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)     

Potassium, sodium and Chloride in the protoplasm of characeae

Using vacuolar perfusion, which enabled us to replace the cellsap with a solution containing no k⁺, Na⁺ and Cl^–, theconcentrations of these ions in the protoplasm of three speciesof fresh water Characeae were determined. They were respectively,78, 2 and 27 mM in Nitella flexilis, 101, 9 and 31 mM in Nitellapulchella, and 112, 3 and 21 mM in Chara australis. Our previouslyreported results (3) indicating that the chloroplast layer containedmuch more Na⁺ and Cl^– than the endoplasm has been questionedin the light of the present results. ¹Present address: Department of Biology, College of GeneralEducation, Osaka University. (Received September 5, 1973; )     

Effect of Ca2+ on Tonoplast Potential of Permeabilized Characeae Cells

Using permeabilized characean cells in which the ionic conditionsat the cytoplasmic side of the tonoplast are easily controlled,effects of Ca²⁺ ion on tonoplast potential were examined. Whenthe cell was treated with 1 µM Ca²⁺, the tonoplast potential(E_M became positive in a complicated manner in Chara corallinawhile it simply became negative in Nitella axilliformis. Whenthe cell was treated with 9-antracenecarboxylic acid, a Cl^–-channelinhibitor, E_m became more negative and the response of E_m toCa²⁺ was significantly suppressed. It is suggested that Ca²⁺activates Cl^–-channel at a low concentration and inactivatesat a higher one in C. corallina while it simply inactivate Cl^–-channelin N. axilliformis. ¹Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Wakaba, 260 Japan. (Received August 22, 1988; Accepted December 26, 1988)     

Electrophysiological characterization of the node in Chara corallina: functional differentiation for wounding response

Electrical characteristics of the node were analyzed in comparisonwith those of the flank of the internodal cell in Chara corallina.The dependence of the membrane potential of the node on pH andK⁺ concentration was almost the same as that of the flank. Inthe flank, the increase in the Ca²⁺ concentration stopped thedepolarization in the presence of 100 mM KCl. In the node, however,Ca²⁺ could not stop the depolarization induced by 100 mM KCl.It has been reported that the node has a function to tranducethe signal of osmotic shock into a transient depolarization.In combination with osmotic shock, 10 mM K⁺ could induce a long-lastingdepolarization of the node. These electrical characteristicsof the node were suggested to be responsible for the electricalresponse to wounding in Characeae.     

Cytoplasmic Hydration Triggers a Transient Increase in Cytoplasmic Ca2+ Concentration in Nitella flexilis

Cells of Nitella flexilis were made inexcitable by treatmentwith 10 mM KCl for more than 24 h. A Ca²⁺-sensitive photoproteinaequorin was injected into the cytoplasm of such cells. Forvacuolar per fusion, the central part of an aequorin-loadedcell was immersed in silicone oil, and both cell ends bathedin the perfusion medium were cut off. A large light emissionfrom aequorin was observed when the vacuole was perfused witha hypotonic medium whose osmotic pressure was adjusted to halfof the osmotic pressure of the cell sap. This shows that hydrationof the cytoplasm triggers release of Ca²⁺ from internal stores,since influx of Ca²⁺ from silicone oil is excluded. Hydration of cells was induced in another way. Cells were firstdehydrated by transferring them from 10 mM KCl solution to thatwith 250 mM sorbitol added. This procedure did not affect thecytoplasmic streaming. When cells were rehydrated by transferringthem to 10 mM KCl solution, cytoplasmic streaming was eitherstopped or slowed down in a few seconds. A quick light emissionfrom aequorin was observed in the rehydration, evidence thatcytoplasmic streaming was inhibited by an increase in the cytoplasmicCa²⁺ concentration. Both streaming cessation and aequorin lightemission were observed even in KCl-treated cells which werefurther treated with 5 mM EGTA. Thus, the increase in Ca²⁺ isconcluded to be caused by the release of Ca²⁺ from internalstores. These results support our previous hypothesis [Tazawa et al.(1994) Plant Cell Physiol. 35:63] that, in Nitella flexilis,the increase in the concentration of Ca²⁺ in the cytoplasm whichoccurs on the endoosmotic side of the cell during transcellularosmosis is caused by hydration of the cytoplasm. (Received June 6, 1994; Accepted December 26, 1994)     

