

Effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Catalpalactone at 5–30 μM decreased intracellular dopamine content with the IC 50 value of 22.1 μM. Catalpalactone at 5–20 μM, but not 30 μM, did not alter cell viability. Catalpalactone at 20 μM inhibited tyrosine hydroxylase (TH) and aromatic- l-amino acid decarboxylase (AADC) activities. Catalpalactone also decreased cyclic AMP levels and inhibited TH phosphorylation. In addition, catalpalactone at 20 μM reduced the increases in dopamine levels induced by L-DOPA (20–50 μM). Catalpalactone (5–30 μM) associated with L-DOPA (50–100 μM) enhanced L-DOPA-induced cytotoxicity at 48 h, which was prevented by N-acetyl- l-cysteine. These results suggest that catalpalactone inhibited dopamine biosynthesis by reducing TH and AADC activities and enhanced L-DOPA-induced cytotoxiciy in PC12 cells.

Haplotype analysis indicates an association between the DOPA decarboxylase (DDC) gene and nicotine dependence

DOPA decarboxylase (DDC; also known as L-amino acid decarboxylase; AADC) is involved in the synthesis of dopamine, norepinephrine and serotonin. Because the mesolimbic dopaminergic system is implicated in the reinforcing effects of many drugs, including nicotine, the DDC gene is considered a plausible candidate for involvement in the development of vulnerability to nicotine dependence (ND). Further, this gene is located within the 7p11 region that showed a 'suggestive linkage' to ND in our previous genome-wide scan in the Framingham Heart Study population. In the present study, we tested eight single nucleotide polymorphisms (SNPs) within DDC for association with ND, which was assessed by smoking quantity (SQ), the heaviness of smoking index (HSI) and the Fagerstrom test for ND (FTND) score, in a total of 2037 smokers and non-smokers from 602 nuclear families of African- or European-American (AA or EA, respectively) ancestry. Association analysis for individual SNPs using the PBAT-GEE program indicated that SNP rs921451 was significantly associated with two of the three adjusted ND measures in the EA sample (P=0.01-0.04). Haplotype-based association analysis revealed a protective T-G-T-G haplotype for rs921451-rs3735273-rs1451371-rs2060762 in the AA sample, which was significantly associated with all three adjusted ND measures after correction for multiple testing (min Z=-2.78, P=0.006 for HSI). In contrast, we found a high-risk T-G-T-G haplotype for a different SNP combination in the EA sample, rs921451-rs3735273-rs1451371-rs3757472, which showed a significant association after Bonferroni correction with the SQ and FTND score (max Z=2.73, P=0.005 for FTND). In summary, our findings provide the first evidence for the involvement of DDC in the susceptibility to ND and, further, reveal the racial specificity of its impact.

The ability of grafted human sympathetic neurons to synthesize and store dopamine: a potential mechanism for the clinical effect of sympathetic neuron autografts in patients with Parkinson's disease

We have investigated the potential of autologous sympathetic neurons as a donor for cell therapy of Parkinson's disease (PD). Our recent study demonstrated that sympathetic neuron autografts increase the duration of levodopa-induced “on” periods with consequent reduction in the percent time spent in “off” phase. We also found that human sympathetic neurons grown in culture have the ability to convert exogenous levodopa to dopamine and to store the synthesized dopamine. This may explain the clinically observed prolongation in the duration of levodopa effects. To further analyze the mechanism for the graft-mediated effect, the present study investigated the metabolic function of human sympathetic ganglionic neurons xenografted into the dopamine (DA)-denervated striatum of rats by monitoring striatal levels of DA and its primary metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), after systemic administration of levodopa. We also explored whether the graft-mediated effect above may last in four PD patients who had been given the grafts and followed for 12–36 months postgrafting. Clinical evaluations showed that an increase in the duration of levodopa-induced “on” phase is detected during a follow-up period of 12–36 months postgrafting in all the four patients tested. Accordingly, the percent time spent in “off” phase exhibited a 30–40% reduction as compared to the pregrafting values. The animal experiment showed that a significant increase in striatal DA levels is noted after systemic levodopa treatment, and that the DA levels remain high for longer periods of time in the grafted rats than in control animals. When given reserpine pretreatment, the levodopa-induced rise of striatal DA levels was significantly attenuated with concomitant increase in DOPAC levels. Histological examinations demonstrated that the grafts contain some tyrosine hydroxylase (TH)-positive cells. These cells were also found to express aromatic- l-amino acid decarboxylase (AADC) and vesicular monoamine transporter-2 (VMAT), both of which are important molecules for the synthesis and the storage of DA, respectively. These results indicate that grafted sympathetic neurons can provide a site for both the conversion of exogenous levodopa to DA and the storage of the synthesized DA in the DA-denervated striatum, explaining a mechanism by which sympathetic neuron autografts can increase the duration of levodopa-induced “on” phase in PD patients.

