Delta-9-tetrahydrocannabinol depresses inward sodium current in mouse neuroblastoma cells.

Whole-cell voltage-clamp techniques were used in order to define the effects of delta-9-tetrahydrocannabinol (THC) on the voltage-gated sodium current in neuroblastoma cells. With regard to the inward sodium current, THC decreased the peak amplitude and increased both the time to peak and tau for recovery. The reversal potential was unchanged, suggesting that channel selectivity for sodium was not altered by the drug. With regard to the outward sodium current, THC had no effect on the peak amplitude, time to peak or tau for recovery. This functional alteration of the voltage-gated sodium channel may contribute to the depressant effects of the cannabinoid.     

丁酸钠诱导Raji细胞表达DAPK的研究

目的探讨丁酸钠(SB)对人淋巴瘤Raji细胞生长和DAPK表达变化的影响。方法用丁酸钠作用于Raji细胞株,观察形态、增殖情况、细胞周期分布变化、死亡相关蛋白激酶(DAPK)基因启动子甲基化的变化及该酶表达的变化。结果成团悬浮生长的Raji细胞呈现贴壁生长,细胞增殖速度明显被抑制,细胞阻滞在G0/G1期;DAPK启动子去甲基化,DAPK表达量增加,细胞凋亡增加。结论丁酸钠具有诱导Raji细胞DAPK基因启动子去甲基化,使DAPK重新表达,诱导细胞凋亡的作用。     

Effects of avermectins on neurite outgrowth in differentiating mouse neuroblastoma N2a cells

Avermectins (AVMs) are macrocyclic lactone compounds that have been widely used as parasiticides in veterinary and human medicine and as pesticides in agriculture and horticulture. The multidrug resistance transporter, P-glycoprotein (P-gp), is associated with the efflux transport of AVMs and other drugs across the blood–brain and placental barrier, and plays an important role in attenuating the neurotoxicity and developmental toxicity of AVMs. In this study, the mouse neuroblastoma N2a cell line was used to investigate the neurotoxicity of two AVM derivatives: abamectin (ABM) and doramectin (DOR). We found that both these compounds caused significant dose-dependent inhibition of neurite growth in differentiating N2a cells. In addition, Western blotting analysis showed that ABM and DOR significantly inhibited the expression of not only P-gp but also the cytoskeletal proteins, β-actin and β-tubulin. This suggests ABM and DOR may inhibit neurite growth by down-regulating the expression of P-gp and cytoskeletal proteins. Furthermore, knockdown of P-gp expression by RNA interference in N2a cells reduced neurite growth even in the absence of ABM and DOR, and reduced it even more in the presence of low levels of these compounds. These results suggest that even subcytotoxic levels of ABM and DOR can be neurotoxic in differentiating cells and that this neurotoxicity may, at least in part, be the result of the down-regulation of P-gp and cytoskeletal proteins.     

Effects of toxic chemicals on the respiratory activity of cultured mouse neuroblastoma cells

Twenty common toxic chemicals were tested for their ability to inhibit respiratory activity in cultured mouse neuroblastoma C1300 cells, clone 41A3. Pentachlorophenol and hexachlorophene exhibited the properties of uncouplers of oxidative phosphorylation, whereas for KCN, pyridine, 2,5-hexandione, NaAsO2, K2Cr2O7, HgCl2, methylmercury and triethyltin more simple time-courses of inhibition were obtained. Ethanol, methanol, dimethyl sulphoxide, benzidine, nickel acetate, MnCl2, phenol, CoCl2, Na2SeO3 and CdCl2 did not cause any significant changes in respiratory activity. Among the effective compounds, those with well-known neurotoxic properties were the most potent in inhibiting respiration in 41A3 cells.     

Effects of toxic chemicals on the respiratory activity of cultured mouse neuroblastoma cells

1. Twenty common toxic chemicals were tested for their ability to inhibit respiratory activity in cultured mouse neuroblastoma C1300 cells, clone 41 A₃.

2. Pentachlorophenol and hexachlorophene exhibited the properties of uncouplers of oxidative phosphorylation, whereas for KCN, pyridine, 2,5-hexandione, NaAsO₂, K₂Cr₂O₇, HgCl₂, methylmercury and triethyltin more simple time-courses of inhibition were obtained.

