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.     

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.     

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.     

Increased chloride conductance as the proximate cause of hydrogen ion concentration effects in Aplysia neurons

A fall in extracellular pH increased membrane conductance of the giant cell in the abdominal ganglion of Aplysia californica. Chloride conductance was trebled whereas potassium conductance was increased by 50%. Half the giant cells were hyperpolarized (2–8 mv) and half were depolarized (3–10 mv) by lowering the pH. The hyperpolarizing response always became a depolarizing response in half-chloride solutions. When internal chloride was increased electrophoretically, the hyperpolarization was either decreased or changed to depolarization. The depolarizing response was reduced or became a hyperpolarizing response after soaking the cell in 10.0 mM chloride, artificial seawater solution for 1 hr. Depolarization was unaffected when either external sodium, calcium, or magnesium was omitted. A glass micropipette having an organic liquid chloride ion exchanger in its tip was used to measure intracellular chloride activity in 14 giant cells; 7 had values of 27.7 ± 1.8 mM (SEM) and 7 others 40.7 ± 1.5 mM. Three of the first group were hyperpolarized when pH was lowered and three of the second group were depolarized. In all six cells, these changes of membrane potential were in the direction of the chloride equilibrium potential. Intracellular potassium activity was measured by means of a potassium ion exchanger microelectrode.     

Ionic interconversion of pacemaker and nonpacemaker cultured chick heart cells

Trypsin-dispersed cells from hearts (ventricles) of 7 to 8 day chick embryos were cultured 3 to 21 days. The cells became attached to the culture dish and assembled into monolayer communities. By means of a bridge circuit, one microelectrode was used for simultaneously passing current and recording membrane potentials (V_m). The input resistance, calculated by the measured ΔV_m for a known step of current, averaged 10 MΩ. Electrotonic depolarization of nonpacemaker cells had no effect on frequency of firing. Within 2 min after addition of Ba⁺⁺ (5 to 10 mM) to the Tyrode bath, the cells became partially depolarized and quiescent nonpacemaker cells developed oscillations in V_m which led to action potentials. With time, the depolarization became nearly complete and the input resistance increased 2 to 10 times. During such sustained depolarizations, action potentials were no longer produced and often tiny oscillations were observed; however, large action potentials developed during hyperpolarizing pulses. Thus, the automaticity of the depolarized cell became apparent during artificial repolarization. Sr⁺⁺ (5 to 10 mM) initially produced hyperpolarization and induced automaticity in quiescent nonpacemaker cells. Elevated [K⁺]_o (20 to 30 mM) suppressed automaticity of pacemaker cells and decreased R_m concomitantly. Thus, Ba⁺⁺ probably converts nonpacemaker cells into pacemaker cells independently of its depolarizing action. Ba⁺⁺ may induce automaticity and depolarization by decreasing g _K, and elevated [K⁺]_o may depress automaticity by increasing g _K. The data support the hypothesis that the level of g _K determines whether a cell shall function as a pacemaker.     

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 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.     

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 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.     

Calcium exchange and contraction strength of guinea pig atrium in normal and hypertonic media

A Krebs-Henseleit (KH) medium made hypertonic by adding nonpermeant molecules substantially increased the isometric peak tension at steady-state contractions below 3 per sec in guinea pig atrium at 27°C. Action potential durations were decreased. KH plus 100 mM raffinose or sucrose resulted in similar and nearly maximal changes which were essentially reversible upon return to normal KH. When one active contracting atrium was used to passively stretch a second atrium, the difference in Ca ion exchange (1 min exchange with the extracellular space) between active and stretched atria significantly increased at 1 per sec and at 2 per sec in going from normal to 100 mM hypertonic KH. The calculated mean Ca ion cellular exchange per beat per 100 g of cells (a) doubled in changing from normal to 100 mM hypertonic KH, and (b) decreased slightly in changing from contractions of 1 per sec to 2 per sec in normal KH. These data are consistent with the hypothesis (a) that Ca ion entry per beat from the extracellular space is proportional to membrane depolarized time with a constant medium and a steady-state condition, and the hypothesis (b) that 100 mM hypertonicity doubles the Ca ion entry rate during depolarization. These data enable rejection of the hypothesis that the peak tension is proportional to the Ca ion entry per beat from the extracellular space under steady-state conditions, and suggest that any additional Ca ion involved in the larger contractions at higher frequencies comes from an increase in Ca ion available from intracellular stores.     

