Nernst Potential Measurements on the Leaf Cells of the Moss, Hookeria lucens

A study has been made of the ion distributions in the leaf cellsof the moss, Hookeria lucens. The cells were subjected to chemicalanalysis and electrical potential measurements. The observedinternal concentrations were 210 mM/1 for potassium, 3.9 mM/1for sodium, 22 mM/1 for calcium, and 27 mM/1 for chloride. Awide range of electrical potential values were obtained. Whilea majority of these values were grouped around —145 mV,a significant number were found at —200 mV. Nernst potentialsfor the various ions were calculated and compared with the observedelectrical potentials.     

Influence of silicon on microdistribution of mineral ions in roots of salt-stressed barley as associated with salt tolerance in plants

Two contrasting barley (Hordeum vulgare L.) cultivars: Kepin No.7 (salt sensitive), and Jian 4 (salt tolerant) were grown hydroponically to investigate the microdistribution of mineral ions in roots as affected by silicon (Si) with respect to salt tolerance. The experiment was undertaken consisting of two treatments with 3 replicates: (i) 120 mmol · L⁻¹ NaCl alone (referred to as Si-NaCl+), (ii) 120 mmol · L⁻¹ NaCl + 1.0 mmol · L⁻¹ Si (as potassium silicate) (referred to as Si+NaCl+). Plant root tips were harvested for microanalysis using an energy dispersive X-ray microanalyzer (EDX) 30 d after transplanting. Higher Cl and Na X-ray peaks were recorded in the root epidermal, cortical and stelar cells of roots for the treatment Si-NaCl+ with the majorities of Na and Cl being accumulated in epidermal and cortical cells, while relatively low K peaks were observed regardless of the barley cultivars used. By contrast, considerably higher K peaks were detected in the epidermal, cortical and stelar cells of the roots for the treatment Si+NaCl+, but lower Cl and Na peaks were also observed for this treatment with both Na and Cl ions being evenly distributed in the epidermal, cortical and stelar cells. These findings directly support our previous finding, which showed that Si depressed the uptake of sodium but enhanced the uptake of potassium by salt-stressed barley. We believe that one of the possible mechanisms involved in Si-enhancement of salt tolerance in barley is attributed to the Si-induced changes in the uptake and microdistribution of mineral ions in plants.     

Active Transport of Ions across the Root of Ricinus communis

The electrical potential difference between the exuding sapof detopped castor oil plants and the external I mM KC1 bathingsolution has been measured, together with the concentrationsof potassium and chloride. A typical value for the electricalpotential of the sap with respect to the external solution was—56 mV, while the sap concentrations of potassium andchloride were 8 mM and 3.5 mM respectively. The total cationconcentration, made up of K, Na, Ca, and Mg, was about Io m.equiv.1.;the anion deficit is made up by SO₄, NO₃, and PO₄. A comparisonof the measured potential difference with the Nernst potentialsfor potassium and chloride suggests that the movement of potassiuminto the sap is a passive process while the movement of chlorideis an active process against the electrochemical potential gradient.It is suggested that the potassium and chloride ions pass throughthe diffusion barrier at which active transport takes placebefore they exchange with the other ions which appear in thesap. On the basis of this assumption it is shown that potassiumis close to passive equilibrium while chloride is accumulatedagainst an electrochemical potential difference of 110 mV or2, 500 cal.mole^–1.     

The effect of CCCP on ion fluxes in the stele and cortex of maize roots

Summary The accumulation of ⁸⁶Rb labelled potassium by isolated stelar and cortical tissues from 7-day-old roots of Zea mays has been compared with the levels accumulated by these tissues in the intact root. Cortical tissues have similar uptake eapacities in these two conditions whereas stelar tissues only exhibit an uptake capacity in the intact root system. The uncoupler carbonylcyanide m-chlorophenylhydrazone caused a considerable decrease in the uptake of potassium by these tissues. In the intact root system it prevented ions from the bathing medium reaching the stelar tissues. The efflux pattern from preloaded isolated stelar and cortical tissues was considerably altered by the inhibitor, a promotion of the efflux occurring in both of these tissues.It is concluded that stelar tissues only accumulated ions when these are supplied through the root symplasm and that the stelar plasmalemma has only a limited uptake capacity per se. Stelar uptake is thus a reflection of vacuolar accumulation across the tonoplast. There is no evidence in the present study of a carrier-mediated active secretion of ions across the stelar plasmalemma. The fact that the efflux was promoted rather than depressed by the uncoupler supports the postulate that a passive leakage is the final stage in the transport of ions across the plant root.     