Chronic administration with nandrolone decanoate induces alterations in the gene-transcript content of dopamine D 1- and D 2-receptors in the rat brain

Some adolescent and young males are engaged in misuse of anabolic–androgenic steroids (AASs) in connection with multiple drug use, in order to become intoxicated and brave, apart from currently known motives connected to sports performance and physical appearance. Recent studies suggest that alterations in neurobiological circuits implicated in the regulation of reward-related learning, aggression and motoric behavior underlie the behavioral changes associated with AAS misuse. We have previously shown that AASs induce alterations in dopamine receptor densities. The aim of the present study was to investigate if these effects could be attributed to altered mRNA content for tyrosine hydroxylase, l-amino acid decarboxylase, dopamine D 1- and dopamine D 2-receptor as measured by in situ hybridisation. Male Sprague–Dawley rats were subjected to 2 weeks of treatment with daily intramuscular injections of the AAS nandrolone decanoate at three different doses (1, 5 and 15 mg/kg/day). Results of the in situ hybridization showed that the mRNA content of the dopamine D 1-receptor subtype was significantly reduced at all doses in the caudate putamen and at the highest doses in the nucleus accumbens shell. The mRNA expression of the dopamine D 2-receptor was significantly increased at the two lowest doses in the caudate putamen and the nucleus accumbens shell. In conclusion, nandrolone has been shown to affect the expression of gene transcripts of dopaminergic receptors possibly implicated in underlying mechanisms of reward-related behavioral changes among AAS misusers.

Spectroscopic analysis of recombinant rat histidine decarboxylase.

Mammalian histidine decarboxylases have not been characterized well owing to their low amounts in tissues and instability. We describe here the first spectroscopic characterization of a mammalian histidine decarboxylase, i.e. a recombinant version of the rat enzyme purified from transformed Escherichia coli cultures, with similar kinetic constants to those reported for mammalian histidine decarboxylases purified from native sources. We analyzed the absorption, fluorescence and circular dichroism spectra of the enzyme and its complexes with the substrate and substrate analogues. The pyridoxal-5'-phosphate-enzyme internal Schiff base is mainly in an enolimine tautomeric form, suggesting an apolar environment around the coenzyme. Michaelis complex formation leads to a polarized, ketoenamine form of the Schiff base. After transaldimination, the coenzyme-substrate Schiff base exists mainly as an unprotonated aldimine, like that observed for dopa decarboxylase. However, the coenzyme-substrate Schiff base suffers greater torsion than that observed in other L-amino acid decarboxylases, which may explain the relatively low catalytic efficiency of this enzyme. The active center is more resistant to the formation of substituted aldamines than the prokaryotic homologous enzyme and other L-amino acid decarboxylases. Characterization of the similarities and differences of mammalian histidine decarboxylase with respect to other homologous enzymes would open new perspectives for the development of new and more specific inhibitors with pharmacological potential.

