Levo-tetrahydropalmatine inhibits α4ß2 nicotinic receptor response to nicotine in cultured SH-EP1 cells.

Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4ß2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1-100 µM) induced inward currents, co-application of l-THP (3 µM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 µM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4ß2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4ß2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42ß23-nAChR) and cytisine-activated, low-sensitive one (α43ß22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4ß2-nAChR function, which may underlie its inhibition on nicotine addiction.

Myricetin reduces cytotoxicity by suppressing hepcidin expression in MES23.5 cells.

Multiple studies implicate iron accumulation in the substantia nigra in the degeneration of dopaminergic neurons in Parkinson's disease. Indeed, slowing of iron accumulation in cells has been identified as the key point for delaying and treating Parkinson's disease. Myricetin reportedly plays an important role in anti-oxidation, anti-apoptosis, anti-inflammation, and iron chelation. However, the mechanism underlying its neuroprotection remains unclear. In the present study, MES23.5 cells were treated with 1 × 10-6 M myricetin for 1 hour, followed by co-treatment with 400 nM rotenone for 24 hours to establish an in vitro cell model of Parkinson's disease. Our results revealed that myricetin alleviated rotenone-induced decreases in cell viability, suppressed the production of intracellular reactive oxygen species, and restored mitochondrial transmembrane potential. In addition, myricetin significantly suppressed rotenone-induced hepcidin gene transcription and partly relieved rotenone-induced inhibition of ferroportin 1 mRNA and protein levels. Furthermore, myricetin inhibited rotenone-induced phosphorylation of STAT3 and SMAD1 in MES23.5 cells. These findings suggest that myricetin protected rotenone-treated MES23.5 cells by potently inhibiting hepcidin expression to prevent iron accumulation, and this effect was mediated by alteration of STAT3 and SMAD1 signaling pathways.

Cocaine potently blocks neuronal α3ß4 nicotinic acetylcholine receptors in SH-SY5Y cells.

Cocaine is one of the most abused illicit drugs worldwide. It is well known that the dopamine (DA) transporter is its major target; but cocaine also acts on other targets including nicotinic acetylcholine receptors (nAChRs). In this study, we investigated the effects of cocaine on a special subtype of neuronal nAChR, α3ß4-nAChR expressed in native SH-SY5Y cells. α3ß4-nAChR-mediated currents were recorded using whole-cell recordings. Drugs were applied using a computer-controlled U-tube drug perfusion system. We showed that bath application of nicotine induced inward currents in a concentration-dependent manner with an EC50 value of 20 µM. Pre-treatment with cocaine concentration-dependently inhibited nicotine-induced current with an IC50 of 1.5 µM. Kinetic analysis showed that cocaine accelerated α3ß4-nAChR desensitization, which caused a reduction of the amplitude of nicotine-induced currents. Co-application of nicotine and cocaine (1.5 µM) depressed the maximum response on the nicotine concentration-response curve without changing the EC50 value, suggesting a non-competitive mechanism. The cocaine-induced inhibition of nicotine response exhibited both voltage- and use-dependence, suggesting an open-channel blocking mechanism. Furthermore, intracellular application of GDP-ßS (via recording electrode) did not affect cocaine-induced inhibition, suggesting that cocaine did not alter receptor internalization. Moreover, intracellular application of cocaine (30 µM) failed to alter the nicotine response. Finally, cocaine (1.5 µM) was unable to inhibit the nicotine-induced inward current in heterologous expressed α6/α3ß2ß3-nAChRs and α4ß2-nAChRs expressed in human SH-EP1 cells. Collectively, our results suggest that cocaine is a potent blocker for native α3ß4-nAChRs expressed in SH-SY5Y cells.

L-type Calcium Channels are Involved in Iron-induced Neurotoxicity in Primary Cultured Ventral Mesencephalon Neurons of Rats.

