Treatment effects of EGb 761 and cholinesterase inhibitors – why available studies do not demonstrate superiority of the latter

Based on simple comparisons of drug-placebo differences found in clinical trials, it has repeatedly been concluded that EGb 761 is less effective in the treatment of Alzheimer's disease than cholinesterase (ChE) inhibitors. However, the data of pivotal studies with both types of drugs show that drug-placebo differences in cognitive outcomes are more influenced by the degrees of deterioration of the placebo groups than by changes of the actively treated groups. Since the former are determined by characteristics of the patient samples and the therapeutic environment, but not by drug effects, it is concluded that direct comparisons of drug-placebo differences are inappropriate to assess the relative potencies of anti-dementia drugs. Comparisons have to take into account the different unspecific influences. The currently available data do not support the notion of superiority of ChE inhibitors over EGb 761.     

The Ginkgo biloba extract (EGb 761) protects and rescues hippocampal cells against nitric oxide-induced toxicity: involvement of its flavonoid constituents and protein kinase C

An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. Numerous studies have shown that the Ginkgo biloba extract EGb 761 is a NO scavenger with neuroprotective properties. However, the mechanisms underlying its neuroprotective ability remain to be fully established. Thus, we investigated the effect of different constituents of EGb 761, i.e., flavonoids and terpenoids, against toxicity induced by NO generators on cells of the hippocampus, a brain area particularly susceptible to neurodegenerative damage. Exposure of rat primary mixed hippocampal cell cultures to either sodium nitroprusside (SNP; 100 microM) or 3-morpholinosydnonimine resulted in both a decrease in cell survival and an increase in free radical accumulation. These SNP-induced events were blocked by either EGb 761 (10-100 microg/ml) or its flavonoid fraction CP 205 (25 microg/ml), as well as by inhibitors of protein kinase C (PKC; chelerythrine) and L-type calcium channels (nitrendipine). In contrast, the terpenoid constituents of EGb 761, known as bilobalide and ginkgolide B, as well as inhibitors of phospholipases A [3-[(4-octadecyl)benzoyl]acrylic acid (OBAA)] and C (U-73122), failed to display any significant effects. Moreover, EGb 761 (50 microm) CP 205 (25 microg/ml), and chelerythrine were also able to rescue hippocampal cells preexposed to SNP (up to 1 mM). Finally, EGb 761 (100 microg/ml) was shown to block the activation of PKC induced by SNP (100 microM). These data suggest that the protective and rescuing abilities of EGb 761 are not only attributable to the antioxidant properties of its flavonoid constituents but also via their ability to inhibit NO-stimulated PKC activity.     

Depressant Effect of Mitochondrial Respiratory Complex Inhibitors on Proteasome Inhibitor-Induced Mitochondrial Dysfunction and Cell Death in PC12 Cells

The addition of rotenone (inhibitor of respiratory complex I), 3-nitropropionic acid (complex II inhibitor), harmine (inhibitor of complexes I and II) and cyclosporin A (CsA, an inhibitor of the mitochondrial permeability transition) reduced the nuclear damage, loss 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 in differentiated PC12 cells treated with MG132, a proteasome inhibitor. Meanwhile, rotenone, 3-nitropropionic acid and harmine did not affect the inhibitory effect of CsA or trifluoperazine (an inhibitor of the mitochondrial permeability transition and calmodulin antagonist) on the cytotoxicity of MG132. The results suggest that proteasome inhibition-induced mitochondrial dysfunction and cell injury may be attenuated by the inhibitions of respiratory chain complex I and II. The cytoprotective effect of the mitochondrial permeability transition prevention not appears to be modulated by respiratory complex inhibition.     

Variation of ginkgolides and bilobalide contents in leaves and cell cultures of<Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

Ginkgolides (GK) and bilobalide are valuable compounds that belong to the lactone terpene. The contents of these metabolites were determined by HPLC from female and male tree ofGinkgo biloba L. The productivity ofG. biloba cells was also compared with the corresponding individual trees. High variations in the ginkgolides and bilobalide were observed from different individuals, plant parts, and cultured cells. The ginkgolides and bilobalide contents were different depending on the plant parts. Callus was obtained from various plant tissues, and NAA was better at callogenesis than 2,4-D in both the female and male trees. The plants and their corresponding cells showed considerable variation in their ginkgolides and bilobalide concentrations. The ginkgolides and bilobalide contents were not correlated with the production between dominant trees and their corresponding cells. Light irradiation enhanced the production of GK-A and GK-B, however, the concentration of bilobalide decreased under dark conditions.     

