OBJECTIVETo set the measuring method of tyrosine hydroxylase activity in the brain of conscious rats.

METHODBy using microdialysis and High Performance Liquid Chromatography-Electrochemical Detector system, the 3, 4-dihydrioxphenylalanine (DOPA) formation in the striatum of 6-hydroxdopamine-pretreated rats during infusion of an L-aromatic amino-acid decarboxylase inhibitor (NSD1015) was monitored.

RESULTThe absence of DOPA in dialysates of 6-hydroxdopamine-pretreated rats, the measurable DOPA and the steady decreasing of 3,4-dihydroxyphenylacetic acid(DOPAC) during infusion of NSD1015 and the disappearance of DOPA after administration of alpha-methyl-rho-tyrosine indicated that the dialyzed DOPA was derived from dopaminergic nerve terminals. After intraperitoneal administration of dopamine receptor agonist apomorphine the DOPA output was deseased. After intraperitoneal administration of dopamine recepter antagonist haloperidol, the DOPA output was increased. The study showed that twenty-four hours ofter implantation of the probe with infusion of 0.01 mmol.L-1 NSD1015, the DOPA level in the striatum of 6-hydroxdopamine-pretreated rats was 0.39 +/- 0.12 pmol/min (X +/- S, n = 5).

CONCLUSIONThe DOPA concentration in striatal dialysates could be considered as an index of tyrosion hydroxlase activity during infusion of 0.01 mM NSD1015. The method in vivo to monitor tyrosine hydroxlase activity in the brain is reliable.

Animals ; Apomorphine ; pharmacology ; Brain ; enzymology ; Dihydroxyphenylalanine ; metabolism ; Dopamine Agonists ; pharmacology ; Dopamine Antagonists ; pharmacology ; Female ; Haloperidol ; pharmacology ; Male ; Microdialysis ; Rats ; Rats, Sprague-Dawley ; Tyrosine 3-Monooxygenase ; metabolism

OBJECTIVE: To explore whether the using of mimetic peptide Gap27, a selective inhibitor of connexin 43 (Cx43), could block the death of dopamine neurons and influence the expression of Cx43 in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease mouse models. METHODS: Eighteen C57BL/6 mice were randomly divided into control group, 6-OHDA group and 6-OHDA+Gap27 group, with 6 mice in each group. Bilateral substantia nigra stereotactic injection was performed. The control group was injected with ascorbate solution, 6-OHDA group was injected with 6-OHDA solution, and 6-OHDA+Gap27 group was injected with 6-OHDA and Gap27 mixed solution. Immuno-histochemical staining was used to detect the number of dopamine neurons, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of Cx43 messenger ribonucleic acid (mRNA), immuno-fluorescence staining was used to detect the distribution of Cx43 protein, the contents of Cx43 protein and Cx43 phosphorylation at serine 368 (Cx43-ps368) in mouse midbrain were detected by Western blot. RESULTS: After injection of 6-OHDA, numerous dopamine neurons in substantia nigra died as Cx43 content increased, Cx43-ps368 content decreased. Mixing Gap27 while injecting 6-OHDA could reduce the number of death dopamine neurons and weaken the changes of Cx43 and Cx43-ps368 content caused by 6-OHDA. The number of tyrosine hydroxylase (TH) immunoreactive positive neurons in 6-OHDA group decreased to 27.7% ± 0.02% of the control group (P < 0.01); The number of TH immunoreactive positive neurons in 6-OHDA+Gap27 group was (1.64±0.16) times higher than that in 6-OHDA group (P < 0.05); The content of total Cx43 protein in 6-OHDA group was (1.44±0.07) times higher than that in 6-OHDA+Gap27 group (P < 0.05) while (1.68±0.07) times higher than that in control group (P < 0.01). In 6-OHDA group, the content of Cx43-ps368 protein and its proportion in total Cx43 protein were significantly lower than that in 6-OHDA+Gap27 group (P < 0.05). CONCLUSION In 6-OHDA mouse models, mimetic peptide Gap27 played a protective role in reducing the damage to substantia nigra dopamine neurons, which was induced by 6-OHDA. The overexpression of Cx43 protein might have neurotoxicity to dopamine neuron. Meanwhile, decreasing Cx43 protein level and keeping Cx43-ps368 protein level may be the protective mechanisms of Gap27.
Animals ; Connexin 43/pharmacology* ; Disease Models, Animal ; Dopaminergic Neurons/metabolism* ; Mice ; Mice, Inbred C57BL ; Oxidopamine/metabolism* ; Parkinson Disease/metabolism* ; Peptides/pharmacology* ; Tyrosine 3-Monooxygenase/pharmacology*

