Studies on Mechanoperception in Characean Cells: Pharmacological Analysis

The mechanisms for generating receptor potentials and actionpotentials upon mechanical stimulation were studied in internodalcells of Chara. Receptor potentials and the subsequent actionpotentials could be generated even when the electrogenic protonpump was inhibited, indicating that the proton pump does notplay a central role in generating receptor potentials and actionpotentials. The involvement of Ca²⁺ and/or Cl^– channelsin both receptor and action potentials was suggested, basedon the equilibrium potentials of these ions across the plasmamembrane. Inhibitors of the Ca²⁺ channel, Cl^– channeland stretch-activated channel could not inhibit generation ofthe receptor potential. These findings suggested that the channelsinvolved in generating the receptor potential are insensitiveto these channel inhibitors, although all inhibitors significantlyinhibited the action potential. (Received July 26, 1996; Accepted November 19, 1996)     

Movement of Ions and Electrogenesis in Higher Plant Cells

During the past 10 years considerable information has accumulatedon the electrochemical relationships of higher plant cells duringtransport of mineral ions. Using the Nernst equation as a criterion,none of eight ions (K⁺, Na⁺, Ca⁺⁺, Mg⁺⁺, NO₃^–, Cl^–,H₂PO₄^–, and SO₄^–) is in a passive equilibrium. Na⁺,Ca⁺⁺, and Mg⁺⁺ are subject to an exclusion mechanism, and allof the anions appear to be pumped inwardly. K⁺ apparently approachesan electrochemical balance under certain conditions but probablyis actively accumulated. Compartmental analyses giving estimatesof amounts in the cytoplasm and vacuole and of unidirectionalfluxes permit application of the Ussing flux-ratio equation.The criterion in oat coleoptile cells suggests that at the plasmalemmaNa⁺ is pumped out while K⁺ and Cl^– are pumped in. K⁺ andCl^– appear to be coupled in active transport across thetonoplast into the vacuole. Good evidence has been found thatthe cell's electropotential arises from an electrogenic pump:CN^– (cyanide) and DNP (dinitrophenol) reversibly blockthe potential and ionic transport; cell potentials are higherthan can be accounted for by diffusion; the responses of respirationand potential to the concentration of CN^– are nearly parallel;and CN^– inhibited tissue approaches a fit to the Goldmanconstant field equation. Future objectives should be identificationof the ion, or ions, subject to the electrogenic pump and discoveryof the immediate energy source.     

Sulphate Influx in Characean Cells: I. GENERAL CHARACTERISTICS

There is a significant sulphate influx in Chara australis, Nitellatranslucens, and Tolypella intricata but not in Nitellopsisobtusa. The uptake is active in Chara, the ions moving intothe vacuole against an electrochemical potential gradient. Influxrate in the light remains constant whereas a transient increasein rate occurs upon transfer to the dark. Both the light andthe dark influx rate respond similarly to temperature, and toexternal sulphate ion concentration. It is proposed that sulphateis pumped actively across the plasmalemma and that the increasein influx rate following transfer to the dark reflects a transientincrease in the rate at which the pump operates. There is anapparent light stimulation of the tonoplast flux.     

Inhibition of Brain Glycolysis by Aluminum

Abstract: Aluminum inhibited both the cytosolic and mitochondrial hexokinase activities in rat brain. The IC₅₀ values were between 4 and 9 μ M . Aluminum was effective at mildly acidic (pH 6.8) or slightly alkaline (pH 7.2–7.5) pH, in the presence of a physiological level of magnesium (0.5 m M ). However, saturating (8 m M ) magnesium antagonized the effect of aluminum on both forms of hexokinase activity. Other enzymes examined were considerably less sensitive to inhibition by aluminum. The IC₅₀ of aluminum for phosphofructokinase was 1.8 m M and for lactate dehydrogenase 0.4 m M . At 10–600 μ M , aluminum actually stimulated pyruvate kinase. Aluminum also inhibited lactate production by rat brain extracts: this effect was much more marked with glucose as substrate than with glucose-6-phosphate. However, the IC₅₀ for inhibiting lactate production using glucose as substrate was 280 μ M , higher than that required to inhibit hexokinase. This concentration of aluminum is comparable to those reportedly found in the brains of patients who had died with dialysis dementia and in the brains of some of the patients who had died with Alzheimer disease. Inhibition of carbohydrate utilization may be one of the mechanisms by which aluminum can act as a neurotoxin.     

