Ca Fluxes and Membrane Potentials in Nitella translucens

The concentrations of Ca have been measured in the flowing cytoplasmand the vacuole of the single cells of Nitella translucens,the cells being immersed in an artificial pond Water (composition:NaCl, 1.0 mM; KCl, 0.1 mM; CaCl₂, 0. mM). In the flowing cytoplasmthe total concentration is 8 mM and in the vacuole 12 mM. Measurementsof the electrical potential differences across the plasmalemmaand tonoplast membranes show that the cytoplasm is at a potentialof —134 mV with respect to the bathing medium and —24mV with respect to the vacuole. An attempt has been made tomeasure the tracer fluxes of Ca and it is shown that the cellsare not in flux equilibrium. The influx is 0.046 µµmoles cm^–2 sec^–1; the efflux was too small to measurewith any degree of accuracy. The observed potential differences across both membranes arecompared with the Nernst potentials for Ca. This analysis showsthat Ca is not in electrochemical equilibrium across eithermembrane and that the driving forces on Ca are directed fromthe bathing medium and the vacuole into the cytoplasm. It issuggested that there is no necessity for a metabolically drivenCa pump at the plasmalemma because the low cytoplasmic Ca contentcould be due to the low permeability of the plasmalemma; theGoldman flux equation gives a value of P_Ca = 4.3x10^–8cm sec^–1. A Ca pump at the tonoplast appears to be necessaryto explain the steep electrochemical potential gradient fromthe vacuole to the cytoplasm. The efflux of Ca from the isolated cell wall has been measured.From these measurements it was possible to estimate the concentrationof indiffusible anions in the Donnan Free Space; the value obtainedwas 0.74 equiv. 1.^–1.     

Chloride Electrochemical Potentials and Membrane Resistances in Nitella translucens

The chloride electrochemical potential difference between theinside of cells of Nitella translucens and the bathing mediumhas been measured by a direct electrical method employing Ag/AgClelectrodes. The membrane potential has been measured by meansof conventional salt bridge microelectrodes. These data havebeen used to calculate the internal chloride concentration ofthe cells; the mean value obtained was 39 mM. This chlorideelectrochemical potential difference has been short-circuitedthus causing an outward (depolarizing) electric current to flowthrough the cell membrane. The resulting membrane depolarizationhas been measured at two points along the length of the cellenabling the membrane resistance and space constant to be deduced;the respective values obtained were 24.8 Kcm² and 3.0 cm. Itis suggested that these experiments lend additional supportto the hypothesis that during the action potential in the Characeaethere occurs a transient increase in the chloride conductanceof the plasmalemma.     

The Control of Na Uptake into Nitella translucens

Movement of Na into cells of Nitella translucens is a ‘downhill’process; the ions move across the plasmalemma down an electrochemicalpotential gradient. Nevertheless, measurements of Na influxesunder a wide range of experimental conditions have shown thatthere must be links between Na uptake and processes controlledby metabolism. When Ca ions are present in the bathing solution,Na influxes are greatly increased by light under conditionswhere photosynthesis can proceed (i.e. when both photosystemsare active). In the presence of Ca, the influx of Na increasesonly slightly when the external Na concentration is raised above1 mM, and the light-promoted Na influx is considerably inhibitedwhen Cl is removed from the bathing solution. When the Cl concentrationis kept constant, the Na influx in light is determined by theconcentrations of other cations present in solution (K, Ca,or NH₄). In the absence of Ca from the cell wall and solution,the influx is stil enhanced by light, but does not saturatewhen the external Na concentration is raised above 1 mM. Itis suggested that the Na influx in light is partly linked tothe inward Cl pump, but there is also a separate (Cl-independent)effect of light on the permeability of the plasmalemma to Na.Links between Na and Cl uptake could be maintained by effectsof Cl on electrochemical driving forces controlling Na entry;alternatively, chemical coupling between the two processes maybe involved.     

