Analysis of the electrical coupling of root cells: implications for ion transport and the existence of an osmotic pump

An equivalent circuit was developed to model the radial electrical coupling between root cells. The results of several experiments were analysed using the circuit to determine whether the electrogenic pumps of the inner cortical cells were active. This analysis indicated that, while in some roots electrogenic pumps appear to be active in all cortical layers, they may be inactive in the inner cortical cells in some cases. The circuit was used to show that, in spite of intercellular symplasmic coupling, if the inner cortical cells have inactive electrogenic pumps, their membrane potentials can be significantly less negative than those of the epidermal cells. The radial difference in membrane potential may in part account for observations that the uptake of ions occurs primarily on the root periphery. This implication is developed in an appendix to show that an osmotic water pump may exist in roots to allow them to extract water from soil at a lower water potential. It is hypothesized that, as a result of a radial difference in membrane potential, there is an efflux of solutes from the inner cortical cells as the symplastic solution moves inward. As a result, the water potential of the root interior is elevated with respect to its exterior.     

HvLsi1 is a silicon influx transporter in barley

Most plants accumulate silicon in their bodies, and this is thought to be important for resistance against biotic and abiotic stresses; however, the molecular mechanisms for Si uptake and accumulation are poorly understood. Here, we describe an Si influx transporter, HvLsi1, in barley. This protein is homologous to rice influx transporter OsLsi1 with 81% identity, and belongs to a Nod26-like major intrinsic protein sub-family of aquaporins. Heterologous expression in both Xenopus laevis oocytes and a rice mutant defective in Si uptake showed that HvLsi1 has transport activity for silicic acid. Expression of HvLsi1 was detected specifically in the basal root, and the expression level was not affected by Si supply. There was a weak correlation between Si uptake and the expression level of HvLsi1 in eight cultivars tested. In the seminal roots, HvLsi1 is localized on the plasma membrane on the distal side of epidermal and cortical cells. HvLsi1 is also located in lateral roots on the plasma membrane of hypodermal cells. These cell-type specificity of localization and expression patterns of HvLsi1 are different from those of OsLsi1. These observations indicate that HvLsi1 is a silicon influx transporter that is involved in radial transport of Si through the epidermal and cortical layers of the basal roots of barley.     

Solute transport process in intestinal epithelial cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na+ and K+ concentrations, significant gains of Na+ and K+ occurred with glucose transport. The quantitative relationships among net gains of Na+, K+ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na+ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na+-efflux and K+-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

The influence of plasma membrane ion permeability modulators on superoxide formation and bioelectrical characteristics of wheat root cells

Changes in superoxide radical formation and bioelectrical characteristics of excised wheat root cells under modification of plasma membrane ion permeability were studied. It was shown that a 2 h treatment of excised roots with valinomycin (Val, 20 microM), N, N'-dicyclohexylcarbodimide (DCCD, 100 microM), gramicidin S (Gr, 20 microM), chlorpromazine (CPZ, 100 microM) caused an increased loss of potassium by cells, lowering of membrane potential (MP) and electrical input resistance (Rin) of the cells. The superoxide formation by excised root cells diminished (under DCCD) or remained at the control level (under Val), which was accompanied by a minor decrease of MP and Rin of the cells, a small increase in potassium loss by excised roots, and in no change of pH of incubation medium. Significant depolarization of plasma membrane, dropping of Rin and essential loss of potassium ions by the cells correlated with a rise in the medium alkalinization and superoxide formation by excised roots (in the presence of Gr, CPZ). Ion channel blocker gadolinium (Gd3+, 200 microM) caused an increase of MP and Rin reduction of potassium loss by cells, and a decrease of pH of the incubation medium, and also enhancement of superoxide formation by excised root cells. It is suggested that upon plasma membrane ion permeability modification the activity of superoxide generating systems depends on the specificity and mechanisms of action of modulators, and is determined by their influence on redox state of plasma membrane as well as by peculiarities of ion transport disturbance.     

