Inhibition of MG132-induced mitochondrial dysfunction and cell death in PC12 cells by 3-morpholinosydnonimine

The effect of 3-morpholinosydnonimine (SIN-1) against the cytotoxicity of MG132, a proteasome inhibitor, in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with MG132 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS), and depletion of GSH. Addition of SIN-1, a producer of nitric oxide (NO) and superoxide, differentially reduced the MG132-induced cell death and GSH depletion concentration dependently with a maximal inhibitory effect at 150 microM. Carboxy-PTIO, superoxide dismutase, Mn-TBAP, and ascorbate prevented the inhibitory effect of SIN-1 on the cytotoxicity of MG132. SIN-1 inhibited the MG132-induced change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents in PC12 cells. S-nitroso-N-acetyl-DL-penicillamine reduced the MG132-induced cell death in PC12 cells, whereas peroxynitrite and H2O2 did not affect the cytotoxicity of MG132. The results suggest that NO and superoxide liberated from SIN-1 exert an inhibitory effect against the cytotoxicity of MG132. SIN-1 may inhibit the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability that is associated with oxidative damage.     

Prevention of 7-ketocholesterol-induced mitochondrial damage and cell death by calmodulin inhibition

Oxysterols such as 7-ketocholesterol and 25-hydroxycholesterol formed under enhanced oxidative stress in the brain are suggested to induce neuronal cell death. The present study investigated the effect of calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) against the cytotoxicity of 7-ketocholesterol in relation to the mitochondria-mediated cell death process and oxidative stress. PC12 cells exposed to 7-ketocholesterol revealed nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. N-Acetylcysteine, trolox, carboxy-PTIO and Mn-TBAP reduced the cytotoxic effect of 7-ketocholesterol. Calmodulin antagonists attenuated the 7-ketocholesterol-induced nuclear damage, formation of the mitochondrial permeability transition and cell viability loss in PC12 cells. The results suggest that calmodulin antagonists may prevent the 7-ketocholesterol-induced viability loss in PC12 cells by suppressing formation of the mitochondrial permeability transition, leading to the release of cytochrome c and subsequent activation of caspase-3. The effects seem to be ascribed to their depressant action on the formation of reactive oxygen species and depletion of GSH. The findings suggest that calmodulin inhibition may exhibit a protective effect against the neurotoxicity of 7-ketocholesterol.     

Econazole attenuates cytotoxicity of 1-methyl-4-phenylpyridinium by suppressing mitochondrial membrane permeability transition

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The effect of econazole against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells was assessed in relation to the mitochondrial membrane permeability changes. Treatment of PC12 cells with MPP(+) resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. Econazole (0.25-2.5 microM) inhibited the cytotoxicity of MPP(+) or rotenone. The addition of econazole (0.5 microM) significantly attenuated the MPP(+)-induced mitochondrial damage, elevation of intracellular Ca(2+) level and cell death. However, because of the cytotoxicity, econazole at 5 microM did not attenuate the toxicity of MPP(+). The results show that econazole at the low concentrations may reduce the MPP(+)-induced viability loss in PC12 cells by suppressing the mitochondrial permeability transition, leading to activation of caspase-3 and the elevation of intracellular Ca(2+) levels, which are associated with the increased formation of ROS and depletion of GSH.     

How does conserved dopamine neurotrophic factor protect against and rescue neurodegeneration of PC12 cells?

Conserved dopamine neurotrophic factor protects and rescues dopaminergic neurodegeneration induced by 6-hydroxydopamine in vivo,but its potential value in treating Parkinson’s disease remains controversial.Here,we used the proteasome inhibitors lactacystin and MG132 to induce neurodegeneration of PC12 cells.Afterwards,conserved dopamine neurotrophic factor was administrated as a therapeutic factor,both pretreatment and posttreatment.Our results showed that(1)conserved dopamine neurotrophic factor enhanced lactacystin/MG132-induced cell viability and morphology,and attenuated alpha-synuclein accumulation in differentiated PC12 cells.(2)Enzyme linked immunosorbent assay showed up-regulated 26S proteasomal activity in MG132-induced PC12 cells after pre-and posttreatment with conserved dopamine neurotrophic factor.Similarly,26S proteasome activity was upregulated in lactacystin-induced PC12 cells pretreated with conserved dopamine neurotrophic factor.(3)With regard proteolytic enzymes(specifically,glutamyl peptide hydrolase,chymotrypsin,and trypsin),glutamyl peptide hydrolase activity was up-regulated in lactacystin/MG132-administered PC12 cells after pre-and posttreatment with conserved dopamine neurotrophic factor.However,upregulation of chymotrypsin activity was only observed in MG132-administered PC12 cells pretreated with conserved dopamine neurotrophic factor.There was no change in trypsin expression.We conclude that conserved dopamine neurotrophic factor develops its neurotrophic effects by modulating proteasomal activities,and thereby protects and rescues PC12 cells against neurodegeneration.     