Objective: To explore the clinical and genetic characteristics of children with dopa-responsive dystonia (DRD) caused by tyrosine hydroxylase (TH) gene variations. Methods: Clinical data of 9 children with DRD caused by TH gene variations diagnosed in the Department of Children Rehabilitation, the Third Affiliated Hospital of Zhengzhou University from January 2017 to August 2022 were retrospectively collected and analyzed, including the general conditions, clinical manifestations, laboratory tests, gene variations and follow-up data. Results: Of the 9 children with DRD caused by TH gene variations, 3 were males and 6 were females. The age at diagnosis was 12.0 (8.0, 15.0) months. The initial symptoms of the 8 severe patients were motor delay or degression. Clinical symptoms of the severe patients included motor delay (8 cases), truncal hypotonia (8 cases), limb muscle hypotonia (7 cases), hypokinesia (6 cases), decreased facial expression (4 cases), tremor (3 cases), limb dystonia (3 cases), diurnal fluctuation (2 cases), ptosis (2 cases), limb muscle hypertonia (1 case) and drooling (1 case). The initial symptom of the very severe patient was motor delay. Clinical symptoms of the very severe patient included motor delay, truncal hypotonia, oculogyric crises, status dystonicus, hypokinesia, decreased facial expression, and decreased sleep. Eleven TH gene variants were found, including 5 missense variants, 3 splice site variants, 2 nonsense variants, and 1 insertion variant, as well as 2 novel variants (c.941C>A (p.T314K), c.316_317insCGT (p.F106delinsSF)). Nine patients were followed up for 40 (29, 43) months, and no one was lost to follow-up. Seven of the 8 severe patients were treated by levodopa and benserazide hydrochloride tablets and 1 severe patient was treated by levodopa tablets. All the severe patients responded well to levodopa and benserazide hydrochloride tablets or levodopa tablets. Although the weight of the patients increased and the drug dosage was not increased, the curative effect remained stable and there was no obvious adverse reaction. One severe patient developed dyskinesia in the early stage of treatment with levodopa and benserazide hydrochloride tablets and it disappeared after oral administration of benzhexol hydrochloride tablets. Until the last follow-up, motor development of 7 severe patients returned to normal and 1 severe patient still had motor delay due to receiving levodopa and benserazide hydrochloride tablets for only 2 months. The very severe patient was extremely sensitive to levodopa and benserazide hydrochloride tablets and no improvement was observed in this patient. Conclusions: Most of the DRD caused by TH gene variations are severe form. The clinical manifestations are varied and easily misdiagnosed. Patients of the severe patients responded well to levodopa and benserazide hydrochloride tablets or levodopa tablets, and it takes a long time before full effects of treatment become established. Long-term effect is stable without increasing the drug dosage, and no obvious side effect is observed.
Female ; Humans ; Infant ; Male ; Benserazide/therapeutic use* ; Dystonia/genetics* ; Hypokinesia/drug therapy* ; Levodopa/pharmacology* ; Muscle Hypotonia ; Retrospective Studies ; Tyrosine 3-Monooxygenase/genetics*