Membrane ATPase Activity of Barley Roots and Potassium Uptake by Isolated Stele and Cortex

Uptake of potassium ions by isolated stelar tissues of barley from 0.5 and 10 mM K⁺ was respectively 13 and 3.6% of that of the cortical tissues. 0.1 mM H₂PO₄, LO mM ATP and 10 mM Ca(NO₃)₂ did not increase the potassium uptake of either stele or cortex during 5 h of uptake period. A time-course incubation for histological demonstration of the ATPase activity of the plasmalemma and tonoplast of the matured sections of the roots demonstrated a greater activity for the cortical than the stelar tissue. In the stelar parenchyma cells, the plasma lemma showed a higher activity than the tonoplast. These results, which support the “leakiness hypothesis” of the stele, are discussed in relation to the proposed mechanisms of radial ion transport in roots.     

Influence of silicon on microdistribution of mineral ions in roots of salt-stressed barley as associated with salt tolerance in plants

Two contrasting barley (Hordeum vulgare L.) cultivars: Kepin No.7 (salt sensitive), and Jian 4 (salt tolerant) were grown hydroponically to investigate the microdistribution of mineral ions in roots as affected by silicon (Si) with respect to salt tolerance. The experiment was undertaken consisting of two treatments with 3 replicates: (i) 120 mmol · L-1 NaCl alone (referred to as Si-NaCl+), (ii) 120 mmol·L-1 NaCl + 1.0 mmol · L-1 Si (as potassium silicate) (referred to as Si+NaCl+). Plant root tips were harvested for microanalysis using an energy dispersive X-ray microanalyzer (EDX) 30 d after transplanting. Higher Cl and Na X-ray peaks were recorded in the root epidermal, cortical and stelar cells of roots for the treatment Si-NaCl+ with the majorities of Na and Cl being accumulated in epidermal and cortical cells, while relatively low K peaks were observed regardless of the barley cultivars used. By contrast, considerably higher K peaks were detected in the epidermal, cortical and stelar cells of th     

Perfusion of charophyte cells: a critical analysis of the method

The data obtained by different types of intracellular perfusionwere compared. As the ligated cells cannot be space-clamped,the efficiency of compartment-clamping was evaluated, showingthat the difference I/V (current-voltage) profile between space-clampedand compartment-clamped data could be approximated by a straightline. The time-dependence of the clamp currents was not affectedby the clamp technique. The comparison of different sets of data was quantified by fittingthe I/V curves with a mathematical model (Beilby and Walker,1996). The I/V curves of ligated cells perfused with 1 mM ATPshowed the closest similarity to intact cells with resting potentialsof –22010 mV (7 cells) and similar model parameter values.The cells under open-end perfusion with ATP showed less hyperpolarizedresting p.d.s (potential differences): –17512 mV (4 cells).For both preparations the —ATP data were similar withresting p.d.s at –80 12 mV (5 ligated cells, 7 open-endcells). The excited state was more pronounced in open-end cells(resting level: –5912 mV, 5 cells) than in ligated cells(resting level: –6512 mV, 7 cells). In open-end cellsthe pump responded faster to changes of ATP concentration thanthe excitation channels. The cells stabilized with Pb(NO₃)₂were strongly depolarized both with ATP: –8010 mV (6cells) and without: 010 mV (6 cells). Most model parametersdiffered from those in the intact cells. The excited state wasabolished. Key words: Intracellular perfusion, current-voltage characteristics, Chara, stabilization with Pb(NO₃)₂, ATP effects, voltage clamp techniques     

An electrogenic NA+/K+ pump in the choroid plexus.

Intracellular electrical potential and potassium activity was measured by means of microelectrodes in the epithelial cells of choroid plexus from bullfrogs (Rana catesbeiana). Ouabain applied from the ventricular side caused an abrupt depolarisation of 10 mV but only a gradual loss of potassium from the cells. Readministration of potassium to the ventricular solution of plexuses which were previously depleted of potassium, caused a hyperpolarisation of about 4 mV. These two experiments are consistent with the notion of an electrogenic Na+/K+ pump situated at the ventricular membrane and which pumps potassium into the cell and sodium into the ventricle. The numerical values obtained suggest that 3 sodium ions are pumped for 2 potassium ions. The permeability coefficient for potassium exit from the cell is calculated to be 1.24 . 10(-5) cm-1 . s-1 expressed per cm2 of flat epithelium.     