Modulation of 1-methyl-4-phenylpyridinium-induced mitochondrial dysfunction and cell death in PC12 cells by K<Subscript>ATP</Subscript> channel block

The present study investigated the effect of 5-hydroxydecanoate, a selective mitochondrial K(ATP) channel blocker, on the cytotoxicity of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells. 5-Hydroxydecanoate and glibenclamide (a cell surface and mitochondrial K(ATP) channel inhibitor) reduced the MPP(+)-induced cell death and GSH depletion and showed a maximal inhibitory effect at 5 and 10 microM, respectively. Addition of 5-hydroxydecanoate attenuated the MPP(+)-induced nuclear damage, changes in the mitochondrial membrane permeability and increase in the reactive oxygen species formation in PC12 cells. The results show that 5-hydroxydecanote may prevent the MPP(+)-induced viability loss in PC12 cells by suppressing formation of the mitochondrial permeability transition, leading to the cytochrome c release and caspase-3 activation. This effect appears to be accomplished by the inhibitory action on the formation of reactive oxygen species and the depletion of GSH. The blockade of mitochondrial K(ATP) channels seems to prevent the MPP(+)-induced neuronal cell damage.     

Comparison of the Protective Effect of Indole ��-carbolines and R-(-)-deprenyl Against Nitrogen Species-Induced Cell Death in Experimental Culture Model of Parkinson's Disease

Background

The membrane permeability transition of mitochondria has been suggested to be involved in toxic and oxidative forms of cell injury. Mitochondrial dysfunction is considered to play a critical role in neurodegeneration in Parkinson''s disease. Despite the suggestion that indole β-carbolines may be neurotoxic, these compounds provide a protective effect against cytotoxicity of other neurotoxins. In addition, the effect of indole β-carbolines on change in the mitochondrial membrane permeability due to reactive nitrogen species (RNS), which may lead to cell death, has not been clarified.

Methods

Differentiated PC12 cells were used as the experimental culture model for the investigation of neuronal cell injury, which occurs in Parkinson''s disease. The effect of indole β-carbolines (harmalol and harmine) on differentiated PC12 cells against toxicity of S-nitroso-N-acetyl-DL-penicillamine (SNAP) was determined by measuring the effect on the change in transmembrane potential, cytochrome c release, formation of ROS, GSH contents, caspase-3 activity and cell viability, and was compared to that of R-(-)-deprenyl.

Results

Specific inhibitors of caspases (z-LEHD.fmk, z-DQMD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol, melatonin, carboxy-PTIO and uric acid) depressed cell death in PC12 cells due to SNAP. β-Carbolines and R-(-)-deprenyl attenuated the SNAP-induced cell death and GSH depletion concentration dependently with a maximal inhibitory effect at 25-50 µM. The compounds inhibited the nuclear damage, decrease in mitochondrial transmembrane potential, cytochrome c release and formation of reactive oxygen species caused by SNAP in PC12 cells. β-Carbolines and R-(-)-deprenyl attenuated the H₂O₂-induced cell death and depletion of GSH.

Conclusions

The results suggest that indole β-carbolines attenuate the SNAP-induced viability loss in PC12 cells by inhibition of change in the mitochondrial membrane permeability, which may be caused by free radicals. Indole β-carbolines appear to exert a protective effect against the nitrogen species-mediated neuronal cell injury in Parkinson''s disease comparable to R-(-)-deprenyl.     