Carbachol and Bradykinin Increase the Production of Diacylglycerol from Sources Other than Inositol-Containing Phospholipids in PC12 Cells

Both carbachol and bradykinin increased diacylglycerol formation in PC12 pheochromocytoma cells. The effect of carbachol was apparent only in cells that had been treated with nerve growth factor. Incubation of the cells in Ca2(+)-free medium attenuated carbachol-stimulated diacylglycerol formation but did not reduce the response to bradykinin. Pretreatment of the cells with pertussis toxin did not affect either carbachol- or bradykinin-stimulated diacylglycerol formation; therefore, the inhibitory guanine nucleotide Gi probably does not mediate this response. The time course of carbachol-stimulated diacylglycerol accumulation did not coincide with the time course of inositol 1,4,5-trisphosphate (IP3) production. IP3 was elevated at the earliest time measured, 15 s, and then slowly declined so that by 5 min IP3 levels were only 50% of maximal. Diacylglycerol levels, in contrast, were not elevated for the first 2 min and then peaked at 5 min. These data indicate that hydrolysis of phosphatidylinositol 4,5-bisphosphate was not the major source of the diacylglycerol peak at 5 min. To investigate the source of diacylglycerol, I examined the fatty acid composition of the diacylglycerol by prelabeling the cells with [3H]palmitic acid and [14C]stearic acid. The 14C/3H ratio in diacylglycerol should reflect the phospholipid(s) from which it is derived. The 14C/3H ratio of the increment in diacylglycerol produced by carbachol and bradykinin was intermediate between the 14C/3H ratios of phosphatidylcholine and phosphatidylinositol. The 14C/3H ratio in triacylglycerol was similar to that of phosphatidylcholine. These data indicate that carbachol and bradykinin stimulate the formation of diacylglycerol from sources other than inositol-containing phospholipids; phosphatidylcholine and triacylglycerol are two possible sources of this diacylglycerol.     

Cytokinins in the leaves of Ginkgo biloba. II. Metabolism and transport of 8 (14C) zeatin applied to shoot explants

Irrespective of their age, leaves of Ginkgo biloba metabolised applied 8 (¹⁴C) zeatin to compounds of similar chromatographic properties. Glucosylation is apparently not a normal feature of cytokinin metabolism in immature leaves. However, the application of zeatin to these leaves did result in the formation of metabolites which co-chromatographed with glucosylated cytokinins. As far as cytokinin metabolism is concerned therefore, this application of excess zeatin allowed immature leaves to behave as mature or senescing leaves. Overall metabolism was fastest in immature leaves. From the metabolites formed it would appear as if oxidation, which resulted in the formation of a metabolite which co-eluted with N-(purin-6-yl)glycine, was also important in immature leaves. In senescing leaves glucosylation was the major form of metabolism. Extraction and re-application of the polar metabolites (formed from zeatin) to mature leaves resulted in the formation of compounds which co-chromatographed with zeatin. This suggests that these compounds could serve as precursors for zeatin or could be hydrolysed to form zeatin.Very little of the applied radioactivity was exported from the leaves irrespective of their physiological age. When the metabolites, obtained after zeatin application to mature leaves, were extracted and reapplied to the leaves, export of radioactive material was much improved. The results suggest that should cytokinins such as zeatin be translocated to mature leaves of this deciduous gymnosperm their export from the leaves would be unlikely unless first metabolised. In all probability the metabolites concerned are cytokinin glucosides.The financial support of the C.S.I.R., Pretoria, is gratefully acknowledged.     

胰岛素对PC12细胞神经型一氧化氮合酶表达及活性的影响

为探讨胰岛素对神经细胞中神经型一氧化氮合酶（nNOS）的表达及活性的影响,应用流式细胞术、原位杂交、电子自旋共振等技术方法研究胰岛素对PC12细胞中神经型一氧化氮合酶的影响.胰岛素作用PC12细胞9 h 后,神经型一氧化氮合酶的免疫荧光强度显著升高,且呈浓度依赖关系,其最大效应为对照的(155±13)%(P＜0.01, n=3, t-test).加入胰岛素(16 mU/L, 6 h)也能够显著上调nNOS mRNA的表达,为对照的(182±13)%(P＜0.01, n=3, t-test).另外加入胰岛素(16 mU/L)作用9 h后,神经型一氧化氮合酶的活性也显著升高,为对照的(167±15)%(P＜0.01, n=4, t-test).由上述结果可知,胰岛素对PC12细胞的神经型一氧化氮合酶的表达及活性有上调作用.     

