Nature of the Schwann cell electrical potential. Effects of the external ionic concentrations and a cardiac glycoside

The effects on the Schwann cell electrical potential of external ionic concentrations and of K-strophanthoside were investigated. Increasing (K)_o depolarized the cell. The potential is related to the logarithm of (K)_o in a quasi-linear fashion. The linear portion of the curve has a slope of 45 mv/ten-fold change in (K)_o. Diminutions of (Na)_o and (Cl)_o produced only small variations in the potential. Calcium and magnesium can be replaced by 44 mM calcium without altering the potential. Increase of (Ca)_o to 88 mM produced about 10 mv hyperpolarization. The cell was hyperpolarized by 11 mv and 4 mv within 1 min after applying K-strophanthoside at concentrations of 10^-3 and 10^-5 M, respectively. No variations of cellular potassium, sodium, or chloride were observed 3 min after applying the glycoside. The hyperpolarization caused by 10^-3 M K-strophanthoside was not observed when (K)_o was diminished to 1 or 0.1 mM or was increased to 30 mM. At a (K)_o of 30 mM, 10^-2 M strophanthoside was required to produce the hyperpolarizing effect. In high calcium, the cell was further hyperpolarized by the glycoside. The initial hyperpolarization caused by the glycoside was followed by a gradual depolarization and a decrease of the cellular potassium concentration. The results indicate that the Schwann cell potential of about -40 mv is due to ionic diffusion, mainly of potassium, and to a cardiac glycoside-sensitive ion transport process.     

Rapid response of the plasma-membrane potential in oat coleoptiles to auxin and other weak acids

We have compared the effects of the auxin, indole-3-acetic acid (IAA) with that of other weak acids on the plasma-membrane potential of oat (Avena sativa L.) coleoptile cells. Cells treated with 1 M IAA at pH 6 depolarize 20–25 mV in 10–12 min, but they then repolarize, until by 20–25 min their potentials are about 25 mV more negative than the initial value. Similar concentrations of benzoic and butyric acids cause the initial depolarization, but not the subsequent hyperpolarization. The hyperpolarization is therefore specific to IAA. All the weak acids, including IAA, evoke a rapid hyperpolarization when their concentrations are raised to 10 mM. This result indicates that at high concentrations, the uptake of undissociated weak acids activates electrogenic proton pumping, most likely by lowering cytoplasmic pH. In contrast, the hyperpolarization observed with concentrations of IAA four orders of magnitude lower appears to be a specific hormonal effect. This specific, auxin-induced hyperpolarization occurs at the same time as the initiation of net proton secretion and supports the hypothesis that auxin initiates extension growth by increasing proton pumping.Abbreviations FC fusicoccin - IAA indole-3-acetic acid     

Responses of Avena coleoptiles to suboptimal fusicoccin: kinetics and comparisons with indoleacetic Acid

Proton excretion induced by optimal concentrations of indoleacetic acid (IAA) and fusicoccin (FC) differs not only in maximum rate of acidification but also in the lag before onset of H⁺ excretion and in sensitivity to cycloheximide. Because these differences might simply be a consequence of the difference in rate of proton excretion, FC and IAA have now been compared using oat coleoptiles (cv. Victory) under conditions where the rates of acidification are more similar, i.e. suboptimal FC versus optimal IAA. As the concentration of FC is reduced, the rate of H⁺ excretion decreases, the final equilibrium pH increases, and the lag before detectable acidification increases up to 7-fold. This enhanced lag period is not primarily a consequence of wall buffering, inasmuch as it persists when a low concentration of FC is added to sections which were already excreting H⁺ in response to IAA. An extended lag also occurs, upon reduction of FC levels, in the hyperpolarization of the membrane potential, before enhancement of O₂ uptake and before the increased rate of Rb⁺ uptake. The presence or absence of a lag is not a distinguishing feature between FC and IAA actions on H⁺ excretion and cannot be used to discriminate between their sites of action. In contrast, the insensitivity of FC-induced H⁺ excretion to cycloheximide, as compared with the nearly complete inhibition of this auxin effect by cycloheximide, persists even at dilute concentrations of FC. This seems to be a basic difference in H⁺ excretion by IAA and FC.     