Protective effect of harmaline and harmalol against dopamine- and 6-hydroxydopamine-induced oxidative damage of brain mitochondria and synaptosomes, and viability loss of PC12 cells

The present study elucidated the protective effect of beta-carbolines (harmaline, harmalol and harmine) against oxidative damage of brain mitochondria, synaptosomes and PC12 cells induced by either dopamine or 6-hydroxydopamine. Harmaline, harmalol and antioxidant enzymes (superoxide dismutase/SOD and catalase) decreased the alteration of mitochondrial swelling and membrane potential induced by 200 microM dopamine or 100 microM 6-hydroxydopamine. Deprenyl attenuated the dopamine-induced mitochondrial dysfunction but did not reduce the effect of 6-hydroxydopamine. While beta-carbolines inhibited the electron flow in mitochondria, they did not enhance the depressant effect of catecholamines. beta-Carbolines and antioxidant enzymes reversed the depression of synaptosomal Ca2+ uptake induced by 10 microM catecholamines. The compounds inhibited the catecholamine-induced thioredoxin reductase inhibition, thiol oxidation and carbonyl formation in mitochondria and synaptosomes. beta-Carbolines decreased the reactive species-induced deoxyribose degradation. Harmaline and harmalol reduced the catecholamine-induced loss of the transmembrane potential and of cell viability in PC12 cells. beta-Carbolines alone did not show a significant cytotoxic effect on PC12 cells. The results suggest that beta-carbolines may attenuate the dopamine- or 6-hydroxydopamine-induced alteration of brain mitochondrial and synaptosomal functions, and viability loss in PC12 cells, by a scavenging action on reactive oxygen species and inhibition of thiol oxidation.     

Proteasome inhibition results in increased poly-ADP-ribosylation: implications for neuron death

This study demonstrates the ability of proteasome inhibitors (lactacystin, MG 115, MG 132) adenosine diphosphate to induce a time- and dose-dependent increase in poly-ADP-ribosylation (PAR) in the neural PC6 cell line, a subclone of PC12 cells. Elevated levels of PAR contribute to the toxicity associated with impaired proteasome activity, based on the ability of PAR inhibitors to ameliorate the toxicity associated with the application of lactacystin, MG 115, and MG 132. Proteasome inhibitors induced the accumulation of PAR and neuron death in primary hippocampal neuron cultures, which were both ameliorated by treatment with PAR inhibitors. Together, these data indicate a role for increased PAR in the toxicity associated with proteasome inhibition, and suggest that inhibitors of PAR may provide neuroprotection in conditions where proteasome inhibition occurs.     

Oxidative stress, induced by 6-hydroxydopamine, reduces proteasome activities in PC12 cells

Mutations in familial Parkinson’s disease (PD) have been associated with the failure of protein degradation through the ubiquitin-proteasome system (UPS). Impairment of proteasome function has also been suggested to play a role in the pathogenesis of sporadic PD. We examined the proteasome activity in PC12 cells treated with 6-hydroxydopamine (6-OHDA), the dopamine synthetic derivate used in models of PD. We found that 6-OHDA treatment increased protein oxidation, as indicated by carbonyl group accumulation, and increased caspase-3 activity. In addition, there was an increase in trypsin-, chymotrypsin-, and postacidic-like proteasome activities in cells treated with 10–100 μM 6-OHDA, whereas higher doses caused a marked decline. 6-OHDA exposure also increased mRNA expression of the 19S regulatory subunit in a dose-dependent manner, whereas the expression of 20S- and 11S-subunit mRNAs did not change. Administration of the antioxidant N-acetylcysteine to 6-OHDA-treated cells prevented the alteration in proteasome functions. Moreover, reduction in cell viability owing to administration of proteasome inhibitor MG132 or lactacystin was partially prevented by the endogenous antioxidant-reduced glutathione. In conclusion, our data indicate that mild oxidative stress elevates proteasome activity in response to increase in protein damage. Severe oxidative insult might cause UPS failure, which leads to protein aggregation and cell death. Moreover, in the case of UPS inhibition or failure, the blockade of physiological reactive oxygen species production during normal aerobic metabolism is enough to ameliorate cell viability. Control of protein clearance by potent, brain-penetrating antioxidants might act to slow down the progression of PD.     

促红细胞生成素对1-甲基-4-苯基吡啶损伤的PC12细胞的保护作用(英文)

目的探讨促红细胞生成素(erythropoietin,EPO)对1-甲基-4-苯基吡啶离子(MPP+)诱导的PC12细胞变性损伤的保护作用及机制。方法用MPP+处理PC12细胞制作帕金森病细胞模型,采用四甲基偶氮唑蓝法检测暴露于不同浓度EPO后细胞的活性;流式细胞术与DNA断端原位标记法(terminal deoxynucleotidyl transferase dUTPnick end labeling, TUNEL)检测各组的细胞凋亡率;免疫印迹法检测不同处理组PC12细胞Bcl-2和Bax的表达,并采用荧光法观察不同处理组PC12细胞活性氧(reactive oxygen species,ROS)与线粒体膜电位水平以及caspase-3活性的变化。结果 MPP+可以使PC12细胞存活率下降,凋亡率增高;同时PC12细胞内ROS增多,线粒体膜电位下降。MPP+还可以明显地提高Bax/Bcl-2比值并激活caspase-3。而EPO可以抑制这些由MPP+引发的改变,并在1 U/mL时发挥最大保护作用。结论 EPO可抑制MPP+诱导的PC12细胞死亡,其作用机制可能与其自身抗氧化和抗凋亡的特性有关。     

