The inhibitory effects of deltamethrin on dopamine biosynthesis in rat PC12 cells

The inhibitory effects of deltamethrin (DM) on dopamine biosynthesis in PC12 cells were investigated. Levels of dopamine in PC12 cells were detected after treatment with different doses from 10(-4)M to 10(-6)M deltamethrin or combined with 20 microM or 50 microM l-3,4-dihydroxyphenylalanine (L-Dopa) for 3 h or 12 h by HPLC. Flow cytometry (FCM) and RT-PCR technology were used to detect the levels of tyrosine hydroxylase (TH) mRNA and protein in PC12 cells treated with different doses of deltamethrin for 3 h or 12 h. Deltamethrin decreased the levels of dopamine, TH mRNA and TH protein in PC12 cells, and the increased dopamine levels induced by l-Dopa at 20-50 microM were also decreased by deltamethrin for 3 h or 12 h. These results indicate that deltamethrin decreases dopamine content by the inhibition of TH mRNA and protein level in PC12 cells and maybe also have inhibitory effect on aromatic l-amino acid decarboxylase.     

Inhibition of phospholipase A(2) activity by S-nitroso-cysteine in a cyclic GMP-independent manner in PC12 cells

Arachidonic acid and nitric oxide (NO) act as retrograde and intercellular messengers in the nervous system. Regulation of cyclooxygenase is well established, but regulation of phospholipase A(2), the enzyme responsible for the liberation of arachidonic acid, by NO has not been thoroughly investigated. Using the PC12 cell line as a neuronal model, we studied the effects of exogenous NO compounds on arachidonic acid release. Incubation with Ca(2+) ionophores or mastoparan (wasp venom peptide) stimulated [3H]arachidonic acid release from prelabeled PC12 cells. [3H]Arachidonic acid release was inhibited by cytosolic phospholipase A(2) inhibitors, but not by dithiothreitol. A cytosolic phospholipase A(2) protein band with a molecular mass of approximately 100 kDa was detected by immunoblotting. S-Nitroso-cysteine inhibited basal and stimulated [3H]arachidonic acid release in concentration-dependent manners. Other NO compounds such as sodium nitroprusside and S-nitroso-N-acetylpenicillamine did not affect [3H]arachidonic acid release. N-Ethylmaleimide also inhibited [3H]arachidonic acid release. The inhibitory effects of S-nitroso-cysteine and N-ethylmaleimide were irreversible, because [3H]arachidonic acid release from PC12 cells preincubated with S-nitroso-cysteine or N-ethylmaleimide was much lower than that from nontreated cells. These findings suggest (a) cytosolic phospholipase A(2) is activated by Ca(2+) or mastoparan, and inhibited by S-nitroso-cysteine in a cyclic GMP-independent manner, (b) N-ethylmaleimide also inhibits cytosolic phospholipase A(2) and arachidonic acid release in PC12 cells. S-Nitroso-cysteine can regulate the production of other retrograde messenger arachidonic acid.     

白屈菜赤碱对PC12细胞乙酰胆碱诱发电流的快速抑制作用

采用全细胞膜片钳技术研究蛋白激酶C(PKC)选择性抑制剂白屈菜赤碱(CHT)对PC12细胞上乙酰胆碱(30 μmol·L^-1)诱发电流(I_ACh)的影响. 研究表明CHT(0.1-10 μmol·L^-1)预温育细胞5 min可使I_ACh峰值受抑制,此作用呈浓度依赖性,可逆性和非电压依赖性. CHT(5 μmol·L^-1)温育细胞6 min内对I_ACh的抑制作用呈时间依赖性. 通过微电极将更为有效的PKC抑制剂PKCI 19-31(0.1-5 μmol·L^-1)透析入细胞内以阻断PKC,并不影响CHT抑制I_ACh的作用. 以上结果提示：CHT对PC12细胞I_ACh有快速抑制作用,此作用与抑制PKC无关,而可能是一种新的药理作用.     