Ion Efflux during a Single Action Potential of Nitella axilliformis in Medium Lacking Ca2-

Ion efflux during excitation of Nitella axilliformis was measuredconductometrically. In medium lacking Ca²⁺ but with 0.1 mM MgCl₂,the duration of the action potential and the total efflux weremuch larger than those in APW, while the efflux rate, givenas the total efflux divided by the duration, was about halfof that in APW. (Received September 4, 1986; Accepted November 25, 1986)     

The light-dependent effect of external pH on the membrane potential of Nitella

In the light the membrane potential of Nitella flexilis andNitella axilliformis was hyperpolarized by raising the externalpH above pH 5.5, at the rate of 30–40 mV/pH below pH 8.This hyperpolarization was largely reduced in the dark. The membrane potential was sensitive to the external pH of mediawith a low potassium concentration, where it was relativelyinsensitive to potassium concentration. In media of a high concentrationwhere it was sensitive to the potassium concentration, the membranebecame insensitive to the external pH. The transition from apH-sensitive to a pH-insensitive state occurred rather abruptlyon increasing the external potassium concentration. (Received September 1, 1972; )     

Nuclear crystals, lampbrush-chromosome-like structures, and perinuclear cytoskeletal elements associated with nuclear fragmentation in characean internodal cells

Summary This article gives a survey of nucleus-associated structures and inclusions in a diverse range of characean algae includingChara braunii Gm.,Chara corallina Klein ex Willd., em. R.D.W.,Nitella cristata A.Br., em. R.D.W.,Nitella flexilis (L.) Ag.,Nitella furcata (Roxb. ex Bruz.) Ag. em. R.D.W.,Nitella hyalina (DC.) Ag.,Nitella pseudoflabellata A.Br., em. R.D.W.,Nitella pseudoflabellata var.imperialis T.F.A.,Nitella translucens var.axillaris (A.Br.) R.D.W. andNitellopsis obtusa (Desv. in Lois.) J.Gr. Lampbrushchromosome-like structures were found in nuclei ofNitella flexilis andNitellopsis obtusa and seem to be involved in the distribution of genetic material during nuclear fragmentation. Intranuclear tubular crystals of unknown protein composition were present in all species, especially in young, elongating cells, and could be important for establishing the main axis of the nuclei. Spindle-shaped protein crystals that originate in the nucleus and are released into the cytoplasm upon nuclear degeneration were observed in branchlet internodal cells of one population ofNitella flexilis. Perinuclear microtubules were present in all species, but perinuclear actin fibrils were hitherto only found in mostNitella species and inNitellopsis obtusa. None of these nucleus-associated structures seems to be responsible for the formation of constrictions leading to nuclear fragmentation. These constrictions were perpendicular to the main axis of the nucleus and symmetrical in theNitella species but asymmetric inC. braunii, C. corallina, and inNitellopsis obtusa. Statistical analysis of nuclear size, number and constriction sites indicate that fragmentation is a nonsynchronous process independent of the light-dark cycle.Abbreviations CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - LCLS lampbrush chromosome-like structure(s) Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Involvement of Water Channels in the Hydration-Induced Calcium Release in Nitella flexilis

An internodal cell of Nitella flexilis treated with 10 mM KC1was vacuole-perfused with an isotonic solution containing ethyleneglycol-bis-(rß-aminoethylether)N,N,N,N-tetraaceticacid (EGTA) and its content including cytoplasm was squeezedout into a vessel and covered with silicone oil. When the hypotonicsolution was added into the cytoplasmic drop which had beenmixed with aequorin, a significant increase in the light emissionfrom aequorin was detected with the photomultiplier, indicatinga release of Ca²⁺ from some cell organdies storing Ca²⁺. Thisincrease in the light emission was strongly inhibited by treatingcells with 0.1 mM HgCl₂ which is known to inhibit water channelsin the plasma membrane. The inhibition was completely recoveredby washing HgCl₂ with 2-mer-captoethanol. This suggests thatwater channels may exist in the membrane of Ca²⁺ stores andplay an essential role in the hydration-induced Ca²⁺ release. (Received February 12, 1998; Accepted May 21, 1998)     

Effect of Divalent Cations on Na+ Permeability of Chara corallina and Freshwater Grown Chara buckellii