In the present study, we investigated the mechanisms underlying the mediation of iron transport by L-type Ca2+ channels (LTCCs) in primary cultured ventral mesencephalon (VM) neurons from rats. We found that co-treatment with 100 µmol/L FeSO4 and MPP+ (1-methyl-4-phenylpyridinium) significantly increased the production of intracellular reactive oxygen species, decreased the mitochondrial transmembrane potential and increased the caspase-3 activation compared to MPP+ treatment alone. Co-treatment with 500 µmol/L CaCl2 further aggravated the FeSO4-induced neurotoxicity in MPP+-treated VM neurons. Co-treatment with 10 µmol/L isradipine, an LTCC blocker, alleviated the neurotoxicity induced by co-application of FeSO4 and FeSO4/CaCl2. Further studies indicated that MPP+ treatment accelerated the iron influx into VM neurons. In addition, FeSO4 treatment significantly increased the intracellular Ca2+ concentration. These effects were blocked by isradipine. These results suggest that elevated extracellular Ca2+ aggravates iron-induced neurotoxicity. LTCCs mediate iron transport in dopaminergic neurons and this, in turn, results in elevated intracellular Ca2+ and further aggravates iron-induced neurotoxicity.

Isradipine attenuates MPTP-induced dopamine neuron degeneration by inhibiting up-regulation of L-type calcium channels and iron accumulation in the substantia nigra of mice.

The aim of this study is to investigate the effects of L-type calcium channels (LTCCs) on MPTP-induced dopamine (DA) neuron degeneration and iron accumulation in the substantia nigra (SN) of mice. By real-time PCR and western blots, we first quatified expressions of L-type Cav1.2 and Cav1.3 calcium channel α1 subunits in the SN of experimental mice treated with MPTP. We found that the expressions of Cav1.2 and Cav1.3 calcium channel α1 subunits markedly increased after MPTP treatment for 2 and 3 weeks. Secondly, we observed the effects of isradipine, a LTCC antagonist, on MPTP-induced DA neuron degeneration and iron accumulation in the SN. Our results showed that isradipine treatment prevented against MPTP-induced Cav1.2 and Cav1.3 calcium channel α1 subunits up-regulation in the SN. We also found that isradipine prevented against MPTP-induced DA neuron depletion in the SN and partly restored the DA content in the striatum. Moreover, we found that isradipine inhibited the increase of iron positive cells in the SN of the MPTP-treated mice. Finally, we investigated the effects of isradipine on cellular iron accumulation in the dopaminergic MES23.5 cell line. Our studies showed that MPP+ treatment accelerated iron influx in the MES23.5 cells. Treatment with Bayk8644 further aggravated iron accumulation. Treatment with isradipine prevented against MPP+-induced iron influx in the MES23.5 cells. These results suggest that up-regulation of LTCCs may be responsible for the DA neuron degeneration in the MPTP-treated mice, The LTCCs may directly contribute to iron influx into DA neurons, and isradipine may suppress cellular iron accumulation and prevents neurodegeneration.

Protective effects of myricetin on chronic stress-induced cognitive deficits.

The aim of the present study is to investigate the possible effects of chronic administration of myricetin, a natural flavonoid, on chronic stress-induced learning and memory deficits in mice. The mice were restrained daily 4 h/day for 21 days in well-ventilated plexiglass tubes without access to food and water. These animals were injected with myricetin or vehicle 40 min before each restraint stress over a period of 21 days. Then, spatial learning and memory of the mice were evaluated by the Morris water maze task. We did not observe a significant difference in the escape latency in mice subjected to repeated restraint stress, which indicates that learning ability was not affected by restraint stress. However, the spatial memory ability was significantly impaired in the repeatedly restrained mice. Myricetin administration specifically increased the time spent in the target quadrant in mice exposed to chronic stress in the probe trial as tested in the Morris water maze task. Further studies showed that myricetin treatment decreased plasma adrenocorticotrophic hormone levels of those mice subjected to repeated restraint stress. The effect of myricetin on the levels of brain-derived neurotrophic factor (BDNF) in hippocampus was also investigated. The result showed that myricetin normalized the decreased BDNF levels in mice subjected to repeated restraint stress. These findings provide more evidence that chronic administration of myricetin improves spatial memory in repeatedly restrained mice and BDNF signaling in the hippocampus may be involved in the protective effects of myricetin.

Quantitative assessment of the association between GAK rs1564282 C/T polymorphism and the risk of Parkinson's disease.

