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.     

An active electrical response in fibroblasts

L cells have a resting potential of about -16 mv (internal negative) at 37°C in Dulbecco''s modified Eagle''s medium containing 10% fetal calf serum and a potassium concentration of 5.4 mM. Membrane resistivity is about 20,000 Ωcm² when the surface filopodia described by others are taken into account. Mechanical and electrical stimuli can evoke an active response from mouse L cells, cells of the 3T3 line, and normal fibroblasts which we have termed hyperpolarizing activation or the H.A. response. This consists of a prolonged (3–5 sec) increase in the membrane permeability by a factor of 2–10 with a parallel increase in membrane potential to about -50 mv. The reversal potential for the H.A. response is -80 mv. The resting cells are depolarized to about -12 mv when the external medium contains 27 mM potassium, and the potential reached at the peak of the H.A. response is about -30 mv. The reversal potential for the H.A. response is about -40 mv in 27 mM external potassium. This effect of potassium ions on the reversal potential of the H.A. response leads us to conclude that the response represents an increase in membrane permeability, predominantly to potassium, by at least a factor of five. This increase must be greater than 20-fold if previous measurements of the ratio of potassium permeability to chloride permeability in L cells are valid for the preparation used in the present study.     

Electrical properties and excitation-contraction coupling in skeletal muscle treated with ethylene glycol

The contractility of the frog sartorius muscle was suppressed after treatment with a Ringer solution added with ethylene glycol (EGR). No contraction was elicited by nerve stimulation when the muscle was brought back to normal Ringer solution after having been soaked in 876 mM EGR for 4 hr or in 1095 mM EGR for 2 hr. However, the action potential of normal amplitude was generated and followed by a depolarizing afterpotential. The resting membrane potential was slightly decreased from the mean normal value of –91.1 mv to –78.8 mv when 1095 mM EGR was used, and to –82.3 mv when 876 mM EGR was used, but remained almost constant for as long as 2 hr. The afterpotential that follows a train of impulses and a slow change in membrane potential produced by a step hyperpolarizing current (so-called "creep") were suppressed after treatment with ethylene glycol. The specific membrane capacity decreased to about 50% of the control values while the specific membrane resistance increased to about twice the control values Therefore, the membrane time constant remained essentially unchanged. The water content of the muscle decreased by about 30% during a 2 hr immersion in 1095 mM EGR, and increased by about 30% beyond the original control level after bringing the muscle back to normal Ringer. The intracellular potassium content did not change significantly during these procedures. Some differences between the present results and those obtained with glycerol are discussed.     

Biphasic potassium contractures in frog muscle fibers

Potassium-induced contractures were studied in single fibers from the semitendinosus muscle of Rana pipiens. Contractures elicited by solutions containing 60–117 mM potassium and 120 mM chloride were biphasic, consisting of a rapid initial contraction with a duration at 23°C of less than 1 sec followed by a slow response with a duration of many seconds. At 13°C, the initial response was greatly prolonged so that the two responses virtually fused into a single smooth contracture. Membrane potential in high potassium, high chloride solutions underwent a transient peak depolarization, probably as a result of time-dependent changes in membrane conductance during depolarization. It is proposed that this complex time course of depolarization gives rise to the biphasic contracture response.     

The effect of calcium on the desensitization of membrane receptors at the neuromuscular junction

Desensitization, as represented by the progressive decline in the electromotive effects of depolarizing agents at the neuromuscular junction, was studied by observing the time course of changes in effective transmembrane resistance during the prolonged application of 0.27 mM carbamylcholine to the postjunctional region of frog skeletal muscle fibers. The effective transmembrane resistance was measured by means of two intracellular microelectrodes implanted in the junctional region of single muscle fibers. When carbamylcholine was applied to the muscle there was an immediate decrease in the effective membrane resistance followed by a slower return toward control values which was identified as the phase of desensitization. When the calcium concentration was increased from 0 to 10 mM there was an approximately sevenfold increase in the rate of desensitization. On the other hand, an increase in the concentration of sodium from 28 to 120 mM caused a slowing of the rate of desensitization. Even in muscles depolarized by potassium sulfate, calcium increased the rate of desensitization while high concentrations of potassium tended to prolong the process. Some mechanisms by which calcium might exert these effects are discussed.     