The effect of elevated Na⁺ concentration on Na⁺ permeability(P_Na) and Na⁺ influx in the presence of two levels of externaldivalent cations was determined in Chara corallina and freshwater-culturedChara buckellii. When Na⁺ in the medium was increased from 1.0to 70 mol m^–3, Na⁺ influx increased in both species ifCa²⁺ was low (0.1 mol m–3). If Ca²⁺ was increased to 7.0mol m^–3 when Na⁺ was increased, Na⁺ influx remained atthe low control level in C. corallina, and showed only a temporaryincrease in C. buckellii. Mg²⁺ was a better substitute for Ca²⁺in C. buckellii than in C. corallina. Na⁺ permeability data suggest that when the external Ca²⁺ concentrationis low, P_Na does not increase in the presence of elevated NaCl;the increase in Na⁺ influx appears to be due to the increasein external Na⁺ concentration alone. Ca^{2 +} supplementation appearsto decrease P_Na whereas supplemental Mg²⁺ has no effect. Na⁺ effluxes were computed from previously determined net fluxesand the influxes. It was found that for both species, fluxesin both directions were stimulated in response to all experimentaltreatments, but Na⁺ influx always exceeded efflux. This resultedin net Na⁺ accumulation in the vacuoles of both species. The results are discussed with reference to net flux and electrophysiologicaldata obtained previously under identical conditions, as wellas the comparative salinity tolerance of both species and theNa⁺/divalent cation ratio. Key words: Na⁺ influx, Na⁺ tolerance, membrane potential, permeability, Chara     

The Measurements of the Membrane Resistance of Characeae by Alternating and Direct Currents

The internodal cells of Nitellopsis obtusa, Chara brauni, andNitella flexilis were investigated quasi-simultaneously by directand alternating currents with a frequency range from 1 to 1000Hz. The membrane resistance obtained with a.c. was significantlylower than that obtained with d.c. We suggest that the discrepancies in the results obtained canbe accounted for in terms of a three-layer model of the cellmembrane.     

Further Studies of Electro-Osmosis in Nitella in Relation to Pores in Membranes

By measuring the number of water molecules per ion which weremoved electro-osmotically through cells of Nitella translucensand N. flexilis it has been shown that a significant differenceexisted between samples of these species in 1965. In each speciesthe electro-osmotic efficiency was greater with Na⁺ than withK⁺⁺. Also 10^-4 to 10^-5 M IAA tended to decrease electro-osmoticefficiencies while IAA, after 30 min. treatment, produced asignificant increase in water flow into the treated end of aliving cell. Calculations based on this work suggest that about10⁸–10⁹ pore sites per cm² exist on the surface membranesfor Na⁺ or K⁺ ion transport.     

Respiration-Dependent Membrane Hyperpolarization in Tonoplast-Free Cells of Nitella axilliformis

Cells of Nitella axilliformis were made tonoplast-free by intracellularperfusion of media containing ethyleneglycol-bis-(ß-aminoethylether)N,N'-tetraaceticacid (EGTA). When the perfusion medium contained ADP as wellas ATP, the membrane hyperpolarized in darkness in a mannersimilar to light-induced hyperpolarization. This light-independenthyperpolarization seems to be due to activation of the electrogenicion pump in the plasma membrane because the hyperpolarized valueof the membrane potential was more negative than the equilibriumpotential for K^$, the most negative ion equilibrium potentialin Nitella. The hyperpolarization was inhibited by the respiratory chaininhibitors NaCN (1 mM), antimycin A (10 µM) and rotenone(10 µM). NaCN slightly decreased the ATP concentrationin the cell perfused with medium containing 1 mM ATP and 1 mMADP; but, even after treatment with NaCN, the cell had about80% of the ATP value for the control. ^* This study is dedicated to the late Professor J. Ashida. (Received June 24, 1982; Accepted October 15, 1982)     