<Emphasis Type="Italic">Ginkgo biloba</Emphasis> extract (EGb761) and FK506 preserve energy metabolites in the striatum during focal cerebral ischemia and reperfusion in gerbils monitored by microdialysis

Cell death after cerebral ischemia is mediated by the accumulation of excitatory amino acids, calcium influx into cells and the generation of free radicals. The aim of this study was to evaluate changes in energy-related metabolites in the striatum of gerbils subjected to focal cerebral ischemia after pretreatment withGinkgo biloba extract (EGb761), a well-known antioxidant, and FK506, a calcium-dependent phosphatase calcineurin inhibitor. Ischemia was induced by occlusion of the right common carotid artery and the right middle cerebral artery for 60 min. A microdialysis probe was inserted into the right striatum to monitor extracellular glucose, lactate and pyruvate levels. This study showed decreases in glucose (10% of the baseline), pyruvate (20% of the baseline) and lactate (60% of the baseline), and a 5-fold increase in the lactate to pyruvate ratio during ischemia in the control group. Both EGb761 treatment and the combination (EGb761 and FK506) therapy significantly preserved glucose (50% of the baseline) and pyruvate (60% of the baseline) levels during ischemia. The marked increase in the lactate to pyruvate ratio was not observed in the combination group. These results suggest that preservation of cellular energy metabolism during cerebral ischemia and after restoration with reperfusion may contribute to the neuroprotective effects of EGb761 and FK506.     

Hydrogen Peroxide Induces Lysosomal Protease Alterations in PC12 Cells

Alterations in lysosomal proteases have been implicated in many neurodegenerative diseases. The current study demonstrates a concentration-dependent decrease in PC12 cell viability and transient changes in cystatin C (CYSC), cathepsin B (CATB), cathepsin D (CATD) and caspase-3 following exposure to H₂O₂. Furthermore, activation of CATD occurred following exposure to H₂O₂ and cysteine protease suppression, while inhibition of CATD with pepstatin A significantly improved cell viability. Additionally, significant PARP cleavage, suggestive of caspase-3-like activity, was observed following H₂O₂ exposure, while inhibition of caspase-3 significantly increased cell viability compared to H₂O₂ administration alone. Collectively, our data suggest that H₂O₂ induced cell death is regulated at least in part by caspase-3 and CATD. Furthermore, cysteine protease suppression increases CATD expression and activity. These studies provide insight for alternate pathways and potential therapeutic targets of cell death associated with oxidative stress and lysosomal protease alterations.     

Nocardia asteroides culture filtrates cause dopamine depletion and cytotoxicity in PC12 cells

Experimental infection of BALB/c mice with the gram-positive bacterium Nocardia asteroides produces marked loss of nigrostriatal dopamine neurons, resulting in striatal dopamine depletion. To investigate the mechanism(s) responsible for this neuronal loss, we evaluated the influence of N. asteroides cell-free culture filtrates on rat pheochromocytoma PC12 cells, an in vitro model for dopamine neurons. Changes in cell viability and cell numbers were minimal after 24 h, but increased with longer incubation. In contrast, dopamine depletion occurred after 30 min incubation, and was greater with GUH-2 filtrate than with filtrate from the less virulent strain 10905. Incubation with the culture filtrate decreased viability in neuroblastoma and glioma cell lines, indicating that cytotoxic effects were not limited to dopaminergic cells. These findings suggest that the loss of nigrostriatal dopamine neurons and concomitant striatal dopamine depletion in Nocardia-infected mice may be due, at least in part, to the neurotoxicity of nocardial secretory products.     

Effect of Agmatine Against Cell Death Induced by NMDA and Glutamate in Neurons and PC12 Cells

1. Aims: Agmatine is an endogenous guanido amine and has been shown to be neuroprotective in vitro and in vivo. The aims of this study are to investigate whether agmatine is protective against cell death induced by different agents in cultured neurons and PC12 cells.2. Methods: Cell death in neurons, cultured from neonatal rat cortex, was induced by incubating with (a) NMDA (100 M) for 10 min, (b) staurosporine (protein kinase inhibitor, 100 nM) for 24 h, and (c) calcimycin (calcium ionophore, 100 nM) for 24 h in the presence and absence of agmatine (1 M to 1 mM). Cell death in PC12 cells was induced by exposure to glutamate (10 mM), staurosporine (100 nM), and calcimycin (100 nM). The activity of lactate dehydrogenase (LDH) in the medium was measured as the marker of cell death and normalized to cellular LDH activity.3. Results: Agmatine significantly reduced the medium LDH in NMDA-treated neurons but failed to reduce the release of LDH induced by staurosporin or calcimycin. In PC12 cells, agmatine significantly reduced LDH release induced by glutamate exposure, but not by staurosporine or calcimycin. Agmatine itself neither increased LDH release nor directly inhibited the enzyme activity.4. Conclusion: We conclude that agmatine protects against NMDA excitotoxicity in neurons and PC12 cells but not the cell death induced by protein kinase blockade or increase in cellular calcium.     