Expression of tyrosine hydroxylase in magnocellular hypothalamic neurons of obese ( fa/fa) and lean heterozygous ( Fa/fa) Zucker rats

In the magnocellular hypothalamic neurons (MCN) of normal rats, tyrosine hydroxylase (TH) is expressed in response to hyperosmotic stimulation and co-exists with vasopressin. The present study shows that both Zucker obese ( fa/fa) and heterozygous lean ( Fa/fa) rats express TH in MCN independently of an osmotic challenge. The lack of L-DOPA and aromatic- L-aminoacid decarboxylase in the MCN showed the absence of mechanisms necessary for catecholamine synthesis in these cells. Therefore, TH in MCN seems to be functionally inactive and is not involved in catecholamine abnormalities observed in these rats. All TH-immunoreactive MCN co-expressed vasopressin mRNA while a part of them co-expressed oxytocin mRNA. This suggests a mechanism of regulation of TH expression in MCN which is different in Zucker rats and in dehydrated normal rats.

Elevated aromatic- l-amino acid decarboxylase in human carcinoid tumors

The carcinoid neoplasm is marked by excessive serotonin, synthesized by the conversion of tryptophan (Trp) to 5-hydroxytryptophan by tryptophan hydroxylase (TPH) (EC 1.14.16.4) and decarboxylation of 5-hydroxytryptophan by aromatic- l-amino acid decarboxylase (AAAD) (EC 4.1.1.28). Because almost no biochemical data were available on human carcinoid TPH and AAAD, we have characterized these enzymes as a preliminary step to developing mechanism-based agents selective against carcinoid tumors. TPH was detected in all fourteen Carcinoids analyzed [ K m = 185 ± 17 μM (mean ± SEM); V max = 2.4 ± 1.2 nmol/hr/mg protein]. AAAD was detected in thirteen tumors ( K m = 45 ± 6.7 μM; V max = 11 ± 2.0 nmol/min/mg protein). In a subset of hepatic metastatic tumors obtained with adjacent normal liver, the K m and V max of TPH (N = 6) and the K m of AAAD (N = 7) were comparable in both tissues. However, the V max of carcinoid AAAD was 50-fold higher ( P < 0.002) than that in normal liver (13 ± 3.1 vs 0.26 ± 0.04 nmol/min/mg protein). Western immunoblot analysis indicated that AAAD polypeptide content of carcinoid tumor was > 20-fold higher than in adjacent normal liver. These results suggest that AAAD might be an appropriate target for enzyme-activated cytotoxic agents for carcinoid tumors.

Defective 3,4-dihydroxyphenylalanine decarboxylation to dopamine in hydralazine-treated hypertensive patients may be pyridoxine remediable.

The previously observed defective dopamine (DA) generation from 3,4-dihydroxyphenylalanine (DOPA) can also be seen in patients treated for many years by hydralazine. This may be due to a hydralazine-induced depletion of pyridoxine, an essential coenzyme of the aromatic L-amino acid decarboxylase (LAAD). Eleven hydralazine-treated stable essential hypertensive (EH) patients, initially found to have a defect in the DOPA decarboxylation to DA, tested by a single DOPA administration (500 mg, orally), were retested by the same test 4 days after pyridoxine pretreatment (100 mg/day) for data on blood pressure (BP), pulse rate, and renal and plasma catecholamines and their metabolites, as well as plasma atrial natriuretic factor (ANF), cyclic GMP (cGMP), plasma renin activity (PRA), and plasma aldosterone (PA). Initially, hydralazine-treated stable EH patients manifested, following DOPA administration, lower DOPA decarboxylation to DA than control subjects. Pyridoxine pretreatment accelerated DA generation from exogenous DOPA and attenuated the DOPA-induced increases in plasma and urinary DOPA and its metabolite 3-O-methyl-DOPA, but accentuated the increase in free DA and its main metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), while BP, ANF, cGMP, PRA, and PA remained unaffected. The DOPA-induced increments of urinary DA were, in contrast to plasma DA changes, blunted by pyridoxine pretreatment. The attenuation of the sodium excretion by pyridoxine pretreatment exceeded that of the DA excretion, suggesting that pyridoxine suppressed a natriuretic factor, other than ANF, or activated a sodium-retaining factor, other than renin or aldosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of high salt intake on dopamine production in rat kidney