The aim of this meta-analysis was to evaluate the association between cyclin G-associated kinase (GAK) rs1564282 C/T polymorphism and Parkinson's disease (PD) susceptibility. GAK modifies α-synuclein expression levels and affects susceptibility to PD. Genetic variation in GAK may influence the risk of occurrence and progression of PD. Many studies have evaluated the association between GAK rs1564282 C/T polymorphism and the risk of PD. However, published data are still controversial. Nine case-control studies with a total of 8159 PD patients and 12,747 controls were included in the meta-analysis. The summary odds ratio with 95% confidence interval was calculated to estimate this association. Both the minor allele frequencies and the genotype distributions of rs1564282 within GAK were different between the two groups when all studies were pooled. Subgroup analysis by ethnicity showed GAK rs1564282 C/T polymorphism was significantly associated with increased risk in both Asian and Caucasian populations. This meta-analysis suggests that GAK rs1564282 C/T polymorphism is associated with increased susceptibility to PD.

HLA-DRA rs3129882 A/G polymorphism was not a risk factor for Parkinson's disease in Chinese-based populations: a meta-analysis.

PURPOSE: Many studies have evaluated the association between the HLA-DRA rs3129882 A/G polymorphism and risk for Parkinson's disease (PD) in Chinese-based populations, however, published data remain inconclusive. Therefore, we performed a meta-analysis from all relevant studies to evaluate an association of HLA-DRA rs3129882 A/G polymorphism with susceptibility to PD. METHODS: The summary odds ratio (OR) with its 95% confidence interval (CI) was calculated to evaluate the association. The Q statistic was used to evaluate homogeneity and funnel plots were used to assess publication bias. The minor A allele frequencies, additive, dominant as well as recessive genetic models were examined in the analyses. RESULTS: Five case-control studies with a total of 2230 PD cases and 2262 controls from Mainland China, Taiwan, Singapore, and Malaysia were included in the final meta-analysis. Neither the minor A allele frequencies nor the genotypic distributions were significantly different between PD cases and controls when all studies were pooled into this meta-analysis. CONCLUSION: The results of this meta-analysis suggest that HLA-DRA rs3129882 A/G polymorphism was not responsible for PD in Chinese-based populations.

Quantitative assessment of the association between fibroblast growth factor 20 rs1721100 C/G polymorphism and the risk of sporadic Parkinson's diseases: a meta-analysis.

Fibroblast growth factor 20 (FGF20) is a neurotrophic factor which enhances the survival of rat midbrain dopamine neurons. Genetic variation in FGF20 may influence the risk of occurrence and development in Parkinson's diseases (PD). Many studies have evaluated the association between FGF20 rs1721100 C/G polymorphism and the risk of sporadic PD; however, published data are still controversial. The aim of the present meta-analysis was to evaluate the association of FGF20 rs1721100 C/G polymorphism with susceptibility of PD. The summary odds ratio (OR) with its 95 % confidence interval (CI) was calculated to estimate the association. Five case-control studies with a total of 3,463 sporadic PD cases and 4,606 controls were finally included into this meta-analysis. Neither the basic allele frequencies nor the genotypic distributions of rs1721100 C/G within FGF20 were different between two groups when all studies were pooled into the meta-analysis. Subgroup analysis by ethnicity showed FGF20 rs1721100 C/G polymorphism was significantly associated with increased risk in the heterozygote comparison model (CG versus GG: OR = 0.83, 95 % CI, 0.72-0.95, P = 0.009) in Asians but not in Caucasians. Overall, this meta-analysis suggests that FGF20 rs1721100 C/G polymorphism is associated with sporadic PD in Asians.

Transplantation of mouse CGR8 embryonic stem cells producing GDNF and TH protects against 6-hydroxydopamine neurotoxicity in the rat.

Embryonic stem cells (ESCs)-based therapies have been increasingly recognized as a potential tool to replace or support cells and their function damaged by the neurodegenerative process that underlies Parkinson's disease (PD). In this study, we implanted engineered mouse embryonic stem (ES) CGR8 cells, which stably co-express glial cell line-derived neurotrophic factor (GDNF) and tyrosine hydroxylase (TH), into striatum (Str) or both Str and substantia nigra (SN) of parkinsonian rats lesioned by 6-hydroxydopamine (6-OHDA). We found that cell transplantation into Str or both Str and SN rescued behavioral abnormalities and striatal DA depletion associated with 6-OHDA lesion. Our findings suggested that the profound functional impairment in nigrostriatal circuitry could be at least partially restored by ESCs-based expression of TH and GDNF, which may be developed into a useful tool for PD therapy.

