Effect of changes in pH of the medium on electrical activity of the Ranvier node

The effect of changes in pH of the medium from 4 to 10 on the action potential and its first derivative was studied at the original resting potential and during hyperpolarization of the membrane in experiments on single nodes of Ranvier. Raising the pH of the medium from 7 to 9 led to a decrease in amplitude of the action potential and of its derivative (V_max). During hyperpolarization of the membrane these parameters were fully restored. Lowering the pH of the solution led to an increase in the action potential and a decrease in V_max. During hyperpolarization of the membrane the action potential and its derivative were not completely restored. Under the influence of solutions with low and high pH values the duration of the action potential was increased. Changes in the action potential and in V_max with an increase in pH can be attributed to increased inactivation of the sodium permeability of the membrane, and in solutions with low pH to a decrease in the maximal sodium permeability and to weakening of its inactivation.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 6, No. 2, pp. 205–210, March–April, 1974.     

The pH-dependent rate of action of local anesthetics on the node of Ranvier

Local anesthetic solutions were applied suddenly to the outside of single myelinated nerve fibers to measure the time course of development of block of sodium channels. Sodium currents were measured under voltage clamp with test pulses applied several times per second during the solution change. The rate of block was studied by using drugs of different lipid solubility and of different charge type, and the external pH was varied from pH 8.3 to pH 6 to change the degree of ionization of the amine compounds. At pH 8.3 the half-time of action of amine anesthetics such as lidocaine, procaine, tetracaine, and others was always less than 2 s and usually less than 1 s. Lowering the pH to 6.0 decreased the apparent potency and slowed the rate of action of these drugs. The rate of action of neutral benzocaine was fast (1 s) and pH independent. The rate of action of cationic quaternary QX-572 was slow (greater than 200 s) and also pH independent. Other quaternary anesthetic derivatives showed no action when applied outside. The result is that neutral drug forms act much more rapidly than charged ones, suggesting that externally applied local anesthetics must cross a hydrophobic barrier to reach their receptor. A model representing diffusion of drug into the nerve fiber gives reasonable time courses of action and reasonable membrane permeability coefficients on the assumption that the hydrophobic barrier is the nodal membrane. Arguments are given that there may be a need for reinterpretation of many published experiments on the location of the anesthetic receptor and on which charge form of the drug is active to take into account the effects of unstirred layers, high membrane permeability, and high lipid solubility.     

Ionic movements across the chorion in newly shed salmon eggs (Salmo salar L.)

Summary Sodium and chloride exchange and trans-chorion potentials were investigated in newly shed eggs from Atlantic salmon. Exposure of eggs to pH 3.5 caused the non-labile sodium fraction at pH 7.0 to become labile and lost from the egg. Chloride fluxes appear unaffected by the pH of the external medium. Trans-chorion potential was inside negative (about –100 mV) in dilute media (10^–5 mol·l^–1 NaCl or KCl) and immediately decreased as external cation concentration was increased, by about 46 mV/decade change in cation concentration, reaching about 0 mV in 10^–2 mol·l^–1 cation. Return to dilute cation solutions resulted in a slow increase in potential (repolarisation) and the time course of these potential changes was paralleled by the rate of sodium efflux, although chloride efflux was very rapid. After exposure to acid conditions repolarisation of the egg on return to dilute cation concentrations was preceeded by a phase when the pvp became inside positive. The results are discussed in terms of chorion structure, anionic charge on the perivitelline molecules and unstirred layers within the chorion.Abbreviations pvp perivitelline potential     

The effects of pH and calcium concentrations on gill potentials in the Brown Trout,Salmo trutta

Summary Measurements of the transepithelial potential (V_int-V_ext) across the gills of Brown Trout,Salmo trutta, were made in solutions of a range of pH and calcium concentrations. The potential was strongly dependent on external pH, being negative in neutral solutions but positive in acid solutions. The addition of calcium to the external medium produced a positive shift in potential in all but very acid media (pH 4.0–3.5), where very little change was seen. The gill membrane appears to act as a hydrogen electrode having a very high permeability to H⁺ ions, and the potential behaves as a diffusion potential. The presence of calcium reduced the permeability to both H⁺ and Na⁺ ions but even at a calcium concentration of 8.0 mM/l the permeability ratio H⁺/Na⁺ was still more than 900. The transepithelial potential is shown to be diffusional in origin and is discussed in terms of the relative permeability of the gill to H⁺, Na⁺ and Cl^– ions. Sodium fluxes across the gills were measured and provide the basis for a theoretical consideration of Na⁺, Cl^– and H⁺ fluxes across the gills in neutral and acid solutions. The positive potential at low pH largely accounts for the increased loss of sodium from fish in these conditions.     

