Masashi Tazawa

Control of membrane potential and excitability ofChara cells with ATP and Mg2

SummmaryElectric characteristics of internodalChara australis cells, from which the tonoplast had been removed by vacuolar perfusion with media containing EGTA, were studied in relation to intracellular concentrations of ATP and Mg2+ using the ordinary microelectrode method and the open-vacuole method developed by Tazawa, Kikuyama and Nakagawa (1975.Plant Cell Physiol.16:611). The concentration of ATP was decreased by introducing hexokinase and glucose into the cell and that of Mg2+ by introducing EDTA or CyDTA. The membrane potential decrease and the membrane resistance increase were both significant when the ATP or Mg2+ concentration was decreased. An ATP-dependent membrane potential was also found in other species of Characeae,Nitella axillaris andN. pulchella. Excitability of the membrane was also completely lost by reducing the ATP or Mg2+ concentration. Both membrane potential and excitability were recovered by introducing ATP or Mg2+ into ATP- or Mg2+-depleted cells.The time course of membrane potential recovery was followed by the open-vacuole method. Recovery began as soon as intracellular perfusion with medium containing ATP and Mg2+ was started. Reversible transition of the membrane potential between polarized and pepolarized levels by controlling the intracellular concentration of ATP or Mg2+ could be repeated many times by the open-vacuole method, when the excitability was suppressed by addition of Pb2+ to the external medium.The ineffectiveness of an ATP analog, AMP-PNP, and the synergism of ATP and Mg2+ in maintaining the membrane potential and excitability strongly suggest that ATP act via its hydrolysis by Mg2+-activated ATPase. The passive nature of the membrane, as judged from responses of the membrane potential to changes of the external K+ concentration, was not altered by lowering the ATP concentration in the cell. The mechanism of membrane potential generation dependent on ATP is discussed on the basic of an electrogenic ion pump. Involvement of the membrane potential generated by the ion pump in the action potential is also discussed.

Demonstration of two stable potential states of plasmalemma ofChara without tonoplast

SummaryThe tonoplast of cells ofChara australis was removed by replacement of the cell sap with a medium containing 5 mM EGTA (ethyleneglycol-bis-(β-aminoethyl ether) N, N′-tetraacetic acid). Such cells without tonoplast could generate an action potential of rectangular shape. In the present paper characteristics of the action potential were studied under various external ionic conditions.Action potentials could be elicited without refractory period and the peak of the action potential was constant among action potentials.Duration of the action potential decreased under repeated excitations, but recovered after pause. Increase in concentrations of alkali metal cations, Li+, Na+, K+, Rb+ and Cs+, resulted in prolongation of the action potential.At proper concentrations of monovalent cations the membrane potential could stay either at the resting level or at the depolarized level and could be shifted reversibly from the former level to the latter one orvice versa by applying outward or inward current. Further increase in concentrations of monovalent cations resulted in arrest of the membrane potential at the depolirized level. The critical concentrations of the monovalent cations to hold the membrane potential at the depolarized level were about 10 mM irrespective of the cation species.Divalent cations, Ca2+, Mg2+, Sr2+, Ni2+ and Mn2+, added to the bathing medium suppressed the effect of monovalent cations to prolong the action potential.Ca2+ and Mg2+ added to the bathing medium caused repolarization of the plasmalemma which had been depolarized by application of high concentrations of K+ to the bathing medium. The antagonism between monovalent and divalent cations on the state of the plasmalemma ofChara cells was discussed based on the two stable states hypothesis proposed by Tasaki (Tasaki, I. 1968. Nerve Excitation. Charles C. Thomas, Springfield, Illinois).

The relation of turgor pressure to cell volume inNitella with special reference to mechanical properties of the cell wall