Flavonoid Myricetin Modulates GABA(A) Receptor Activity through Activation of Ca(2+) Channels and CaMK-II Pathway.

The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN) by increasing the decay time and frequency of the inhibitory currents mediated by GABA(A) receptor. This effect of myricetin was not blocked by the GABA(A) receptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca(2+)/calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca(2+) imaging studies found that myricetin could increase Ca(2+) current and intracellular Ca(2+) concentration, respectively, via T- and L-type Ca(2+) channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABA(A) receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABA(A) receptor.

Mitochondria dysfunction was involved in copper-induced toxicity in MES23.5 cells.

OBJECTIVE: To investigate the toxicity of copper on MES23.5 dopaminergic cells and the probable mechanisms involved in this process. METHODS: MES23.5 dopaminergic cells were selected as our experimental model. [3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide] (MTT) assay was used to detect the influence of copper on the cell viability. The semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blotting and the high performance liquid chromatography-electrochemical detection (HPLC-ECD) have been used to detect the tyrosine hydroxlase (TH) mRNA and protein expression and the dopamine content in MES23.5 cells. The flow cytometry have been used to detect the changes of mitochondrial transmembrane potential. RESULTS: 100 and 200 mumol/L copper had no effect on the MES23.5 cell viability, whereas 400 and 800 mumol/L of copper could decrease the cell viability (P < 0.01). Treating cells with 200 mumol/L copper for 24 h decreased the TH mRNA expression, the TH expression and the dopamine content compared with the control (P < 0.01, P < 0.01, P < 0.05, respectively). Besides, the mitochondrial transmembrane potential also decreased with the treatment of 200 mumol/L copper for 24 h (P < 0.01). CONCLUSION: Copper could exert the toxic effects on MES23.5 dopaminergic cells and decrease the cell function. The dysfunction of mitochondria may be the mechanism of this toxicity effect.

Myricetin reduces 6-hydroxydopamine-induced dopamine neuron degeneration in rats.

The effects of myricetin on 6-hydroxydopamine (6-OHDA)-induced neurodegeneration in the substantia nigra-striatum system were investigated. By high-performance liquid chromatography electrochemical detection, we showed that the dopamine content in the striatum decreased after 6-OHDA treatment, which could be restored by myricetin. The immunohistochemistry and semiquantitative reverse transcription-PCR studies showed that myricetin could prevent the 6-OHDA-induced decrease of tyrosine hydroxylase positive neurons and the tyrosine hydroxylase mRNA expression in the substantia nigra. Perls' iron staining study further demonstrated that myricetin prevented the 6-OHDA-induced increase of iron-staining cells in the substantia nigra. These results suggested that the protective effects of myricetin on the toxicity of 6-OHDA could be attributed to the myricetin-suppressed iron toxicity.

Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel, mutations of which cause cystic fibrosis, a disease characterized by defective Cl(-) and HCO(3)(-) transport. Although >95% of all CF male patients are infertile because of congenital bilateral absence of the vas deferens (CBAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense debates. Here we report that CFTR is detected in both human and mouse sperm. CFTR inhibitor or antibody significantly reduces the sperm capacitation, and the associated HCO(3)(-)-dependent events, including increases in intracellular pH, cAMP production and membrane hyperpolarization. The fertilizing capacity of the sperm obtained from heterozygous CFTR mutant mice is also significantly lower compared with that of the wild-type. These results suggest that CFTR in sperm may be involved in the transport of HCO(3)(-) important for sperm capacitation and that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CBAVD.

C31 enhances voltage-gated calcium channel currents in undifferentiated PC12 cells.

C31, consisting of 31 amino acid residues, is generated from the carboxyl terminal fragments (CTFs) of amyloid precursor protein (APP). It has been shown that C31 causes apoptosis in neurons and is present in brains of Alzheimer disease (AD) patients. Using whole-cell patch clamp techniques, we investigated effects of C31 on voltage-gated calcium channel (VGCC) currents and the protective effects of beta-estradiol on PC12 cells. The results demonstrated that C31 induced a significant increase of the VGCC currents in PC12 cells, which was blocked by beta-estradiol. These results suggest that modulation of intracellular calcium levels by VGCC may in part be involved in C31 induced neuronal death associated with AD.