Electrical Properties of Neurospora crassa : Effects of external cations on the intracellular potential

Glass micropipette electrodes have been employed to study the transsurface potential difference of Neurospora crassa. For mature hyphae grown in agar cultures, the internal potential is large and negative, often exceeding -200 mv. The potential is sensitive to the concentrations of extracellular potassium, sodium, hydrogen, and calcium ions, but does not vary in a manner which is readily explained by ionic diffusion potentials. With extracellular solutions containing only potassium chloride (or sulfate) and sucrose, the internal potential shifts toward zero (becomes less negative) at 45 mv per tenfold increase of potassium, over the range 0.1 to 10 mM. A similar result has been found with sodium, though the slope is only 33 mv/log unit. Calcium (1 mM) diminishes the influence of potassium and sodium by 60 to 70 per cent. As potassium or sodium is raised above 20 mM, the slope of the internal potential increases sharply to 85 to 90 mv/log unit, both in the presence and absence of calcium. With increasing hydrogen ion concentration, too, the internal potential shifts toward zero; in this case the slope is about 12 mv/pH unit at pH 9 and rises smoothly to 33 mv/pH unit at pH 3. All these phenomena are probably properties of the plasma membrane. The polysaccharide cell wall contains few fixed negative charges, has a low transverse resistance, and supports very little potential difference when separated from the plasma membrane.     

Characteristics of electrogenic sodium pumping in rat myometrium

Sodium-rich myometrium, obtained from the uteri of pregnant rats, rapidly hyperpolarized when 4.6–120 mM potassium was added to the bathing medium at 37°C. Hyperpolarization was due to sodium pumping since the process was markedly temperature dependent, was abolished by ouabain, and required both intracellular sodium and extracellular potassium. The observed membrane potential exceeded the calculated potassium equilibrium potential during hyperpolarization providing evidence that sodium pumping was electrogenic. Hyperpolarization was reduced in the presence of chloride. The rate of sodium pumping may influence potassium permeability since potassium apparently did not short-circuit the pump during hyperpolarization.     

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.     

Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

Electrical properties of Neurospora crassa. Respiration and the intracellular potential

The internal potential of Neurospora appears to have two components, one (a) which is reduced by anoxia or abolished by respiratory inhibitors such as azide and 2,4-dinitrophenol, and (b) a fraction that remains in the presence of respiratory inhibitors and is sensitive to the external potassium concentration. Under standard conditions 1 mM azide or dinitrophenol diminishes internal potentials from near -200 mv to about -30 mv within 1 minute and at a maximal rate of 20 mv/second. The internal potential usually recovers within 10 minutes after the inhibitor has been removed. The effect of carbon monoxide on the internal potential is similar to that of azide or dinitrophenol, but can be reversed by visible light, specifically of the wavelengths (430 mµ and 590 mµ) known to decompose cytochrome-CO complexes in yeast. Respiration and internal potentials vary proportionally with azide concentration, but dinitrophenol at low (3 x 10^-6 M) concentrations enhances oxygen consumption without affecting the internal potential. In the presence of 0.1 mM calcium, the fraction of the internal potential which persists during respiratory inhibition increases (becomes more negative) about 30 mv for each tenfold decrease of external potassium over the range 10 to 0.1 mM. The surface resistivity of Neurospora, normally about 5000 ohm.cm², is unchanged by respiratory inhibitors during the period of rapid potential shift.     

Effects of External Potassium and Strophanthidin on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes in isolated fibers from the frog''s semitendinosus muscle were measured in the presence of strophanthidin and increased external potassium ion concentrations. Strophanthidin at a concentration of 10^-5 M inhibited about 80 per cent of the resting Na efflux without having any detectable effect on the resting Na influx. From this it is concluded that the major portion of the resting Na efflux is caused by active transport processes. External potassium concentrations from 2.5 to 7.5 mM had little effect on resting Na efflux. Above 7.5 mM and up to 15 mM external K, the Na efflux was markedly stimulated; with 15 mM K the Na influx was 250 to 300 per cent greater than normal. On the other hand, Na influx was unchanged with 15 mM K. The stimulated Na efflux with the higher concentrations was not appreciably reduced when choline or Li was substituted for external Na, but was completely inhibited by 10^-5 M strophanthidin. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of K. It is postulated that this effect on the Na "pump" is produced as a result of the depolarization of the muscle membranes and is related to the increased metabolism and heat production found under conditions of high external K.     

Some factors influencing sodium extrusion by internally dialyzed squid axons

Squid giant axons were internally dialyzed by a technique previously described. In an axon exposed to cyanide seawater for 1 hr and dialyzed with an ATP-free medium, the Na efflux had a mean value of 1.3 pmole/cm²sec when [Na]_i was 88 mM, in quantitative agreement with flux ratio calculations for a purely passive Na movement. When ATP at a concentration of 5–10 mM was supplied to the axoplasm by dialysis, Na efflux rose almost 30-fold, while if phosphoarginine, 10 mM, was supplied instead of ATP, the Na efflux rose only about 15-fold. The substitution of Li for Na in the seawater outside did not affect the Na efflux from an axon supplied with ATP, while a change to K-free Na seawater reduced the Na efflux to about one-half. When special means were used to free an axon of virtually all ADP, the response of the Na efflux to dialysis with phosphoarginine (PA) at 10 mM was very small (an increment of ca. 3 pmole/cm²sec) and it can be concluded that more than 96% of the Na efflux from an axon is fueled by ATP rather than PA. Measurements of [ATP] in the fluid flowing out of the dialysis tube when the [ATP] supplied was 5 mM made it possible to have a continuous measurement of ATP consumption by the axon. This averaged 43 pmole/cm²sec. The ATP content of axons was also measured and averaged 4.4 mM. Estimates were made of the activities of the following enzymes in axoplasm: ATPase, adenylate kinase, and arginine phosphokinase. Values are scaled to 13°C.     