Inhibitory effects of noscapine on dopamine biosynthesis in PC12 cells

The effects of noscapine, a phthalide isoquinoline alkaloid, on dopamine biosynthesis and tyrosine hydroxylase (TH) activity in PC12 cells were investigated. Noscapine showed 74.6% inhibition on dopamine content in PC12 cells at a concentration of 20 μM. IC₅₀ of noscapine was 6.8 μM. TH activity was inhibited by the treatment of noscapine in PC12 cells (20.9% inhibition at 20 μM). Therefore, the inhibition of TH activity by noscapine might be involved in at least one component of the reduction of dopamine biosynthesis in PC12 cells.     

Effects of hydrastine derivatives on dopamine biosynthesis in PC12 cells

The effects of hydrastine derivatives on dopamine biosynthesis in PC12 cells were investigated. Treatments of PC12 cells with (1R,9S)-beta-hydrastine hydrochloride [(+)-beta-hydrastine HCl] and (1R,9S)-beta-hydrastine [(-)-beta-hydrastine] showed 50.6 % and 33.1 % inhibition of dopamine content at a concentration of 10 microM for 48 h. However, (1S,9R)-beta-hydrastine [(+)-beta-hydrastine] and hydrastinine hydrochloride did not reduce dopamine content. The IC(50) values of (1R,9S)-beta-hydrastine hydrochloride and (1R,9S)-beta-hydrastine were 9.3 microM and 20.7 microM , respectively. Next, the intracellular mechanisms of (1R,9S)-beta-hydrastine hydrochloride in PC12 cells were investigated. Dopamine content decreased at 6 h and reached a minimal level at 24 h after the exposure of PC12 cells to 20 microM (1R,9S)-beta-hydrastine hydrochloride. Tyrosine hydroxylase (TH) activity was inhibited at 6 h following the treatment with (1R,9S)-beta-hydrastine hydrochloride, and was maintained at a reduced level for up to 36 h in PC12 cells (17 - 27 % inhibition at 20 microM), whereas TH mRNA level was not found to alter for 24 h. However, the level of intracellular Ca++ concentration decreased by treatment with (1R,9S)-beta-hydrastine hydrochloride at 20 microM by 18.4 % inhibition relative to the control level in PC12 cells. These results suggest that (1R,9S)-beta-hydrastine hydrochloride contributes partially to the decrease in dopamine content by the inhibition of TH activity in PC12 cells.     

Effects of tributyltin chloride on L-DOPA-induced cytotoxicity in PC12 cells

Tributyltin chloride (TBTC) at concentrations of 0.5-1.0 microM inhibits dopamine biosynthesis in PC12 cells. In this study, the effects of TBTC on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced cytotoxicity in PC12 cells were investigated. TBTC at concentrations up to 1.0 microM neither affected cell viability, nor induced apoptosis after 24 or 48 h in PC12 cells. However, TBTC at concentrations higher than 2.0 microM caused cytotoxicity through an apoptotic process. In addition, exposure of PC12 cells to non-cytotoxic (0.5 and 1.0 microM) or cytotoxic (2.0 microM) concentrations of TBTC in combination with L-DOPA (20, 50 and 100 microM) resulted in a significant increase in cell loss and the percentage of apoptotic cells after 24 or 48 h compared with TBTC or L-DOPA alone. The enhancing effects of TBTC on L-DOPA-induced cytotoxicity were concentration- and treatment time-dependent. These data demonstrate that TBTC enhances L-DOPA-induced cytotoxicity in PC 12 cells.     

Protective effects of puerarin on alcohol-induced oxidative injury in PC12 cells

OBJECTIVE The aim of this study was to investigate the protective effect of puerarin on alcoholtoxicity in rat pheochromocytoma cell line(PC12). METHODS The PC12 cells were incubated with different concentrations of puerarin in advance. The protective effects of the puerarin on alcohol induced PC12 cel impairment were evaluated according to the fol owing approach: the viability of PC12 cel was determined by MTT assay and the impairment level was evaluated by analysis the leakage content of the lactate dehydrogenase(LDH). The cel apoptosis degree and the pro-apoptotic p53 protein expression were measured by flow cytometry. RESULTS Alcohol significantly impaired PC12 cel viability(P<0.05),and increased LDH leakage(P<0.05),induced cell apoptosis and upregulated expression of p53(P<0.05).While Puerarin significantly reversed these changes(P<0.05). CONCLUSION Puerarin might exert protection effect against ethanol-induced neurotoxicity via inhibition the expression of p53 protein.     