Effects of Sodium Azide on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes from frog''s striated muscle were measured in the presence of 0 to 5 mM sodium azide. With azide concentrations of 2 and 5 mM the Na efflux was markedly stimulated; the Na efflux with 5 mM azide was about 300 per cent greater than normal. A similar increase was present when all but the 5.0 mM sodium added with azide was replaced by choline. 10^-5 M strophanthidin abolished the azide effect on Na²⁴ efflux. Concentrations of azide of 1.0 mM or less had no effect on Na efflux. The Na influx, on the other hand, was only increased by 41 per cent in the presence of 5 mM NaN₃. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of azide. The hypothesis is advanced that the active transport of Na is controlled by the transmembrane potential and that the stimulation of Na efflux is produced as a consequence of the membrane depolarization caused by the azide.     

Sodium Movements in Perfused Squid Giant Axons : Passive fluxes

Sodium movements in internally perfused giant axons from the squid Dosidicus gigas were studied with varying internal sodium concentrations and with fluoride as the internal anion. It was found that as the internal concentration of sodium was increased from 2 to 200 mM the resting sodium efflux increased from 0.09 to 34.0 pmoles/cm²sec and the average resting sodium influx increased from 42.9 to 64.5 pmoles/cm²sec but this last change was not statistically significant. When perfusing with a mixture of 500 mM K glutamate and 100 mM Na glutamate the resting efflux was 10 ± 3 pmoles/cm²sec and 41 ± 10 pmoles/cm²sec for sodium influx. Increasing the internal sodium concentration also increased both the extra influx and the extra efflux of sodium due to impulse propagation. At any given internal sodium concentration the net extra influx was about 5 pmoles/cm²impulse. This finding supports the notion that the inward current generated in a propagated action potential can be completely accounted for by movements of sodium.     

The ionic basis of electrical activity in embryonic cardiac muscle

The intracellular sodium concentration reported for young, embryonic chick hearts is extremely high and decreases progressively throughout the embryonic period, reaching a value of 43 mM immediately before hatching. This observation suggested that the ionic basis for excitation in embryonic chick heart may differ from that responsible for electrical activity of the adult organ. This hypothesis was tested by recording transmembrane resting and action potentials on hearts isolated from 6-day and 19-day chick embryos and varying the extracellular sodium and potassium concentrations. The results show that for both young and old embryonic cardiac cells the resting potential depends primarily on the extracellular potassium concentration and the amplitude and rate of rise of the action potential depend primarily on the extracellular sodium concentration.     

Further studies of the effect of calcium on the time course of action of carbamylcholine at the neuromuscular junction

The rate at which the postjunctional membrane of muscle fibers becomes desensitized to the action of carbamylcholine is increased after the muscle has been soaked in solutions containing increased concentrations of calcium. Some further aspects of this effect of calcium were investigated by measuring changes in the input resistance of single fibers of the frog sartorius during local perfusion of the neuromuscular junction with 2.73 x 10^-3 M carbamylcholine in isolated muscles immersed in 165 mM potassium acetate. It was found that (a) sudden changes in the local concentration of calcium brought about by perfusing fibers with carbamylcholine solutions containing 20 mM calcium, 40 mM oxalate, or 40 mM EDTA were followed within 20 sec by marked changes in the rate of desensitization; (b) prior to 13 sec after the introduction of carbamylcholine, however, no effect on the input resistance could be detected even though the muscle had been presoaked in 10 mM calcium; (c) the ability of high concentrations of calcium to bring about rapid desensitization disappears when a lower concentration of carbamylcholine (0.137 x 10^-3 M) is applied to the muscle fiber. These findings suggest that calcium present in the extracellular fluid can act directly on the postjunctional membrane to promote the desensitization process and that an increased permeability of the membrane to calcium brought about by the presence of carbamylcholine is a factor which contributes to this action.     

Net uptake of potassium in Neurospora. Exchange for sodium and hydrogen ions

Net uptake of potassium by low K, high Na cells of Neurospora at pH 5.8 is accompanied by net extrusion of sodium and hydrogen ions. The amount of potassium taken up by the cells is matched by the sum of sodium and hydrogen ions lost, under a variety of conditions: prolonged preincubation, partial respiratory inhibition (DNP), and lowered [K]_o. All three fluxes are exponential with time and obey Michaelis kinetics as functions of [K]_o. The V _max for net potassium uptake, 22.7 mmoles/kg cell water/min, is very close to that for K/K exchange reported previously (20 mmoles/kg cell water/min). However, the apparent K_m for net potassium uptake, 11.8 mM [K]_o, is an order of magnitude larger than the value (1 mM) for K/K exchange. It is suggested that a single transport system handles both net K uptake and K/K exchange, but that the affinity of the external site for potassium is influenced by the species of ion being extruded.     

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.     