PC12 Cells Overexpressing Tissue Plasminogen Activator Regenerate Neurites to a Greater Extent and Migrate Faster than Control Cells in Complex Extracellular Matrix

Abstract: PC12 cells were stably transfected with expression vectors containing rat tissue plasminogen activator (tPA) under control of either a cytomegalovirus or rous sarcoma virus promoter. Cell lines were characterized using protease assays, ELISAs, immunoblots, northern blots, and Southern blots. Control PC12 cells or cells containing vectors alone released about 1 pg tPA/cell/24 h, whereas cells stably transfected with a tPA cDNA released 2–5 pg tPA/cell/24 h. A strong correlation existed between the amount of tPA released and the ability of cells to degrade extracellular matrix. Experiments with protease inhibitors and antibodies against tPA and plasminogen indicated that degradation of matrix involved tPA-generated plasmin and that the amount of matrix degraded was dependent on the amount of tPA released. Cells expressing high levels of tPA migrated on a three-dimensional matrix about twice as fast as control cells and regenerated neurites within three-dimensional gels of Matrigel to a greater extent than control cells. Antibodies that inhibited tPA and plasminogen decreased migration and neurite regeneration, indicating that tPA was involved in both events. PC12 cells overexpressing tPA should provide a useful model system for investigating neural functions of tPA including its role in migration and regeneration.     

Inhibition of Dopamine Biosynthesis by Protoberberine Alkaloids in PC12 Cells

Berberine and palmatine exhibit a mild and competitive inhibition on bovine adrenal tyrosine hydroxylase (EC 1.14.16.2; TH). In this study, the inhibitory effects of protoberberine alkaloids (such as berberine, palmatine and coptisine) on dopamine biosynthesis in PC12 cells were investigated. Treatment with berberine and palmatine showed 53.7% and 61.0% inhibition of dopamine content in PC12 cells at a concentration of 20 M for 24 hr, respectively. However, coptisine did not reduce dopamine content. The IC₅₀ values of berberine and palmatine were 18.6 M and 7.9 M. Dopamine content was lowered at 6 hr and reached the minimal level at 24 hr after exposure to berberine and palmatine at 20 M. The decreased dopamine level was maintained up to 48 hr, and then recovered to the control level at about 72 hr. TH activity was inhibited at 6 hr following treatment with berberine and palmatine, and was maintained at a reduced level up to 36 hr in PC12 cells (21–27% inhibition at 20 M), whereas TH mRNA level was not found to alter for 24 hr. However, the intracellular Ca²⁺ concentration decreased by treatment with berberine and palmatine at 20 M by 22–26% inhibition relative to the control level in PC12 cells. These results give evidence that berberine and palmatine lead to decreased dopamine content by inhibition of TH activity but not by regulation of TH gene expression in PC12 cells.     

Antioxidant Effect of Phenelzine on MPP+-Induced Cell Viability Loss in Differentiated PC12 Cells

Phenelzine, deprenyl, and antioxidants (SOD, catalase, ascorbate, or rutin) reduced the loss of cell viability in differentiated PC12 cells treated with 250 M MPP⁺, whereas N-acetylcysteine and dithiothreitol did not inhibit cell death. Phenelzine reduced the condensation and fragmentation of nuclei caused by MPP⁺ in PC12 cells. Phenelzine and deprenyl prevented the MPP⁺-induced decrease in mitochondrial membrane potential, cytochrome c release, formation of reactive oxygen species, and depletion of GSH in PC12 cells. Phenelzine revealed a scavenging action on hydrogen peroxide and reduced the hydrogen peroxide–induced cell death in PC12 cells, whereas deprenyl did not depress the cytotoxic effect of hydrogen peroxide. Both compounds reduced the iron and EDTA-mediated degradation of 2-deoxy-d-ribose degradation. The results suggest that phenelzine attenuates the MPP⁺-induced viability loss in PC12 cells by reducing the alteration of mitochondrial membrane permeability that seems to be mediated by oxidative stress.     