葛根素对二氯化钴造成PC12细胞缺氧损伤的保护作用

目的研究葛根素对PC12细胞缺氧损伤的影响。方法使用氯化钴对PC12细胞制备缺氧损伤模型,通过测定受损细胞给药后的活力、受损细胞LDH泄漏量和受损细胞SOD活力,研究葛根素对受损细胞的保护作用。结果在5×10-4~2×10-11mg.mL-1范围内,葛根素显著地增加模型受损细胞的活力;降低细胞LDH的泄露量;并增强受损细胞的SOD活性。结论葛根素对二氯化钴造成PC12细胞缺氧损伤具有修复作用。     

Inhibitory effects of higenamine on dopamine content in PC12 cells.

The inhibitory effects of (+/-)-higenamine, a benzylisoquinoline alkaloid, on dopamine biosynthesis in PC12 cells were investigated. Higenamine decreased the intracellular dopamine content dose-dependently and showed 55.2% inhibition of dopamine content in PC12 cells at a concentration of 20 microM with 24 h incubation. The IC50 value of higenamine was 18.2 microM. Dopamine content was lowered and reached minimal level at 12-24 h after exposure to higenamine. In this condition, tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, was also inhibited by the treatment of higenamine in PC12 cells (21.9% inhibition at 20 microM). Higenamine at 20 microM lowered the intracellular Ca2+ concentration by 33.1% inhibition relative to control in PC12 cells. These results suggest that the inhibition of tyrosine hydroxylase activity by higenamine might partially contribute to the decrease in dopamine content in PC12 cells.     

贯叶连翘对PC12细胞缺血性损伤的保护作用

目的:研究贯叶连翘对PC12细胞缺血性损伤的保护作用。方法:在离体培养的PC12细胞,用NaCN加缺糖造成拟缺血损伤模型,噻唑蓝(MTT)、LDH活力测定、细胞内肌酸激酶(CK)活力测定、流式细胞仪测定细胞凋亡率和线粒体跨膜电位。结果:在10-7~10-5mol.L-1范围内,贯叶连翘浓度依赖地增加MTT比色值,降低缺血性损伤所致培养介质内LDH的释放,增加细胞内CK活力。贯叶连翘还可剂量相关性地降低PC12细胞的凋亡百分率和稳定细胞线粒体跨膜电位。结论:贯叶连翘对PC12细胞缺血性损伤具有保护作用,其机制可能与增加细胞内CK活力和稳定细胞线粒体跨膜电位而抑制缺血性损伤诱导的PC12细胞的凋亡有关。     

Effects of asimilobine on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells

The effects of asimilobine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Asimilobine at concentration ranges of 0.05-0.2 microM showed a significant inhibition of intracellular dopamine levels for 24 h in a concentration-dependent manner with an IC50 value of 0.13 microM. Asimilobine at 0.15 microM inhibited tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities at 24 h (73.2% inhibition of TH activity): the inhibition of TH activity was stronger and longer than that of AADC activity. Asimilobine also decreased TH mRNA levels and intracellular cyclic AMP levels, but not the basal Ca2+ concentrations. In addition, asimilobine at 0.05-5.0 microM, but not 10 microM, did not alter cell viability toward PC12 cells. A non-cytotoxic asimilobine (0.15 microM) associated with l-DOPA (20, 50, and 100 microM) for 24 h inhibited L-DOPA-induced increases in dopamine levels and enhanced L-DOPA-induced cell death when compared with L-DOPA alone. These results suggest that asimilobine inhibits dopamine biosynthesis by mainly reducing the TH activity and TH mRNA expression, and enhances L-DOPA-induced cytotoxicity in PC12 cells.     

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 IC₅₀ 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.     

