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.     

The effect of ammonium ions on membrane potential and anion flux in roots of barley and tomato

The effects of ammonium (0–5 mol m^?3) on root hair membrane potential and on the influx of nitrate and phosphate were investigated in roots of intact barley and tomato plants. In both species, addition of ammonium to the medium bathing the roots caused an almost immediate depolarization of the membrane potential; the depolarization was greater at higher concentrations of ammonium. Influx of ¹³NC₃^? and ³²Pi was inhibited over the same time scale and concentration range. In tomato roots, there was little further depolarization of the membrane potential or inhibition of anion influx at ammonium concentrations above 0.4 mol m^?3. In barley roots, the inhibition of nitrate influx and the depolarization of the membrane potential did not saturate below 5 mol m^?3 ammonium.     

Photogeneration of membrane potential hyperpolarization and depolarization in non-excitable cells

We monitored femtosecond laser induced membrane potential changes in non-excitable cells using patchclamp analysis. Membrane potential hyperpolarization of HeLa cells was evoked by 780 nm, 80 fs laser pulses focused in the cellular cytoplasm at average powers of 30–60 mW. Simultaneous detection of intracellular Ca²⁺ concentration and membrane potential revealed coincident photogeneration of Ca²⁺ waves and membrane potential hyperpolarization. By using non-excitable cells, the cell dynamics are slow enough that we can calculate the membrane potential using the steady-state approximation for ion gradients and permeabilities, as formulated in the GHK equations. The calculations predict hyperpolarization that matches the experimental measurements and indicates that the cellular response to laser irradiation is biological, and occurs via laser triggered Ca²⁺ which acts on Ca²⁺ activated K⁺ channels, causing hyperpolarization. Furthermore, by irradiating the cellular plasma membrane, we observed membrane potential depolarization in combination with a drop in membrane resistance that was consistent with a transient laser-induced membrane perforation. These results entail the first quantitative analysis of location-dependent laser-induced membrane potential modification and will help to clarify cellular biological responses under exposure to high intensity ultrashort laser pulses.     

Temperature-dependent changes of electric potential differences on opposite sides of the tepals of Eranthis in relation to thermonastic responses

Electric potential differences (PDs) across the plasma membrane in cells of tepals of Eranthis hyemalis (L.) Salisb. were recorded under a regime in which the temperature was changed rapidly between two levels of approximately 8 and 19°C. The warming of an intact tepal resulted in a transient small increase in the magnitude of PD followed by a substantial long-lasting depolarization. Upon cooling the reverse response occurred: a small transient depolarization was followed by a substantial hyperpolarization. These responses were more pronounced in the epidermis on the abaxial side of the tepal than in that on the adaxial side, indicating an electrophysiological dorsiventrality of the tepals which may be related to thermonastic movements through differential proton secretion on the two sides.     

Evidence for cotransport of nitrate and protons in maize roots : I. Effects of nitrate on the membrane potential

The electrical response of nitrate-grown maize (Zea mays L.) roots to 0.1 millimolar nitrate was comprised of two sequential parts: a rapid and transient depolarization of the membrane potential, followed by a slower, net hyperpolarization to a value more negative than the original resting potential. The magnitude of the response was smaller in roots of seedlings grown in the absence of nitrate, but, within 3 hours of initial exposure to 0.1 millimolar nitrate, increased to that of nitrate-grown roots. Chloride elicited a separate electrical response with a pattern similar to that of the nitrate response. However, the results presented in this study strongly indicate that the electrical response to nitrate reflects the activity of a nitrate-inducible membrane transport system for nitrate which is distinct from that for chloride. Inhibitors of the plasmalemma H⁺-ATPase (vanadate, diethylstilbestrol) completely inhibited both parts of the electrical response to nitrate, as did alkaline external pH. The magnitude of the initial nitrate-dependent, membrane potential depolarization was independent of nitrate concentration, but the subsequent nitrate-dependent hyperpolarization showed saturable dependence with an apparent K_m of 0.05 millimolar. These results support a model for nitrate uptake in maize roots which includes a depolarizing NO₃⁻/H⁺ symport. The model proposes that the nitrate-dependent membrane potential hyperpolarization is due to the plasma membrane proton pump, which is secondarily stimulated by the operation of the NO₃⁻/H⁺ symport.     