Studies on Mechano-Perception in Characean Cells: Development of a Monitoring Apparatus

An apparatus to monitor the electrical signal generated in Characells upon mechanical stimulation was developed. An internodalcell was stimulated by dropping a glass tubing with the intensityof the stimulus being controlled by changing either the weightof the glass tubing or the height from which it was dropped.Upon stimulation, a receptor potential was generated with theamplitude being dependent on the stimulus intensity. When thereceptor potential reached a threshold value, an action potentialwas generated. The receptor potential and action potential werecharacterized. The usefulness of this apparatus for analyzingreceptor potentials is discussed. (Received January 29, 1996; Accepted April 16, 1996)     

Light-Induced Membrane Hyperpolarization and Adenine Nucleotide Levels in Perfused Characean Cells

This study examines the relationship between light-induced membranehyperpolarization and changes in adenine nucleotide levels intonoplast-free characean cells. When cells were perfused witha medium containing 1 mM ATP in the dark, the plasma membranedepolarized, the cytosolic ATP level decreased, and the ADPand AMP levels increased. Under light, the membrane hyperpolarized,the ATP level increased, and the ADP and AMP levels decreased.These changes in the adenine-nucleotide levels could partiallyexplain the membrane hyperpolarization. When cells were perfusedwith a medium containing an ATP-regenerating system consistingof phosphoenolpyruvate and pyruvate kinase, the membrane potentialremained in the hyperpolarized state, the ATP level remainedat a high level and no light-induced hyperpolarization was observed.The intracellular adenine nucleotide levels were also controlledby continuous perfusion. The membrane potential was determinedonly by the adenine nucleotide levels of perfusion media, irrespectiveof the light condition. Chloroplast-free Nitellopsis cells into which isolated Pisumchloroplasts were introduced also showed light-induced membranehyperpolarization. Pretreatment of chloroplasts with dicyclohexylcarbodiimide(DCCD) completely abolished the hyperpolarization with parallelinhibition of photophosphorylation. These results strongly suggestthat changes in adenine nucleotide levels caused by photophosphorylationare responsible for light-induced membrane hyperpolarizationin perfused cells. (Received August 17, 1985; Accepted December 13, 1985)     

Aluminum Uptake by Neuroblastoma Cells

Aluminum uptake studies in viable neuroblastoma cells were performed. Aluminum uptake was largely dependent on the pH of the suspension medium. At physiological pH values, cells were apparently unable to incorporate detectable amounts of aluminum in the absence of proper mediators. Aluminum uptake was enhanced as the pH decreased, attaining a plateau at about pH 6.0. In experiments with 2 x 10(6) cells/ml, pH 6.0, and 25 microM aluminum in the medium, aluminum incorporation reached saturation at 5 nmol of aluminum/mg of cellular protein, accounting for 60-70% of aluminum added. At pH 6.0, cells showed a large capacity for accumulating aluminum; about 70% of intracellular aluminum was associated with the postmitochondrial fraction. At neutral pH, application of apotransferrin seemed to facilitate aluminum translocation into cells via membrane receptors. Fatty acids were also capable of mediating aluminum uptake at neutral pH, probably by forming aluminum-fatty acid complexes. Low molecular weight aluminum chelators, e.g., citrate, inhibited aluminum uptake. Treatment of cells with energy metabolism blockers had virtually no influence on aluminum uptake, indicative of passive mechanisms. The results suggest that aluminum uptake occurs via different modes dependent on growth conditions, such as medium pH.     