7-Ketocholesterol enhances 1-methyl-4-phenylpyridinium-induced mitochondrial dysfunction and cell death in PC12 cells

Summary. The present study investigated the promoting effect of oxysterol 7-ketocholesterol against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP⁺) in differentiated PC12 cells. 7-Ketocholesterol significantly enhanced the MPP⁺-induced nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. N-Acetylcysteine, ascorbate, trolox, carboxy-PTIO and Mn-TBAP reduced the cytotoxic effect of MPP⁺ in the presence of 7-ketocholesterol. The results indicate that 7-ketocholesterol shows a synergistic effect against the cytotoxic effect of MPP⁺. 7-Ketocholesterol may enhance the MPP⁺-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of reactive oxygen species and depletion of GSH. The findings suggest that 7-ketocholesterol as a promoting agent for the formation of mitochondrial permeability transition may enhance the toxic neuronal cell injury.     

Regulation of Wallerian degeneration and nerve growth factor withdrawal-induced pruning of axons of sympathetic neurons by the proteasome and the MEK/Erk pathway

Treatment of transected distal axons of rat sympathetic neurons in compartmented cultures with MG132 (5 microM) and other inhibitors of proteasome activity, preserved axonal mitochondrial function, assessed by Mitotracker-Orange and MTT staining, for at least 24 h. MG132 similarly protected axons from undergoing branch elimination (pruning) in response to local NGF deprivation. Axons protected by MG132 displayed persistent phosphorylation of Erk1/2, and pharmacological inhibition of MEK activity with U0126 (50 microM) restored rapid axonal degeneration. Therefore, the proteasome does not appear to be necessary as a general effector of protein degradation during axonal degeneration. Rather, the proteasome functions in the regulation of signaling pathways that control axonal survival and degeneration. Specifically, the down-regulation of the MEK/Erk pathway by the proteasome plays roles in Wallerian degeneration of severed axons and axonal pruning in response to local NGF deprivation. Identification of the pathways that regulate axonal survival and degeneration will provide possible target sites for pharmacological treatments of neurodegenerative diseases and traumatic injury.     

PINK1 mutants associated with recessive Parkinson's disease are defective in inhibiting mitochondrial release of cytochrome c

Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinson's disease (PARK6). We investigated molecular mechanisms underlying PINK1 neuroprotective function and PARK6 mutation-induced loss of PINK1 function. Overexpression of wild-type PINK1 blocked mitochondrial release of apoptogenic cytochrome c, caspase-3 activation and apoptotic cell death induced by proteasome inhibitor MG132. N-terminal truncated PINK1 (NDelta35), which lacks mitochondrial localization sequence, did not block MG132-induced cytochrome c release and cytotoxicity. Despite mitochondrial expression, PARK6 mutant (E240K), (H271Q), (G309D), (L347P), (E417G) and C-terminal truncated (CDelta145) PINK1 failed to inhibit MG132-induced cytochrome c release and caspase-3 activation. Overexpression of wild-type PINK1 blocked cytochrome c release and cell death caused by atractyloside, which opens mitochondrial permeability transition pore (mPTP). PARK6 PINK1 mutants failed to inhibit atractyloside-induced cytochrome c release. These results suggest that PINK1 exerts anti-apoptotic effect by inhibiting the opening of mPTP and that PARK6 mutant PINK1 loses its ability to prevent mPTP opening and cytochrome c release.     