Microelectrode study of K+ accumulation by tight epithelia: I. Baseline values of split frog skin and toad urinary bladder

Toad bladder and split frog skin were impaled with fine-tipped single- and double-barrelled K+-selective microelectrodes. In order to circumvent membrane damage induced by impaling toad bladder, a null point method was developed, involving elevations of mucosal potassium concentration. The results suggest that intracellular potassium activity of short-circuited toad bladder is approximately 82 mM, twice as large as earlier estimates. Far more stable and rigorously defined intracellular measurements were recorded from short-circuited split frog skins. The intracellular positions of the micropipette and microelectrode tips were verified by transient hyperpolarizations of the membrane potential with mucosal amiloride or by transient depolarizations with serosal barium or strophanthidin. Simultaneous impalement of distant cells with separate micropipettes demonstrated that both the baseline membrane potentials and the responses to depolarizing agents were similar, further documenting that frog skin is a functional syncytium. Measurements with double-barrelled microelectrodes and simultaneous single-barrelled microelectrodes and reference micropipettes suggest that the intracellular potassium activity is about 104 mM, lower than previously reported. Taken together with measurements of intracellular potassium concentration, this datum suggests that potassium is uniformly distributed within the epithelial cells.     

Regulation of cytosolic sodium ion activity in frog sartorius

To explore the regulation of cytosolic sodium ion activity in the frog sartorius, we used Na(+)-selective microelectrodes to monitor intracellular sodium ion activity in situations of lowering external sodium concentration and elevating external potassium concentration. Reductions of 20%, 40%, 60% and 80% in extracellular sodium concentration produced slight but statistically insignificant changes in the membrane potential of the muscle. However, cytosolic sodium ion activity decreased significantly from 10.0 +/- 1.1 mM to 7.8 +/- 1.1 mM, 7.1 +/- 1.4 mM, 6.5 +/- 1.2 mM and 5.9 +/- 1.1 mM, respectively. In addition, elevation of the external potassium concentration from 2 mM to 12 mM, 32 mM and 62 mM caused respective stepwise depolarization of membrane potential from -87.2 +/- 1.6 mV to -62.4 +/- 3.6 mV, -45.4 +/- 3.0 mV, -27.2 +/- 1.8 mV. Under these conditions, the cytosolic sodium ion activity decreased from 10.5 +/- 1.4 mM to 7.3 +/- 1.6 mM, 6.4 +/- 1.1 mM and 5.2 +/- 0.8 mM, respectively. The results illustrate that the net sodium flux is out of cell either in the reduction of sodium chemical gradient or in the potassium depolarization across the cell membrane.     

Immunofluorescence Microscopy of Microtubule Arrangement in Root Cells of Pisum sativum L. var Alaska

The arrangement of wall microtubules (MTs) in Pisum sativumroots was viewed immunofluorescently using cryosectioning. Mostcells in the tip region of pea roots (0–2 mm from tip)had wall MTs arranged transversely to the root axis. In theregion elongating at a higher rate (2–4 mm), wall MTsof epidermal, cortical and stelar cells were all transverselyarranged. In the region of about 5 mm from the tip, in whichcell elongation had already ceased, wall MTs in cortical cellschanged from a transverse to an oblique arrangement in relationto the root axis. Some cells had a crossed arrangement of wallMTs, which was interpreted as representing two sets of unidirectional,oblique wall MTs in opposite cell cortices of a single cell.This change was completed within a region of 1-mm width. Sinceroots elongated at a rate of 0.6 mm h^–1, it means thatthe arrangement of wall MTs changed within 2 h. An oblique arrangementof wall MTs was also observed in stelar cells. As the cellsaged, the oblique arrangement tended to change to a steeperor even a longitudinal one. (Received January 24, 1986; Accepted May 15, 1986)     