Membrane potential of rat hepatoma cells in culture: Influence of factors affecting amino acid transport

The effect has been studied of various media, hormones and of amino acids on the membrane potential of rat hepatoma cells in culture measured by microelectrode impalement. Cells in Eagle's minimal essential medium plus 5% serum had a value which varied daily from about 5–8 mV, inside negative. The membrane potential of rat hepatocytes was measured to be 8.7 ± 0.2mV, inside negative. The membrane potential of the hepatoma cells was decreased by insulin and increased by glucagon. Membrane potential was unaffected by change of medium to Hanks' or Earle's balanced salt solutions or deprivation of serum. It was, however, reduced in cells in phosphate-buffered saline and by reduction of pH. The former effect was shown to be due to the higher [Na⁺] of phosphat-buffered saline as opposed to the other media. Addition of alanine, glycine, serine, proline and methylaminoisobutyrate all reduced membrane potential by 2–3 mV. Smaller decreases were seen with methionine, leucine and phenylalanine, but none with glutamine, threonine, BCH (2-aminonorborane-2-carboxylic acid) and D-alanine. The results are compared with the effects of similar conditions on aminoisobutyrate uptake. Whilst there was a correlation under some conditions there was not under others. It is concluded that for the hepatoma cells factors additional to the membrane potential must exert some influence on the capacity for amino acid transport.     

Effect of pH changes on after potentials of single frog Ranvier nodes

In experiments on single nodes of Ranvier of isolated frog nerve fibers a decrease in pH of the medium to 5 caused a small increase in after-depolarization but an increase in pH to 9 caused virtually no change in it. The after-hyperpolarization arising in potassium-free Ringer's solution was abolished by a decrease in pH but remained substantially unchanged as a result of an increase in pH; posttetanic hyperpolarization was reduced in amplitude and slightly increased in duration by a decrease in pH, but not appreciably changed by an increase in pH. It is concluded that the character of changes in after-potentials of single nodes of Ranvier of isolated nerve fibers in medium of different pH is entirely determined by the influence of this factor on the kinetics of the potassium permeability of the excitable membrane.I. N. Ul'yanov Pedagogic Institute, Ul'yanovsk. Translated from Neirofiziologiya, Vol. 8, No. 1, pp. 62–66, January–February, 1976.     

Correlations between changes in membrane capacitance induced by changes in ionic environment and the conductance of channels incorporated into bilayer lipid membranes

The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd²⁺, La³⁺ or Tb³⁺ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8–9.0. A pH shift from 7.4 to 3–4 or 11–12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested.     

Lipid-polypeptide interactions in bilayer lipid membranes

Summary The modifications of the electrical properties of bilayer lipid membranes (BLM) composed of cholesterol and an ionic surfactant upon interaction with charged polypeptides were studied. The addition of 10^–8 m polylysine (Ps⁺) to one side of anionic cholesterol dodecylphosphate BLM increases the specific membrane conductance over 1000-fold (from 10^–8 to 10^–5 mho/cm²) and develops a cationic transmembrane potential larger than 50 mV. This potential is reverted by addition of polyanions such as RNA, polyglutamic or polyadenilic acid to the same side on which Ps⁺ is present, by addition of Ps⁺ to the opposite side, or by addition of trypsin to either side. Both conductance and potential changes are hindered by increasing the ionic strength or by raising the pH of the bathing medium, disappearing above pH 11.5 where it is known that Ps⁺ folds into an -helix. The interaction of polyglutamic acid (PGA) with a cationic cholesterol-hexadecyltrimethylammonium bromide BLM results in increased membrane conductance and development of an anionic transmembrane potential which is reverted by addition of polycations to the same aqueous phase where PGA is present. Addition of either Ps⁺ or PGA to one or both sides of a neutral BLM composed of 7-dehydrocholesterol induces no significant change. The observations suggest the formation of a lipid polymer membrane resultant from the interaction, predominantly electrostatic, of the isolated components. The implications of these results are discussed in terms of the current models of membrane structure.     

Effect of histrionicotoxin on ion channels in synaptic and conducting membranes of electroplax ofElectrophorus electricus

Histrionicotoxin (HTX) at low concentrations of 5-10 microM blocks the postsynaptic potential of the electroplax of Electrophorus electricus. At 100-fold higher concentrations, HTX blocks the directly evoked action potentials of the conducting membrane. The pH dependence of the blockade by HTX at synaptic channels is different from that at the conducting membrane. At the synapse HTX is more potent at acid pH, while at the conducting membrane it is more potent at basic pH. HTX at high concentrations antagonizes the effects of batrachotoxin, indicative of an effect on the batrachotoxin-sensitive sodium channels involved in action potential generation. While the effects of HTX on the synaptic channels are concentration, time, and pH dependent, the effects on the channels of the conducting membrane are, in addition, use dependent, suggesting interactions of HTX with the activated forms of these channels.     