Differential susceptibility of naive and differentiated PC-12 cells to methylglyoxal-induced apoptosis: influence of cellular redox

Neuropathologies have been associated with neuronal de-differentiation and oxidative susceptibility. To address whether cellular states determines their oxidative vulnerability, we have challenged naive (undifferentiated) and nerve growth factor-induced differentiated pheochromocytoma (PC12) with methylglyoxal (MG), a model of carbonyl stress. MG dose-dependently induced greater apoptosis (24 h) in naive (nPC12) than differentiated (dPC12) cells. This enhanced nPC12 susceptibility was correlated with a high basal oxidized cellular glutathione-to-glutathione disulfide (GSH/GSSG) redox and an MG-induced GSH-to-Disulfide (GSSG plus protein-bound SSG) imbalance. The loss of redox balance occurred at 30 min post-MG exposure, and was prevented by N-acetylcysteine (NAC) that was unrelated to de novo GSH synthesis. NAC was ineffective when added at 1h post-MG, consistent with an early window of redox signaling. This redox shift was kinetically linked to decreased BcL-2, increased Bax, and release of mitochondrial cytochrome c which preceded caspase-9 and -3 activation and poly ADP-ribose polymerase (PARP) cleavage (1-2 h), consistent with mitochondrial apoptotic signaling. The blockade of apoptosis by cyclosporine A supported an involvement of the mitochondrial permeability transition pore. The enhanced vulnerability of nPC12 cells to MG and its relationship to cellular redox shifts will have important implications for understanding differential oxidative vulnerability in various cell types and their transition states.     

Beta-estradiol attenuate amyloid beta-peptide toxicity via nicotinic receptors

A number of epidemiological studies suggest that estrogen therapy is linked to a reduced risk of developing Alzheimer's disease (AD). The present study was conducted to evaluate the effect of 17beta-estradiol on beta-amyloid (Abeta)-induced toxicity and was performed in rat pheochromocytoma PC 12 cells by measuring the mitochondrial activity. 17Beta-estradiol (10(-5), 10(-6) and 10(-8) M) attenuated Abeta(25-35)-induced toxicity in PC 12 cells. The neuroprotective effect of 17beta-estradiol (10(-5) M) was prevented in the presence of the nicotinic antagonists methyllycaconitine (MLA) and mecamylamine, suggesting an interaction probably via the alpha7 nicotinic receptor subtype. Chronic treatment with 17beta-estradiol (10(-10)-10(-5) M) alone did not change the number of [3H]epibatidine binding sites in human neuroblastoma SH-SY5Y cells and rat PC 12 cells, but significantly prevented the enhanced [3H]epibatidine binding in nicotine-treated PC 12 cells. This study demonstrates that 17beta-estradiol exerts neuroprotective effects which might involve interaction with the alpha7 nicotinic receptor subtype.     

Low concentrations of 1-methyl-4-phenylpyridinium ion induce caspase-mediated apoptosis in human SH-SY5Y neuroblastoma cells

There is growing evidence that apoptotic mechanisms underlie the neurodegeneration leading to Parkinson's disease. 1-Methyl-4-phenylpyridinium ion (MPP(+)), the active metabolite of the parkinsonism-inducing drug MPTP, induced apoptosis in cultures of human SH-SY5Y neuroblastoma cells. Nuclear fragmentation, DNA laddering, and a 20% decrease in viability were seen after a 4-day incubation with 5 microM MPP(+). Cell viability decreased by 40% at 100 microM MPP(+), but the degree of apoptosis was not correlatively increased. The MPP(+)-induced apoptosis was completely prevented by the broad caspase inhibitor zVAD.fmk but not by the caspase-8 inhibitor IETD.fmk. Furthermore, MPP(+) had no effect on the levels of Fas or Fas-L, suggesting lack of activation of the Fas-L/Fas/caspase-8 pathway of apoptosis. There was no evidence of mitochondrial dysfunction at 5 microM MPP(+): No differences were seen in transmembrane potential or in cytochrome c release from controls. At 100 microM MPP(+), the mitochondrial potential decreased, and cytoplasmic cytochrome c and caspase-9 activation increased slightly. At both low and high concentrations of MPP(+), VDVADase and DEVDase activities increased. We conclude that MPP(+) can induce caspase-mediated apoptosis, which is prevented by caspase inhibition, at concentrations lower than those needed to trigger mitochondrial dysfunction and closer to those found in the brains of MPTP-treated animals.     