Membrane hyperpolarization by sperm-activating and -attracting factor increases cAMP level and activates sperm motility in the ascidian Ciona intestinalis.

In the ascidian Ciona intestinalis (and C. savignyi), sperm-activating and -attracting factor (SAAF) is released from the egg at fertilization and stimulates both Ca(2+) influx and a transient increase in cAMP level of the sperm, leading to the activation of sperm motility (M. Yoshida et al., 1994, Dev. Growth Differ. 36, 589-595). In this paper we show in C. intestinalis that valinomycin, a potassium-selective ionophore, as well as SAAF, activated sperm motility, and this activation was suppressed by extracellular high K(+). Membrane potential measurements showed that both SAAF and valinomycin increase K(+) permeability of sperm and induce membrane hyperpolarization, the amplitude of which depends on the external K(+) concentration. The membrane potential and intracellular K(+) concentration of Ciona sperm without SAAF were estimated to be about -50 mV and 560 +/- 40 mM, respectively. After treatment with SAAF or valinomycin the membrane potential became almost equal to the equilibrium potential of K(+) (-100 mV), and the cAMP level increased in artificial seawater. A potent voltage-dependent K(+) channel blocker, MCD peptide, at the concentration of 10 microM blocked SAAF-induced hyperpolarization of the cells, increase in cAMP, and sperm motility. These results suggest that membrane hyperpolarization produced by the opening of K(+) channels elevates cAMP synthesis and leads to the activation of sperm motility in Ciona.     

Transmembrane electrical potentials in growing maize roots

The anti-auxin 4-chlorophenoxyisobutyric acid (PCIB) applied at a concentration of 10^-2 mol m^-3 to maize root segments was found to induce a transmembrane electrical potential of up to-130 mV (pd of 30 mV). The kinetics of this response were comparable to the time scale for PCIB-stimulated H⁺-extrusion. Both effects are eliminated by the addition of p-fluoromethoxycarbonyl cyanide phenylhydrazone (FCCP). Treatment with fusicoccin (FC) and PCIB together does not result in a hyperpolarization greater than with FC alone. Benzoic acid (10^-2 mol m^-3) had no effect on the transmembrane electrical potentials. These results are discussed in relation to a possible electrogenic proton pump which may be regulated by perturbations in the cellular auxin content or activity.Abbreviations ATPase adenosine triphosphatase - FC fusicoccin - FCCP p-fluoromethoxy carbonylcyanide phenylhydrazone - IAA indole-3yl-acetic acid - NAA naphthyl-lylacetic acid - PCIB 4-chlorophenoxyisobutyric acid - PD potential difference     

Inwardly rectifying K(+) channels in esophageal smooth muscle

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.     

Effect of temperature on growth,proton extrusion and membrane potential in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) coleoptile segments

The effects of temperature (5–45°C) on endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC), and proton extrusion in maize coleoptile segments were studied. In addition, membrane potential changes at some temperatures were also determined. It was found that in this model system endogenous growth exhibits a clear maximum at 30°C, whereas growth in the presence of IAA and FC shows the maximum value in the range 30–35°C and 35–40°C, respectively. Simultaneous measurements of growth and external medium pH indicated that FC at stressful temperatures was not only much more active in the stimulation of growth, but was also more effective in acidifying the external medium than IAA. Also the addition of either IAA or FC to the bathing medium at 30 and 40°C did not change the kinetic characteristic of membrane potential changes observed for both substances at 25°C. However, the increased temperature significantly decreased IAA and FC-induced membrane hyperpolarization. IAA in the incubation medium, at 10°C, brought about additional membrane depolarization (apart from the one induced by low temperature). In contrast to IAA, FC at 10°C caused gradual repolarization of membrane potential, which correlated with both FC-induced growth and FC-induced proton extrusion. A plausible interpretation for temperature-induced changes in growth of maize coleoptile segments is that, at least in part, these changes were mediated via a PM H⁺-ATPase activity.     