3. Ethanol, methanol, dimethyl sulphoxide, benzidine, nickel acetate, MnCl₂, phenol, CoCl₂, Na₂SeO₃ and CdCl₂ did not cause any significant changes in respiratory activity.

4. Among the effective compounds, those with well-known neurotoxic properties were the most potent in inhibiting respiration in 41A₃ cells.     

Mechanism of action of a polypeptide neurotoxin from the coral Goniopora on sodium channels in mouse neuroblastoma cells

Goniopora toxin (GPT), a polypeptide toxin of 9700 Da isolated from coral, markedly slows inactivation of sodium currents recorded under voltage clamp in mouse neuroblastoma cells. The voltage dependence of sodium channel activation is shifted to more negative membrane potentials by 9.8 +/- 2.1 mV, and the voltage dependence of channel inactivation is shifted to more positive membrane potential by 6.0 +/- 2.5 mV. These actions of GPT are voltage dependent with an e-fold increase in K0.5 for toxin action for each 48.3-mV depolarization between -80 and +40 mV. GPT requires Na+ or another alkali metal cation in the extracellular medium for its effect on sodium channels. The relative effectiveness of the different cations tested is Na+ greater than K+ greater than Rb+ greater than Li+ greater than Cs+ much greater than choline+. Like other polypeptide neurotoxins that slow inactivation of sodium channels, GPT enhances persistent activation of sodium channels by veratridine. However, GPT does not block the binding of 125I-labeled Leiurus scorpion toxin to neurotoxin receptor site 3 on sodium channels at concentrations which effectively slow channel inactivation. Therefore, our results define a new site on the sodium channel at which specific effects on inactivation can occur.     

Effects of benzimidazoles on mouse and rat limb bud cells in culture

Benzimidazoles exhibited an inhibitory action on the chondrogenesis in a mouse limb bud cell culture system; further, nitro and chloro groups at the 5 position in 2-(2-pyridyl)benzimidazole were found to be potent substituents. However, 2-(2-pyridyl)benzoxazole and 5-methoxy-2-(2-pyridyl)benzothiazole exhibited no inhibitory action under our experimental conditions. Thus, imidazole NH proton seemed to be important in the inhibitory action in the mouse cell system. The concentrations of thiabendazole (TBZ), 5-hydroxy-TBZ (5-HY-TBZ) and N-methyl-TBZ necessary to reduce the amounts of cartilage proteoglycan by 50% (TP50) were estimated to be about 0.35, 0.25 and 0.70 mM, respectively, in the rat limb bud cell culture system. The TP50 of TBZ and 5-HY-TBZ in the rat cell system were 3- to 4-fold higher than those in the mouse cell system.     

Lithium transport by mouse neuroblastoma cells

Lithium entry and its distribution into veratridine-activated cells of an electrically excitable clone (N1E-115) of mouse neuroblastoma differed from that observed in resting cells. At a constant external lithium concentration, increasing the concentration of veratridine (an alkaloid which selectively activates sodium channels responsible for the generation of action potentials), resulted in a corresponding increase of both the rate of entry as well as the intracellular steady state concentration of lithium. Lithium entry occurred by a temperature dependent (Q₁₀ ～- 1.5), formally saturable process having an apparentK_T of about 20 mM and an apparent V_max which increased from 3 nmol · (mg protein) ^?1 · (min)^?1 in the absence of veratridine to 14 nmol · (mg protein)^?1 · (min)^?1 in the presence of 0.67 mM veratridine. In both the veratridine-activated and resting states, mouse neuroblastoma cells concentrated lithium from the external medium, achieving steady state levels within 60 min; thus, in the absence of veratridine. neuroblastoma cells exhibited an intra: extracellular lithium distribution ratio of about 1.5 compared to a distribution ratio of 3.3 in the presence of 0.1 mM veratridine. Lithium accumulation in the presence of supranormal concentrations of extracellular potassium was not different from that found in controls. In addition to characterizing the differences between lithium transport found in veratridine-activated and resting cells, these results suggest that electrically active tissue may accumulate lithium to an extent proportional to the activity of the tissue.     