Hypoxia-induced inhibition of whole cell membrane currents and ion transport of A549 cells

In excitable cells, hypoxia inhibits K channels, causes membrane depolarization, and initiates complex adaptive mechanisms. It is unclear whether K channels of alveolar epithelial cells reveal a similar response to hypoxia. A549 cells were exposed to hypoxia during whole cell patch-clamp measurements. Hypoxia reversibly inhibited a voltage-dependent outward current, consistent with a K current, because tetraethylamonium (TEA; 10 mM) abolished this effect; however, iberiotoxin (0.1 microM) does not. In normoxia, TEA and iberiotoxin inhibited whole cell current (-35%), whereas the K-channel inhibitors glibenclamide (1 microM), barium (1 mM), chromanol B293 (10 microM), and 4-aminopyridine (1 mM) were ineffective. (86)Rb uptake was measured to see whether K-channel modulation also affected transport activity. TEA, iberiotoxin, and 4-h hypoxia (1.5% O(2)) inhibited total (86)Rb uptake by 40, 20, and 35%, respectively. Increased extracellular K also inhibited (86)Rb uptake in a dose-dependent way. The K-channel opener 1-ethyl-2-benzimidazolinone (1 mM) increased (86)Rb uptake by 120% in normoxic and hypoxic cells by activation of Na-K pumps (+60%) and Na-K-2Cl cotransport (+170%). However, hypoxic transport inhibition was also seen in the presence of 1-ethyl-2-benzimidazolinone, TEA, and iberiotoxin. These results indicate that hypoxia, membrane depolarization, and K-channel inhibition decrease whole cell membrane currents and transport activity. It appears, therefore, that a hypoxia-induced change in membrane conductance and membrane potential might be a link between hypoxia and alveolar ion transport inhibition.     

Synaptic inhibition and cell communication; impairment of cell-to-cell coupling produced by gamma-aminobutyric acid (GABA) in the somatic musculature of Ascaris lumbricoides

The influence of gamma-aminobutyric acid (GABA) (10(-5) M) on the electrical coupling of giant somatic muscle cells of Ascaris lumbricoides was investigated. GABA enhanced the resting potential of the cells and abolished the spike activity. The coupling coefficient (V2/V1) was reduced by 58.8% while the input resistance (Rin) was decreased by 38.8%. The decline in Rin was not related to unlinearity of the current-voltage relation. As the time constant of cell membrane was reduced by 28.4% by the addition of GABA the effect of the neurotransmitter on cell-to-cell coupling seems to be mainly related to a decrease in resistance of the non-junctional membrane due to an increase in chloride conductance.     

Gravity-induced changes in intracellular potentials in elongating cortical cells of mung bean roots

Gravity-induced changes in intracellular potentials in primary roots of 2-day-old mung bean (Vigna mungo L. cv. black matpe) seedlings were investigated using glass microelectrodes held by 3-dimensional hydraulic micro-drives. The electrodes were inserted into outer cortical cells within the elongation zone. Intracellular potentials, angle of root orientation with respect to gravity, and position within the root of the impaled cortical cell were measured simultaneously. Gravistimulation caused intracellular potential changes in cortical cells of the elongation zone. When the roots were oriented vertically, the intracellular potentials of the outer cortical cells (2 mm behind the root apex) were approximately - 115 mV. When the roots were placed horizontally cortical cells on the upper side hyperpolarized to - 154 mV within 30 s while cortical cells on the lower side depolarized to about - 62 mV. This electrical asymmetry did not occur in cells of the maturation zone. Because attempts to insert the electrode into cells of the root cap were unsuccessful, these cells were not measured. The hyperpolarization of cortical cells on the upper side was greatly reduced upon application of N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of respiratory energy coupling. When stimulated roots were returned to the vertical, the degree of hyperpolarization of cortical cells on the previous upper side decreased within 30 s and approached that of cortical cells in non-stimulated roots. This cycle of hyperpolarization/loss of hyperpolarization was repeatable at least ten times by alternately turning the root from the vertical to the horizontal and back again. The very short (<30 s) lag period of these electrical changes indicates that they may result from stimulus-perception and transduction within the elongation zone rather than from transmission of a signal from the root cap.     