An electrogenic Na+/K+ pump in the choroid plexus

Intracellular electrical potential and potassium activity was measured by means of microelectrodes in the epithelial cells of choroid plexus from bullfrogs (Rana catesbeiana). Ouabain applied from the ventricular side caused an abrupt depolarisation of 10 mV but only a gradual loss of potassium from the cells. Readministration of potassium to the ventricular solution of plexuses which were previously depleted of potassium, caused a hyperpolarisation of about 4 mV. These two experiments are consistent with the notion of an electrogenic Na⁺/K⁺ pump situated at the ventricular membrane and which pumps potassium into the cell and sodium into the ventricle. The numerical values obtained suggest that 3 sodium ions are pumped for 2 potassium ions. The permeability coefficient for potassium exit from the cell is calculated to be 1.24 · 10^?5 cm^?1 · s^?1 expressed per cm² of flat epithelium.     

The Radial Exchange of Labelled Water in Isolated Steles of Maize Roots

The kinetics of the efflux of labelled water from isolated stelesof maize roots have been studied.Analysis of the efflux kineticsindicated that the rate-limiting step was possibly permeationacross cell ‘membranes‘. On this basis we have computedfrom the efflux curve that the ‘typical‘ membranepermeability of stelar cells to water is about 1.8x10^-5 cm s^-1;thus, the cells in the stele are about four times more permeableto water than the cortical cells. The water permeability ofthe stelar cells, however, may have been substantially alteredby the extraction of steles from the cortical sleeves. Similarefflux studies were performed on ‘dead‘ steles preparedby boiling the normal steles for a short time. These experimentsdemonstrated that diffusion within the tissue and external ‘unstirredlayer‘ was rate-controlling the initial stage of the effluxfrom ‘dead‘ steles.A common feature of both effluxcurves for normal and ‘dead‘ steles was that eachdeviated from theoretical cylindrical diffusion curves for largevalues of time. Certain experimental evidence suggested thatthis deviation resulted from the existence of a slowly exchangingunidentified compartment occupying at least 8 per cent of thestelar volume.     

Repolarization of striated muscle fibers by the antifatigue agent,K-Mg-aspartate

Resting membrane potentials of isolated frog sartorius muscles were measured under a variety of conditions using intracellular glass microelectrodes. Muscle cells depolarized by the addition of 5.0 or 10.0 mM KCl to the bathing Ringer solution can be repolarized some 5 to 10 mV by the substitution of an equivalent amount of K-aspartate for KCl in the presence of 2.0 mM Mg⁺⁺. The repolarization produced by this method persists when the muscle is again placed in the initial KCl solution, thus eliminating the possibility that the hyperpolarization is due to the reduction of chloride in the bathing medium. If for some reason the resting membrane potential of the muscle fibers is considerably below (less negative than) the normal level of 92 mV reported for muscles bathed in 2.5 mM Ringer solution, the substitution of 2.5 mM K-aspartate for the 2.5 mM KCl and the addition of 2.0 mM Mg-aspartate to the Ringer solution will, within 15 minutes, repolarize the fiber to the normal level. Magnesium ions alone will not produce the observed repolarization nor can it be attributed to a reduction in the activity of the potassium in the Ringer solution.     

Fast inactivating potassium current in cultured hippocampal neurons

Using a voltage-clamp whole-cell technique, we studied transmembrane currents in hippocampal neurons after their long-lasting cultivation. Based on the activational characteristics and data on pharmacological sensitivity, we isolated and described an A-type voltage-activated fast inactivating potassium current (FIPC). This transient FIPC was activated at −50… −40 mV and was rather sensitive to 4-aminopyridine (4-AP). Extracellular application of 5 mM 4-AP decreased the FIPC amplitude by 75%, while application of 10 mM tetraethylammonium evoked no significant changes in it. Kinetics of FIPC activation could be described by one exponent in the fourth degree. With variations of the membrane potential from −40 to 30 mV, the activation time constant varied from 2.8 to 1.5 msec. Inactivation kinetics was described by one exponent with the time constant varying from 37 msec at −45 mV to 18 msec at 40 mV. Stationary activation and inactivation curves could be described by Boltzmann's equation; a half value of the level of stationary inactivation was reached at −80 mV, while stationary activation was attained at −25 mV. Kinetics of deinactivation (from stationary inactivation) was monoexponential with the time constant of 41 msec. It is supposed that FIPC through the membrane of hippocampal neurons is provided by the type Kv4.2 potassium channels.     