Lipid factor (bVLF) from bovine vitreous body evokes in EGFR-T17 cells a Ca2+-dependent K+ current associated with inositol 1,4,5-trisphosphate-independent Ca2+ mobilization

Bovine vitreous lipid factor (bVLF) is a complex phospholipid isolated from bovine vitreous body with strong Ca(2+)-mobilizing activity. In this study, the effects of bVLF on membrane potential were investigated in EGFR-T17 fibroblasts with the whole-cell patch clamp technique on monolayer cells, as well as with the fluorescent dye bis-oxonol as membrane potential-sensitive probe on monolayer and suspension cells. bVLF induced a transient hyperpolarization characterized by an initial peak and subsequent return to resting membrane potential levels within 1-2 min. The increase of [Ca(2+)](i) was concomitant with an outward current responsible for the hyperpolarizing response. Results with: (a) high [K(+)](o) media; (b) the monovalent cation ionophore gramicidin; and (c) substitution of K(+) with Cs(+) in the intracellular solution were consistent with the involvement of K(+) channels. The bVLF-induced hyperpolarization was blocked by the K(+) channel blockers, quinine and tetraethylamonium chloride, and partially affected by 4-aminopyridine. The calcium ionophore ionomycin caused a similar hyperpolarization as bVLF. When intracellular calcium was buffered by adding BAPTA to the pipette solution, bVLF-activated outward current was prevented. Moreover, the hyperpolarization response was strongly reduced at low doses (3 nM) of specific Ca(2+)-activated K(+) channel blockers, charybdotoxin and iberiotoxin. Based on these observations we conclude that bVLF hyperpolarizes the cells via the activation of a Ca(2+)-dependent K(+) current. In addition, it was observed that bVLF did not have a significant effect on intercellular communication measured by a single patch-electrode technique. Thus, membrane potential changes appeared to belong to the earliest cellular responses triggered by bVLF, and are closely associated with phosphatidic acid-dependent [Ca(2+)](i) mobilization.     

Potassium currents regulating secretion from Brunner's glands in guinea pig duodenum

This study examined the role of outward K(+) currents in the acinar cells underlying secretion from Brunner's glands in guinea pig duodenum. Intracellular recordings were made from single acinar cells in intact acini in in vitro submucosal preparations, and videomicroscopy was employed in the same preparation to correlate these measures with secretion. Mean resting membrane potential was -74 mV and was depolarized by high external K(+) (20 mM) and the K(+) channel blockers 4-aminopyridine (4-AP), quinine, and clotrimazole. The cholinergic agonist carbachol (60-2,000 nM; EC(50) = 200 nM) caused a concentration-dependent initial hyperpolarization of the membrane and an associated decrease in input resistance. This hyperpolarization was significantly decreased by 20 mM external K(+) or membrane hyperpolarization and increased by 1 mM external K(+) or membrane depolarization. It was blocked by the K(+) channel blockers tetraethylammonium (TEA), 4-AP, quinine, and clotrimazole but not iberiotoxin. When videomicroscopy was employed to measure dilation of acinar lumen in the same preparation, carbachol-evoked dilations were altered in a parallel fashion when external K(+) was altered. The dilations were also blocked by the K(+) channel blockers TEA, 4-AP, quinine, and clotrimazole but not iberiotoxin. These findings suggest that activation of outward K(+) currents is fundamental to the initiation of secretion from these glands, consistent with the model of K(+) efflux from the basolateral membrane providing the driving force for secretion. The pharmacological profile suggests that these K(+) channels belong to the intermediate conductance group.     

