Relationship between light-induced potential change and internal ATP concentration in tonoplast-free Chara cells

The effect of the intracellular concentration of ATP ([ATP]₁)on the light-induced potential change (LPC) in tonoplast-freeChara cells was studied. The LPC was hardly affected by loweringthe [ATP]₁ by about 1/10 or by raising it to about 10 timesthe normal cytoplasmic concentration (0.5–1.3 mM). Theinsensitivity of LPC to [ATP]₁ excludes the possibility thatan increase in [ATP]₁ due to photosynthesis may induce the LPC.However, extreme lowering of the [ATP]₁ to about 1–2 µMcompletely inhibited LPC, although photosynthetic O₂ evolutionwas not significantly inhibited. This fact supports the hypothesisthat light stimulates the putative H⁺pump fueled by ATP. Theuncoupling agents DNP and CCCP greatly depolarized the membrane,and inhibited LPC strongly, but they did not decrease [ATP]₁.Photosynthetic O₂ evolution was inhibited to some extent by2 µM CCCP and strongly inhibited by 0.1 mM DNP. Sincethe membrane resistance increased significantly, these chemicalsare believed to act on the membrane as an inhibitor of the electrogenicH⁺ pump not as an H⁺conductor. Introduction of 1 mM ATP intocells treated with uncouplers, to a large extent restored theirability to produce LPC although the membrane potential in darknesswas maintained at a low level. ¹Present address: Niigata College of Pharmacy, 5829 Kamishinei-cho,Niigata 950-21, Japan. ²Present address: Department of Agricultural Chemistry, Collegeof Agriculture, Kyoto University, Kyoto 606, Japan. ³Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received March 9, 1979; )     

Demonstration of light-induced potential change in Chara cells lacking tonoplast

Chara cells without tonoplasts, prepared by replacing the cellsap with EGTA-containing media, showed essentially the samepattern of light-induced changes in membrane potential and membraneresistance as normal cells although the concentrations of ionsand ATP in the cytoplasm decreased considerably (1/3–1/10)after loss of the tonoplast. Removal of the tonoplast reducedthe rate of photosynthetic O₂ evolution to about 50% of thatof normal cells but did not affect the magnitude of light-inducedpotential change. Not a full but a certain level of electronflow seems necessary to activate the putative electrogenic H⁺-pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Japan. ² Present address: Niigata College of Pharmacy, Niigata 950-21,Japan. (Received September 4, 1978; )     

Dependence of the membrane potential of Chara cells on external pH in the presence or absence of internal adenosinetriphosphate

The dependence of the membrane potential (E_m) and the membrane resistance (R_m) of Chara australis R. Brown on the pH of the external medium (pH₀) was studied by controlling the activity of the plasmamembrane H⁺ pump under both light and dark conditions. The activity of the pump was controlled by regulating the internal ATP or Mg²⁺ concentration in tonoplast-free cells prepared by vacuolar perfusion. In these cells, which contained Mg · ATP (mgATP cells), E_m and R_m were very sensitive to pH₀, as in normal cells. E_m was more negative in light than in the dark at all pH₀ values tested. Tonoplast-free cells with very low [ATP]_i (-ATP cells) or [Mg²⁺]_i (-Mg cells) showed very weak dependence of E_m and R_m on pH₀. Thus, the active and not the passive component of E_m was sensitive to pH₀. At the same time, the high permeability of the plasma membrane to H⁺ was questioned. In both-ATP cells and-Mg cells, E_m was scarcely affected and R_m markedly decreased on illumination.Abbreviations CyDTA 1,2-cyclohexanediamine-N,N-tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether)N,N-tetraacetic acid - HK hexokinase     

An assay for leukoagglutinating lectins using suspension cultured mouse lymphoma cells (BW5147) stained with neutral red

A sensitive and rapid assay for leukoagglutinating lectins has been developed. This assay utilizes neutral red-stained mouse lymphoma cells from the suspension cultured cell line BW5147. The agglutination of the stained cells can be monitored visually in a manner similar to that for conventional assays for erythroagglutinating lectins using erythrocytes. The activity of lekoagglutinating lectins that are not capable of agglutinating erythrocytes can be quantified by this assay. The utility of the assay was demonstrated using leukoagglutinating and erythroagglutinating lectins from the seeds of Phaseolus vulgaris and Maackia amurensis.     