Water channels in Chara corallina

Water relations parameters ofChara corallina inter-nodes weremeasured using the single cell pressure probe. The effect ofmercurials, which are recognized as non-specific water channelinhibitors, was examined. HgCl₂ concentrations greater than5 mmol m^–3 were found to inhibit hydraulic conductivity{Lp) close to 90%, whereas pCMPS was found to have no effecton Lp. The activation energy of water flow was increased significantlyfrom 21.0 kJ mol^–1 to 45.6 kJ mol^–1, following theapplication of HgCl₂. These results are in accordance with evidencefor Hg²⁺sensitive water channels in the plasma membrane of charophytes(Henzler and Steudle, 1995; Tazawa et al., 1996). The metaboliceffects must, however, be considered in view of the rapid inhibitionof respiration and the depolarization of the membrane potentialwith HgCl₂ concentrations lower than those found to affect Lp.It was possible to measure simultaneously water relations andmembrane PD, in order to examine the contribution of potassiumchannels to Lp. Cells were induced into a K⁺ permeable state.The K⁺ channels, assumed to be open, were subsequently blockedby various blockers. No significant difference in Lp was foundfor any of these treatments. Finally, the permeability of C.corallina membranes to ethanol was examined. HgCl₂ was foundto cause a decrease in reflection coefficient, coinciding witha decrease in Lp, but there was no change in the ethanol permeabilitycoefficient. This has been interpreted in terms of both thefrictional model and composite model of non-electrolyte membranetransport. Key words: Water channels, Chara, hydraulic, conductivity, membrane transport models, reflection coefficient     

Osmotic and ionic regulation in Nitella

When the osmotic value of an internodal cell of Nitella flexiliswas modified by the method of transcellular osmosis, the normalosmotic value was chiefly restored by the release or absorptionof K⁺. The release or uptake of Na⁺ was observed only when themodification of osmotic value was significant. Both the uptakeand release of K⁺ were linearly dependent on the degree of modificationof the osmotic value. The effectiveness of alkali metal cationsin restoring the osmotic value in cells of lower osmotic valueswas in the order K⁺>Rb⁺>Na⁺, Cs⁺>Li⁺. The absorptionof K⁺ by cells of lower osmotic values depended strongly ontemperature, while the release of K⁺ from cells of higher osmoticvalues did not. To clarify whether the Nitella cell regulates the osmotic valueor regulates the concentration of K⁺ in the vacuole, the cellsap was exchanged for artificial cell saps whose osmotic valuesand ionic concentrations were varied independent of each other.It was shown that in Nitella two regulating mechanisms are operating,one which regulates the osmotic value of the cell sap irrespectiveof the level of vacuolar K⁺ (0.1–140 mM) and another whichregulates the vacuolar K⁺-level when it is abnormaly high (>160mM). Both mechanisms are assumed to operate in order to keepthe concentration of K⁺ in the cytoplasm at a constant level.The presence of Na⁺ (0–100 mM) and Ca²⁺ (5–40 mM)did not affect the movement of K⁺ during osmoregulation. ¹Present address: Sanki Engineering Limited, Nagaokakyo, Kyoto,Japan. (Received December 19, 1973; )     

Effect of Sodium, Potassium, Calcium, Magnesium and Tetraethylammonium on the Transient Voltage Response to a Galvanostatic Step and of the Temperature on the Steady Membrane Conductance of Chara corallina: a Further Evidence for the Involvement of Potassium Channels in the Fast Time Variant Conductance

Homble F. 1985. Effect of sodium, potassium, calcium, magnesiumand tetraethylammonium on the transient voltage response toa galvanostatic step and of the temperature on the steady membraneconductance of Chara corallina: A further evidence for the involvementof potassium in the fast time variant conductance.—J.exp. Bot. 36: 1603–1611. Potassium channels of Chara corallina have an activation energyof 36±1 kJ mol^–1 and 50±2 kJ mol^–1at temperatures higher and lower than 15°C respectively.The fast time variant conductance property of potassium channelsis insensitive to sodium and magnesium ions and is depressedby the presence of calcium, potassium and tetraethylammoniumions. It is suggested that in Chara two different kinds of potassiumchannels exist, each kind being distinguished by their kineticsand their response to calcium and magnesium ions. Key words: —Chara corallina, membrane conductance, potassium channels, temperature     