Effects of asimilobine on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells

The effects of asimilobine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells were investigated. Asimilobine at concentration ranges of 0.05-0.2 muM showed a significant inhibition of intracellular dopamine levels for 24 h in a concentration-dependent manner with an IC(50) value of 0.13 muM. Asimilobine at 0.15 muM inhibited tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC) activities at 24 h (73.2% inhibition of TH activity): the inhibition of TH activity was stronger and longer than that of AADC activity. Asimilobine also decreased TH mRNA levels and intracellular cyclic AMP levels, but not the basal Ca(2 + ) concentrations. In addition, asimilobine at 0.05-5.0 muM, but not 10 muM, did not alter cell viability toward PC12 cells. A non-cytotoxic asimilobine (0.15 muM) associated with l-DOPA (20, 50, and 100 muM) for 24 h inhibited l-DOPA-induced increases in dopamine levels and enhanced l-DOPA-induced cell death when compared with l-DOPA alone. These results suggest that asimilobine inhibits dopamine biosynthesis by mainly reducing the TH activity and TH mRNA expression, and enhances l-DOPA-induced cytotoxicity in PC12 cells.     

Effects of asimilobine on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells

The effects of asimilobine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells were investigated. Asimilobine at concentration ranges of 0.05–0.2 μM showed a significant inhibition of intracellular dopamine levels for 24 h in a concentration-dependent manner with an IC₅₀ value of 0.13 μM. Asimilobine at 0.15 μM inhibited tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC) activities at 24 h (73.2% inhibition of TH activity): the inhibition of TH activity was stronger and longer than that of AADC activity. Asimilobine also decreased TH mRNA levels and intracellular cyclic AMP levels, but not the basal Ca^2 +  concentrations. In addition, asimilobine at 0.05–5.0 μM, but not 10 μM, did not alter cell viability toward PC12 cells. A non-cytotoxic asimilobine (0.15 μM) associated with l-DOPA (20, 50, and 100 μM) for 24 h inhibited l-DOPA-induced increases in dopamine levels and enhanced l-DOPA-induced cell death when compared with l-DOPA alone. These results suggest that asimilobine inhibits dopamine biosynthesis by mainly reducing the TH activity and TH mRNA expression, and enhances l-DOPA-induced cytotoxicity in PC12 cells.     

异丙酚对PC12细胞和脂质体膜流动性的影响

目的：研究异丙酚对PC12细胞和脂质体膜流动性的影响，探讨其全麻作用机理，方法：以PC12细胞作为神经细胞模型，以脂质体作为生物膜模型，利用荧光偏振技术离体动态地检测30min内异丙酚对PC12细胞各向异性r、荧光偏振度P和微粘度η及脂质体微度η的影响，分析细胞膜流动性的改变。结果：各浓度的异丙酚均可引起PC12细胞膜的各向异性r、荧光偏振度P和微粘度η的显性降低，尤其在5min内r,p,η值下降迅速，5min后各指标下降的趋势渐渐平缓。与PC12细胞相类似，除了1mg.L^-1组外，10、100mg.L^-1的异丙酚均显降低脂质体微粘度η值。结论：异丙酚能显增加PC12细胞和脂质体的膜流动性，提示其全麻作用原理可能是通过改变神经细胞的功能或结构产生。     

表没食子儿茶素没食子酸酯对鱼藤酮诱导PC12细胞损伤的保护作用

目的探讨表没食子儿茶素没食子酸酯(EGCG)对鱼藤酮诱导的嗜铬细胞瘤PC12细胞损伤的保护作用及其可能机制。方法将培养的大鼠PC12细胞分别加入EGCG1,5和10μmol.L-1预处理30 min后加入鱼藤酮250 nmol.L-1继续作用24 h。MTT比色法检测细胞存活率,Hoechst33258染色观察细胞核形态的变化,流式细胞仪检测细胞凋亡率,JC-1染色检测线粒体膜电位变化。结果与鱼藤酮模型组存活率(77.0±1.3)%相比,EGCG1,5和10μmol.L-1组细胞存活率显著增加(P<0.05),分别为(79.8±2.3)%,(82.4±2.2)%和(88.3±2.0)%。Hoechst33258染色发现,EGCG组细胞核形态明显改善。与正常对照组比较,模型组细胞凋亡率增加8.2倍;与鱼藤酮模型组比较,EGCG组细胞凋亡率分别下降46%,25%和63%,差异具有统计学意义(P<0.01)。流式细胞仪检测鱼藤酮组凋亡率为33.8%,EGCG 1,5和10μmo.lL-1组的凋亡率下降至30.6%,14%和15.7%。与模型组相比,EGCG1,5和10μmol.L-1组线粒体膜分别增高了2.48,3.96和4.04倍,差异具有统计学意义(P<0.01)。结论 EGCG具有抑制鱼藤酮诱导的细胞凋亡作用,其机制可能与其稳定线粒体膜电位有关。     