Effect of intracellular pH on the light-induced potential change and electrogenic activity in tonoplast-free cells of Chora australis

Effects of the intracellular H+ concentration on the membranepotential and membrane resistance of tonoplast-free cells ofChara australis were examined under light and dark conditions.Cells were made tonoplast-free by perfusing the vacuole withmedia containing 5 mM EGTA and 30 mM buffers of various pH values.The electrogenic pump was stopped by removing the intracellularATP. The ATP-dependent part of the membrane potential was largeat pH₁ 6.2 and 6.9, but decreased in both the acidic (pH₁ 5.1,5.7) and alkaline (pH₁ 7.9, 8.7) ranges. Assuming that the putativeelectrogenic H+ pump acts solely as a current source, we estimatedthe current and the work done by the pump. Both the currentand the work were at a maximum at pH₁ 6.9, but decreased tosome extent at pH₁ 7.9–8.7 and decreased greatly at pH₁5.7–5.1. Light caused a significant membrane hyperpolarization at pH₁6.2–7.9, but only a slight hyperpolarization at pH₁ 5.1,5.7 and 8.7. The presence of light-induced hyperpolarizationat pH₁ lower than 6.2 suggests that acidification of the cytoplasmupon illumination does not cause activation of the putativeH+ pump. Membrane resistance was very high at pH₁ 5.1 and 5.7 and verylow at pH₁ 8.7. The very high R_m values at pH₁ 5.1 and 5.6 arebelieved to be related to inhibition of the activity of thepump. (Received June 20, 1979; )     

Far-red light-induced changes in intracellular potentials of spinach mesophyll cells: interaction with red light

In green plants, the large bioelectric changes that photosynthetically active light stimulates make it difficult to observe electrical potential changes related to phytochrome photoconversion. As a first step towards distinguishing between photosynthetic and phytochrome effects, we showed that red light enhances far-red stimulated intracellular potential changes in spinach (Spinacia oleracea) leaf mesophyll cells.

For a dark-adapted leaf, the response to far-red light increased during the first 10 to 30 exposures of 2.5 minutes, after which it was constant. The intracellular potential depolarized by an average of 0.3 millivolts during each 2.5-minute far-red light period, and returned to the resting value during each subsequent dark period. Continuous supplementary red light (at 1-5% of the fluence rate of the far-red light that stimulated the depolarizations) increased the response to far-red 2- to 3-fold. Supplementary red light did not amplify the response to alternating 702 nanometers light and dark periods. The Emerson enhancement effect thus does not seem to explain amplification of the response to 730 nanometers light by supplementary red light. This does not prove that photosynthetic pigments are not involved in some other way.

     

Simulation of the light-induced oscillations of the membrane potential in Potamogeton leaf cells

Summary An attempt has been made to simulate the light-induced oscillations of the membrane potential of Potamogeton lucens leaf cells in relation to the apoplastic pH changes. Previously it was demonstrated that the membrane potential of these cells can be described in terms of proton movements only. It is hypothesized that the membrane potential is determined by an electrogenic H⁺-ATPase with a variable H⁺/ATP stoichiometry. The stoichiometry shifts from a value of two in the dark to a value of one in the light. Moreover, this H⁺ pump shows the characteristics of either a pump or a passive H⁺ conductance: the mode of operation of the H⁺ translocator is considered to be regulated by the external pH. The pump conductance is assumed to be dominant at low or neutral pH, while the passive H⁺ conductance becomes more significant at alkaline pH. The pH dependence of the transport characteristic is expressed by protonation reactions in the plasma membrane. The proposed model can account for most features of the light-induced oscillations but not for the absolute level of the membrane potential.This research was supported by the Foundation of Biophysics, part of the Dutch Organization for Scientific Research (NWO) ECOTRANS publication No. 34.     

油红O染色原代未活化胰腺星状细胞脂肪滴的实验观察

目的观察油红O染色原代培养的未活化胰腺星状细胞脂肪滴,并予鉴定。方法选取接种于6孔板中培养4天的原代未活化胰腺星状细胞,予100g/L甲醛液固定,磷酸盐缓冲液漂洗后,加入5g/L油红O饱和液染色,镜下动态观察。胞浆内的脂肪滴一旦着色,即可洗去油红,苏木素衬染胞核,漂洗分化脱色后镜下成像。结果未活化的胰腺星状细胞脂肪滴被油红0染成鲜艳的红色,大小不一的串珠样"油珠子"分散于胞质中,簇集成"环状",呈戒环样包绕在细胞核周围。结论油红染色原代未活化胰星状细胞脂肪滴是鉴定该细胞的重要方法之一。     

Magnetic detection of a single action potential in Chara corallina internodal cells.

The electrical activity that occurs in plants has not yet been detected magnetically. Magnetic detection of electrical activity in some animal as well as in human cells and organs, on the other hand, is an established research method. Our experiments demonstrate the propagation of a single action potential in the internodal cell of the green algae Chara corallina, measured magnetically. The propagation velocity and the intracellular current were determined.     