The electrical activity of embryonic chick heart cells isolated in tissue culture singly or in interconnected cell sheets

Embryonic chick heart cells were cultured on a plastic surface in sparse sheets of 2–50 cells mutually in contact, or isolated as single cells. Conditions are described which permitted conjoint cells to be impaled with recording microelectrodes with 75 % success, and isolated single cells with 8 % success. It is proposed that cells in electrical contact with neighbors are protected from irreversible damage by the penetrating electrode, by a flow of ions or other substances from connected cells across low-impedance intercellular junctions. Action potentials recorded from conjoint and isolated single cells were similar in form and amplitude. The height or shape of the action potential thus appears not to depend upon spatial relationships of one cell to another. As the external potassium concentration was increased from 1.3 mM to 6 mM, cells became hyperpolarized while the afterhyperpolarization was reduced. At higher potassium levels, the afterhyperpolarization disappeared, the slope of the slow diastolic depolarization decreased, and resting potential fell along a linear curve with a slope of 61 mv per 10-fold increase in potassium. In pacemaker cells the diastolic depolarization consists of two phases: (a) recovery from the afterpotential of the previous action potential and (b) the pacemaker potential. These phases are separated by a point of inflection, and represent manifestations of different mechanisms. Evidence is presented that it is the point of inflection (PBA) rather than the point of maximal diastolic potential, that should be taken as the resting potential.     

Effects of caffeine on crayfish muscle fibers. I. Activation of contraction and induction of Ca spike electrogenesis

Contractions are evoked in single muscle fibers of crayfish by intracellular as well as extracellular applications of caffeine. Responses to external applications in concentrations above 2 mM could be induced indefinitely. With concentrations above 5 mM the caffeine-induced responses were highly repeatable. Tensions were transient even when the caffeine remained in the bath. There was no change in resting potential, but during the contraction the effective resistance decreased about 10%. A number of factors (change in pH, Ca, K, and Cl) modified the responses. The time course of the tension was greatly prolonged when the transverse tubular system (TTS) was s swollen and was again shortened when the TTS was caused to shrink. An increased permeability to Ca induced by caffeine was evidenced by the transformation of the normally graded electrical responses to Ca spikes, which are insensitive to tetrodotoxin. The overshoot is a function of both external Ca and caffeine. A 10-fold change in Ca changed the overshoot by 19 mv in the presence of 10 mM caffeine and by 29 mv in 80 mM caffeine. The role of the increased permeability to Ca for caffeine-induced contractions will be analyzed in the accompanying paper.     

Choline permeability in cardiac muscle cells of the cat

Permeability of the cardiac cell membrane to choline ions was estimated by measuring radioactive choline influx and efflux in cat ventricular muscle. Maximum values for choline influx in 3.5 and 137 mM choline were respectively 0.56 and 9 pmoles/cm²·sec. In 3.5 mM choline the intracellular choline concentration was raised more than five times above the extracellular concentration after 2 hr of incubation. In 137 mM choline, choline influx corresponded to the combined loss of intracellular Na and K ions. Paper chromatography of muscle extracts indicated that choline was not metabolized to any important degree. The accumulation of intracellular choline rules out the existence of an efficient active pumping mechanism. By measuring simultaneously choline and sucrose exchange, choline efflux was analyzed in an extracellular phase, followed by two intracellular phases: a rapid and a slow one. Efflux corresponding to the rapid phase was estimated at 16–45 pmoles/cm²·sec in 137 mM choline and at 1.3–3.5 pmoles/cm²·sec in 3.5 mM choline; efflux in 3.5 mM choline was proportional to the intracellular choline concentration. The absolute figures for unidirectional efflux were much larger than the net influx values. The data are compared to Na and Li exchange in heart cells. Possible mechanisms for explaining the choline behavior in heart muscle are discussed.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