Membrane potential,pH and the activation of surf clam oocytes

Unfertilized oocytes of the surf clam, Spisula solidissima, have resting membrane potentials of ?18 ± 7 mV (n = 20). Within five seconds of sperm addition, an electrophysiologically detectable response was apparent, which was characterized by a rapid and prolonged depolarrization depolarization followed four to five minutes post-insemination by the beginning of the beginning of a steady hyperpolarization to approximatelv ?70 mV. This final hyperpolarization was completed within ten minutes of sperm addition. The initial rapid depolarization following insemination may result from a transient increase in sodium conductance, and it may be crucial in preventing polyspermy, since the degree of polyspermy in Spisula oocytes was sensitive to external sodium ion concentrations. Evidence was obtained that changes in intracellular pH are essential for oocvte activation. Using germinal vesical breakdown (GVB) as a marker for activation, it was shown that agents that raise intracellular pH (ammonia and procaine) induced GVB, whereas agents that lower intracellular pH pH (Na-acetate or Na-propionate seawater) inhibited GVB.     

Membrane potential, lipid regulation and adenylate energy charge in acyl chain modified Acholeplasma laidlawii

In Acholeplasma laidlawii variations induced in the transmembrane electrical potential have been shown to affect the membrane lipid composition. Particularly the molar ratio between the predominant glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol, decreases upon hyperpolarization and increases upon depolarization (Clementz et al. (1986) Biochemistry 25, 823-830). Upon variation of the degree of membrane fatty acyl chain unsaturation, known to affect the passive permeability for a number of small molecules, there was no significant correlation between acyl chain composition and the magnitude of the electrical potential. Hyperpolarization by valinomycin decreased the glucolipid ratio for all kinds of membranes, but the size of the decrease was not correlated to the acyl chain composition. However, a clear relationship, independent of acyl chain composition, was found between the extent of hyperpolarization and the size of the decrease in the glucolipid ratio. The adenylate energy charge value (Ec) of the cells was affected by the acyl chain composition, although not exclusively by the proportion of unsaturation. Furthermore, a larger hyperpolarization upon valinomycin addition was accompanied by a stronger reduction in Ec.     

Glial origin of negative shifts of cortical surface potential during tetanic stimulation: Microelectrode study and mathematical analysis

Experiments on anesthetized cats showed that a negative shift of potential on the surface of the cerebral cortex caused by its tetanic stimulation is similar in shape and time course to the depolarization shift of membrane potential of the glial cells, but has a more rapid decline. The hyperpolarization shifts of membrane potential of neurons differed in shape and time course from the negative shift of cortical surface potential. It is concluded that the contribution of hyperpolarization of neurons to the surface-negative potential shift during tetanic stimulation may be manifested visibly only at the beginning (the first 200–300 msec) of such stimulation. The negative potential shift on the cortical surface is due mainly to depolarization of glial cells under the influence of K⁺ secreted from excited nerve cells.     

Sequential depolarization of root cortical and stelar cells induced by an acute salt shock - implications for Na(+) and K(+) transport into xylem vessels

Early events in NaCl-induced root ion and water transport were investigated in maize (Zea mays L) roots using a range of microelectrode and imaging techniques. Addition of 100 mm NaCl to the bath resulted in an exponential drop in root xylem pressure, rapid depolarization of trans-root potential and a transient drop in xylem K(+) activity (A(K+) ) within ～1 min after stress onset. At this time, no detectable amounts of Na(+) were released into the xylem vessels. The observed drop in A(K+) was unexpected, given the fact that application of the physiologically relevant concentrations of Na(+) to isolated stele has caused rapid plasma membrane depolarization and a subsequent K(+) efflux from the stelar tissues. This controversy was explained by the difference in kinetics of NaCl-induced depolarization between cortical and stelar cells. As root cortical cells are first to be depolarized and lose K(+) to the environment, this is associated with some K(+) shift from the stelar symplast to the cortex, resulting in K(+) being transiently removed from the xylem. Once Na(+) is loaded into the xylem (between 1 and 5 min of root exposure to NaCl), stelar cells become more depolarized, and a gradual recovery in A(K+) occurs.     