Biochemical effects of chlorpromazine on mouse neuroblastoma cells.

Chlorpromazine and other phenothiazine derivatives are neuroleptic drugs of widespread use for clinical situations beyond the realm of psychiatry, such as to control nausea, vomiting and intractable hiccups. The present study investigated in vitro different cytotoxic effects of chlorpromazine in cultures of mouse neuroblastoma cell line Neuro-2a exposed to different concentrations of this compound. Indicators assessed were cell proliferation by quantification of total protein content of the cell culture, lysosomal function evaluated by the relative uptake of neutral red cytosolic phosphofructokinase (PFK) and enolase (ENL) activities in glycolysis, mitochondrial succinate dehydrogenase (SDH) activity in the citric acid cycle, lysosomal beta-galactosidase (GAL) activity, and neuronal acetylcholinesterase activity. Marked inhibitory effects were found for cell proliferation and relative neutral red uptake; PFK, ENL and GAL activities had no significant differences from control. Stimulation was specifically detected on SDH and the Krebs cycle at concentrations up to 30 microM. Chlorpromazine did not have high toxicity for cytotoxic effects on lysosomes.     

Interaction of phencyclidine with mouse neuroblastoma cells

Growth of mouse neuroblastoma (Nb) cells (clone Ml) was not affected by phencyclidine (PCP) concentrations of 10^?6M up to 2 × 10^?4M, whereas 10^?3 PCP caused a 100% inhibition of cell growth. Several PCP analogs, including the quaternary PCP methiodide, exerted effects similar to those of PCP. The uptake of [piperidyl-3,4-³H]PCP ([³H]PCP) by the Nb cells was studied using cell monolayers in Petri dishes. Non-specific entry of PCP into the cells was linear with added substrate but specific uptake exhibited saturation kinetics. The concentration for half-maximum specific uptake was 2 × 10^?5 M, and the capacity of the cells at saturation was 2–3 nmoles [³H]PCP/mg protein, at 22°. The uptake rate constant was 0.2 ± 0.05 × 10⁵ (M^?1min^?1) and the dissociation constant was 0.25 ± 0.05 (min^?1). Uptake was temperature dependent and was inhibited by 2,4-dinitrophenol (DNP). This may indicate that this binding represents (at least in part) an active uptake process of PCP into the cells.     

Characterization of palytoxin-induced channels in mouse neuroblastoma cells.

Palytoxin-induced whole-cell and single channel currents were recorded in mouse neuroblastoma cells. Palytoxin-induced single channel currents had a slope conductance of 26 pS (20-22 degrees C). Palytoxin-induced channels were permeable to sodium and potassium and slightly permeable to calcium, choline and tetramethylammonium. They did not seem to be significantly permeable to chloride or protons. Both the steady-state and the rate of the dose-dependent effects of palytoxin could be accounted for if one assumed that a palytoxin-induced channel resulted from the binding of two palytoxin molecules to a membrane receptor with respective dissociation constants of 5 nM and 10 pM. In the continued presence of low palytoxin concentrations (less than 1 nM) the effect was maintained. Higher palytoxin concentrations induced a transient and irreproducible effect. The effect of palytoxin was decreased when either external sodium was replaced by potassium or in the absence of calcium in external and/or internal media. The results suggest that ionic currents result from the binding of palytoxin molecules to a membrane receptor and that receptor-toxin complexes can be internalized.     

丁酸钠对乳腺癌细胞MCF-7生长抑制的影响

目的探讨丁酸钠对乳腺癌细胞MCF-7生长的抑制作用。方法将乳腺癌细胞MCF-7常规培养至对数生长期,采用浓度为0、2．5、5．0、10．0mmol／L的丁酸钠处理后,观察对MCF生长的抑制作用。结果浓度为2．5、5．0、10．0mmol／L的丁酸钠处理的MCF-7细胞生长速度明显慢于亲本细胞,随着浓度的增加呈减慢趋势,差异有统计学意义（P〈0．05）。浓度为5mmol／L的丁酸钠处理后的MCF-7细胞克隆形成率明显低于MCF亲本细胞,差异有统计学意义（P〈0．05）。丁酸钠处理的细胞G1明显高于亲本细胞,S期及G2／M期明显低于亲本细胞,凋亡细胞百分比明显高于亲本细胞,差异有统计学意义（P〈0．05）。结论丁酸钠可能是通过诱导MCF-7细胞凋亡而起到逆转其恶性生物学行为的作用。     