Analogue Inhibition of the Active HCO-3 Transport Site in the Characean Plasma Membrane

Competitive inhibition of the HCO^–₃ transport site, atthe plasmalemma of Chara coraUina, by the CO^2–₃ ion isdemonstrated. This CO^2–₃ inhibition was used to demonstratethat HCO^–₃ ions enter the cell by facilitated ‘diffusion’when the HCO^–₃ transport system has been inactivated bytreatment with 10 mM K⁺. Use of CO^2–₃ as a HCO^–₃analogue is limited, however, because of the necessity to employsolutions of high pH. Inhibition was not observed in the presenceof a range of organic and inorganic acid anions. These resultsdemonstrate the stereo-specific nature of the HCO^–₃ bindingsite. A variety of amino compounds were found to inhibit H¹⁴CO^–₃influx. Inhibition appeared to be competitive, being completelyrelieved at higher substrate (HCO^–₃) concentrations. Asimple correlation was not found between the degree of inhibitionand the concentration of neutral base. A combination of thepresence of neutral base and experimental pH values of at least8·0 was required to produce the reactive species thatinhibited HCO^–₃ transport. This species is consideredto be the amino carbamate. These results are discussed withrespect to further HCO^–₃ analogue experiments.     

Temporal Relationship between Action Potential and Ca2+ Transient in Characean Cells

Temporal relationship between the action potential and the changein cytosolic Ca²⁺ concentration was investigated in cells offour species of Characeae, Chara corallina, Nitellopsis obtusa,Nitella flexilis and Nitella axilliformis. The Ca²⁺ transientwas detected by light emission from Ca²⁺-sensitive photoproteinaequorin injected into the cytoplasm. Action potential was triggeredby an outward or sometimes inward electric current pulse of20–50 ms in most cases. In all species the action potentialstarted at almost the same time as the time at which the lightemission from aequorin began to increase. Also the peak of actionpotential almost coincided with that of light emission, whichis in contrast with the slower Ca²⁺ transient in Chara reportedby Thiel et al. [(1997) J. Exp. Bot. 48: 609]. A discussionwas made on the origin of Ca²⁺ transient and the ionic processesduring membrane excitation. (Received July 2, 1998; Accepted October 5, 1998)     

Synergistic Inhibition of Growth by Aluminum and Iron of Tobacco (Nicotiana tabacum L.) Cells in Suspension Culture

Growth of tobacco cells at the logarithmic phase was inhibitedafter treatment with both AlCl₃ and FeSO₄ in modified Murashige-Skoogmedium prepared without P_i and EDTA at pH 5.0 for up to 20 h,whereas cells treated with either AlCl₃ or FeSO₄ (at concentrationsup to 100 µM, respectively) grew normally. These resultssuggest the synergistic inhibition of growth by Al³⁺ and Fe²⁺ions. During the exposure to both salts, there was a lag timeof about 10 h before growth inhibition became apparent in culturesof cells upon treatment with the two salts together. Then theextent of inhibition increased and reached a maximum value afterexposure for 18 h. After the lag period, cells treated withboth salts, but not cells treated with either salt alone, exhibitedsignificant increases in the amounts of both aluminum and ironin the cells and could be stained with hematoxylin, with thenuclei staining especially strongly. Cells treated with bothsalts also exhibited a decrease in the number of cells thatcould be plasmolyzed in 1 M mannitol, a decrease in the numberof cells that could be stained with fluorescein diacetate, adecrease in the amount of potassium in cells, and an increasein the extent of lipid peroxidation. These results suggest thataluminum causes the peroxidation of lipids in the presence ofiron and that lipid peroxidation alters the permeability ofthe plasma membrane and leads to cell death. (Received April 18, 1994; Accepted November 11, 1994)     

Studies on Electrogenesis under High K+ Concentrations in Internodal Cells of Chara corallina

