Decrease in acetylcholine-induced current by neomycin in PC12 cells

The effects of neomycin, one of the aminoglycoside antibiotics, on the acetylcholine (ACh)-induced current (I(ACh)) were studied in pheochromocytoma cells by using the whole-cell clamp technique. The I(ACh) proved to be generated through neuronal nicotinic receptor. ACh (30 microM) induced an inward current at a holding potential of -80 mV. When cells were treated with neomycin (0.01-1 mM) and ACh (30 microM) simultaneously, an inhibitory effect of neomycin on the peak of I(ACh) was found. This effect was fast, reversible, and concentration dependent. Pretreatment with neomycin for 3-8 min had no effect on the inhibition of I(ACh) induced by neomycin. External application of 0.1 mM neomycin neither shifted the dose-response curve of the peak I(ACh) to the right (dissociation constant (K(d)) = 16.5 microM) nor affected its coefficient (1.8) but inhibited the curve amplitudes by approximately 33%. Stimulated protein kinase C activation by using an exogenous activator produced inhibition of I(ACh), while using protein kinase C inhibitor (PKCI 19-31) had no effect on the inhibition of I(ACh) induced by neomycin. These results suggest that neomycin has an inhibitory effect on I(ACh) without the involvement of phospholipase C. It indicates that neomycin binds to a specific site on the cell membrane, probably on the neuronal nicotinic receptor-coupled channel, and inhibits the I(ACh) in a noncompetitive manner, thus controlling the immediate catecholamine release from the sympathetic cells.     

雷公藤内酯醇对 PC12细胞增殖的抑制作用及机制初探

目的:研究雷公藤内酯醇(triptolide)对PC12细胞增殖的影响及其作用的机制,为其在临床上治疗肿瘤提供实验依据.方法:利用形态学观察、四甲基偶氮唑(MTT)比色分析、流式细胞术和逆转录聚合酶链式反应(RTPCR)检测雷公藤内酯醇对体外培养的嗜铬细胞瘤细胞(PC12 cell)增殖的影响.结果:雷公藤内酯醇(5×103、25×103 g/L)与PC12细胞作用24 h、48 h或72 h均可抑制PC12细胞的增殖,并且这种抑制作用可随着雷公藤内酯醇浓度的增加而增强.但低浓度的雷公藤内酯醇(1×103g/L)对PC12细胞增殖无明显影响.5×103 g/L雷公藤内酯醇与PC12细胞作用24 h后,可使细胞周期中的G0～G1期比例增加,S期比例下降.PC12细胞与雷公藤内酯醇作用后,细胞的翻译延伸因子2A3-2的表达减弱,而且作用48 h与作用24 h相比,2A3-2的表达减弱更为明显.结论:雷公藤内酯醇可抑制PC12细胞的增殖,该抑制可能是通过改变2A3-2基因的表达从而阻止细胞的G0～G1期向S期过渡来实现的.     

Inhibitory effect of Artemisia asiatica alkaloids on acetylcholinesterase activity from rat PC12 cells

We screened 42 Korean traditional tea plants to determine the inhibitory effect of acetylcholinesterase and attenuation of toxicity induced by amyloid-beta peptide, which were related to the treatment of Alzheimer's disease (AD). The methanolic extract from Artemisia asiatica among tested 42 tea plants, showed the highest inhibitory effect (48%) on acetylcholinesterase in vitro. The methanolic extract was further separated with n-hexane, chloroform, and ethyl acetate of water, in order. The chloroform solubles, which were high in inhibitory effect of acetylcholinesterase, were repeatedly subjected to open column chromatography on silica gel. From the highest inhibitory fraction (78%) on acetylcholinesterase, the single compound was obtained by the Sep-Pak Cartridge (C18: reverse phase column). This compound was found to react positively on Dragendorff's reagent (potassium bismuth iodide), which typically reacted with the alkaloid. This compound was purified by HPLC (mu-bondapack C18 reverse phase column: 3.9 x 150 mm). The IC50 (the concentration of 50% enzyme inhibition) value of this compound was 23 micrograms/ml and the inhibitory pattern on acetylcholinesterase was mixed with competitive/non-competitive type. We examined the effects of this compound on toxicity induced by A beta (25-35) in rat pheochromocytoma PC12 cells. Pretreatment of the PC12 cells for 2 h with an alkaloid of Artemisia asiatica (1200 microg/ml) reduced the toxicity induced by A beta. This study demonstrated that an alkaloid of Artemisia asiatica, which was metabolized to small molecule in digestive tract and then could pass through the blood-brain barrier, appeared to be an acetylcholinesterase inhibitor with a blocker of neurotoxicity induced by A beta in human brain causing Alzheimer's disease.     