The role of BKCa channels on hyperpolarization mediated by hyperosmolarity in human articular chondrocytes

Chondrocytes, the only cell in cartilage, are subjected to hyperosmotic challenges continuously since extracellular osmolarity in articular cartilage increases in response to mechanical loads during joint movement. Hyperosmolarity can affect membrane transport, and it is possible that load modulates matrix synthesis through alterations in intracellular composition. In the present study, the effects of hyperosmotic challenges were evaluated using the whole-cell patch clamp technique, whole cell mode on freshly isolated human and bovine articular chondrocytes. In human chondrocytes, hypertonicity induced the activation of outward Ca(2+)-sensitive K(+) currents, which were inhibited by iberiotoxin and TEA-Cl. The current induced by hypertonic switching (osmolarity from 300 to 400 mOsm/l) caused cell hyperpolarization (from -39 mV to -70 mV) with a reversal potential of -96 ± 7 mV. These results suggest a role for Ca(2+)-activated K(+) channels in human articular chondrocytes, leading to hyperpolarization as a consequence of K(+) efflux through these channels. These channels could have a role in the articular chondrocyte's response to a hyperosmotic challenge and matrix metabolism regulation by load.     

Disorders of supporting mechanisms of resting membrane potentials of the cardiomyocytes during stress and their prevention

Incubation of the rats' hearts in low temperature (4 degrees C) in the course of 1.5 h reduced the rest membrane potential (RP) of the cardiomyocytes from 86 +/- 2 to 60 +/- 2 mv. Rapid rewarming to 36 degrees C resulted in hyperpolarization of the membrane potential to 103 +/- 3 mv with maximal speed of restitution of RP about 0.35 mv/sec. These results were determined by electrogenic Na-pump. In the heart of rats, exposed to 6-hour immobilization stress, the speed of restitution of RP decreased to 0.15 +/- 0.01 mv/sec and the hyperpolarization effect was eliminated. This was indicated on the disturbance of Na-pump of the cardiomyocytes membranes. Preliminary treatment with gamma-hydroxybutyric acid or antioxidants alpha-tocopherol and ionol prevented stress-induced disturbances of the cardiomyocytes.     

Inwardly rectifying K(+) channels in spermatogenic cells: functional expression and implication in sperm capacitation

To fertilize, mammalian sperm must complete a maturational process called capacitation. It is thought that the membrane potential of sperm hyperpolarizes during capacitation, possibly due to the opening of K(+) channels, but electrophysiological evidence is lacking. In this report, using patch-clamp recordings obtained from isolated mouse spermatogenic cells we document the presence of a novel K(+)-selective inwardly rectifying current. Macroscopic current activated at membrane potentials below the equilibrium potential for K(+) and its magnitude was dependent on the external K(+) concentration. The channels selected K(+) over other monovalent cations. Current was virtually absent when external K(+) was replaced with Na(+) or N-methyl-D-glucamine. Addition of Cs(+) or Ba(2+) (IC(50) of approximately 15 microM) to the external solution effectively blocked K(+) current. Dialyzing the cells with a Mg(2+)-free solution did not affect channel activity. Cytosolic acidification reversibly inhibited the current. We verified that the resting membrane potential of mouse sperm changed from -52 +/- 6 to -66 +/- 9 mV during capacitation in vitro. Notably, application of 0.3-1 mM Ba(2+) during capacitation prevented this hyperpolarization and decreased the subsequent exocytotic response to zona pellucida. A mechanism is proposed whereby opening of inwardly rectifying K(+) channels may produce hyperpolarization under physiological conditions and contribute to the cellular changes that give rise to the capacitated state in mature sperm.     

Analysis of K Inactivation and TEA Action in the Supramedullary Cells of Puffer

Under the voltage clamp condition, the K inactivation was analyzed in cells bathed in the isosmotic KCl Lophius-Ringer solution. After conditioning hyperpolarization, the cells respond to depolarizations with increased K permeability, which in turn is decreased during maintained depolarizations. The steady-state levels of the K inactivation as a function of the membrane potential are related by an S-shaped curve similar to that which describes the steady-state Na inactivation in the squid giant axon. TEA reduced the K conductance by a factor which is independent of the potential, and without a shift of the inactivation curve along the voltage axis. The rapid phase of the K activation is less susceptible to TEA than the slow phase of the K activation. Hyperpolarizing steps remove the K inactivation, the rate of the removal being faster the larger the hyperpolarization from the standard potential of about -60 mv.     