Surface charge of mammalian neurones as revealed by microelectrophoresis

Summary The surface charge of isolated rat dorsal root ganglion neurones was studied by microelectrophoresis technique. The increase of Ca concentration caused greater reduction of the electrophoretic mobility compared to that produced by an equivalent amount of divalent organic cations, dimethonium or hexamethonium. No charge reversal for Ca concentrations up to 80 mM was observed. These data fit the suggestion that two anion groups of the outer membrane surface can bind one Ca ion with apparent binding constant of about 50 M^–1. In solutions of low pH the electrophoretic mobility of cells decreased corresponding to titration of acidic groups with apparent pK=4.2. Trypsin treatment in mild conditions markedly reduced the surface charge: however, neuraminidase and hyaluronidase did not change it. N-bromosuccinimide (a specific reagent for carboxylic groups of proteins) decreased the electrophoretic mobility about 60%. However, no increase of the surface charge after the action of specific reagents for amino groups (2,4,6-trinitrobenzenesulfonic acid and maleic anhydride) was observed. It was shown that the surface charge depends also on the intracellular metabolism. If 1 mM dibutyryl cAMP or theophilline was added to the culture medium (thus, raising the concentration of cAMP inside the cell) the surface charge increased. This effect developed slowly and reached its maximum on the third day of incubation. Treatment of cells by 5 mM tolbutamide (an inhibitor of some protein kinases) did not change cell mobility. Addition of 5 mM N-ethylmaleimide (an inhibitor of adenylate cyclase) to the culture medium produced some decrease of the surface charge. On the basis of data obtained it is suggested that the charge of the outer membrane surface of neurones studied is mainly determined by carboxylic groups of membrane proteins, and changes in intracellular cAMP concentration influence the synthesis and reconstruction of these membrane components.     

Differences in Na and Ca Spikes As Examined by Application of Tetrodotoxin, Procaine, and Manganese Ions

The effects of tetrodotoxin, procaine, and manganese ions were examined on the Ca spike of the barnacle muscle fiber injected with Ca-binding agent as well as on the action potential of the ventricular muscle fiber of the frog heart. Although tetrodotoxin and procaine very effectively suppress the "Na spike" of other tissues, no suppressing effects are found on "Ca spike" of the barnacle fiber, while the initiation of the Ca spike is competitively inhibited by manganese ions. The initial rate of rise of the ventricular action potential is suppressed by tetrodotoxin and procaine but the plateau phase of the action potential is little affected. In contrast the suppressing effect of manganese ions is mainly on the plateau phase. The results suggest that the plateau phase of the ventricular action potential is related to the conductance increase in the membrane to Ca ions even though Na conductance change may also contribute to the plateau.     

Competitive Action of Calcium and Procaine on Lobster Axon : A study of the mechanism of action of certain local anesthetics

Voltage clamp studies with the squid giant axon have shown that changes in the external calcium concentration (Frankenhaeuser and Hodgkin, 1957) shift the sodium and potassium conductance versus membrane potential curves along the potential axis. Taylor (1959) found that procaine acts primarily by reducing the sodium and, to a lesser extent, the potassium conductances. Both procaine and increased calcium also delay the turning on of the sodium conductance mechanism. Calcium and procaine have similar effects on lobster giant axon. In addition, we have observed that the magnitude of the response to procaine is influenced by the external calcium concentration. Increasing external calcium tends to reduce the effectiveness of procaine in decreasing sodium conductance. Conversely, procaine is more effective in reducing the membrane conductance if external calcium is decreased. The amplitude of the nerve action potential reflects these conductance changes in that, for example, reductions in amplitude resulting from the addition of procaine to the medium are partially restored by increasing external calcium, as was first noted by Aceves and Machne (1963). These phenomena suggest that calcium and procaine compete with one another with respect to their actions on the membrane conductance mechanism. The fact that procaine and its analogues compete with calcium for binding to phospholipids in vitro (Feinstein, 1964) suggests that the concept of competitive binding to phospholipids may provide a useful model for interpreting these data.     