Regulation of protein kinase activities in PC12 pheochromocytoma cells.

Stimulation of serine protein kinase activity (referred to as S6 kinase) occurs within minutes of addition of nerve growth factor (NGF) to PC12 rat pheochromocytoma cells. This enzyme activity is not related to the cAMP-dependent protein kinase (protein kinase A) or the Ca2+- and phospholipid-dependent protein kinase (protein kinase C), two other protein kinases potentially involved in signal transduction. Two peaks of NGF-stimulated S6 phosphotransferase activity are observed upon ion exchange chromatography; one that comigrates with the serine kinase previously described in chicken embryo fibroblasts and another with distinct elution properties. Several other factors are also found to regulate S6 phosphotransferase activity in PC12 cells including epidermal growth factor, insulin, and phorbol myristate acetate. Dibutyryl cAMP stimulates S6 phosphotransferase activity; however, this activity is strongly inhibited by the protein kinase A heat stable inhibitor. At least two mechanisms exist through which the NGF-stimulated S6 kinase activity can be regulated, one that apparently can use protein kinase C whereas the other(s) does not. The potential roles of these protein kinase activities in signal transduction and regulation of cell growth and differentiation is discussed.     

Identification of a MAP kinase kinase kinase in phaeochromocytoma (PC12) cells.

A MAP kinase kinase kinase (MAPKKK) was identified in phaeochromocytoma (PC12) cells which reactivated homogeneous MAP kinase kinase (MAPKK) from rabbit skeletal muscle that had been inactivated by incubation with protein phosphatase 2A. Reactivation was accompanied by stoichiometric phosphorylation of MAPKK on a serine residue(s). Following stimulation of PC12 cells with nerve growth factor and chromatography of the extracts on Mono Q, MAP kinase and MAPKK were detected as active phosphorylated enzymes, whereas MAPKKK was inactive and only activated after prolonged storage at 4 degrees C. The results suggest that the activation of MAPKKK by growth factors is likely to occur by a non-covalent mechanism.     

The neurite-initiating effect of a tripeptide aldehyde protease inhibitor on PC12h cells

We report here the possible involvement of a new protease in neurite initiation by PC12h cells. Addition of a leupeptin analogue (Ac-Leu-Leu-Nle-al, ALLNal) to PC12h cells on culture plates coated with collagen type I caused de novo neurite outgrowth. Other protease inhibitors (Ac-Leu-Leu-Met-al, leupeptin, E64c, E64d, soybean trypsin inhibitor, hirudin, aprotinin, diisofluorophosphate, 6-aminocapric acid, and pepstatin A) could not mimic this neurite-initiating action. ALLNal induced the initiation of one or two long neurites from the cell body, and increased the cellular level of acetylcholinesterase to an extent similar to nerve growth factor (NGF). However, ALLNal-induced neuritogenesis is different from that induced by NGF, in which many neurites are induced from a single cell body. In addition, in contrast to neurons induced by NGF, which survive for a long time, ALLNal-induced differentiation was transient, and after 48 h percentage of cells bearing neurites started to decrease. After about 120 h exposure to ALLNal, neurites had mostly disappeared and the acetylcholinesterase activity level was not as great as that produced by NGF. These results provide evidence that ALLNal and NGF elicit neurite initiation by different mechanisms, and suggest the existence of a regulatory system of neuronal differentiation through specific protease-protease inhibitor interaction.     

Effects of myosin light-chain kinase inhibitor on catecholamine secretion from rat pheochromocytoma PC12h cells

Release of dopamine from rat pheochromocytoma PC12h cells by high K+ (50 mM) was inhibited by a specific inhibitor of myosin light-chain kinase (ML-9) dose-dependently. The myosin light-chain kinase inhibitor also specifically inhibited the phosphorylation of a 20 KDa protein by myosin light-chain kinase. Myosin light chain kinase may play a stimulatory role in the release reaction of catecholamines from the rat pheochromocytoma cells.     