Measurements of Electro-osmosis in Nitella translucens

Determinations of the electro-osmotic efficiency (models H₂Oper Faraday) in Nitella translucens have been made under differentconditions. Associated tracer measurements show that the mainpath of current flow ins through the cell, and not in the cellwall external to the membrane, but that the measured influxesof Na and K can account for only a small fraction of the current.The electro-osmotic coefficient increases markedly at very lowcurrents in the light (to as high as 700 H₂O/F), is decreasedin the dark, in the presence of low concentrations of N-dichlorophenyl-N',N' -dimethyl urea (DCMU) or of carbonyl cyanide m-chlorophenylhydrazone(CCCP) and on the addition of I mM NaHCO₃ to the bathing solution;the efficiency is higher at 10.5 °C than at 22 °C. Electro-osmosis,generated by a difference in membrane potential at the two endsof the cell bathed by different solutions, gave values of theelectro-osmotic coefficient similar to those characteristicof low applied currents.The results suggest that the cell membrane,and not only the cell wall, shows electro-osmotic properties.A simplified analysis of the system shows that the electro-osmoticcoefficient measured depends on conditions at each end of thecell and is not simply a property of the cation added at theend of the cell at which positive current enters.     

Electrophysiological Techniques and the Magnitudes of the Membrane Potentials and Resistances of Nitella translucens

The membrane resistance of internodal cells of Nitella translucensincreased by 50 per cent during the first 5 h after insertionof two microelectrodes into the vacuole even when precautionswere taken to eliminate external disturbances. The insertionof a third microelectrode into the cytoplasm did not affectthe resistance. In artificial pond water the final value forthe plasmalemma resistance was 112 k cm² and that for the tonoplastwas 6 k cm². The increase in the membrane potential after thefirst hour was less than 10 per cent. A recent suggestion that accurate measurements of the plasmalemmaresistance can be made with a microelectrode outside the plasmalemma,but in close contact with it, is criticized. Tests were made of the claim that leakage of current at thepoint where microelectrodes enter the cytoplasm gives rise toa local increase in current density at the tonoplast and henceleads to an overestimate of the tonoplast resistance. Valuesfor the tonoplast resistance obtained when the cytoplasmic microelectrodewas inserted through the plasmalemma were similar to those observedwhen it was pushed across the cell and inserted through thetonoplast at a point remote from the postulated current leakage.Furthermore, the tonoplast resistance stayed remarkably constantwhen the plasmalemma resistance varied in a way which wouldcause different proportions of the applied current to pass throughthe leak resistance and produce variations in the apparent tonoplastresistance. It is concluded that published values of the tonoplastresistance are not grossly inaccurate.     

Ion Fluxes to the Vacuole of Nitella translucens

The time course of the appearance in the vacuole of Nitellatranslucens and of Tolypella intricata of tracer from the outsidesolution has been studied over short periods of uptake. Thereare two components of chloride transfer to the vacuole, a fastcomponent linear with time and a second component at longertimes whose behaviour is reasonably well described in termsof a single rate constant for exchange; a constant fractionof the total entry is in the fast component and the apparentrate constant for the second component is proportional to theinflux. In Nitella the path of rapid transfer involves chlorideand sodium, and may also involve a small but variable amountof potassium, but in Tolypella potassium has a significant componentof rapid transfer; these correspond to the cations for whichchloride-linked components of cation influx have been shownby another worker. Over both parts of the time course the level of activity inthe cytoplasm specifies, not the rate of transfer to the vacuoleas would be expected, but the rate as a fraction of the influx;the processes of influx to the cell and transfer to the vacuoleare intimately linked. It is difficult to explain the results in terms of static membranesand fixed compartments. An explanation in terms of the sequence,entry of salt by pino-cytotic vesicles at the plasmalemma, fusionof these vesicles with the endoplasmic reticulum after someloss of tracer to the surrounding cytoplasm, and transfer tothe vacuole in minivacuoles formed from the endoplasmic reticulum,is consistent with the time course found. A model of this kind,involving transport by a dynamic membrane system, seems necessaryto explain the results.     