Purkinje cell protein 4 positively regulates neurite outgrowth and neurotransmitter release

Purkinje cell protein 4 (PCP4), also called brain-specific polypeptide 19 (PEP19), is a neurospecific, small calmodulin-binding protein that binds both calcium-free and calcium-binding calmodulin to regulate the calmodulin-mediated signal. The expression level of this molecule is decreased in the brain in Alzheimer's disease, Huntington's disease, and alcoholism. However, little is known of the function of PCP4 regarding neuronal or neuroendocrine cell differentiation and neurotransmitter release. To address this, we established a PCP4 tetracycline-inducible rat chromaffin cell line, PC12. When PCP4 expression was induced with doxcycline, neurite outgrowth was significantly advanced in the presence of nerve growth factor (NGF) and dibutyryl cAMP, which was inhibited by W-7, a calmodulin inhibitor, and PD98059, an ERK inhibitor. In addition, size of the cell body also was increased by treatment with NGF in the PCP4-induced PC12 cells. Constitutive and potassium-evoked release of acetylcholine and dopamine was increased and apoptosis induced by hydrogen peroxide (H(2)O(2)) was inhibited in PCP4-induced PC12 cells. On the other hand, knockdown of PCP4 by siRNA transfection decreased neurite outgrowth and dopamine release and increased H(2)O(2)-induced apoptosis in PC12 cells. These results indicate that PCP4 promotes neuroendocrine cell differentiation and neurotransmitter release by activating calmodulin function.     

Lamotrigine inhibition of rotenone- or 1-methyl-4-phenylpyridinium-induced mitochondrial damage and cell death

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the effect of antiepileptic lamotrigine against the cytotoxicity of mitochondrial respiratory complex I inhibitors rotenone and 1-methyl-4-phenylpyridinium (MPP+) in relation to the mitochondria-mediated cell death process and oxidative stress. Both rotenone and MPP+ induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Lamotrigine significantly attenuated the rotenone- or MPP+-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. The preventive effect of lamotrigine against the toxicity of rotenone was greater than its effect on that of MPP+. The results show that lamotrigine seems to reduce the cytotoxicity of rotenone and MPP+ by suppressing the mitochondrial permeability transition formation, leading to cytochrome c release and subsequent activation of caspase-3. The preventive effect may be ascribed to its inhibitory action on the formation of reactive oxygen species and depletion of GSH. Lamotrigine seems to exert a protective effect against the neuronal cell injury due to the mitochondrial respiratory complex I inhibition.     

Catecholamines in murine bone marrow derived mast cells

Cultured murine bone marrow derived mast cells (BMMC) were found to store high levels of dopamine (3753+/-844 pg/10(7) cells) and occasionally produce norepinephrine and epinephrine. The catecholamine synthesis inhibitor, alpha-methyl-para-tyrosine, decreased intracellular catecholamine concentrations, and activation with ionomycin stimulated dopamine release. Neither dopaminergic receptor antagonists nor exogenous dopamine < or =10 microM affected IL-3-induced cell proliferation. High exogenous dopamine (20-100 microM) decreased proliferation and increased apoptosis, and the anti-oxidant ascorbic acid prevented these effects. Increased expression of the anti-apoptotic factor Bcl-2 or loss of pro-apoptotic Bax expression attenuated dopamine-induced apoptosis, suggesting the apoptosis proceeds through a mitochondrial pathway.     

丹参川芎嗪注射液对Aβ损伤的PC12细胞保护作用及机制研究

目的 探讨丹参川芎嗪注射液对Aβ损伤的PC12细胞可能的保护作用及机制。方法 将PC12细胞分为5组:空白对照组(未加任何处理药物)、Aβ诱导组(20 μmol/L Aβ处理组)和预处理组(分别加入浓度为5 ml/L、10 ml/L、20 ml/L的丹参川芎嗪注射液孵育24 h后加20 μmol/L Aβ),通过CCK-8法检测细胞增殖活性,流式细胞术(FCM)检测细胞凋亡率,Hoechst 33258染色观察PC12细胞核的改变,荧光分光光度计测定LDH、SOD、GSH及caspase-3活性水平,免疫组织化学方法观察细胞色素C(Cyt-C)蛋白释放水平,Western Blot检测Bcl-2的表达水平。结果 丹参川芎嗪注射液(5、10、20 ml/L)预处理对Aβ诱导的PC12细胞损伤有较好的保护作用,其保护作用随着药物浓度的增加而增强。它能增加Aβ损伤的PC12细胞增殖活力,减少Aβ诱导的PC12细胞凋亡,降低细胞核凝聚现象,抑制Aβ损伤的PC12细胞LDH释放,增强SOD和GSH活性,促进Cyt-C在细胞内表达,降低caspase-3活性,促进Bcl-2的表达。结论 丹参川芎嗪注射液对Aβ诱导的PC12细胞损伤具有与线粒体通路相关的保护作用,其保护作用与它抑制细胞凋亡、抗氧化应激、维持线粒体正常功能、抑制caspase-3的激活、促进抗凋亡因子Bcl-2的表达有关。