Solute Transport Process in Intestinal Epithelial Cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na⁺ and K⁺ concentrations, significant gains of Na⁺ and K⁺ occurred with glucose transport. The quantitative relationships among net gains of Na⁺, K⁺ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na⁺ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na⁺-efflux and K⁺-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

Homeostasis in the vertebrate lens: mechanisms of solute exchange

The eye lens is avascular, deriving nutrients from the aqueous and vitreous humours. It is, however, unclear which mechanisms mediate the transfer of solutes between these humours and the lens' fibre cells (FCs). In this review, we integrate the published data with the previously unpublished ultrastructural, dye loading and magnetic resonance imaging results. The picture emerging is that solute transfer between the humours and the fibre mass is determined by four processes: (i) paracellular transport of ions, water and small molecules along the intercellular spaces between epithelial and FCs, driven by Na(+)-leak conductance; (ii) membrane transport of such solutes from the intercellular spaces into the fibre cytoplasm by specific carriers and transporters; (iii) gap-junctional coupling mediating solute flux between superficial and deeper fibres, Na(+)/K(+)-ATPase-driven efflux of waste products in the equator, and electrical coupling of fibres; and (iv) transcellular transfer via caveoli and coated vesicles for the uptake of macromolecules and cholesterol. There is evidence that the Na(+)-driven influx of solutes occurs via paracellular and membrane transport and the Na(+)/K(+)-ATPase-driven efflux of waste products via gap junctions. This micro-circulation is likely restricted to the superficial cortex and nearly absent beyond the zone of organelle loss, forming a solute exchange barrier in the lens.     

Trans-root potential, xylem pressure, and root cortical membrane potential of 'low-salt' maize plants as influenced by nitrate and ammonium

Upon addition of nitrate and ammonium, respectively, to the bath of intact ‘low salt’ maize plants, the cortical membrane potential and the trans-root potential changed in a similar and synchronous way as revealed by applying conventional microelectrode techniques and the xylem pressure-potential probe ( Wegner & Zimmermann 1998). Upon addition of nitrate, a hyperpolarization response was observed which was frequently preceded by a short depolarization phase. In contrast, addition of ammonium resulted in an overall depolarization response both of the cortical membrane potential and the trans-root potential. The nitrate-induced hyperpolarization response and the depolarization following the addition of ammonium were concentration-dependent. The data suggest that a tight electrical coupling exists between the cellular and tissue level in the root of the intact plant and that the resistance of the cellular (symplastic) space is much less than the resistance of the apoplast.     

Gradient of electrical characteristics along roots of wheat seedlings under oxygen deprivation

The current-response method was used to characterize effect of oxygen deficiency on functional state of membranes along the roots of wheat seedlings. The results show apical parts of older roots being most affected by hypoxia, while the youngest roots behaved as effectively adapted.     

Intercellular communication in Chara: factors affecting transnodal electrical resistance and solute fluxes

The permeability of plasmodesmata in the nodal complex of branch cells of Chara corallina was examined by measuring both the transnodal electrical resistance and transnodal fluxes of ³⁶CI and ¹⁴C-buty-rate. Under normal circumstances, the resistance across the node was low, but increased rapidly in response to metabolic inhibition, pressure gradients across the node or excision of one of the cells. For each of these treatments, there was a substantial reduction in solute transport between the cells. Acidification of the cytoplasm by weak acids or alkalinization by amines did not affect either the electrical resistance or the flux of solutes through the node between whorl cells. The transnodal resistance was significantly higher in older cell pairs, but was unaffected by large transnodal voltage differences or by the passage of action potentials. There was no evidence that short-term increases in cytoplasmic calcium have any effect on plasmodesmatal permeability.     

Hypoxia/reoxygenation reduces microvascular endothelial cell coupling by a tyrosine and MAP kinase dependent pathway