l-NAME has Opposite Effects on the Productions of S-adenosylhomocysteine and S-adenosylmethionine in V12-H-Ras and M-CR3B-Ras Pheochromocytoma Cells

Homocysteine is a sulfur-containing, nonproteinogenic, neurotoxic amino acid biosynthesized during methyl cycles after demethylation of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) and subsequent hydrolysis of SAH into homocysteine and adenosine. Formed homocysteine is either catabolized into cystathionine (transsulfuration pathway) by cystathionine β-synthase, or remethylated into methionine (remethylation pathway) by methionine synthase. To demonstrate the specificity of Ras-elicited effects on the activity of methyl cycles, wild-type pheochromocytoma PC12, mutant oncogenic rasH gene (MVR) expressing PC12 pheochromocytoma and normal c-rasH stably transfected M-CR3B cells were incubated with the N^ω-nitro-l-arginine methyl ester (l-NAME), and manumycin, (inhibitors of nitric oxide synthase and farnesyltransferase, respectively). We have found that l-NAME significantly changes the SAM/SAH ratio in both MCR and MVR cells. Moreover, these alterations have reciprocal character; in the MCR cells, the SAM/SAH ratio was raised, whereas in the MVR cells this ratio was decreased. We conclude that depletion of endogenous NO with l-NAME increased the production of SAH only in cells with mutated oncogenic RasH, possibly through enhancement of production of reactive oxygen species (ROS). Oxidative stress can increase cystathionine β-synthase activity that switches methyl cycles from remethylation into transsulfuration pathway to maintain the intracellular glutathione pool (essential for the redox-regulating capacity of cells) via an adaptive process.     

Respiration and Mitochondrial Membrane Potential Are not Required for Apoptosis and Anti-apoptotic Action of Bcl-2 in HeLa Cells

The release of cytochrome c from intermembrane space of mitochondria into cytosol is one of the critical events in apoptotic cell death. The important anti-apoptotic oncoprotein Bcl-2 inhibits this process. In the present study it was shown that apoptosis and release of cytochrome c induced by staurosporine or by tumor necrosis factor- in HeLa cells were not affected by inhibitors of respiration (rotenone, myxothiazol, antimycin A) or by uncouplers (CCCP, DNP) that decrease the membrane potential at the inner mitochondrial membrane. The inhibitors of respiration and the uncouplers did not affect also the anti-apoptotic activity of Bcl-2.     

菟丝子提取物在PC12细胞株中的神经营养因子样活性

以常用的PC12细胞株为实验模型,考察了菟丝子提取物诱导PC12细胞分化及对相关激酶活性的影响.发现该提取物在诱导PC12细胞分化的同时,可明显提高有丝分裂原激活的蛋白激酶(MAPK)磷酸化.MAPK激酶的特异抑制剂PD98059,能够有效地抑制菟丝子提取物诱导PC12细胞中MAPK的磷酸化和突起的延伸,表明该提取物诱导PC12细胞分化可能与MAPK途径有关.同时还发现,该提取物能一定程度地抑制去血清引起的细胞凋亡,表明它具有一定的神经营养因子样作用.     

Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms

Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

Differential Modulation of 7-Ketocholesterol Toxicity Against PC12 Cells by Calmodulin Antagonists and Ca<Superscript>2+</Superscript> Channel Blockers

The present study assessed the influence of intracellular Ca²⁺ and calmodulin against the neurotoxicity of oxysterol 7-ketocholesterol in relation to the mitochondria-mediated cell death process and oxidative stress in PC12 cells. Calmodulin antagonists calmidazolium and W-7 prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death, whereas Ca²⁺ channel blocker nicardipine, mitochondrial Ca²⁺ uptake inhibitor ruthenium red, and cell permeable Ca²⁺ chelator BAPTA-AM did not reduce it. Exposure of PC12 cells to 7-ketocholesterol caused elevation of intracellular Ca²⁺ levels. Unlike cell injury, calmodulin antagonists, nicardipine, and BAPTA-AM prevented the 7-ketocholesterol-induced elevations of intracellular Ca²⁺ levels. The results show that the cytotoxicity of 7-ketocholesterol seems to be modulated by calmodulin rather than changes in intracellular Ca²⁺ levels. Calmodulin antagonists may prevent the cytotoxicity of 7-ketocholesterol by suppressing the mitochondrial permeability transition formation, which is associated with the increased formation of reactive oxygen species and the depletion of GSH.