Choline uptake into the malaria parasite is energized by the membrane potential

The uptake by the intraerythrocytic malaria parasite of the phospholipid precursor choline was investigated in parasites 'isolated' from their host cells by saponin permeabilization of the erythrocyte membrane. Choline is transported across the parasite plasma membrane then phosphorylated and thereby trapped within the parasite. Choline influx was inhibited competitively by quinine. It increased with increasing extracellular pH, decreased on depolarization of the parasite plasma membrane with a protonophore or by increasing extracellular [K+], and increased in response to hyperpolarization of the membrane by decreasing extracellular [K+] or by addition of the K+ channel blocker Cs+. In ATP-depleted parasites choline was taken up but not phosphorylated. Under these conditions, imposition of an inwardly negative membrane potential using the K+ ionophore valinomycin resulted in the accumulation of choline to an intracellular concentration more than 15-fold higher than the extracellular concentration. Choline influx is therefore an electrogenic process, energized by the parasite plasma membrane potential.     

Physiological characteristics of the synaptic response of an identified sensory nonspiking interneuron in the crayfish Procambarus clarkii girard

Voltage-dependent variability in the shape of synaptic responses of the LDS interneuron, an identified nonspiking cell of crayfish, to mechanosensory stimulation was studied using intracellular recording and current injection techniques. Stimulation of the sensory root ipsilateral to the interneuron soma evoked a large depolarizing synaptic response. Its peak amplitude was decreased and the time course was shortened when the LDS interneuron was depolarized by current injection. When the cell was hyperpolarized, the peak amplitude was increased and the time course was prolonged. Upon large hyperpolarization, however, the amplitude did not increase further while the time course showed a slight decrease. The dendritic membrane of the LDS interneuron was found to show an outward rectification upon depolarization and an inward rectification upon large hyperpolarization. Current injection experiments at varying membrane potentials revealed that the voltage-dependent changes in the shape of the synaptic response were based on an increase in membrane conductance due to the rectifying properties of the LDS interneuron. Stimulation of the contralateral root evoked a small depolarizing potential comprising an early excitatory response and a later inhibitory component. Its shape also varied depending on the membrane potential in a manner similar to that of the synaptic response evoked ipsilaterally.     

去甲肾上腺素引起蟾蜍背根神经节神经细胞膜电位变化的离子机制

本文应用细胞内记录方法,对去甲肾上腺素(NA)引起蟾蜍背根神经节(DRG)神经细胞膜电位去极化或超极化反应时的膜电导及翻转电位值进行了测量,并观察了钾和钙离子通道阻断剂灌流DRG对NA引起膜电位反应的影响。当NA引起去极化反应时,15个细胞的膜电导减小32.6％。少数细胞膜电导开始增加,继而减小(n=4)。NA超极化反应时膜电导增加13.2％(n=8)。NA去极化反应的翻转电位值为-88.5±0.9mV((?)±SE,n=4),NA超极化反应在膜电位处于-89至-92mV时消失。 钾通道阻断剂四乙铵可使NA去极化幅值增加73.7±11.9％((?)±SE,n=7),并使NA超极化幅值减小40.5％(n=4)。细胞内注入氯化铯使苯肾上腺素去极化幅值增加34.5％(n=4)。钙通道阻断剂氯化锰使NA去极化及超极化反应分别减小50.5±9.9％((?)±SE,n=10)和89.5±4.9％((?)±SE,n=7)。结果提示,NA引起DRG神经细胞膜电位的去极化或超极化反应,可能与膜的钾及钙通道活动的改变有关。     

Membrane potentials in respiring and respiration-deficient yeasts monitored by a fluorescent dye

Changes in fluorescence of 3,3′-dipropylthiodicarbocyanine iodide which had been equilibrated with suspensions of the wild-type yeast Saccharomyces cerevisiae and of respiration-deficient mutants were followed. The changes have been attributed to changes of yeast membrane potentials, since the fluorescence with wild-type yeast could be affected in a predictable manner by uncouplers and the pore-forming agent nystatin. As in other systems, a rise of steady-state fluorescence was ascribed to depolarization and a drop of the fluorescence to hyperpolarization. (1) A considerable rise in steady-state fluorescence was brought about by addition of antimycin A or some other mitochondrial inhibitors to respiring cells. A major part of the composite membrane potential monitored in intact yeast cells appeared to be represented by the membrane potential of mitochondria. (2) Addition of D-glucose and of other substrates of hexokinase, including non-metabolizable 2-deoxy-D-glucose, induced a two-phase response of fluorescence, indicating transient depolarization followed by repolarization. Such a response was not elicited by other sugars which had been reported to be transported into the cells by a glucose carrier or by D-galactose in galactose-adapted cells. The depolarization was explained by electrogenic ATP exit from mitochondria to replenish the ATP consumed in the hexokinase reaction and the repolarization by subsequent activation of respiration. (3) In non-respiring cells only a drop in fluorescence was induced by glucose and this was ascribed to an ATP-dependent polarization of the plasma membrane. (4) Steady-state fluorescence in suspensions of respiration-deficient mutants, lacking cytochrome a, cytochrome b, or both, was high and remained unaffected by uncouplers and nystatin. This indicates that membranes of the mutants may have been entirely depolarized. A partial polarization, apparently restricted to the plasma membrane, could be achieved by glucose addition.     