Communication of electrical signals along the microvascular endothelium plays a key role in integrating microvascular function required for local regulation of blood flow. The aim of the present study was to examine the effect of a short-term hypoxia (0.1% O(2), 1 h) plus reoxygenation (H/R) on electrical coupling in cultured monolayers of microvascular endothelial cells (rat skeletal muscle origin). To assess coupling, we used a current injection technique and a Bessel function model to compute the intercellular resistance (an inverse measure of coupling) and cell membrane resistivity (a measure of resistance to current leakage across the cell membrane). H/R resulted in rapid (within 4 min after reoxygenation) and sustained (up to 100 min) reduction in intercellular coupling, but it did not alter membrane resistivity. H/R did not alter gap junction protein connexin 43 expression nor its tyrosine phosphorylation as determined by immunoblot and immunoprecipitation analyses. Inhibition of mitochondrial respiration (1 mM NaCN) did not mimic the effect of H/R. However, pre-treatment of monolayers with tyrphostin A48 (1.5 microM), PP2 (10 nM) (tyrosine kinase inhibitors), U 0126 (20 microM), and PD 98059 (5 microM) (MEK1/2 inhibitors) inhibited the H/R-induced reduction in coupling. These results indicate that endothelial cell coupling was reduced quickly after reoxygenation, via activation of a tyrosine and MAP kinase dependent pathway. We predict that a short-term H/R can rapidly compromise microvascular function in terms of reduced cellular communication along the vascular wall.     

Ubiquitin-Directed Antibodies Inhibit Neuronal Transporters in Rat Brain Synaptosomes

Affinity-purified antibodies specific for ubiquitin were found to inhibit the sodium-dependent uptake of [3H]choline, gamma-[3H]aminobutyric acid [( 3H]GABA), [3H]glutamate, [3H]norepinephrine, [3H]aspartate, and [3H]serotonin in rat cerebral cortical synaptosomes at a low concentration (10 micrograms/ml). These antibodies (termed anti-Ub) had no effect on the sodium-independent uptake of these substances or their calcium-dependent efflux. Synaptosomal [3H]deoxyglucose uptake was not affected in normal Krebs Ringer buffer containing 10 mM glucose, but was inhibited in glucose-free medium. Other nonneuronal sodium-dependent transport processes were found to be unaffected by 10 micrograms/ml anti-Ub, suggesting that anti-Ub does not bind indiscriminantly to sodium-binding sites on sodium-dependent organic solute transporters. Finally, anti-Ub inhibited sodium-dependent [3H]GABA and [3H]glutamate uptake in plasma membrane ghosts, devoid of membrane potential, which were derived from rat cerebral cortical synaptosomes. These results suggest that neuronal transporters or sites proximal to them may be ubiquitinylated on the plasma membrane surface.     

The electrical structure of normal liver tissue]

The electrical coupling between cells of mouse liver was investigated in vitro. The membrane potential of liver cells is 15--30 mv immediatly after dissection, and increases to 40--48 mv within 4--7 hours. This level of membrane potential is constant during the next 2--3 hours. The mean input resistance varies within 189+/-9 and 613+/-+25 kohm to be higher in preparations examined in summer than in winter time. The cytoplasm of liver cells is equipotential. The reducing of potential from intracellular source is not exponential. This potential distribution is well approximated by a solution for the two-dimentional model of the liver lamella, when characteristic length is 500 mcm. On the basis of this model the outher membrane resistance and functional membrane resistance were found to be 700-2100 and 1.2 ohm-cm2, resp.     

Electrical properties and active solute transport in rat small intestine

Summary The transepithelial resistance, the cell membrane resistance and the ratio of resistances of the serosal (baso-lateral) to the mucosal (brush border) cell membrane were measured in rat duodenum, jejunum and ileum by means of microelectrode techniques. These measured values were not affected in the presence of actively transported solutes in the mucosal bathing fluid.Contribution of an electrical conductance through the extracellular shunt pathway to the total transepithelial conductance was quantitatively estimated using an electrically equivalent circuit analysis. These values estimated in respective tissues of small intestine were approx. 95% of the total transepithelial conductance, remaining unaffected by an active solute transport.From these data, the changes in emf's of the mucosal and serosal membrane induced byd-glucose or glycine were separately evaluated.     

Pressure regulation of the electrical properties of growing Arabidopsis thaliana L. root hairs.