Summary1.A method was developed, by which it was possible to measure the volume of an internodal cell ofNitella flexilis as a function of interior pressure. For this to be done, the cell wall tube, closed at one end with the natural septum, was filled with mercury and pressure was applied to the mercury. The accompanying change in volume of the cell wall tube was measured simultaneously with the applied pressure.2.The time course of volume change of the cell in response to change in interior pressure indicates that cell wall elasticity is composed of at least two components, an instantaneous elastic component, and a retarded elastic component with a retardation time of about 1–5 minutes.3.Both instantaneous and slow processes in volume change vary according to the level of the pressure applied and to the direction of the pressure change.4.The volume of the cell can be kept at different values, under the same interior pressure, according to the direction of the pressure change; in other words, the interior pressure-volume relation shows a hysteresis.5.Taking into consideration the hysteresis character in mechanical properties of the cell wall, the osmotic pressure, turgor pressure and suction force (diffusion pressure deficit) of an internodal cell ofNitella flexilis was illustrated in relation to the cell volume in an osmotic diagram after Höfler. A characteristic of the diagram is that the cell can have different turgor pressures and suction forces within certain limits even though the volume of the cell is the same.6.The length of the living cell was measured under different turgor pressures. The facts that the pressure-cell length relation showed also a distinct hysteresis character and that the wall elasticity of the living cells was in the same order as that of the cells filled with mercury, indicate that the results obtained with cell wall tubes were also true of the living cells.7.The cell wall of the internodal cell ofNitella flexilis extends more in the direction of transverse axis of the cell than in the direction of longitudinal axis under the influence of turgor pressure. When equal tensions in the respective direction are considered, however, the cell wall extends to the same extent in each direction.8.The uniaxial longitudinal tension, caused by loading, elongates the cell about 3–4 times more than does the longitudinal component of the equivalent tension caused by turgor pressure.

Motive force of the cytoplasmic streaming in nitella.

SummaryBy modifying the technique of vacuolar perfusion, a new method for measuring the motive force of the rotational cytoplasmic streaming inNitella internodes was developed. The motive force, being almost independent of temperature in the range of 10–30‡ C, amounts to 1.4–2.0 dynes/cm2. The decrease in the rate of streaming with decrease in temperature is therefore explained in terms of the increase of cytoplasmic viscosity. Lowering of the tonicity of the cell sap decreases both motive force and rate of streaming, while heightening of tonicity increases both. The cell is without turgor pressure throughout. Inhibition of streaming byp-chloromerocuribenzoate is accounted for by an effect on the motive force, not on the cytoplasmic viscosity.

Hydraulic conductivity of tonoplast-freeChara cells

SummaryThis study is the first trial to measure the osmotic water permeability or the hydraulic conductivity of the plasmalemma alone of a plant cell. For this purpose tonoplast-free cells were prepared from intenodal cells ofChara australis and their hydraulic conductivities were measured by the transcellular osmosis method.The transcellular hydraulic conductivity did not change after removing the tonoplast. The transcellular hydraulic conductivity of the tonoplast-free cells was dependent on the internal osmotic pressure as is the case in the tonoplast-containing normal cells. The hydraulic conductivities for both endosmosis and exosmosis of the tonoplast-free cells were equal to respective values of the normal cells. Consequently the ratio between the inward and outward hydraulic conductivities did not change due to the loss of the tonoplast. The results indicate that the resistance of the tonoplast to water flow is negligibly small as compared with that of the plasmalemma and further that the tonoplast is not a factor responsible for the direction-dependency of hydraulic conductivity. The hydraulic conductivity of the plasmalemma is invariable for wide variations of K+ and Ca2+ in the cytoplasm.

Tonoplast action potential inNitella in relation to vacuolar chloride concentration

SummaryThe action potential ofNitella internode was studied in relation to K+ and Cl− concentrations in the vacuole. When the vacuole ofNitella pulchella was filled with an artificial solution with extremely low Cl− concentration, a diphasic action potential (DAP) was observed. The first phase consists of a rapid depolarization followed by a relatively rapid repolarization, and the second one consists of a strong hyperpolarization followed by a gradual return to the resting potential.When the cell was stimulated immediately after the generation ofDAP, a monophasic action potential which resembles an action potential of the natural cell was observed, indicating that theDAP consists of two components with different refractory periods. The refractory period of the component responsible for the depolarizing phase is shorter than that of a component responsible for the hyperpolarizing phase. Measuring the plasmalemma potential and vacuolar potential separately, it was demonstrated that the hyperpolarizing component ofDAP originates from the tonoplast.The action potential of the tonoplast, in contrast with that of the plasmalemma, could be generated independently of concentration of K+ in the vacuole. Since the maximum amplitude of hyperpolarization decreased significantly by increasing Cl− concentration of the vacuole, it is concluded that the tonoplast is very sensitive to Cl− during excitation.