Changes in apoplastic pH and membrane potential in leaves in relation to stomatal responses to CO2, malate, abscisic acid or interruption of water supply

Low CO2 concentrations open CO2-sensitive stomata whereas elevated CO2 levels close them. This CO2 response is maintained in the dark. To elucidate mechanisms underlying the dark CO2 response we introduced pH- and potential-sensitive dyes into the apoplast of leaves. After mounting excised leaves in a gas-exchange chamber, changes in extracellular proton concentration and transmembrane potential differences as well as transpiration and respiration were simultaneously monitored. Upon an increase in CO2 concentration transient changes in apoplastic pH (occasionally brief acidification, but always followed by alkalinization) and in membrane potential (brief hyperpolarization followed by depolarization) accompanied stomatal closure. Alkalinization and depolarization were also observed when leaves were challenged with abscisic acid or when water flow was interrupted. During stomatal opening in response to CO2-free air the apoplastic pH increased while the membrane potential initially depolarized before it transiently hyperpolarized. To examine whether changes in apoplastic malate concentrations represent a closing signal for stomata, malate was fed into the transpiration stream. Although malate caused apoplastic alkalinization and membrane depolarization reminiscent of the effects observed with CO2 and abscisic acid, this dicarboxylate closed the stomata only partially and less effectively than CO2. Apoplastic alkalinization was also observed and stomata closed partially when KCl was fed to the leaves. Respiration increased on feeding of malate or KCl, or while abscisic acid closed the stomate. From these results we conclude that CO2 signals modulate the activity of plasma-membrane ion channels and of plasmalemma H+-ATPases during changes in stomatal aperture. Responses to potassium malate and KCl are not restricted to guard cells and neighbouring cells.     

Effects of membrane potential on calcium fluxes of Pelvetia eggs

⁴⁵Ca²⁺ fluxes across the plasma membrane of zygotes of the fucoid alga, Pelvetia fastagiata (J. Ag.) De Toni, were studied in artificial sea waters of various potassium concentrations. Except for two cases, hyperpolarization of the cell membrane (with low [K⁺]) increases, and depolarization (with high [K⁺]) decreases the influx of Ca²⁺ over the range of [K⁺] studied (1–100 mM). The fractional increases of influx during hyperpolarization are close to the fractional increases in membrane potential but the decreases during depolarization are much smaller than those in membrane potential. In two anomalous cases, the influxes of ⁴⁵Ca²⁺ at a potassium concentration of 30 mM were about 20% higher than the control value instead of being 10% lower.The effluxes of ⁴⁵Ca²⁺ are increased by both hyperpolarization and by depolarization. On balance (and excepting the two anomalous cases) the net result of hyperpolarization should be to increase and that of depolarization to decrease intracellular [Ca²⁺].     

Negative surface potential shift and responses of neurons and glial cells during tetanic stimulation of the cortical surface

The negative potential shift in response to tetanic stimulation of the surface of the cortex or thalamic nucleus was recorded from the cortical surface in cats lightly anesthetized with pentobarbital. Parallel intracellular recordings were obtained of activity of neurons and glial cells. Glial cells responded to this stimulation by slow depolarization, which, under certain conditions of stimulation, was followed by slow hyperpolarization; hyperpolarization shifts were observed in neurons. Depolarization and hyperpolarization of glial cells, like hyperpolarization of neurons, did not correlate in time with the development of a negative shift of the surface potential. It is postulated that this shift is a response of complex origin involving the participation not only of glial cells, but also of cortical neurons.I. S. Beritashvili Institute of Physiology, Academy of Sciences of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 248–253, May–June, 1982.     