Cortical Spreading Depolarization (CSD) Recorded from Intact Skin,from Surface of Dura Mater or Cortex: Comparison with Intracortical Recordings in the Neocortex of Adult Rats

In cerebral cortex of anesthetized rats single waves of spreading depolarization (CSD) were elicited by needle prick. CSD-related changes of DC (direct current) potentials were either recorded from the intact skin or together with concomitant changes of potassium concentration with K⁺-selective microelectrodes simultaneously at the surface of the dura mater or of the cortex ([K⁺]_s) and in the extracellular space at a cortical depth of 1200 µm. At the intact skin CSD-related DC-shifts had amplitudes of less than 1 mV and had only in a minority of cases the typical CSD-like shape. In the majority these DC-shifts rose and recovered very slowly and were difficult to identify without further indicators. At dura surface CSD-related DC shifts were significantly smaller and rose and recovered slower than intracortically recorded CSD. Concomitant increases in [K⁺]_s were delayed and reached maximal values of about 5 mM from a baseline of 3 mM. They rose and recovered slower than simultaneously recorded intracortical changes in extracellular potassium concentration ([K⁺]_e) that were up to 65 mM. The results suggest that extracellular potassium during CSD is diffusing through the subarachnoid space and across the dura mater. In a few cases CSD was either absent at the dura or at a depth of 1200 µm. Even full blown CSDs in this cortical depth could remain without concomitant deflections at the dura. Our data confirmed in principle the possibility of non-invasive recordings of CSD-related DC-shifts. For a use in clinical routine sensitivity and specificity will have to be improved.

     

Transmembrane electrical characteristics of cultured human skeletal muscle cells

Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3-4 days, and fusion of myoblasts was observed in 5-10 days. Multinucleated myotubes were impaled under high optical magnification, at 37 degrees C, with conventional glass microelectrodes. The mean resting potential was -44.4 mV +/- 2.4 (n = 399); -33 +/- 2.3 mV at 9 days (n = 10) vs -48 +/- 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M omega (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 +/- 2.1 mV with a mean maximum rate of rise (Vmax) of 72.1 +/- 7.5 V/s. The mean overshoot was 13.9 +/- 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5-100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.     

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.     

Membrane potential measurements during rat liver regeneration

Membrane potential was measured in perfused rat liver and was shown to increase from ?33 ± 1.0 mV in livers from normal rats to ?50 ± 1.1 mV in livers from rats 12 hr after partial hepatectomy. The hyperpolarization of the membrane in regenerating liver was no longer evident after perfusion with 1 mM ouabain for 5 min. Ouabain had a small (4 mV) depolarizing effect on membrane potential in normal liver. The potential measured in normal and regenerating liver decreased as a function of the external potassium concentration above 5 mM; however, the potential was more electronegative in regenerating liver compared to normal liver at all values of external potassium concentration, and the differences in potential between the two kinds of cells did not decrease at higher concentrations of external potassium. Thus, a plot of membrane potential vs external potassium concentration resulted in approximately parallel curves for the two different cell types. We conclude that hyperpolarization of the liver cell membrane is an early event during rat liver regeneration and results from an electrogenic Na-K pump.     

Intracellular Na+, K+, and C1- activities in Balanus photoreceptors

Ion-sensitive microelectrodes were used to measure intracellular activities (aix) of Na+, K+, and C-1 in Balanus photoreceptors. Average values of aiNa, aiK, and aiCl were 28 mM, 120 mM, and 65 mM, respectively. Equilibrium potentials calculated from these average values were: Na+ +64 mV, K+ - 77 mV, and and Cl- -42 mV; ther average value of the resting potential for all cells examined was -41 mV. Long exposure to intense illumination produced measurable increases in aiNa. Classical Na+ - K+ reciprocal dilution experiments were analyzed with and without observed changes in aiK. As aoK was increased, the membrane depolarized, and aiK increased. Better agreement was found between the membrane potential and the directly determined EK than expected from the standard relation between Em and aoK. The latter produced pNa:pK estimates of the resting photoreceptor membrane that were higher than estimates based on data from the ion electrodes. Generally, Em was more negative than EK as aoK was increased. This is consistent with a significant chloride permeability in the dark-adapted photoreceptor.