4-Chloro-L-phenylalanine

Background: The intestinal mucosa recovers from injury by accelerating enterocyte proliferation resulting in villus growth. A similar phenomenon is seen after massive bowel resection. Serotonin (5-HT) has been implicated as an important regulator of mucosal homeostasis by promoting growth in the epithelium. The impact of 5-HT on other components of growing villi is not known. We hypothesized that 5-HT-stimulated growth in the intestinal epithelium would be associated with growth in other components of the villus such as enteric neural axonal processes. Materials and methods: Enteric serotonergic signaling is inactivated by the serotonin reuptake transporter, or SERT, molecule. Enhanced serotonin signaling was achieved via SERT knockout (SERTKO) and administration of selective serotonin reuptake inhibitors (SSRI) to wild-type mice (WT-SSRI). 5-HT synthesis inhibition was achieved with administration of 4-chloro-L-phenylalanine (PCPA). Intestinal segments from age-matched WT, SERTKO, WT-SSRI, and corresponding PCPA-treated animals were assessed via villus height, crypt depth, and crypt proliferation. Gap 43, a marker of neuroplasticity, was assessed via immunofluorescence and Western blot. Results: SERTKO and WT-SSRI mice had taller villi, deeper crypts, and increased enterocyte proliferation compared with WT mice. Gap 43 expression via immunofluorescence was significantly increased in SERTKO and WT-SSRI samples, as well as in Western blot analysis. PCPA-treated SERTKO and WT-SSRI animals demonstrated reversal of 5-HT-induced growth and Gap 43 expression. Conclusions: Enhanced 5-HT signaling results in intestinal mucosal growth in both the epithelial cell compartment and the enteric nervous system. Furthermore, 5-HT synthesis inhibition resulted in reversal of effects, suggesting that 5-HT is a critically important regulator of intestinal mucosal growth and neuronal plasticity.

Parkinson's disease (PD) is characterized not only by typical motor symptoms, but also by nonmotor symptoms in the early stages. In addition to the loss of dopaminergic (DAergic) neurons, progressive degenerations of noradrenergic (NA) and serotonergic (5-HT) neurons were also observed. However, the respective effects and interactions of these monoamine depletions on certain nonmotor symptoms are still largely unknown. In the present study, we performed selective depletions of NA, 5-HT and DA in mice by intraperitioneal injection of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4), 4-chloro-L-phenylalanine (pCPA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), respectively. DSP-4 led to a 34% decrease in the number of NAergic neurons in the locus coeruleus, and MPTP led to a 30% decrease in the number of DAergic neurons in the substantia nigra. Although there was no obvious change in the number of 5-HTergic neurons in the dorsal raphe nucleus after pCPA treatment, the levels of 5-HT and its metabolite in the frontal cortex and hippocampus were reduced, respectively. Locomotor activity deficit was induced by DA depletion and a decrease in traveled distance was potentiated by additional NA depletion. Despair-associated depressive-like behavior could be observed in every group. Anxiety states emerged only from the combined depletion of two or three monoamines. However, combined depletion of the three monoamines dramatically induced anhedonia, and it could also aggravate the depressive-like and anxiety behavior. Furthermore, NA depletion significantly reduced spatial learning and memory ability, which was not enhanced by additional 5-HT or DA depletion. Our data highlighted the interactive role of NA, 5-HT and DA in the motor, emotional and cognitive deficits, providing new insight into the complex orchestration of impaired monoaminergic systems that related to the pathology of PD.

Aromatic L-amino acid decarboxylases (AADCs) are ubiquitously found in higher organisms owing to their physiological role in the synthesis of neurotransmitters and alkaloids. However, bacterial AADC has not attracted much attention because of its rather limited availability and narrow substrate range. Here, we examined the biochemical properties of AADC from Bacillus atrophaeus (AADC-BA) and assessed the synthetic feasibility of the enzyme for the preparation of monoamine neurotransmitters. AADC-BA was expressed in Escherichia coli BL21(DE3) and the purified enzyme showed a specific activity of 2.6 ± 0.4 U/mg for 10 mM L-phenylalanine (L-Phe) at 37 °C. AADC-BA showed optimal pH and temperature ranges at 7-8 and 37-45 °C, respectively. The KM and kcat values for L-Phe were 7.2 mM and 7.4 s-1, respectively, at pH 7.0 and 37 °C. Comparison of the kinetic constants at different temperatures revealed that the temperature dependency of the enzyme was mainly determined by catalytic turnover rather than substrate binding. AADC-BA showed a broad substrate scope for various aromatic amino acids, including L-Phe, L-tryptophan (610% relative to L-Phe), L-tyrosine (12%), 3,4-dihydroxyphenyl-L-alanine (24%), 5-hydroxy-L-tryptophan (L-HTP, 71%), 4-chloro-L-phenylalanine (520%), and 4-nitro-L-phenylalanine (450%). Homology modeling and docking simulations were carried out and were consistent with the observed substrate specificity. To demonstrate the synthetic potential of AADC-BA, we carried out the production of serotonin by decarboxylation of L-HTP. The reaction yield of serotonin reached 98% after 1 h at the reaction conditions of 50 mM L-HTP and 4 U/mL AADC-BA. Moreover, we carried out preparative-scale decarboxylation of L-Phe (100 mM in 40-mL reaction mixture) and isolated the resulting 2-phenylethylamine (51% recovery yield). We expect that the broad substrate specificity of AADC-BA can be exploited to produce various aromatic biogenic amines. KEY POINTS: · AADC-BA showed broad substrate specificity for various aromatic amino acids. · The substrate specificity was elucidated by in silico structural modeling. · The synthetic potential of AADC-BA was demonstrated for the production of biogenic amines.