Comparison of tetrodotoxin and procaine in internally perfused squid giant axons

Squid giant axons were internally perfused with tetrodotoxin and procaine, and excitability and electrical properties were studied by means of current-clamp and sucrose-gap voltage-clamp methods. Internally perfused tetrodotoxin was virtually without effect on the resting potential, the action potential, the early transient membrane ionic current, and the late steady-state membrane ionic current even at very high concentrations (1,000–10,000 nM) for a long period of time (up to 36 min). Externally applied tetrodotoxin at a concentration of 100 nM blocked the action potential and the early transient current in 2–3 min. Internally perfused procaine at concentrations of 1–10 mM reversibly depressed or blocked the action potential with an accompanying hyperpolarization of 2–4 mv, and inhibited both the early transient and late steady-state currents to the same extent. The time to peak early transient current was increased. The present results and the insolubility of tetrodotoxin in lipids have led to the conclusion that the gate controlling the flow of sodium ions through channels is located on the outer surface of the nerve membrane.     

Reaction of Local Anesthetics with Phospholipids : A possible chemical basis for anesthesia

Local anesthetics (LA) have been found to interact with phospholipids and lipids extracted from nerve and muscle. This reaction is demonstrated by: (a) Inhibition by LA of phospholipid (and tissue lipid) facilitated transport of calcium from a methanol: water phase into chloroform. This action is dependent upon the cationic form of the LA. (b) LA increase the electrical resistance of "membranes" prepared by impregnating Millipore filters with cephalin:cholesterol or tissue lipid extracts and bathed with NaCl or KCl solutions. (c) LA coagulate aqueous dispersions of cephalin, phosphatidyl serine, phosphatidyl ethanolamine, and inositide, an action shared by calcium. The order of potency in coagulating cephalin sols is tetracaine > calcium > butacaine > procaine. Na⁺ and K⁺ do not coagulate phospholipid dispersions at 0.1 M concentration and antagonize the effect of Ca²⁺. (d) LA produce a marked fall in the pH of cephalin sols equivalent to that produced by calcium, (e) Ca²⁺ and LA form 1:2 molar complexes with phospholipids probably by ion-ion and ion-induced polar type of binding at the phosphate groups of the lipid. It is suggested that such reactions with cell membrane phospholipids may underlie inhibitory effects of LA on cellular ion fluxes and provide a chemical basis for anesthetic action.     

Intracellular compartmentation of Na+, K+ and Cl− in the Ehrlich ascites tumor cell: Correlation with the membrane potential

Summary The intracellular distribution of Na⁺, K⁺, Cl^– and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La³⁺ and mechanical stress indicated that La³⁺ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na⁺ is sequestered within the nucleus, while K⁺ and Cl^– are more highly concentrated in the cell cytoplasm. Reduction of the [Na⁺] of the incubation medium by replacement with K⁺ results in reduced cytoplasmic [Na⁺], increased [Cl^–] and no change in [K⁺]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was –13.7 mV. Reduction of [Na⁺] in the medium caused significant depolarization. The measured potential is describable by the Cl^– equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed.     

Interacting effects of pH,aluminium and base cations on growth and mineral composition of the woodland grasses Bromus benekenii and Hordelymus europaeus

The influence of base cation concentrations on pH and aluminium sensitivity of the woodland grasses Bromus benekenii and Hordelymus europaeus was studied in flowing solution culture experiments. Plants were exposed to low pH (3.9, experiment 1) and Al concentrations of 19 and 37 M (experiment 2) at two base cation (Ca+Mg+K) levels, all within the ranges measured in natural forest soil solutions. Elevated base cation concentrations ameliorated both H and Al toxicity, as indicated by increased root and shoot growth. In the third experiment, interactions between pH (4.3 and 4.0) and Al (0 and 19 M) were investigated. It was shown that the combined toxicity effects of H and Al were not greater than the separate H or Al effects. Tissue concentrations of base cations and Al increased with increasing concentrations in the solution, but were also influenced by the base cation : Al ratio. Relating the experimental evidence with the composition of forest soil solutions suggests an important role of soil pH and Al in controlling the distribution of the two species. Growth conditions also differ at various soil depths. Concentrations of free cationic Al were higher and base cation concentrations lower at 5–10 cm than at 0–5 cm soil depth. Increasing base cation concentrations may protect roots from both H and Al injury during periods of drought when concentrations of most elements increase in the soil solution, whereas molar ratios between base cations, H and Al remain unchanged.     