OBJECTIVES: Nerve growth factor (NGF) induces neuron transdifferentiation of adrenal medulla chromaffin cells (AMCCs) and consequently downregulates the secretion of epinephrine (EPI), which may be involved in the pathogenesis of bronchial asthma. Mammalian achaete scute-homologous 1 (MASH1), a key regulator of neurogenesis in the nervous system, has been proved to be elevated in AMCCs with neuron transdifferentiation in vivo. This study aims to explore the role of MASH1 in the process of neuron transdifferentiation of AMCCs and the mechanisms. METHODS: Rat AMCCs were isolated and cultured. AMCCs were transfected with siMASH1 or MASH1 overexpression plasmid, then were stimulated with NGF and/or dexamethasone, PD98059 (a MAPK kinase-1 inhibitor) for 48 hours. Morphological changes were observed using light and electron microscope. Phenylethanolamine-N-methyltransferase (PNMT, the key enzyme for epinephrine synthesis) and tyrosine hydroxylase were detected by immunofluorescence. Western blotting was used to test the protein levels of PNMT, MASH1, peripherin (neuronal markers), extracellular regulated protein kinases (ERK), phosphorylated extracellular regulated protein kinases (pERK), and JMJD3. Real-time RT-PCR was applied to analyze the mRNA levels of MASH1 and JMJD3. EPI levels in the cellular supernatant were measured using ELISA. RESULTS: Cells with both tyrosine hydroxylase and PNMT positive by immunofluorescence were proved to be AMCCs. Exposure to NGF, AMCCs exhibited neurite-like processes concomitant with increases in pERK/ERK, peripherin, and MASH1 levels (all P<0.05). Additionally, impairment of endocrine phenotype was proved by a signifcant decrease in the PNMT level and the secretion of EPI from AMCCs (all P<0.01). MASH1 interference reversed the effect of NGF, causing increases in the levels of PNMT and EPI, conversely reduced the peripherin level and cell processes (all P<0.01). MASH1 overexpression significantly increased the number of cell processes and peripherin level, while decreased the levels of PNMT and EPI (all P<0.01). Compared with the NGF group, the levels of MASH1, JMJD3 protein and mRNA in AMCCs in the NGF+PD98059 group were decreased (all P<0.05). After treatment with PD98059 and dexamethasone, the effect of NGF on promoting the transdifferentiation of AMCCs was inhibited, and the number of cell processes and EPI levels were decreased (both P<0.05). In addition, the activity of the pERK/MASH1 pathway activated by NGF was also inhibited. CONCLUSIONS MASH1 is the key factor in neuron transdifferentiation of AMCCs. NGF-induced neuron transdifferentiation is probably mediated via pERK/MASH1 signaling.
Animals ; Rats ; Adrenal Medulla ; Cell Transdifferentiation ; Chromaffin Cells ; Dexamethasone ; Epinephrine/pharmacology* ; Mammals ; Nerve Growth Factor ; Neurons ; Peripherins ; Protein Kinases ; Tyrosine 3-Monooxygenase

AIM AND METHODSIn the present study, we investigated the TH immunoreactivity and the expression of angiotensin AT1 receptor in locus coeruleus after intracerebroventricular (i. c. v.) injection of carbachol in conscious SD rats with immunohistochemistry. Meanwhile the effects of blocking AT1 receptor were also observed.

RESULTSBoth mean optical density and number of TH and AT1 immunoreactive positive neurons were markedly increased in locus coeruleus after 40 minutes of i.c.v. injection of carbachol (0.5 microg). The enhancement was significantly reduced by i. c. v. injection of losartan.

CONCLUSIONThe results above suggest that i. c. v. injection of cholinergic agonist carbachol can enhance the activity of adrenergic neurons and the expression of AT1 receptor in locus coeruleus. The blockade of AT1 receptor may down regulate the above action induced by carbachol in locus coeruleus.

Animals ; Brain ; Carbachol ; pharmacology ; Cholinergic Agents ; pharmacology ; Injections, Intraventricular ; Locus Coeruleus ; drug effects ; metabolism ; Losartan ; pharmacology ; Male ; Neurons ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Angiotensin, Type 1 ; metabolism ; Tyrosine 3-Monooxygenase

AIMTo investigate the protective effect of nicotine on dopaminergic neurons and its mechanisms in mice with Parkinson's disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

METHODSC57BL/6J mice were injected with MPTP for 8 days to establish PD model. Nicotine was given for 10 days in the pretreatment group. Animals were examined behaviorally with the pole test and traction test. Tyrosine hydroxylase (TH) and gamma-aminobutyric acid (GABA) were investigated by the immunocytochemistry (ICC) method. The ultrastructural changes of caudate nucleus(CN) were observed by electron microscope.