Intracellular chloride and potassium ions in relation to excitability ofChara membrane

SummaryInternodal cells ofChara australis were made tonoplast-free by replacing the cell sap with EGTA-containing media; then the involvement of internal Cl− and K+ in the excitation of the plasmalemma was studied.[Cl−]i was drastically decreased by perfusing the cell interior twice with a medium lacking Cl−. The lowered [Cl−]i was about 0.01mm. Cells with this low [Cl−]i generated action potential and showed anN-shapedV−I curve under voltage clamped depolarization like Cl−-rich cells containing 13 or 29mm Cl−.Em at the peak of the action potential was constant at [Cl−]i between 0.01 and 29mm. The possibility that the plasmalemma becomes as permeable to other anions as to Cl− during excitation is discussed.At [Cl−]i higher than 48mm, cells were inexcitable. When anions were added to the perfusion medium to bring the K+ concentration to 100mm, NO3−, F−, SO42−, acetate, and propionate inhibited the generation of action potentials like Cl−, while methane sulfonate, PIPES, and phosphate did not inhibit excitability.The duration of the action potential depended strongly on the intracellular K+ concentration. It decreased as [K+]i (K-methane sulfonate) increased. Increase in [Na+]i (Na-methane sulfonate) also caused its decrease, although this effect was weaker than that of K+. The action of these monovalent cations on the duration of the action potential is the opposite of their action on the membrane from the outside (cf. Shimmen, Kikuyama & Tazawa, 1976,J. Membrane Biol.30:249).

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 (Em) and the membrane resistance (Rm) 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), Em and Rm were very sensitive to pH0, as in normal cells. Em 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 Em and Rm on pH0. Thus, the active and not the passive component of Em 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, Em was scarcely affected and Rm markedly decreased on illumination.

Influence of intracellular and extracellular tonicities on water permeability in Characean cells

SummaryThe effect of the concentration of the central vacuolar sap on water permeability previously demonstrated onNitella internode (Tazawa and Kamiya 1966), has been further studied. By using a technique of vacuole perfusion the ionic concentration of the cell sap has been modified independently of its tonicity. Transcellular water permeability has been measured by means of a double-chamber osmometer.When the tonicities of artificial saps were adjusted to that of the natural cell sap, wide variations in the concentration of K+, Na+, or Ca++ in the vacuole did not bring about any change in the magnitude of water permeability. On the other hand, water permeability was strongly influenced by varying the tonicity of the vacuolar medium by addition of mannitol. It increased when the tonicity was lowered from the normal level, while it decreased when tonicity was heightened. Water permeability was also decreased by increase in the tonicity of the external medium.Analysis of the results showed that the specific resistance to water flow across the plasmalemma and the tonoplast in series (the reciprocal of the water permeability k′p) was related to the osmotic pressures of the intracellular (πi) and the extracellular (π0) medium by the empirical formula, l/k′p=0.088 + 0.015 π. + 0.0074π0. Thus, intra- and extracellular tonicities influence the water permeability of theNitella internode independently of each other. The decrease in water permeability by increase in tonicity of the intra- or extracellular medium may be explained in terms of the effect of these tonicities on hydration of the cell membranes.The water permeability ofLamprothamnium, a brackish water Characeae was only one fourth that ofNitella, a fresh water Characeae. The lower permeability inLamprothamnium may be accounted for in terms of the high tonicities of its cell sap and external medium.

Membrane characteristics as revealed by water and ionic relations of algal cells

Membrane characteristics of plant cells have been intensively studied in algal cells. Because of the occurrence of giant cell forms in some algae, algal cells are much appreciated as material for the study of water and ionic relations in a single cell. In addition to the general outstanding merits in using single cells instead of tissues, studies on single giant algal cells have further merits, since for them techniques required for the study of membrane physiology in plant cells such as insertion of microelectrodes, intracellular perfusion, isolation of cytoplasm from the cell sap, fragmentation of a single cell into many parts, etc. are easily applicable. Many reviews treating water and ionic relations of plant cells have therefore been presented mainly on the basis of results accumulated on algal cells, especially on giant algal cells. The reader should refer to reviews and monographs by Be>~N~T-CLaRI~ (1950), DAINTY (1963, 1964), DICK (1966) on water relations of plant cells, DAINTY (1962), SCOTT (1967), SCI-tlLDE (1968), and GUTKI,~ECI-IT and DAINTY (1968), MACROBBIE (1971), and HOPE (1971) on ionic relations and electrophysiology. In the present article the author intends to treat several problems concerning characteristics of the outer and inner membranes of plant cells based on the results obtained recently on algal cells, mainly on giant internodal cells of Characeae.