Light-induced changes of bioelectric potential in Chara

Experiments were performed on light-induced changes of the restingpotential in Chara under various conditions. In the dark-adaptedcells, a slow increase in resting potential, by about 60 mv,appeared in a solution containing 0.5 mM KCl, 0.2 mM NaCl and0.5 mM CaCl₂. On the contrary, a rapid decrease preceded bya small, sharp rise in potential was produced when the cellsbecame adapted to light. On the first illumination, the cellmembrane resistance decreased in dark-adapted cells, but a slightincrease was observed every time on subsequent illuminations.No parallel relation was found between the time course of thechanges of resistance and the potential difference. During severalilluminations, as well as during the short dark periods betweenthem, cells lost their sensitivity to change in a potassiumconcentration. The time courses of photosynthetic oxygen evolutionwere not in accordance with those of changes in the potential.However, 10µM 3-(3,4-dichlorophenyl)-l, l-dimethylureareversibly abolished both the oxygen evolution and the changesin potential. An enhancement of the photoelectric response wasobserved when bicarbonate ions were added in the external solution.On the other hand, the oxygen evolution was not affected bythe bicarbonate ions. On the basis of these observations itwas assumed that some assimilation products of photosynthesiswere responsible for the photoelectric response. (Received February 13, 1968; )     

Cyclic variations in the ruthenium red stained coat of cells from a synchronized human lymphoblastoid line

A human lymphoblastoid cell line derived from a normal donor has been synchronized with a double thymidine block. The affinity of the cell membrane for ruthenium red was examined during different phases of the cell cycle. The thickness of the cell coat increased from G1 to G2. The significance of this finding is discussed.     

Involvement of membrane potential in alkaline band formation by internodal cells of Chara corallina

Internodal cells of Chara corallina form alkaline bands on their surface upon illumination via photosynthesis. In the present study, the effect of KCl on alkaline band formation was analyzed. When the extracellular KCl concentration was increased, alkaline band formation was extensively inhibited. Electrophysiological analysis unequivocally showed the need for inner negative membrane potential for alkaline band formation.     

Dynamics of chloride and potassium currents during the action potential in Chara studied with action potential clamp

TheCl^– and K⁺ currents underlying the action potential (AP) in the giant alga Chara were directly recorded with the action potential clamp method. An electrically triggered action potential was recorded and repetitively replayed as command voltage to the same cell under voltage clamp. The resulting clamp current was close to zero. Only the initial rectangular current used for stimulation was approximately reproduced by the clamp circuit. Inhibition of Cl^– channels with niflumic acid or ethacrynic acid and of K⁺ channels with Ba²⁺ evoked characteristic compensation currents because the amplifier had to add the selectively inhibited currents. Integration of the compensation currents revealed a mean flux through Cl^– and K⁺ channels of 3.3 10^–6 and 2.1 10^–6 mole M^–2 AP^–1 respectively. The dynamics of CI^– and K⁺ channel activation/inactivation were obtained by converting the relevant clamp currents to ionic permeabilities using the Goldman-Hodgkin-Katz current equation. During the AP the Cl^– permeability reaches a peak 370 ms, on average, after termination of the stimulating pulse. The following inactivation proceeds 3.6 times slower than the activation. The increase in K⁺ permeability lags behind the rise in Cl^– permeability, reaching a peak approximately 2 s after the latter.     

The cause of the green polarization color of amyloid stained with Congo red

Summary Experiments done with Congo red crystals and with Congo red deposits polished in a single direction by a glass wheel have shown that the appearance of green polarization color primarily depends on near-perfect parallel alignment of the dye particles. The green polarization color was seen only in the deposits which showed a clear transition from red to colorless when examined for dichroism. Another factor was found to be the thickness of the object, as the green polarization color was not present in too thick or too thin sections of amyloid-containing tissues stained with Congo red.The phenomena can be explained by the assumption that the green polarization color is due to interference between the red ray and the red component of the white ray whenever the retardation by the object approximates half the wavelength of red light.The findings indicate that amyloid differs from other materials which are stained by Congo red in that amyloid deposits bind the dye molecules in a more orderly and parallel fashion. It is suggested that minimal amounts of amyloid which are not visible in Congo red stained sections with ordinary light microscopy and which do not give the green polarization color can best be detected by examination for dichroism in ultraviolet light after having been stained with fluorescent dyes.     

Functional evaluation of cultured cells under hypotonic conditions by intravital staining with neutral red

A high stability of pig embryo kidney-cells to hypotony was shown with neutral red vital staining. In the medium, diluted 1 : 1, the cells kept their ability to make granules during 6 days. In the medium diluted by water 1 : 3, both granular and diffuse staining of cells was shown within 6-12 hours. Later (18-48 hours) the cell ability to make granules was kept, but the neutral red was deposed as large vacuoles. In the normal medium the cell ability to make granules was kept too. In the medium, diluted by water 1 : 7, the cells lost their ability to make granules. The ability was recovered when cells were returned to the normal medium. Thus, after 6-18 hours of incubation in such a hypotonic medium, the cells produced small granules of neutral red, and 24-48 hours later they made large granules.