To examine the origin of increased urinary dopamine excretion (UDAV) during high salt intake, we measured UDAV from the innervated (INN) or the chronically denervated (DEN) kidney in rats fed either a high-salt (HS) or low-salt (LS) diet. UDAV of DEN [3.50 +/- 0.46 ng. min-1.inulin clearance (CIN)-1] and INN (4.00 +/- 0.59 ng. min-1.CIN-1) kidneys in the HS group showed a significant increase compared with that of the respective kidney in the LS group (DEN 1.42 +/- 0.12, INN 1.44 +/- 0.09 ng.min-1.CIN-1), whereas the effect of denervation on UDAV was not significantly different between two groups. We determined aromatic L-amino acid decarboxylase (L-AADC) activity and dopamine degradation rate of microdissected proximal convoluted tubules to study the changes in dopamine metabolism. L-AADC activity in the HS group showed a significant increase compared with that in the LS group, although there was no significant change in dopamine degradation rate. We conclude that the increase in UDAV during high salt intake was mainly caused by the enhancement of extraneural dopamine production by the kidney in rats. Dopamine-producing enzyme, but not its degradation in the tubular cells, plays a role in the regulation of extraneural dopamine production.

Molecular cloning of guinea-pig aromatic-L-amino acid decarboxylase cDNA

Guinea-pig aromatic- l-amino acid decarboxylase (DOPA decarboxylase, DDC), but not rat DDC, reacts with the antibody against rat histidine decarboxylase (HDC). For determination of the molecular reaction for this cross-reactivity, a cDNA clone of guinea-pig DDC was isolated. Guinea-pig DDC consists of 480 amino acids and its molecular weight is 54,148. The sequence identity of guinea-pig DDC with rat DDC is 86%. Guinea-pig DDC has a region showing 100 % sequence identity with rat HDC, but only 67 % sequence identity with rat DDC, suggesting that this region is related with the cross-reactivity of guinea-pig DDC and anti-rat HDC antibody.

The Significance of L-Amino Acid Decarboxylase and DARPP-32 in the Kidney

This study examines the role of endogenous dopamine (DA) for the regulation of renal tubular sodium (Na) transport. The enzyme L-amino acid decarboxylase (L-AADC) that converts L-dopa to DA has been localized to the proximal tubule cells with immunocytochemistry. Locally formed DA will inhibit the activity of Na-K-ATPase, the enzyme that yields energy to active Na transport. The effect is of physiological importance during high salt diet. The phosphoprotein DARPP-32, a DA1 receptor associated third messenger is abundant in the medullary thick ascending limb of Henle (mTAL). DARPP-32 is phosphorylated after activation of DA1 receptors. DARPP-32 is in its phosphorylated form a potent phosphatase inhibitor. Activation of the DA1 receptor in mTAL with the DA1 agonist SKF 82526 causes dose-dependent inhibition of Na-K-ATPase activity. The effect involves activation of cAMP protein kinase. It is likely that this effect is potentiated by DARPP-32.

Aromatic L-amino acid decarboxylase activity along the rat nephron

Extraneural dopamine is thought to be synthesized by an aromatic L-amino acid decarboxylase (L-AADC) activity in tubular cells. However, the previous histochemical studies of this enzyme's localization in the nephron were not consistent. To determine the localization of L-AADC and whether changes in Na intake regulate this enzyme, L-AADC was measured in microdissected nephron segments from rat kidneys. Dopamine formed by isolated tubules incubated with exogenous L-dopa was quantitated by high-performance liquid chromatography (HPLC) and with the more sensitive radioenzyme assay (REA). L-AADC activity was present only in proximal convoluted (PCT, 208 +/- 19 ng.cm-1.h-1) and proximal straight tubules (PST, 81 +/- 9 ng.cm-1.h-1), whereas no significant activity was detected in other nephron segments by either HPLC or REA. Maximal velocity (Vmax) of L-AADC in a low-salt diet group (246 +/- 4 ng.cm-1.h-1) showed a small but a significant decrease (P less than 0.05) compared with control and high-salt diet groups (311 +/- 6 and 293 +/- 4 ng.cm-1.h-1, respectively), whereas the apparent Michaelis constant (Km) was similar in these three groups. These results show that L-AADC is present only in the PCT and PST of the rat nephron, and suggest that the changes in L-AADC activity may contribute to the regulation of extraneural dopamine production in the kidney during low-salt intake.