Selected contribution: effect of the aldehyde acrolein on acetylcholine-induced membrane current in airway smooth muscle cells.

Acrolein administered to isolated airways has been shown to alter airway responsiveness as a consequence of its effect on Ca(2+) signaling. To examine the mechanisms involved, we studied the effect of acrolein on ACh- and caffeine-induced membrane currents (patch-clamp) in myocytes freshly isolated from rat trachea. In cells clamped at -60 mV, ACh (0.1-10 microM) induced a concentration-dependent inward current, which, in approximately 50% of the cells, was followed by current oscillations in response to high concentration of ACh (10 microM). Exposure to acrolein (0.2 microM) for 10 min significantly enhanced the amplitude of the low-ACh (0.1 microM) concentration-induced initial peak of current (318.8 +/- 28.3 vs. 251.2 +/- 40.3 pA; n = 25, P < 0.05). At a high-ACh concentration (10 microM), the frequency at which subsequent peaks occurred was significantly increased (13.2 +/- 1.1 vs. 8.7 +/- 2 min(-1); n = 20, P < 0.05). ACh-induced current was identified as a Ca(2+)-activated Cl(-) current. In contrast, similar exposure to acrolein, which does not alter caffeine-induced Ca(2+) release, did not alter caffeine-induced transient membrane currents (595 +/- 45 and 640 +/- 45 pA in control cells and in cells exposed to acrolein, respectively; n = 15). It is concluded that acrolein alters ACh-induced current as a consequence of its effect on the cytosolic Ca(2+) concentration response and that the protective role of inhibitors of Cl(-) channels in air pollutant-induced airway hyperresponsiveness should be examined.     

Muscarinic activation of mitogen-activated protein kinase in PC12 cells

Muscarinic acetylcholine receptors (mAChRs) activate many downstream signaling pathways, some of which can lead to mitogen-activated protein kinase (MAPK) phosphorylation and activation. MAPKs play roles in regulating cell growth, differentiation, and synaptic plasticity. Here, the activation of MAPK was examined in PC12 cells endogenously expressing mAChRs. Western blot analysis using a phosphospecific MAPK antibody revealed a dose-dependent and atropine-sensitive increase in MAPK phosphorylation in cells stimulated with carbachol (CCh). The maximal response occurred after 5 min and was rapidly reduced to baseline. To investigate the receptors responsible for CCh activation of MAPK in PC12 cells, the mAChR subtypes present were determined using RT-PCR and immunoprecipitation. RT-PCR was used to amplify fragments of the appropriate sizes for m1, m4, and m5, and the identities of the bands were confirmed with restriction digests. Immunoprecipitation using subtype-specific antibodies showed that approximately 95% of the expressed receptors were m4, whereas the remaining approximately 5% were m1 and m5. A highly specific m1 toxin completely blocked MAPK phosphorylation in response to CCh stimulation. The mAChR-induced MAPK activation was abolished by protein kinase C down-regulation and partially inhibited by pertussis toxin. Although m1 represents a small proportion of the total mAChR population, pharmacological evidence suggests that m1 is responsible for MAPK activation in PC12 cells.     

The dual-specificity CLK kinase induces neuronal differentiation of PC12 cells.

CLK is a dual-specificity protein kinase capable of phosphorylating serine, threonine, and tyrosine residues. We have investigated the action of CLK by establishing stable PC12 cell lines capable of inducibly expressing CLK. Expression of CLK in stably transfected PC12 cells mimicked a number of nerve growth factor (NGF)-dependent events, including the morphological differentiation of these cells and the elaboration of neurites. Moreover, CLK expression enhanced the rate of NGF-mediated neurite outgrowth of these cells, indicating that CLK expression and NGF treatment activate similar signal transduction pathways. CLK expression, unlike NGF, was not able to promote PC12 cell survival in serum-free media, demonstrating that CLK only partially recapitulated the actions of NGF on these cells and that the biochemical pathways necessary for morphological differentiation can be stimulated without also stimulating those necessary for survival. Induction of CLK expression also resulted in the selective activation of protein kinases that are components of growth factor-stimulated signal transduction cascades, including ERK1, ERK2, pp90RSK, and S6PKII. Induction of CLK expression, however, did not stimulate pp70S6K or Fos kinase, two NGF-sensitive protein kinases. These data indicate that CLK action mediates the morphological differentiation of these cells through its capacity to independently stimulate signal transduction pathways normally employed by NGF.     