The Electrical Resistance and Capacitance of the Membranes of Nitella translucens

The resistance and capacitance of the membranes of Nitella translucenshave been measured by direct current and alternating currentmethods. Current of the order of 10^-7 amp. was injected intothe cell by means of a conventional Ag, AgCl_-3N KCl glass microelectrodeinserted into the vacuole of the cell. The change of potentialacross the membrane was recorded by two other internal microelectrodeswhich had been inserted into the cell at known distances fromthe current-injecting electrode. In the direct-current experimentsthe input current was in the form of a square pulse, while sinusoidalcurrents of frequency 25 cycles per second were used in thealternating current experiments. The cell was treated as a shortlength of coaxial cable and from the measurements the followingparameters could be obtained: the space constant (), the membraneresistance (R_m) and the membrane capacitance (C_m). The valuesof R_m ranged from 6.7 to 36 K ohm cm.² (mean of 21.4 K ohm cm.²)and those of ranged from 1.5 to 5.7 cm. (mean of 2.6 cm.).The capacitance value was about I µF cm.^-2 These results are discussed within the framework of our knowledgeof these parameters for other cells, particularly plant cells.The measured electrical resistance is shown to be at least tentimes less than the value estimated from the passive fluxesof the principal ions K, Na, and Cl. It is suggested that thisdiscrepancy, which is usually attributed to non-independentmovement of these ions, could be partially explained on electro-osmoticgrounds. The value of the capacitance is very close to thatwhich is usually obtained for other cell membranes. One exceptionallylow value for Nitella has been quoted in the literature. Thereason for the gross error in this particular measurement isgiven.     

Vacuolar Fluxes of Chloride and Bromide in Nitella translucens

The kinetics of halide-ion transfer to the vacuole of Nitellatranslucens have been further examined. The percentage of totaltracer found in the vacuole can be expressed as the sum of twocomponents, a fast component important at short times, and aslow component increasing as the level of tracer in the cytoplasmrises. In the individual cells in any given experiment the rate oftransfer to the vacuole in the fast component is a quantizedfraction of the total influx. This statement expresses the factthat in each experiment the cells can be divided into groups,each covering the whole range of influx values and having thesame mean influx, whose mean vacuolar percentages are in theratio 1:2:3 (with also a few cells having higher values of vacuolarpercentage). There appears therefore to be a process of quantizeddischarge to the vacuole from an internal cytoplasmic compartment. Double-labelling with ³⁶Cl and ⁸²Br was used to measure bothfast and slow components in the same cell. Although the bromideinflux was only one-third of the chloride influx from equalexternal concentrations of O.6 mM, the vacuolar percentagesfor bromide and chloride were equal in cells loaded for thesame time in bromide and chloride, whether the loading was fora short or a longer time. Therefore both slow and fast componentsare equal for the two ions, and in cells labelled for a shorttime in bromide and a longer time in chloride, the vacuolarpercentage for bromide can be used as a measure of the fastcomponent for chloride. The contribution of the slow componentto the vacuolar transfer increases with time, but the slopeof its time dependence is proportional to the influx in thecell. It is not quantized. Hence the rate of transfer to thevacuole can be specified only as a fraction of the total influx,and not as an absolute rate, and the exchange with the slowcytoplasmic compartment is very closely linked to the influx.The equality of the slow components of vacuole transfer forbromide and chloride (in spite of very different influxes) isconsistent with the hypothesis that the slow exchange reflectsexchange with pinocytotic vesicles, but is difficult to explainin terms which do not involve a common entry process for salt,in a unit involving both bromide and chloride. The significance of the quantized discharge, and the relationbetween the fast and slow components, are not yet understood.The increase in the slow component as the cell becomes labelledis very steep, and hence even the slow cytoplasmic compartmentcan represent only a part of the cytoplasm, in which the specificactivity is higher than the rest.     

Quantized Fluxes of Chloride to the Vacuole of Nitella translucens

It has been shown previously that the transfer of tracer chloridefrom the outside solution to the vacuole of Nitella translucensis initially linear with time. In this paper the relations betweenthe initial rate of chloride transfer to the vacuole and thetotal influx to the cell are further examined. In the individualcells in each experiment the ratio (initial rate of transferto the vacuole/rate of entry to the cell), M_ov/M_T, is quantized;that is, in each experiment the ratio takes values close to, 2, 3,... etc. Formation of pinocytotic vesicles is a processwhich could be imagined to be quantized, but the fact that itis a flux ratio, rather than a flux, which is quantized suggeststhat entry to a small cytoplasmic phase, such as the endoplasmicreticulum, must precede a quantized discharge to the vacuole.It is suggested that the kinetics of tracer movement to thevacuole are consistent with transfer in small vacuoles buddedoff the endoplasmic reticulum which then fuse with the centralvacuole.     