Studies on Mechano-Perception in Characeae: Effects of External Ca2+ and Cl-

The effects of modification of extracellular concentrationsof Ca²⁺ and C^– on mechano-perception were studied in internodalcells of Chara corallina. Cells were stimulated by droppinga piece of glass tubing on them, and the resulting receptorpotentials and action potentials were analyzed. When the Ca²⁺concentration was extremely lowered by adding EGTA, the amplitudesof both receptor potentials and action potentials were attenuated,suggesting the involvement of Ca²⁺ channels. However, the possibilityremained that attenuation of the amplitude of the receptor potentialwas caused by modification of membrane characteristics by extremelowering of [Ca²⁺]_o. When the plasma membrane was depolarizedto about 0 mV by adding 100 mM KC1, responses in the negativedirection were induced upon mechanical stimulation. When theplasma membrane was depolarized by adding 50 mM K²SO⁴, responsesin the positive direction were induced. Thus, Cl^– channelsmay be involved in responses induced by mechanical stimulationunder K+-induced depolarization. (Received January 16, 1996; Accepted March 25, 1997)     

Effects of Cations on the Cytoplasmic pH of Chara corallina

Smith, F. A. and Gibson, J.–L. 1985. Effects of cationson the cytoplasmic pH of Chara corallina.—J.exp. Bot.36: 1331–1340 Removal of external Ca²⁺ from cells of Chara corallina lowersthe cytoplasmic pH, as determined by the intracellular distributionof the weak acid 5,5–dimethyloxazolidine2–,4–dione(DM0), when the external pH is below about 60. This effect isreversed, at least partially, by addition of the following cationsto Ca²⁺-free solutions: tetraethylammonium (TEA⁺) and Na⁺ at5 or 10 mol m^-3, Li⁺ and Cs⁺ (10 mol m^-3), or Mg²⁺, Mn²⁺ andLa³⁺ (02 or 05 mol m^-3). Under the same conditions, increasesin pH sometimes, but not always, occur in the presence of 10mol m^-3 K⁺ or Rb⁺ The results are discussed in relation to the major transportprocesses that determine pH and the electric potential differenceacross the plasma membrane, namely fluxes of H⁺ and of K⁺. Thesimplest explanation of the effects of the various cations testedin this study is that they primarily affect pHi_c via changesin influx of H⁺ but direct effects on the H⁺ pump or on K⁺ fluxesmay also be involved Key words: Chara corallina, cytoplasmic pH, cations, H⁺transport     

Slow Onset of Activation and Delay of Inactivation in Transient Current of Nitella axilliformis

Single membrane samples of Nitella axilliformis, in which majorparts of vacuoles were removed, were prepared by centrifugationand ligation with threads. Voltage clamp experiments were madewith the samples, proving that the transient current occursonly after a initial delay as was observed in Chara corallinaby Beilby and Coster [(1979) Aust. J. Plant Physiol. 6 : 337].The membrane potential measured at a corner of the sample exhibitedpractically the same time course as the membrane potential atthe center which changed stepwise, indicating that the delayis not related to the propagation of the potential change. Thedelay did not change sensitively to ionic strength of the externalsolution, suggesting that it is not caused by low electric conductancearound the sample but is related to the gating mechanism ofCl channel. Various models were examined to explain the timecourse of the transient current. The best agreement was obtainedby introducing a delay _h in inactivation with the expressionof Ici=cim⁸h(VM— Vci),where Ici stands for the transient current and VM is the clamppotential, and ci = 15mS.cm^–2,Vci = –31.5 mV. The delay _h decreases similarly to _Tmwith increasing VM, suggesting that inactivation starts afteractivation proceeds. (Received November 4, 1982; Accepted January 8, 1983)     

IONIC COMPOSITION OF THE CYTOPLASM OF NITELLA FLEXILIS

The K, Na and Cl concentrations of the chloroplast layer andthe flowing cytoplasm of Nitella flexilis have been determinedby applying an internal perfusion technique, which enabled usto avoid contamination of ions from the cell sap. K, Na andCl concentrations of the chloroplast layer are 110, 26 and 136mM and those of the flowing cytoplasm are 125, 5 and 36 mM respectively.The cell sap contains 80 mM K, 28 mM Na and 136 mM Cl. Althoughthere are some variations in these values among samples, theflowing cytoplasm is rich in K and poor in Cl and especiallyin Na. The exchange of K and Na across the tonoplasl occursfairly easily (half-time, a few hours), while that of Cl occursextremely slowly (half-time, a few days). ¹This work was supported by Research Grants from the Ministryof Education of Japan