Involvement of tetrahydrobiopterin in trophic effect of erythropoietin on PC12 cells.

Tetrahydrobiopterin (BH(4)) synthesis is reported to be stimulated by nerve growth factor (NGF) in PC12 cells, suggesting involvement of BH(4) in the trophic effect of NGF. We have recently reported that erythropoietin (EPO) and BH(4) enhance survival of PC12 cells. In the present study, we investigated involvement of BH(4) in the trophic effect of EPO on PC12 cells. Cellular BH(4) content was increased by EPO (10(-10) to 10(-8) M) in a dose- and time-related manner. EPO (10(-10) to 10(-8) M) increased the viable cell number of PC12 cells. In addition to EPO, BH(4) (1, 3, and 10 microM) increased the viable cell number of PC12 cells. Administration of 0.3 mM 2,4-diamino-6-hydroxypyrimidine, an inhibitor of BH(4) synthesis, blunted EPO-induced increases in BH(4) content and the viable cell number of PC12 cells. These results taken together suggest that BH(4) is involved in the trophic effects of EPO on PC12 cells.     

Inhibitory effect of zeatin, isolated from Fiatoua villosa, on acetylcholinesterase activity from PC12 cells

Acetylcholinesterase (AChE) inhibitors, which enhance cholinergic transmission by reducing the enzymatic degradation of acetylcholine, are the only source of the compound that is currently approved for the treatment of Alzheimer's disease (AD). The methanol extract from Fiatoua villosa among 100 traditional edible plants that were tested, showed the most potent inhibitory effect (51%) on acetylcholinesterase in vitro. After the sequential solvent fractionation of the methanol extract of Fiatoua villosa, the active fraction was repeatedly subjected to open-column chromatography on silica gel. From the highest inhibitory fraction, the chloroform fraction (75%) on AChE, the single compound, was obtained by the Sep-Pak Cartridge (C18: reverse phase column). This compound was finally purified by HPLC (micro-bondapack C18 reverse phase column: 19 x 300 mm). According to the electron impact mass spectrometry (EI-MS), we confirmed that the molecular mass was 219 m/z. The structure of this compound was identified as zeatin [2-methyl-4-(1H-purine-6-ylamino)-2-buten-1-ol], one of the derivatives of purine adenine. The concentration that was required for 50% enzyme inhibition (IC50 value) was 1.09 x 10(-4) M. This study demonstrated that the zeatin from Fiatoua villosa appeared to be the most potent AChE inhibitor in AD.     

Diverse effect of tributyltin on apoptosis in PC12 cells

It has not been fully elucidated how endocrine-disrupting chemicals disrupt hormone functions or how strong their effects are compared with natural hormones. There is little information concerning the effects of tributyltin (TBT), one of the endocrine disrupters on living organisms. Although TBT at high concentration induced apoptosis in PC12 cells, TBT at low concentration inhibited the DNA fragmentation in the cells cultured in serum-free medium or in medium containing 6-hydroxydopamine. The cell viability grown in both medium conditions increased after treatment with TBT. These findings suggest that TBT exerted a apoptosis-inducing and -inhibiting effect. These diverse effect of TBT on apoptosis would cause serious damages on cell differentiation.     

Multiple Ca2+ signaling pathways converge on CaM kinase in PC12 cells.

The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in mediating various Ca2+ signaling pathways was examined in PC12 cells. Conversion of the kinase to a Ca(2+)-independent form was used to monitor which neurotransmitters activate the enzyme in situ. CaM kinase responds to Ca2+ influx elicited by ligand-gated Ca2+ channels for ATP and acetylcholine. It also responds to Ca2+ mobilization of IP3-sensitive stores elicited by phospholipase C-linked receptors for ATP and acetylcholine as well as by caffeine. CaM kinase mediates the actions of many neurotransmitters and Ca2+ signaling pathways.     

Nerve growth factor stimulates the tyrosine phosphorylation of MAP2 kinase in PC12 cells.