Links between Glucose Uptake and Metabolism in Nitella translucens

The uptake of ¹⁴C-glucose into cells of Nitella translucenshas been investigated under experimental conditions previouslyused in studies of the ionic relations of these cells. Glucoseentry was considerably stimulated by light, and under aerobicconditions the fluxes remained constant for many hours. Theinflux of glucose was inhibited by over 80 per cent at low temperature(4° C) and by over 90 per cent by the uncoupler carbonylcycanide-m-chlorophenylhydrazone. 2-Deoxy-D-glucose was a non-competitiveinhibitor of glucose uptake both in light and darkness. Cyclicphotophosphorylation promoted the influx (with decreasing efficiency)for several hours. It is suggested that an ATP-dependent transportprocess controls glucose entry to the cells, and that passivediffusion is of little significance.     

Intracellular Kinetics of Tracer Chloride and Bromide in Nitella translucens

The kinetics of transfer of ³⁶CI and ₈₂Br to the vacuole ofNitella translucens have been compared with the kinetics oftransfer of the flowing cytoplasm in half-cell labelling experiments.About 40 per cent of the activity entering the cell reachesthe flowing layers, much more than could be accounted for asthe fast cytoplasmic phase seen in vacuolar kinetics. Also thespecific activity in the flowing cytoplasm is proportional totime over the first 20 min of uptake, and is essentially constantduring a 10-min wash period. Hence the major fraction of tracerin the flowing cytoplasm is in the slow phase of the cytoplasm.In spite of this there is ready movement of tracer from theflowing cytoplasm to the stationary cytoplasmic layer in theinactive end. It is concluded that the bulk (watery) phase ofthe cytoplasm is to be identified as the slow phase, and thatthe fast phase (if it exists as a real cytoplasmic compartment)can be only a very small membrane-bound phase within the bulkcytoplasm. It is shown that in the presence of 10 mM Mn²⁺, whichmakes the cells inexcitable, there is little or no fast componentof vacuolar transfer. The alternative explanation of the fastcomponent, as an artefact arising from transfer of tracer associatedwith action potentials on cutting open the cell, may providea more satisfactory explanation of the kinetics. The kineticcharacteristics of the slow phase and the lack of discriminationbetween chloride and bromide in the face of the link betweenvacuolar transfer and influx are still held to suggest ion transferto the vacuole by the creation and discharge of salt-filledvesicles, rather than by processes of single ion transfer atpre-formed tonoplast.     

The Influence of H+ on the Membrane Potential and Ion Fluxes of Nitella

The resting membrane potential of the Nitella cell is relatively insensitive to [K]_o, but behaves like a hydrogen electrode. K⁺ and Cl^- effluxes from the cell were measured continuously, while the membrane potential was changed either by means of a negative feedback circuit or by external pH changes. The experiments indicate that P_K and P_Cl are independent of pH but are a function of membrane potential. Slope ion conductances, G_K, G_Cl, and G_Na were calculated from efflux measurements, and their sum was found to be negligible compared to membrane conductance. The possibility that a boundary potential change might be responsible for the membrane potential change was considered but was ruled out by the fact that the peak of the action potential remained at a constant level regardless of pH changes in the external solution. The conductance for H⁺ was estimated by measuring the membrane current change during an external pH change while the membrane potential was clamped at K⁺ equilibrium potential. In the range of external pH 5 to 6, H⁺ chord conductance was substantially equal to the membrane conductance. However, the [H]_i measured by various methods was not such as would be predicted from the [H]_o and the membrane potential using the Nernst equation. In artificial pond water containing DNP, the resting membrane potential decreased; this suggested that some energy-consuming mechanism maintains the membrane potential at the resting level. It is probable that there is a H⁺ extrusion mechanism in the Nitella cell, because the potential difference between the resting potential and the H⁺ equilibrium potential is always maintained notwithstanding a continuous H⁺ inward current which should result from the potential difference.