Membrane effects of 2,4-dichlorophenoxyacetic acid in motor cells of Mimosa pudica L.

2,4-dichlorophenoxyacetic acid applied to excised leaves of Mimosa pudica L. inhibited in a dose-dependent manner the shock-induced pulvinar movement. This inhibition was negatively correlated with the amount of [(14)C] 2,4-dichlorophenoxyacetic acid present in the vicinity of the motor cells. Although 2,4-dichlorophenoxyacetic acid is a weak acid, its greatest physiological efficiency was obtained with pH values close to neutrality. This observation opens the question of its mode of action which may be through external signaling or following internal transport by a specific anionic form transporter. The effect was related to molecular structure since 2,4-dichlorophenoxyacetic acid>3,4-dichlorophenoxyacetic acid>2,3-dichlorophenoxyacetic acid. An essential target of 2,4-dichlorophenoxyacetic acid action lies at the plasmalemma as indicated by the induced hyperpolarization of the cell membrane. Compared to indole-3-acetic acid and fusicoccin, it induced a complex effect on H(+) fluxes. Applied to plasma membrane vesicles purified from motor organs, 2,4-dichlorophenoxyacetic acid enhanced proton pumping, but, unlike fusicoccin, it did not increase the H(+)-ATPase catalytic activity in our experimental conditions. Taken together, the data suggest that 2,4-dichlorophenoxyacetic acid acts on cell turgor variation and the concomittant ion migration, in particular K(+), by a mechanism involving specific steps compared to indole-3-acetic acid and fusicoccin.     

急性低温/再复温对大鼠心室肌膜电位和钾电流的影响

本文旨在研究急性低温/再复温对大鼠心室肌膜电位和钾电流的影响.膜电位和膜电流分别在全细胞膜片钳的电压钳和电流钳模式下记录.当细胞外灌流液从25℃降低到4℃后,一过性外向电流(transient outward current, Ito)完全消失,膜电位为 60mV时的稳态外向K 电流(sustained outward K current, Iss)和膜电位为-120mV时的内向整流K 电流(inward rectifier K current, IK1)分别降低(48.5±14.1)%和(35.7±18.2)%,同时,膜电位绝对值降低.当细胞外灌流液从4℃再升高到36℃后,膜电位出现一过性超级化,然后恢复到静息电位水平;在58个细胞中,有36个细胞伴随复温出现ATP-敏感性K (ATP-sensitive K , KATP)通道的激活.再复温引起的上述变化可以被Na /K -ATP酶抑制剂哇巴因(100μmol/L)所抑制.再复温引起的KATP通道激活也能被蛋白激酶A抑制剂H-89(100μmol/L)所抑制.在细胞膜电位被钳制在0mV时,当细胞外灌流液温度从25℃降低到4℃后,细胞的体积没有发生明显改变,但当再复温引起KATP通道激活后,细胞很快发生皱缩,同时细胞内部出现许多折光较强的斑点.上述结果表明急性低温/再复温对大鼠心室肌膜电位和K 电流有明显影响,并提示KATP通道激活可能与心肌低温/再复温损伤有关.     

