Voltage-clamp experiments on frog single skeletal muscle fibres: evidence for a tubular sodium current.

1. A late inward current following the initial sodium current is sometimes observed on frog single skeletal muscle fibres studied in voltage-clamp conditions by the double sucrose-gap method. 2. The late inward current is time and voltage dependent; it is sensitive to extracellular sodium concentration and inhibited by tetrodotoxin. Therefore, it is very probably carried by sodium ions. 3. It is assumed that the late current reflects a regenerative increase of sodium conductance in the tubular membrane. The main argument favouring this assumption is that the late current is never observed on detubulated fibres. 4. Inactivation studies show a marked difference between the evolution of the two inward currents when the membrane resting potential is increased by previous hyperpolarizations in a high range of values: the late current is decreased while the initial current remains constant. The decrease of the late current is suppressed by tetraethylammonium ions or in a potassium-free medium. On detubulated fibres, the sodium current is never decreased by conditioning hyperpolarizations. 5. It is postulated that a fast transient potassium current, located in the bubular membrane, should develop contemporaneously with the sodium current. 6. The decay of the sodium inactivation is anaylsed on semilogarithmic plot: on normal fibres, it runs in two phases while on detubulated fibres, one phase only is found.     

The interaction of lithium ions with the sodium-potassium pump in frog skeletal muscle.

1. The effects of external Li on Na and K efflux as well as those on K influx were studied in high Na muscles from Rana pipiens. 2. In the absence of external Ba, substitution of K-free Li for K-free Mg resulted in an increase of both Na and K efflux. The additional of ouabain produced an inhibition of Na efflux and at the same time a marked increase in the efflux of K. 3. K permeability was greatly reduced by adding 2 mM-Ba to the incubation solutions. Under these conditions, Li gave rise to a ouabain sensitive Na efflux which was 57% of that in the absence of Ba. On the other hand, the efflux of K was only slightly increased and was not affected further by ouabain. 4. The activation curves of Na efflux against the stimulating cation concentration in Na-free Mg-Ba Ringer followed a more or less hyperbolic function for both K and Li. While half-maximal activation was attained at higher concentrations of Li than of K, the maximal efflux in Li was smaller than in K. 5. The extra Na efflux produced by K was increased when Li was added to the media. This increment was not a simple additive effect and was independent of the Li concentration. In addition, at some concentrations Li increased the ouabain-sensitive K influx, whereas at others it reduced it. 6. Reversible changes in membrane permeability to monovalent cations were accomplished by incubating the muscles in the presence of Nystatin, 50 mug/ml. When internal K was reduced to values around 1-2 mumole/g (using Li as a replacement), thus minimizing the possibility of K leaking out of the cells, both Ko and Lio were able to promote a ouabain-sensitive extra efflux of Na. 7. The residual Na efflux in (K+Na)-free solutions was not affected by the removal of Ca from the media in either Mg or Li solutions, both in the absence and the presence of Ba. On the other hand, the values for the residual efflux were higher in Mg (0-00228 min-1) than in Li (0.00135 min-1). 8. These results fully support the notion that Li ions have a K-like activating action on the Na pump in muscles. In addition, they suggest that some other kind of interaction may exist between Li and the Na-K pump.     

The components of the sodium efflux in frog muscle

1. In normal Ringer solution containing 2.5 mM-K only 37% of the efflux of labelled sodium from a freshly dissected frog muscle is blocked by treatment with ouabain; in sodium-loaded muscles the ouabain-sensitive fraction of the efflux increases to 75%.2. Under all conditions, the ouabain-insensitive component of the sodium efflux is markedly reduced if the sodium in the external medium is replaced by lithium; at least in sodium-loaded muscles, the ouabain-sensitive component is increased in lithium Ringer.3. Only the ouabain-sensitive component of the efflux is affected by the external potassium concentration.4. In the presence of 2.5 mM-K the sodium influx in a freshly dissected muscle is not significantly altered by ouabain, but in a K-free medium the influx and the efflux are both reduced by nearly 20%.5. The sodium efflux can therefore be regarded as consisting of (1) a sodium-potassium coupled component that is blocked by ouabain and involves a sodium-sodium exchange in the absence of external potassium, and (2) a potassium-insensitive component that is unaffected by ouabain and tends to reach saturation at relatively lower internal sodium concentrations.6. The evidence is considered for attributing component (1) to an efflux of sodium from the sarcoplasm proper, and component (2) to an efflux from the sarcoplasmic reticulum. Although such an interpretation is consistent with many of the observations, a definite identification of the possible sodium compartments in frog muscle cannot yet be made.     