Potassium fluxes in dialyzed squid axons

Measurements have been made of K influx in squid giant axons under internal solute control by dialysis. With [ATP]_i = 1 µM, [Na]_i = 0, K influx was 6 ± 0.6 pmole/cm² sec; an increase to [ATP]_i = 4 mM gave an influx of 8 ± 0.5 pmole/cm² sec, while [ATP]_i 4, [Na]_i 80 gave a K influx of 19 ± 0.7 pmole/cm² sec (all measurements at ∼16°C). Strophanthidin (10 µM) in seawater quantitatively abolished the ATP-dependent increase in K influx. The concentration dependence of ATP-dependent K influx on [ATP]_i, [Na]_i, and [K]_o was measured; an [ATP]_i of 30 µM gave a K influx about half that at physiological concentrations (2–3 mM). About 7 mM [Na]_i yielded half the K influx found at 80 mM [Na]_i. The ATP-dependent K influx responded linearly to [K]_o from 1–20 mM and was independent of whether Na, Li, or choline was the principal cation of seawater. Substances tested as possible energy sources for the K pump were acetyl phosphate, phosphoarginine, PEP, and d-ATP. None was effective except d-ATP and this substance gave 70% of the maximal flux only when phosphoarginine or PEP was also present.     

The ionic requirements for the initiation of action potentials in insect muscle fibers

Electrical properties of locust leg muscle fibers were studied by means of intracellular electrodes. In most fibers, a depolarizing current pulse initiated a local response. A delayed decrease in membrane resistance appeared with more than about 10 mv depolarization. In some fibers a regenerative response also was found. Membrane constants were measured, applying the short cable model. The value of the space constant λ was 1.6 mm and the calculated value of R_m was about 1750 ohm cm². Action potentials could be elicited when the bathing fluid contained more than 2–5 mM Ba or Sr. Similar responses were seen with 2 mM Ca in the presence of tetraethylammonium (TEA). The overshoot of these action potentials increased with increasing [Ca⁺⁺]_o, [Sr⁺⁺]_o, or [Ba⁺⁺]_o, the increment for a 10-fold increase being about 29 mv for Ca and Sr and between 40 and 50 mv for Ba. These action potentials were inhibited by Mn ions but were not affected by tetrodotoxin or procaine. In solutions containing Ba or Sr, action potentials generated were suppressed by addition of Ca. The removal of Na ions did not change the configuration of the action potential. The results suggest that an increase in permeability to Ca, Ba, or Sr ions makes a major contribution to the initiation of action potentials in this tissue.     

Chloride fluxes in isolated dialyzed barnacle muscle fibers

Chloride outflux and influx has been studied in single isolated muscle fibers from the giant barnacle under constant internal composition by means of a dialysis perfusion technique. Membrane potential was continually recorded. The chloride outfluxes and influxes were 143 and 144 pmoles/cm²-sec (mean resting potential: 58 mv, temperature: 22°–24°C) with internal and external chloride concentrations of 30 and 541 mM, respectively. The chloride conductance calculated from tracer measurements using constant field assumptions is about fourfold greater than that calculated from published electrical data. Replacing 97% of the external chloride ions by propionate reduces the chloride efflux by 51%. Nitrate ions applied either to the internal or external surface of the membrane slows the chloride efflux. The external pH dependence of the chloride efflux follows the external pH dependence of the membrane conductance, in the range pH 3.9–4.7, increasing with decreasing pH. In the range pH 5–9, the chloride efflux increased with increasing pH, in a manner similar to that observed in frog muscle fibers. The titration curve for internal pH changes in the range 4.0–7.0 was quantitatively much different from that for external pH change, indicating significant asymmetry in the internal and external pH dependence of the chloride efflux.     

The interaction between caffeine and calcium in the desensitization of muscle postjunctional membrane receptors

The interaction between caffeine and calcium on the rate of desensitization of muscle postjunctional membrane (PJM) receptors during the sustained application of 0.27 mM carbamylcholine (CARB) has been studied in vitro on the sartorius muscle of the frog. The rate of PJM repolarization with CARB added to the solution bathing the muscle or the recovery of the effective transmembrane resistance (EMR) during the microperfusion of CARB directly onto the end-plate region of individual fibers was used as an index of the rate of desensitization. Caffeine (1.5 mM) increased the rate of PJM repolarization with bulk application of CARB in a 1.8 or 10 mM calcium Ringer solution but had no effect on PJM repolarization in a calcium-deficient, 4 mM magnesium Ringer solution. For EMR measurements the preparation was rendered mechanically quiescent by repeated challenges with isotonic KCl during an exposure of several hours to a calcium-free, 4 mM magnesium-1 mM EGTA Ringer solution. In these fibers, the microperfusion of 0.27 mM CARB together with 1.8 mM calcium plus 1.5 mM caffeine significantly increased the rate of EMR recovery above that observed in the absence of caffeine. Raising the calcium concentration to 10 mM had a similar effect; however, no additional increase was noted by the inclusion of 1.5 mM caffeine. It is suggested that the major role of caffeine in PJM desensitization is to increase the calcium permeability of the surface membrane. The transmembrane movement of calcium and the consequent intracellular accumulation of calcium is seen as a critical factor in controlling the rate of PJM desensitization.