蝙蝠葛苏林碱对PC12细胞内游离钙浓度的影响

目的进一步探讨蝙蝠葛苏林碱（Dau）的抗脑缺血／缺氧损伤与其钙拮抗作用间的关系。方法：培养的PC12细胞用Fura－2／AM负载，用AR－CM－MIC阳离子测定系统观测Dau对单细胞内游离钙（[Ca(2+)]_i）升高的影响。结果：Dau（0．1～100μmol·L(－1)）可浓度依赖性抑制高钾和caffeine引起的[Ca(2+)］_i增加，对肌浆网钙泵抑制剂cy－clopiszonicacid引起的[Ca(2+)]_i升高也有抑制作用。结论：Dau不仅抑制电压依赖性钙通道开放引起的细胞外钙内流和caffeine引起的内钙释放．而且对钙泵也可能有影响，这可能是其抗脑缺血／缺氧损伤的重要机制。     

米诺环素调控小胶质细胞激活对PC12细胞生长的影响

目的探讨米诺环素抑制小胶质细胞激活对PC12生长和凋亡的影响。方法 BV2细胞分为对照组、LPS组、LPS加米诺环素组;以LPS刺激激活小胶质细胞系BV2细胞,用米诺环素干预BV2细胞的激活;将各组BV2细胞与PC12细胞进行共培养24h;酶联免疫吸附法检测共培养体系细胞上清液TNF-α、IL-1β的含量,MTT法检测PC12细胞生存率。结果 BV2细胞与PC12细胞共培养24h后,LPS组上清液炎症因子TNF-α、IL-1β表达明显升高,PC12细胞存活率下降,米诺环素能抑制以上趋势。结论 MG激活对PC12细胞存在明显损伤效应,米诺环素可保护MG介导的PC12细胞损伤,可能与通过下调炎症表达有关。     

Modulatory effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Sesamin at concentration ranges of 20-75 μM exhibited a significant increase in intracellular dopamine levels at 24 h: 50 μM sesamin increased dopamine levels to 133% and tyrosine hydroxylase (TH) activity to 128.2% of control levels. Sesamin at 20-100 μM rapidly increased the intracellular levels of cyclic AMP (cAMP) to 158.3%-270.3% of control levels at 30 min. At 50 μM, sesamin combined with L-DOPA (50, 100 and 200 μM) further increased the intracellular dopamine levels for 24 h compared to L-DOPA alone. In the absence or presence of L-DOPA (100 and 200 μM), sesamin (50 μM) increased the phosphorylation of TH, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), as well as the mRNA levels of TH and CREB for 24 h, an effect which was reduced by L-DOPA (100 and 200 μM). In addition, 50 μM sesamin exhibited a protective effect against L-DOPA (100 and 200 μM)-induced cytotoxicity via the inhibition of reactive oxygen species (ROS) production and superoxide dismutase reduction, induction of extracellular signal-regulated kinase (ERK)1/2 and BadSer112 phosphorylation and Bcl-2 expression, and inhibition of cleaved-caspase-3 formation. These results suggested that sesamin enhanced dopamine biosynthesis and L-DOPA-induced increase in dopamine levels by inducing TH activity and TH gene expression, which was mediated by cAMP-PKA-CREB systems. Sesamin also protected against L-DOPA (100-200 μM)-induced cytotoxicity through the suppression of ROS activity via the modulation of ERK1/2, BadSer112, Bcl-2, and caspase-3 pathways in PC12 cells. Therefore, sesamin might serve as an adjuvant phytonutrient for neurodegenerative diseases.