RESULTSThe results showed that pretreatment nicotine could improve the dyskinesia of PD mice markedly. Simultaneously, TH (P < 0.01) neurons and GABA (P < 0.05) neurons were much more in the pretreatment group when compared with those in the model group. The ultrastructural injury of the pretreatment group was also ameliorated.

CONCLUSIONNicotine has a protective effect on the dopaminergic neurons in the MPTP-treated mice.

Animals ; Caudate Nucleus ; ultrastructure ; Disease Models, Animal ; Dopaminergic Neurons ; drug effects ; Female ; Male ; Mice ; Mice, Inbred C57BL ; Neuroprotective Agents ; pharmacology ; Nicotine ; pharmacology ; Parkinson Disease ; drug therapy ; Tyrosine 3-Monooxygenase ; metabolism ; gamma-Aminobutyric Acid ; metabolism

OBJECTIVETo investigate the effect of AT₁ receptor on the changes of tyrosine hydroxylase-immunoreactivity (TH-IR) in rostral ventrolateral medulla (RVLM) induced by brain cholinergic stimuli in rats.

METHODSMale SD rats were randomly divided into 4 groups: NS + CBC group, Los + CBC group, Los + NS group and NS + NS group. AT₁ was blocked by pretreatment of 20 μg losartan in Los + CBC and Los + NS groups; intracerebroventricular injection of 0.5 μg carbachol was used for cholinergic stimuli in NS + CBC and Los + CBC groups; normal saline (NS) was used for control. The output amount of natrium in kidney, glomerular filtration rate (GFR) and renal plasma flow (PRF) were observed. The changes of TH-IR in the RVLM were observed by immunohistochemistry.

RESULTIn NS + CBC group carbachol induced potent natriuresis, after pretreatment of losartan the natriuretic effect was partially inhibited in Los + CBC group. Both the number and optical density of TH-IR positive neurons in NS + CBC group were markedly increased than those in NS + NS group (P < 0.05); while those in Los + CBC group were significantly lower than those in NS+CBC group (P < 0.05). Intracerebroventricular injection of carbachol and losartan had no effect on GFR and RPF(P > 0.05).

CONCLUSIONThe results suggest that cholinergic stimuli can induce potent natriuresis and increase the activity of adrenergic neurons in the RVLM; the above effects can be down regulated by blockade of brain AT₁ receptor.

Animals ; Carbachol ; administration & dosage ; pharmacology ; Drug Antagonism ; Glomerular Filtration Rate ; drug effects ; Losartan ; pharmacology ; Male ; Medulla Oblongata ; drug effects ; metabolism ; Natriuresis ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor, Angiotensin, Type 1 ; physiology ; Tyrosine 3-Monooxygenase ; metabolism

AIMTo provide further evidence for the synthesis of catecholamines (CAs) in lymphocytes and to investigate the effect of the endogenous CAs synthesized by lymphocytes on function of the lymphocytes themselves and the receptor mechanisms involved in the effect.

METHODSRT-PCR was performed to detect the expression of TH mRNA in the lymphocytes from the mesenteric lymph nodes of rats. Different concentrations of pargyline, an inhibitor of monoamine oxydase, and antagonists of alpha1-, alpha2-, beta1-, and beta2-adrenergic receptor (AR) were added to the lymphocyte cultures, and then proliferative response of the lymphocytes to mitogen concanavalin A (Con A) were measured via methyl-thiazole-tetrazolium (MTT) assay.

RESULTSThe lymphocytes could express TH mRNA, and the expression of TH mRNA was significantly higher in the Con A-activated lymphocytes than in the resting ones. The treatment of pargyline of 10(-6) and 10(-5) mol/L (not 10(-7) mol/L) notably attenuated Con A-induced lymphocyte proliferation. Beta2-AR antagonist ICI118551 (10(-7) and 10(-6) mol/L) completely blocked, but alpha1-AR antagonist corynanthine and alpha2-AR antagonist yohimbine (10(-7) and 10(-6) mol/L) partly blocked the suppressive effect of pargyline on the Con A-induced lymphocyte proliferation. Nevertheless, atenolol, an antagonist of beta1-AR, had no blocking effect on pargyline inhibition of lymphocyte proliferation.