Membrane potential modulates divalent cation entry in rat parotid acini

This study examines the effect of membrane potential on divalent cation entry in dispersed parotid acini following stimulation by the muscarinic agonist, carbachol, and during refill of the agonist-sensitive internal Ca2+ pool. Depolarizing conditions (addition of gramicidin to cells in Na(+)-containing medium or incubation of cells in medium with elevated [K+]) prevent carbachol-stimulated hyperpolarization of acini and also inhibit carbachol activation of Ca2+ and Mn2+ entry into these cells. Conditions promoting hyperpolarization (cells in medium with Na+ or with N-methyl-D-glucamine instead of Na+) enhance carbachol stimulation of divalent cation entry. Intracellular Ca2+ release (initial increase in [Ca2+]i) does not appear to be affected by these manipulations. Mn2+ entry into resting and internal Ca2+ pool-depleted cells (10-min carbachol stimulation in a Ca(2+)-free medium) is similarly affected by membrane potential modulations, and refill of the internal pool by Ca2+ is inhibited by depolarization. The inhibitory effects of depolarization on divalent cation entry can be overcome by increasing extracellular [Ca2+] or [Mn2+]. These data demonstrate that the modulation of Ca2+ entry into parotid acini by membrane potential is most likely due to effects on the electrochemical gradient (Em-ECa) for Ca2+ entry.     

Distribution of chemoreceptors to quinine on the cell surface of Paramecium caudatum

The topological distribution of the chemoreceptors to quinine in the membrane of a ciliate Paramecium caudatum were examined by conventional electrophysiological techniques. A CNR-mutant specimen defective in voltage-gated Ca channels produced a transient depolarization followed by a transient hyperpolarization and a sustained depolarization when 1 mM quinine-containing solution was applied to its entirety. A Ni²⁺-paralyzed CNR-mutant specimen produced a simple membrane depolarization in response to a local application of 1 mM quinine-containing solution to its anterior end, whereas it produced a transient membrane hyperpolarization in response to an application to its posterior end. An anterior half fragment of a CNR specimen produced a membrane depolarization whereas a posterior half fragment of the specimen produced a transient hyperpolarization upon application of 1 mM quinine-containing solution. Both anterior depolarization and posterior hyperpolarization took place prior to the contraction of the cell body. It is concluded that Paramecium caudatum possesses two kinds of chemoreceptors or two kinds of coupling of the same receptor to different signal transduction pathways to quinine which are distributed in different locations on the cell surface. Activation of the anterior receptor produces a sustained depolarizing receptor potential while activation of the posterior receptor produces a transient hyperpolarizing receptor potential.Abbreviation CNR caudatum non reversal     

Depletion and accumulation of potassium in the extracellular clefts of cardiac Purkinje fibers during voltage clamp hyperpolarization and depolarization: experiments in sodium-free bathing media

Voltage clamp hyperpolarization and depolarization result in currents consistent with depletion and accumulation of potassium in the extracellular clefts o cardiac Purkinje fibers exposed to sodium-free solutions. Upon hyperpolarization, an inward current that decreased with time (id) was observed. The time course of tail currents could not be explained by a conductance exhibiting voltage-dependent kinetics. The effect of exposure to cesium, changes in bathing media potassium concentration and osmolarity, and the behavior of membrane potential after hyperpolarizing pulses are all consistent with depletion of potassium upon hyperpolarization. A declining outward current was observed upon depolarization. Increasing the bathing media potassium concentration reduced the magnitude of this current. After voltage clamp depolarizations, membrane potential transiently became more positive. These findings suggest that accumulation of potassium occurs upon depolarization. The results indicate that changes in ionic driving force may be easily and rapidly induced. Consequently, conclusions based on the assumption that driving force remains constant during the course of a voltage step may be in error.     

酪氨酸、hCG对蟾蜍卵母细胞膜电位的影响

作者用微电极记录了蟾蜍卵母细胞的膜电位。当用含hCG的溶液培灌时,蟾蜍卵母细胞膜电位呈去极化变化;当用含酪氨酸溶液培灌时膜电位呈超极化变化,并能抑制hCG的去极化作用。超微结构的变化与膜电位变化相一致。因此我们认为,酪氨酸可能在蟾蜍卵母细胞有对抗hCG的作用。