Potassium and sodium transport across single distal tubules of Amphiuma

The transport properties of potassium (K) and sodium (Na) were studied in single distal tubules of Amphiuma using free-flow micropuncture techniques and stationary microperfusion methods. The transepithelial movement of labeled potassium was measured utilizing a three-compartment system in series in which the time course of tracer disappearance from the lumen was followed. Under control conditions, in blood- and doubly-perfused kidneys, extensive active net reabsorption of sodium and potassium obtains along single distal tubules. Tubular potassium reabsorption is abolished by ouabain at a concentration of 5 x 10^-6 M. Significant net secretion of K can be induced by exposing Amphiuma to a high K environment (100 mM KCl) or by adding acetazoleamide (1 x 10^-4 M) to the perfusion fluid. Transepithelial movement of potassium involves mixing with only a small fraction of total distal tubular cell potassium. This transport pool of potassium increases significantly with the transition from tubular net reabsorption to net secretion. Indirect evidence is presented which indicates that increased active K uptake across the peritubular cell boundary may be of prime importance during states of net K secretion.     

Relative Ion Permeabilities in the Crayfish Giant Axon Determined from Rapid External Ion Changes

The changes in membrane potential of isolated, single crayfish giant axons following rapid shifts in external ion concentrations have been studied. At normal resting potential the immediate change in membrane potential after a variation in external potassium concentration is quite marked compared to the effect of an equivalent chloride change. If the membrane is depolarized by a maintained potassium elevation, the immediate potential change due to a chloride variation becomes comparable to that of an equivalent potassium change. There is no appreciable effect on membrane potential when external sodium is varied, at normal or at a depolarized membrane potential. Starting from the constant field equation, expressions for the permeability ratios P _Cl/P _K, P _Na/P _K, and for intracellular potassium and chloride concentrations are derived. At normal resting membrane potential, P _Cl/P _K is 0.13 but at a membrane potential of -53 mv (external potassium level increased about five times) it is 0.85. The intracellular concentrations of potassium and chloride are estimated to be 233 and 34 mM, respectively, and it is pointed out that this is not compatible with ions distributed in a Nernst equilibrium across the membrane. It is also stressed that the information given by a plot of membrane potential vs. the logarithm of external potassium concentrations is very limited and rests upon several important assumptions.     

Effects of batrachotoxin on membrane potential and conductance of squid giant axons

The effects of batrachotoxin (BTX) on the membrane potential and conductances of squid giant axons have been studied by means of intracellular microelectrode recording, internal perfusion, and voltage clamp techniques. BTX (550–1100 nM) caused a marked and irreversible depolarization of the nerve membrane, the membrane potential being eventually reversed in polarity by as much as 15 mv. The depolarization progressed more rapidly with internal application than with external application of BTX to the axon. External application of tetrodotoxin (1000 nM) completely restored the BTX depolarization. Removal or drastic reduction of external sodium caused a hyperpolarization of the BTX-poisoned membrane. However, no change in the resting membrane potential occurred when BTX was applied in the absence of sodium ions in both external and internal phases. These observations demonstrate that BTX specifically increases the resting sodium permeability of the squid axon membrane. Despite such an increase in resting sodium permeability, the BTX-poisoned membrane was still capable of undergoing a large sodium permeability increase of normal magnitude upon depolarizing stimulation provided that the membrane potential was brought back to the original or higher level. The possibility that a single sodium channel is operative for both the resting sodium, permeability and the sodium permeability increase upon stimulation is discussed.     

MAINTENANCE OF IMAGINAL DISCS OF DROSOPHILA MELANOGASTER IN CHEMICALLY DEFINED MEDIA

A phosphate-buffered saline and a chemically defined synthetic medium for in vitro maintenance of imaginal discs of Drosophila melanogaster were developed. The composition of the chemically defined medium was varied in order to optimize the incorporation of tritiated uridine into RNA and tritiated amino acids into acid-insoluble protein. The optimal ranges obtained were: pH, 6.75–7.35; osmolarity, 285–345 milliosmoles/liter; sodium concentration, 40–60 mM/liter; potassium concentration, 40–60 mM/liter; magnesium concentration, 0.5–3.5 mM/liter; calcium concentration, 0.3–1.5 mM/liter; and inorganic phosphate concentration, 1.5–4.0 mM/liter. The phosphate-buffered saline is superior to a commonly used insect Ringer solution in maintaining total RNA and acid-insoluble protein synthesis in culture. The chemically defined synthetic medium permits linear total RNA and acid-insoluble protein synthesis for more than 48 hr, DNA synthesis for several hours, normal differentiation to occur after 74 hr in vitro, and trypsinization of imaginal discs into single cell suspensions without developmental damage.