NGF treatment of PC12 cells results in the rapid activation of MAP2 kinase. We report here that the induction of enzyme activity was correlated with the phosphorylation of MAP2 kinase, detected by metabolic labeling of the enzyme and with anti-phosphotyrosine antibodies. NGF stimulated the phosphorylation of MAP2 kinase on tyrosine, as well as serine and threonine residues. Western blot analysis using a polyclonal anti-phosphotyrosine antibody demonstrated that the tyrosine phosphorylation of MAP2 kinase was maximal within 2 min following NGF exposure and preceded the induction of MAP2 kinase activity. The NGF-stimulated tyrosine phosphorylation of an identified substrate provides direct evidence for the participation of a tyrosine kinase in the mechanism of action of NGF.     

A decrease of neuroprotector effect of ganglioside GM1 on PC12 cells under conditions of oxidative stress in the presence of inhibitor of tyrosine kinase of Trk-receptors

Effects of inhibitors of tyrosine kinases (K-252a, genistein) and of phospholipase A2 (bromophenacetyl bromide) on viability of PC12 cells are studied in the presence of hydrogen peroxide and ganglioside GM1. The degree of inhibition of hydrogen peroxide cytotoxic effect by ganglioside GM1 amounted to 52.8 +/- 4.3 %. However, in the presence in the medium of 0.1 and 1 microM inhibitors of tyrosine kinase of Trk-receptors (K-252a) it was as low as 32.7 +/- 6.5 % and 11.7 +/- 9.8 %, respectively. GM1 prevented Na+, K+-ATPase produced by H2O2, but in the presence of 1 microM K-252a this effect was practically not pronounced. In the presence of another inhibitor of tyrosine kinases--genistein, a tendency for a decrease of the GM1 protective effect was observed at its concentrations 0.1 and 1 microM, whereas at a higher concentration 10 microM genistein depressed the GM1 neuroprotective effect statistically significantly. It was found that inhibitor of phospholipase A2 bromophenacetyl bromide did not affect the action of GM1 aimed at increasing the viability of cells under action of hydrogen peroxide on them. It seems that this enzyme is not involved in the cascade of reactions participating in realization of the ganglioside protective effect. Thus, inhibitor of tyrosine kinase of Trk-receptors K-252 decreases or practically prevents the ganglioside GM1 neuroprotective effect of PC12 cells under stress conditions; the same ability is characteristic of genistein--an inhibitor of tyrosine kinases of the wider spectrum of action.     

A Decrease of neuroprotective effect of ganglioside GM1 on PC12 cells under conditions of oxidative stress in the presence of inhibitor of tyrosine kinase of Trk-receptors

Effects of inhibitors of tyrosine kinases (K-252a, genistein) and of phospholipase A₂ (bromophenacyl bromide) on viability of PC12 cells are studied in the presence of hydrogen peroxide and ganglioside GM1. The degree of inhibition of hydrogen peroxide cytotoxic effects by ganglioside GM1 amounted to 52.8 ± 4.2%. However, in the presence in the medium of 0.1 and 1 μM inhibitors of tyrosine kinase of Trk-receptors (K-252a) it was as low as 32.7 ± 6.5% and 11.7 ± 9.8%, respectively. GM1 prevented Na⁺,K⁺-ATPase oxidative inactivation produced by H₂O₂, but in the presence of 1 μM K-252a this effect was practically not pronounced. In the presence of another inhibitor of tyrosine kinases-genistein, a tendency for a decrease of the GM1 protective effect was observed at its concentrations 0.1 and 1 μM, whereas at a higher concentration 10 μM, genistein depressed statistically significantly the GM1 neuroprotective effect. It was found that inhibitor of phospholipase A₂ bromophenacyl bromide did not affect the action of GM1 aimed at increasing the viability of cells under action of hydrogen peroxide on them. It seems that this enzyme is not involved in the cascade of reactions participating in realization of the ganglioside protective effect. Thus, inhibitor of tyrosine kinase of Trk-receptors K-252a decreases or practically prevents the ganglioside GM1 neuroprotective effect on PC12 cells under stress conditions; the same ability is characteristic of genistein—an inhibitor of tyrosine kinases of the wider spectrum of action.