+ concentration ([K⁺]_o) on the membrane potential (E_m) of Chara corallina was studied. E_m more negative than -100 mV was maintained even at 100 mM [K⁺]_o. Addition of Ca²⁺ to the external medium further increased this tendency. However, E_m responded sensitively to the increase in [K⁺]_o, when the electrogenic proton pump of the plasma membrane was inhibited by treating cells with dicyclohexylcarbodiimide, an inhibitor of proton pump. Analysis using equivalent circuit model of the plasma membrane suggested that the electrogenic proton pump was activated by the increase in [K⁺]_o. In the presence of 100 mM K⁺, action potentials were generated by electric stimuli. The ionic mechanism of generation of action potentials in the presence of K⁺ at high concentration was discussed. Received 3 October 2000/ Accepted in revised form 6 January 2001     

Synaptic Electrogenesis in Eel Electroplaques

Whether evoked by neural or by chemical stimulation, the synaptic membrane of eel electroplaques contributes a depolarizing electrogenesis that is due to an increased conductance for Na and K. The reversal potential (E_S) is the same for the two modes of synaptic activation. It is inside-positive by about 30–60 mv, or about midway between the emf's of the ionic batteries for Na (E_Na) and K(E_K). The total conductance contributed by synaptic activity (G_S) varied over a fivefold range, but the individual ionic branches, G_SSNa, and G_SSK, change nearly equally so that the ratio G_SSNa:G_SSK is near unity. G_SSK increases independently of the presence or absence of Na in the bathing medium, and independently of the presence or absence of the electrically excitable G_K channels. When activated, the synaptic membrane appears to be slightly permeable to Ca and Mg. When the membrane is depolarized into inside positivity the conductance of the synaptic components decreases and approaches zero for large inside-positive values. Thus, the synaptic components become electrically excitable when the potential across the membrane becomes inside-positive, responding as do the nonsynaptic components, with depolarizing inactivation.     

Movement of Ions and Electrogenesis in Microorganisms

The relationship between movement of ions and the electricalproperties of microorganisms (algae, fungi, and bacteria) arereviewed, with particular emphasis on the giant alga, Nitella,and the fungus, Neurospora. The hypothesis is presented thatthere are two basically different components to the membranepotential of both organisms: (1) one arising from the diffusionof sodium, potassium, and hydrogen ions down their chemicalgradients, and (2) one associated with the utilization of energyand the active efflux of hydrogen ions, and attributed to an"electrogenic H⁺ pump." Numerous discrepancies between the measuredelectrical properties of the algae or fungi and the predictionsof ordinary ion-diffusion theory can be accounted for by suchan H⁺ pump, and its existence is further supported by a fewindirect experiments on the bacteria.     

Rates of Photosynthesis in Characean Cells: I. PHOTOSYNTHETIC 14CO2 FIXATION BY NITELLA TRANSLUCENS

A study has been made of photosynthetic ¹⁴CO₂ fixation by isolated‘mature’ internodes of Nitella translucens. Experimentalconditions were similar to those used in studies of the ionicrelations of these cells. Maximum rates of photosynthesis were33–40µµmoles CO₂, fixed per cm² of surfacearea per second (equivalent to 12–15 /xmoles fixed permg chlorophyll per hour). ^l4CO₂ fixation was inhibited to thedark level by 3(3,4,dichlorophenyl)-1, 1-dimethylurea (at 0-6µM or 10µM) and by the uncoupler carbonyl cyanide-m-chlorophenylhydrazone(SµM). The presence of imidazole or ammonium sulphate(both of which uncouple ATP production in vitro) did not resultin an inhibition of 14CO2 fixation. These results are discussedin relation to published work on solute uptake by Nitella translucens.During photosynthesis there was rapid movement of ¹⁴C-labelledorganic compounds out of the chloroplasts. ¹⁴C-labelled sucrose,ammo-acids, and sugar phosphates were found in samples of vacuolarsap.     

Formation of band-type electric patterns in Characean cells

It is observed by experiments that band patterns of alternating acid and alkaline zones are formed on the surface of Characean cells under illumination. In order to understand theoretically such pattern formation, we employ a reaction-diffusion equation model with activator-inhibitor interaction. We study the existence problem of band patterns and hysteresis phenomena such as patterns appearing and disappearing with changing light intensity by using singular perturbation methods.     