Modulation of serotonin-induced currents by metals in mouse neuroblastoma cells

 The effects of several metals on the serotonin receptor-channel complex were studied using mouse neuroblastoma N1E-115 cells which are known to be endowed with the 5-HT₃ subclass of the receptor. The whole-cell patch clamp technique was used to record currents induced by serotonin at a concentration of 3 μM which was equivalent to the apparent dissociation constant. Methylmercury and mercuric chloride suppressed serotonin-induced currents irreversibly, with a 50% suppression being observed at concentrations of 3 μM and 2 μM, respectively. Lead and zinc suppressed the current with IC₅₀s of 80 μM and 50 μM, respectively, and the effects of both metals were reversible after washing with metal-free solution. Lanthanum also suppressed the current with an IC₅₀ of 10 μM, and the effect was partially reversible. Cadmium and cobalt augmented serotonin-induced currents slightly but consistently at a concentration of 100 μM, and the effect was reversible. Aluminum at 100 μM had no effect on serotonin-induced currents. It was concluded that the 5-HT₃ receptor is endowed with a unique property with respect to the actions of metals which is not shared by some other ligand-gated and voltage-gated ion channels. Received: 15 November 1995/Accepted: 18 January 1996     

Acute toxicity testing in cultures of mouse neuroblastoma cells.

Abstract Cultured mouse neuroblastoma cells (C1300) may be used as models for nerve cells since they have a number of properties in common with their normal counterparts in vivo. In order to test the possibility of using C1300 cells as alternativeto experimental animals when testing for acute toxicity, cells (clone 41A₃) were exposed to a number of common chemicals (CH₃HgCl, CdCl₂HgCl₂ ppDDT, n-butanol, benzene, dioxan, n-propanol, aceton and t-butanol). The toxic effect was quantified by measuring the degree of cell detachment in the cultures. The concentrations of chemicals that caused 25% of the total cell number to detach (TD₂₅) were used for comparison with LD₅₀ values. In spite of the very simplified situation in culture, where the toxicity of a substance is little or not at all influenced by factors like penetration, storage, metabolism and excretion a good correlation (corr. coeff. 0,98) was obtained between TD₂₅ values and LD₅₀ values. Good correlations between in vitro and in vivo tests have also been reported by others. One possible explanation to these findings could be simplified in vivo toxicokinetics of these substances when tested in high doses for general effects like animal death. If so, simple in vitro tests may be used for predicting acute toxicity of certain groups of substances.     

Effect of sodium butyrate on metallothionein induction and cadmium cytotoxicity in ROS 17/2.8 cells

ROS 17/2.8 cells, a cloned rat osteosarcoma cell line, are exceptionally sensitive to the cytotoxic effects of cadmium. This sensitivity is associated with the inability of this metal to induce the synthesis of metallothionein, a transition metal-binding protein, which detoxifies this metal by its sequestration. Sodium butyrate induces the synthesis of metallothionein in these cells in a concentration-dependent manner. Treatment with this agent also significantly increases the resistance of these cells to the cytotoxic effects of cadmium and the protective effect of butyrate is reversed upon its removal from culture medium. Butyrate treatment did not significantly alter the accumulation of cadmium by these cells. Hence, the increased synthesis of metallothionein in butyrate-treated cells is not due to increased cellular uptake of cadmium. Inhibition of DNA synthesis due to butyrate was not a sufficient condition to alter metallothionein synthesis or to protect against Cd-induced cytotoxicity. Equivalent inhibition of DNA synthesis with hydroxyurea failed to increase metallothionein synthesis in cadmium-treated cells. These results indicate that modulation of metallothionein gene expression in this cell line is the critical factor in determining cellular sensitivity to the cytotoxic effects of cadmium.