Effect of caffeine on K+ efflux in frog skeletal muscle

 The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K⁺ (⁴²K⁺) efflux rate coefficient (k _K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca²⁺ ([Ca²⁺]_i), because the effect is mimicked by ionomycin (1.25 μM), a Ca²⁺ ionophore. Second, the inhibition of caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (SR) by 40 μM tetracaine significantly reduced the increase in k _K,o (Δk _K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca²⁺-dependent K⁺ channels (BK_Ca channels) reduced Δk _K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca²⁺-dependent K⁺ channels (SK_Ca channels), did not affect Δk _K,o. Fifth, tolbutamide (800 μM), an inhibitor of K_ATP channels, reduced Δk _K,o by about 23%. Sixth, Ba²⁺, a blocker of most K⁺ channels, did not preclude the caffeine-induced Δk _K,o. Seventh, omitting Na⁺ from the external medium reduced Δk _K,o by about 40%. Eight, amiloride (5 mM) decreased Δk _K,o by 65%. It is concluded that the caffeine-induced rise of [Ca²⁺]_i increases K⁺ efflux, through the activation of: (1) two channels (BK_Ca and K_ATP) and (2) an external Na⁺-dependent amiloride-sensitive process. Received: 13 March 1998 / Received after revision: 17 June 1998 / Accepted: 14 September 1998     

Optical evidence for a chloride conductance in the T-system of frog skeletal muscle

T-system action potentials were recorded optically from intact frog skeletal muscle fibers stained with the non-penetrating potentiometric dye NK-2367. The effect of chloride removal on the falling phase of the radially propagating tubular action potential was studied to determine whether a chloride conductance located in the T-system membranes contributes to tubular repolarization during activity. Our results show that, in chloride-free Ringer, repolarization of the tubular action potential is significantly slowed. Moreover, the late phase of tubular repolarization is characterized by a large afterpotential, which is highly temperature-dependent and appears as a secondary peak above 10° C. The optical data were compared with predicted T-system action potentials generated from a radial cable equivalent circuit model of the T-system, in which the effects of a distributed tubular leak conductance were tested. Results of this analysis are consistent with the proposal that some of the outward repolarization current during the T-system action potential is drawn across a chloride conductance located in the T-system membranes.     

Diphenylhydantoin reduces veratridine-induced sodium permeability in frog skeletal muscle

The effect of the anti-epileptic drug 5,5'-diphenylhydantoin (Dilantin) on Na permeability (PNa) in frog skeletal muscle was studied. Because there is no detectable tetrodotoxin-sensitive Na permeability in resting frog muscle, PNa was induced using the alkaloid 'neurotoxin' veratridine. Veratridine has been previously characterized as a useful 'tool' for producing a population of open Na channels in frog muscle. The amount of sodium permeability produced by veratridine was quantified in two ways: by measuring (a) membrane potential (Vm) and (b) 22Na influx. Dilantin was shown to reverse veratridine-induced depolarization by approximately one-third and to inhibit veratridine-induced Na influx in a dose-dependent manner, with an apparent Km of 78 +/- 8 microM.     

Tension development in frog skeletal muscle induced by silver ions

In single fibers of frog toe muscles placed in a Cl- free MOPS solution containing 1.8 mM Ca2+, tension developed slowly in the presence of very low concentrations of Ag+. This tension was not blocked by the administration of Co2+ or Ni2+. On the other hand, two types of transient tensions developed with the application of 5 microM Ag+, in fibers pretreated with 0-Ca2+ MOPS solution, containing either 2 mM Co2+ or 1mM Ni2+, for 10 min. In the presence of divalent cations or TTX, the first repetitive twitch-like contraction disappeared, indicating this tension is induced by action potentials repeatedly generated by the lack of divalent cations. The 2nd subsequent transient tension was caused by 5 microM Ag+ in the presence of various kinds of divalent cations, or TTX. After reversion to the resting tension, the fiber was contracted by adding more than 0.1 mM of Ca2+ or 25 mM caffeine to the external medium. Even when placed in a Ca2+-free solution containing 3 mM EGTA and 3 mM Mg2+ for 30 min, the fiber still developed an appreciable tension in response to 5 microM Ag+. These findings suggest that a transient development of the Ag+-induced tension does not require the presence of external Ca2+. A specific sulfhydryl reagent, pCMPS, did not contract the muscle fiber. Therefore, Ag+ may develop tension by mediating unknown chemical reaction(s) other than the sulfhydryl group on T-tubular membrane proteins.     

The effect of pH on the 36Cl efflux from frog skeletal muscle

1. Frog sartorii and toe muscles were loaded in a 100 mM-K 216 mM-Cl solution at pH 7·4 labelled with either ³⁶Cl, or ⁴²K, or with both isotopes. The efflux of the isotopes into inactive solution of the same composition at pH 5·0 and 9·8 was followed.2. The efflux of ³⁶Cl into acid solution was extremely slow so that the cellular fraction could easily be distinguished. On raising the pH the efflux of ³⁶Cl from sartorii increased 4·5 ± 0·43 (mean ± S.E.) times; from toe muscles 6·7 ± 0·69 (mean ± S.E.) times. The effect was fully reversible.3. The efflux of ⁴²K from sartorii was only slightly increased by alkaline solutions. When the effluxes of ³⁶Cl and ⁴²K were measured simultaneously the increment in chloride efflux was much greater than the increment in ⁴²K efflux.4. Toe muscles depolarized by high potassium solution showed an approximately twofold increase in ⁴²K efflux on transfer from acid to alkaline solution. This effect was independent of the presence of chloride ions.5. The results confirm the identification of chloride as the ion species principally concerned in the pH sensitivity of the membrane conductance.     