Inhibition of Proteasome Activity by Tyropeptin A in PC12 Cells

Tyropeptin A, a potent proteasome inhibitor not reported before, was produced by Kitasatospora sp. MK993-dF2. In this study, we investigated the effects of tyropeptin A on proteasome activity in PC12 cells. Tyropeptin A inhibited the intracellular proteasome activity in a dose-dependent way and seemed to cause neurite outgrowth. As expected, ubiquitinated proteins that should be substrates for the proteasome accumulated in cells treated with tyropeptin A. Hence, it appears that tyropeptin A can permeate into cells and there inhibit the intracellular proteasome activity.     

Electrogenesis in mouse neuroblastoma cells in vitro

Intracellular microelectrode studies of passive membrane properties and action potential generation were carried out on cloned and uncloned mouse neuroblastoma cells in tissue culture. The cloned cells were studied between the eighth and tenth months and the uncloned cells between the third and fifth weeks after primary dissociation. Electrophysiologic measurements of cell membrane properties were made by passing stimulating current pulses across the cell membrane from an intracellular microelectrode and recording simultaneously from the same electrode, by means of a bridge circuit, the changes in membrane potential. The range of responses to electrical stimulation varied from passive increases in membrane potential to repetitive firing of action potentials. A 20 fold range in spike generating capability was found. Passive membrane properties (membrane potential, specific membrane resistivity, and specific membrane capacitance) were similar to those of sympathetic neurons in intact preparations. Seventy-nine percent of the cloned cell line compared to 94% of the uncloned line were capable of generating action potentials. Less than 2% of the cloned cells showed repetitive firing whereas 23% of the uncloned cells had this property. As in several types of normal neurons, the action potential mechanism was largely, although not completely, blocked by iontophoretic and bath applied tetrodotoxin.     

Inhibition of Drug-Induced Apoptosis by Survival Factors in PC12 Cells

Abstract: Pheochromocytoma (PC12) cells have been shown to undergo apoptosis (programmed cell death) when deprived of serum and to be rescued by nerve growth factor, fibroblast growth factor, dibutyryl cyclic AMP, aurintricarboxylic acid, or exogenous expression of bcl-2 . We show here that the cytotoxic drugs cycloheximide, actinomycin D, colchicine, and EGTA also induce apoptosis in PC12 cells. These findings prompted us to investigate whether apoptosis induced by these drugs involves similar pathways in each case, and whether the factors preventing the apoptotic death of serum-deprived PC12 cells can also protect the cells from apoptosis induced by the cytotoxic drugs. Nerve growth factor, dibutyryl cyclic AMP, and expression of bcl-2 inhibited apoptosis induced by all four cytotoxic drugs. Fibroblast growth factor inhibited apoptosis induced by EGTA or colchicine. Aurintricarboxylic acid inhibited apoptosis induced by EGTA. These results suggest that apoptosis induced by treatments with the various drugs is mediated by different initiating pathways, all of which converge into a final, common pathway. Nerve growth factor, dibutyryl cyclic AMP, and bcl-2 appear to affect the final common pathway, whereas fibroblast growth factor and aurincarboxylic acid appear to be more specific and affect only some of the pathways.     

利用RNA干扰抑制HeLa细胞中CTCF基因的表达

目的:CTCF是一种多功能的转录因子,参与启动子调控、绝缘子功能和遗传印记等过程。利用RNA干扰技术抑制CTCF在体细胞中的表达。方法:以CTCF为靶基因,利用ShortCutRNaseⅢ切割体外转录的长双链RNA制备esiRNA,转染HeLa细胞,用RTReal-TimePCR检测CTCF的mRNA水平,用免疫印迹检测CTCF水平。结果:转染CTCFesiRNA后,细胞内的CTCFmRNA被抑制了70%左右,CTCF水平明显下降。结论:CTCF的esiRNA可以明显抑制CTCF在HeLa细胞中的表达,从而为进一步研究CTCF的未知功能打下了基础。