CONCLUSIONLymphocytes have the ability to synthesize CAs and the ability is enhanced in the activated lymphocytes. The endogenous CAs synthesized by lymphocytes can inhibit T cell proliferation and the inhibition of T cells by the CAs is mediated predominantly by beta2-AR on the lymphocytes.

Animals ; Catecholamines ; biosynthesis ; physiology ; Cell Proliferation ; drug effects ; Concanavalin A ; pharmacology ; Female ; Lymphocyte Activation ; Lymphocytes ; metabolism ; Male ; Neuroimmunomodulation ; physiology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, beta ; physiology ; T-Lymphocytes ; cytology ; immunology ; Tyrosine 3-Monooxygenase ; genetics ; metabolism

The protective effect of puerarin on the Parkinson disease (PD) mice with decreased estrogen level was investigated in order to develop a new potential medicine as a substitute for estrogen for preventing and treating PD. By using immunohistochemical method of avidinbiotin peroxidase complex (ABC), the distribution of the cells positive for tyrosine hydroxylase (TH) and fibres in the substantia nigra of the mouse were observed. These mice were divided into three groups randomly: group A, normal-female-mouse models; group B containing three subgroups, B1 (normal saline), B2 (estrogen), B3 (puerarin); group C containing three sub groups, C1 (normal saline), C2 (estrogen), C3 (puerarin). By using TUNEL the numbers of apoptosis cells in every visual field was counted and the difference between the experimental group and control group was compared. The results showed the numbers of the cells positive for TH were more and the numbers of apoptosis cells were less in the normal-female-mouse models group than in the group of model made after ovariosteresis and the group of model made before ovariosteresis (P < 0.05), respectively. However, there was no significant difference, between the group given estrogen/puerarin and the controls, and between the group given estrogen and given puerarin. (P > 0.05). It was suggested that puerarin may have protective effect on the nigral neurons to PD. Moreover, the protective effect might serve as a surrogate of estrogen and be associated with the apoptosis.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Apoptosis ; Estrogens ; blood ; pharmacology ; Female ; Isoflavones ; chemistry ; pharmacology ; Mice ; Mice, Inbred BALB C ; Ovariectomy ; Parkinson Disease ; blood ; prevention & control ; Phytoestrogens ; Plant Preparations ; pharmacology ; Random Allocation ; Substantia Nigra ; metabolism ; Tyrosine 3-Monooxygenase ; metabolism ; Vasodilator Agents ; chemistry ; pharmacology

OBJECTIVETo investigate the effects of total saponins of panax ginseng (TSPG) on proliferation and differentiation of human embryonic neural stem cell (NSC) into dopaminergic neuron.

METHODIsolation, cultivation and identification of human embryonic NSC from cerebral cortex of 7-12 week abortus. By using flow cytometry and MTT assay, the effects of various concentration of TSPG and TSPG cooperating with cytokines( EGF, bFGF) in NSC culture media for 3 days on proliferation of human embryonic NSC has studied. By employing immunocytochemistry assay of the expression of tyrosine hydroxylase (TH), the effect of different dilution of TSPG and TSPG cooperating with IL-1 on induced differentiation of human embryonic NSC into dopaminergic neuron has researched.

RESULTTSPG can significantly promote the proliferation of NSC. When TSPG cooperating with EGF and bFGF, the proliferation of NSC is much stronger than that of only using FGF and bFGF. TSPG also induces NSC to differentiate into dopaminergic neuron, especially when TSPG is cooperating with IL-1.

CONCLUSIONTSPG can not only obviously accelerate the proliferation of NSC, but also significantly induce differentiation of NSC into dopaminergic neuron.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dopamine ; metabolism ; Drug Synergism ; Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Immunohistochemistry ; Interleukin-1 ; pharmacology ; Neurons ; cytology ; drug effects ; metabolism ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Saponins ; isolation & purification ; pharmacology ; Tyrosine 3-Monooxygenase ; metabolism