Effect of monoidoacetic acid on electrophysiological properties of snail giant neurons

Monoiodoacetic acid (MIA) causes depolarization and a decrease in the amplitude of the action potential and resistance of the membrane, and also leads to significant changes in the potassium and sodium concentrations in the neurons of the subesophageal ganglia. All these changes are more marked with a decrease in pH of the Ringer's solution containing the inhibitor. During the action of acid Ringer's solution without an inhibitor the electrophysiological changes in the neurons develop more slowly and to a lesser degree and they are easily reversible. It is concluded that the electrophysiological changes induced in neurons by MIA are due not only to inhibition of active ion transport but also to changes in the ionic permeability of the membrane and, in particular, to an increase in sodium permeability.A. A. Bogomolets' Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 4, No. 1, pp. 97–104, January–February 1972.     

Effects of phenothiazines on inhibition of plasma membrane ATPase and hyperpolarization of cell membranes in the yeast Saccharomyces cerevisiae

The transmembranal potential, in Saccharomyces cerevisiae, has been calculated from the distribution of the lipophilic cation tetraphenylphosphonium (TPP⁺) between the intracellular and extracellular water. Trifluoperazine at concentrations of 10 to 50 μM, caused a substantial increase in the membrane potential (negative inside). This increase was observed only in the presence of a metabolic substrate and was eliminated by the addition of the protonophores 2,4-dinitrophenol and sodium azide, removal of glucose, replacement of glucose by the nonmetabolizable analog 3-O-methyl glucose, or by the addition of 100mM KCl. An increase in ⁴⁵CaCl₂ accumulation from solutions of low concentrations (1 μM) was observed under all conditions where membrane potential was increased. Proton ejection activity was monitored by measurements of the rates of the decrease in the pH of unbuffered cell suspensions in the presence of glucose. Trifluoperazine inhibited the changes in medium pH; this inhibition was not the result of an increase in the permeability of cell membranes to protons since in the absence of glucose, trifluoperazine did not cause a change in the rate of pH change generated by proton influx. The activity of plasma membrane ATPase was measured in crude membrane preparations in the presence of sodium azide to inhibit mitochondrial ATPase. Trifluoperazine strongly inhibited the activity of the plasma membrane ATPase. The effect of phenothiazines on transport and on membrane potential reported in this study and in the previous one (Eilam, Y. (1983) Biochim. Biophys. Acta 733, 242–248) were observed only in the presence of a metabolic substrate. The possibility that energy is required for the uptake of phenothiazines into the cells was eliminated by results showing energy-independent uptake of [³H]chlorpromazine. The results strongly suggest that phenothiazines activate energy-dependent K⁺-extrusion pumps, which lead to increased membrane potential. Increased influx of calcium seems to be energized by membrane potential, and therefore stimulated under all conditions where membrane potential is increased. The analog which does not bind to calmodulin, trifluoperazine sulfoxide, had no effect on the cells, but the involvement of calmodulin in the processes altered by trifluoperazine cannot as yet, be determined.     

Alteration of the action potential of tissue cultured neuronal cells by growth in the presence of a polyunsaturated fatty acid

The effects of alterations in membrane phospholipid fatty acid composition on the excitability of neuroblastoma × glioma hybrid cells, clone NG108-15, were examined using intracellular recording techniques. Cells were grown in the presence of arachidonate (20:4) added to the culture medium as a complex with bovine serum albumin. Exposure of the cells to 20:4 for 3–21 days produced a 40% decrease in the maximum rate of rise of the action potential (dV/dt) with a small change in its amplitude. The resting membrane potential and passive properties of the cells were unaffected. An effect of 20:4 was not observed until 24 hr after treatment and increased over the next 2 days. The phospholipid content of 20:4 and its metabolite 22:4 increased from 6.9% to 25.3% of total fatty acids during approximately the same time span. It is concluded that the action potential dV/dt can be altered by changes in membrane lipid composition.     

Effects of pH,potential, chloride and furosemide on passive Na+ and K+ effluxes from human red blood cells

Summary Ouabain-resistant effluxes from pretreated cells containing K⁺/Na⁺=1.5 into K⁺ and Na⁺ free media were measured.Furosemide-sensitive cation effluxes from cells with nearly normal membrane potential and pH were lower in NO ₃ ^– media than in Cl^– media; they were reduced when pH was lowered in Cl^– media. When the membrane potential was positive inside furosemide increased the effluxes of Na⁺ and K⁺ (7 experiments). With inside-positive membrane potential thefurosemideinsensitive effluxes were markedly increased, they decreased with decreasing pH at constant internal Cl^– and also when internal Cl^– was reduced at constant pH. The correlation between cation flux and the membrane potential was different for cells with high or low internal chloride concentrations. The data with chloride47mm showed a better fit with the single-barrier model than with the infinite number-of-barriers model. With low chloride no significant correlation between flux and membrane potential was found. The data are not compatible with pure independent diffusion of Na⁺ and K⁺ in the presence of ouabain and furosemide.