Actively growing Arabidopsis thaliana L. (Columbia wild type) root hairs were used to examine the interplay between cell turgor pressure and electrical properties of the cell: membrane potential, conductance, cell-to-cell coupling, and input resistance. Pressure was directly modulated using a pressure probe or indirectly by changing the extracellular osmolarity. Direct modulation of pressure in the range of 0 to about 15 x 10(5) Pa (normal turgor pressure was 6.8 +/- 2.0 x 10(5) Pa, n = 29) did not affect the membrane potential, conductance, coupling, or input resistance. Indirect modulation of turgor pressure by adding (hyperosmotic) or removing (hypo-osmotic) 200 mM mannitol/sorbitol affected the potential and conductance but not cell-to-cell coupling. Hypo-osmotic treatment depolarized the potential about 40 mV from an initial potential of about -190 mV and increased membrane conductance, consistent with an increase in anion efflux from the cell. Hyperosmotic treatment hyperpolarized the cell about 25 mV from the same initial potential and decreased conductance, consistent with a decline in cation influx. The results are likely due to the presence of an "osmo-sensor," rather than a "turgor-sensor," regulating the cell's response to osmotic stress.     

Simultaneous recording of xylem pressure and trans-root potential in roots of intact glycophytes using a novel xylem pressure probe technique

The radial electrical potential difference between the root xylem and the bathing solution, i.e. the so-called trans-root potential, was measured in intact maize and wheat plants using a xylem pressure probe into which an Ag/AgCl electrode was incorporated. Besides other advantages (e.g. detection and removal of tip clogging; determination of the radial root resistance), the novel probe allowed placement of the electrode precisely in a single xylem vessel as indicated by the reading of sub-atmospheric or negative pressure values upon penetration. The trans-root potentials were of the order of 0 to – 70 mV and + 40 to – 20 mV for 2- to 3-week-old maize and wheat plants, respectively. Osmotic experiments performed on maize demonstrated that addition of 100 mM mannitol to the solution resulted in a decrease of xylem pressure associated with a slow, but continuous depolarization. The depolarization was reversible upon removal of the mannitol. For wheat plants it could be shown that the oscillations of the xylem pressure described recently by Schneider et al. (1997, Plant, Cell and Environment 20, 221–229) were accompanied by (rectangular, saw-tooth and/or U-shaped) oscillations in the trans-root potential (but not by corresponding changes of the membrane potential of the cortical cells measured simultaneously with conventional microelectrodes). Increase of the light intensity (up to 550 μmol m^–2 s^–1) resulted in a drop of the xylem pressure in wheat, whereas the trans-root potential showed a biphasic response: first hyperpolarization (by about 10 mV) was observed, followed by depolarization (by up to about + 40 mV). Similar light-induced biphasic (but often less pronounced) changes in the trans-root potential were also recorded for maize plants. Most interestingly, the response of the trans-root potential was always faster (by about 1–3 min) than the response of the xylem pressure upon illumination, suggesting that changes in the transpiration rate are reflected very quickly in the electrical properties of the root tissue. The impact of this and other findings on long-distance transport of solutes and water as well as on long-distance signalling is discussed.     

Electrical coupling between cells of higher plants: A direct demonstration of intercellular communication

Summary Electrical coupling between adjacent cells of Elodea canadensis has been demonstrated using a microelectrode technique in which the membrane potentials were recorded during the passage of a current pulse from the vacuole of one cell to the external solution. The changes in membrane potential resulting from the passage of the current may be simulated by an equivalent circuit in which the tonoplast:plasmalemma:plasmodesmata resistances are in the ratio 1.0:5.6:2.2. On this basis, the specific resistances are 3.1 k cm² for the plasmalemma, 1.0 k cm² for the tonoplast and 0.051 k cm² for the junction between the cells. Although the plasmodesmata permit the passage of current, it is estimated that they have a resistance about 60 times higher than would be the case if they were completely open channels. Electrical coupling has also been demonstrated between parenchymal cells in oat coleoptiles and between cortical cells in maize roots. The significance of these findings is discussed in relation to the symplastic transport of ions and other small molecules and in relation to the quantitative measurement of membrane resistance in multicellular tissue.     

Model for the cell biomembrane electric potential during active transport of various ions

A model of a stationary electrical potential on biomembrane was created. This model takes into account conformational changes in transport ATPase. N positive ions are transported simultaneously by the system of active transport. The model allows one to determine independently ion concentrations inside the cell and membrane electrical potential. It is shown that, to obtain the electrical potential, it is necessary to take into account organic negative intracellular ions. The effect of positive ions that are not transported by active transport systems on the potential value is discussed. The results obtained are in a good agreement with experimental data for various cells.