Evidence for the acid-growth theory of fusicoccin action

Three predictions of the acid-growth theory of fusicoccin (FC) action in inducing cell elongation were reinvestigated using abraded segments of maize (Zea mays L.) coleoptiles. i) Quantitative comparison of segment elongation and medium-acidification kinetics measured in the same sample of tissue shows that these FC-induced processes are strictly correlated in time and respond coordinately to cations present in the medium. ii) Fusicoccin (1 mol l^-1) induces a rapid acidification of the cell-wall solution, reaching a final level of pH 3.8–4.0. Exogenous protons are able to substitute quantitatively for FC in causing segment elongation at pH 3.8–4.0. At pH 4, FC has no additional effect on cell elongation. iii) Neutral buffers (pH 7) completely abolish the FC-mediated growth response. iv) Cycloheximide (10 mg l^-1) inhibits both FC-induced and acid-buffer(pH 4)-induced elongation after a lag of 40–45 min, and FC-induced H⁺ excretion after a lag of 2 h. Under the same conditions, indole-3-acetic acid-induced elongation and H⁺ excretion are inhibited without detectable lag. It is concluded that these results are fully compatible with the acid-growth theory of FC action.Abbreviations IAA indole-3-acetic acid - CHI cycloheximide - FC fusicoccin     

Glucose induces a Na(+),K(+)-ATPase-dependent transient hyperpolarization in human sperm. I. Induction of changes in plasma membrane potential by the proton ionophore CCCP

When human sperm was incubated in medium deprived of glucose, glucose restoration caused a transient hyperpolarization of the plasma membrane. This hyperpolarization was also induced by fructose but not by 2-deoxyglucose, a substrate that cannot be metabolized. The hyperpolarization was inhibited by NaF, a glycolysis inhibitor, but not by mitochondrial inhibitors (cyanide, rotenone and antimycin), suggesting that it depended on glycolysis. Furthermore, the hyperpolarization was still induced in medium containing a high concentration of KCl and was insensitive to the K(+) channel blocker TEA and the Cl(-) channel blocker niflumic acid, but it was blocked by ouabain. This suggested that upon glucose addition, there was an increase in the concentration of ATP, that in turns increased the Na(+),K(+)-ATPase activity. Since this pump is electrogenic (2K(+)/3Na(+)) the plasma membrane hyperpolarized. On the other hand, CCCP, a proton ionophore, inhibited the hyperpolarization induced by glucose. When CCCP was added to glucose-treated hyperpolarized sperm, it caused a depolarization that triggered a Ca(2+) influx sensitive to nickel, an inhibitor of voltage-dependent calcium channels. Moreover, CCCP caused hyperpolarization in sperm incubated in medium without calcium, a known condition that depolarizes sperm. This indicated that CCCP induced proton permeability in the plasma membrane that was able to change the membrane potential to a value corresponding to the E(H) and that was also able to clamp it, so that it prevented the hyperpolarization induced by glucose.     

A comparison of the effects of IAA and 4-Cl-IAA on growth, proton secretion and membrane potential in maize coleoptile segments

The physiological activity of exogenous 4-Cl-IAA, as compared to IAA, was examined in maize coleoptile segments. It was found that in this model system 4-Cl-IAA is much more active in the stimulation of elongation than IAA. Simultaneous measurements of growth and external pH indicated that administration of either IAA or 4-Cl-IAA resulted in medium acidification. The kinetics of the pH changes, however, were faster after the addition of 4-Cl-IAA. In contrast to IAA, the coleoptile segments treated with chlorinated auxin were not able to increase medium pH after its initial drop. The re-addition of IAA after 5 h further enhanced growth over the next 2 h by 31%. By contrast, the re-addition of 4-Cl-IAA at the same time protocol as IAA did not cause an additional effect. The administration of 10 microM IAA induced in maize coleoptile cells a transient depolarization followed by a slow hyperpolarization of their membrane potential. In contrast to IAA, 4-Cl-IAA at 1 microM caused an immediate hyperpolarization of the membrane potential which, on average, was 2-fold greater than for IAA. The results reported here provide further evidence that 4-Cl-IAA is much more active, as compared to IAA, in stimulating the growth of maize coleoptile segments. Although it has not been directly demonstrated here, a plausible interpretation for the high 4-Cl-IAA activity is that, at least in part, it might be caused via a reduced metabolism of 4-Cl-IAA. Furthermore, for the first time, the data show that membrane potential responds to 4-Cl-IAA in a qualitatively different fashion than to IAA. These findings may, in turn, suggest a specific signal transduction pathway to 4-Cl-IAA in maize coleoptile cells.