Effect of dantrolene sodium on excitation-contraction coupling in frog skeletal muscle.

The effect of dantrolene sodium, 1-(5-(p-nitrophenyl)furfuryli-deneamino)hydantoin sodium hydrate, on electrical and mechanical response in frog skeltal muscles (whole muscles or single fibers) and on the biochemical properties of contractile proteins and fragmented sarcoplasmic reticulum isolated from frog or rabbit skeletal muscle was investigated. The peak tensions of twitch, tetanus and potassium contracture were significantly inhibited by dantrolene, without affecting the magnitude of resting potential, the amplitude and duration of action potential and the negative afterpotential. On the other hand, ATP-INDUCED SHORTENING OF GLYCEROL-EXTRACTED RABBIT PSOAS MUSCLE FIBERS, ATPase activity of frog myofibrils and Ca release induced by caffeine, Ca uptake and ATPase activity of fragmented sarcoplasmic reticulum of frog or rabbit muscle were not affected by dantrolene. Caffeine contracture was partially inhibited by dantrolene and was almost unchanged by it in potassium-depolarized muscele fiber. Nitrate ions and low concentration of caffeine rapidly recovered the twitch inhibition induced by dantrolene. These results suggested that dantrolene acts on the membrane of transverse tubules and possibly the triadic junction and that it inhibits the inward movement of Ca and subsequently decreases the release of activator Ca from sarcoplasmic reticulum.     

Energy coupling to sodium transport in frog skeletal muscle.

In addition to a strophanthidin-sensitive (SS) sodium efflux, a large component of the sodium efflux in freshly isolated frog skeletal muscle is sodium-activated and strophanthidin-insensitive (SASI). The amount of metabolic energy associated with sodium movement by each of these components was measured and the coupling between sodium movement and adenosine 5'-triphosphate (ATP) hydrolysis in muscle was calculated. Energy production was blocked by iodoacetate and cyanide. Energy turnover was estimated from the change in creatine phosphate (CrP) and ATP contents and expressed as potential energy (PE = CrP + 2ATP). After metabolic poisoning a linear fall of PE occurred (6.3 mumol/g.h). Metabolic poisoning had no effect on the magnitude of the SS or SASI components of sodium efflux. In 2 h the sodium moved, and PE change due to the SS component was 4.35 and 1.66 mumol/g.h, respectively, which gave a coupling factor of 2.6. The amount of sodium moved by the SASI component was similar to that moved by the SS component in 2 h whereas no energy change was observed. It was, therefore, concluded that sodium movement by the SASI component requires no energy input.     

Effect of insulin upon the sodium pump in frog skeletal muscle

1. Insulin increased the rate of net Na extrusion from Na-loaded frog skeletal muscle into glucose-free Na-Ringer. After a 90 min period of efflux, the insulin-treated muscles contained approximately 11% less intracellular water than did their controls. This decrease in intracellular water resulted in an increase in the concentration of intracellular K, [K(+)](i), even though there was no definite effect upon net K flux. In spite of the decrease in intracellular water, [Na(+)](i) was lower in those muscles treated with 500 m-u. insulin/ml. than in the controls.2. Insulin consistently increased (22)Na efflux into Na-Ringer containing either 10 or 2.5 mM-K(+). This effect was reversible and was not produced by other proteins.3. Acetylstrophanthidin (5 x 10(-6)M) blocked all or nearly all net Na efflux even in the presence of insulin. The presence of this concentration of acetylstrophanthidin or of K-free Na-Ringer inhibited the effect of insulin upon (22)Na efflux from Na-loaded muscles.4. All of the above results indicate that insulin in some way increases the activity of the Na pump. The inhibition by K-free Na-Ringer also suggests that this is not due to production of additional pump sites.5. Insulin also increased (22)Na efflux and net sodium efflux into Li-Ringer. When the new steady-state was reached after addition of insulin, the (22)Na kinetics still obeyed a power relation to intracellular (22)Na. However, in every single case, insulin resulted in a decrease of approximately 18% in the exponent, n.6. Curve-fitting of the kinetic data to equations based upon a three-site model of the Na pump suggests that insulin increases the affinity of the sites toward Na(+). In terms of Eisenman's theory of ion selectivity, this would indicate an increase in the anionic field strength of the Na-carrying sites and also predict that the increase in affinity for H(+) would be greater than that for Na(+). This latter prediction is entirely consistent with the observed decrease in n.7. The results suggest that insulin may be increasing H(+) efflux as well as Na(+) efflux and thereby may be increasing intracellular pH. It is suggested that some of the intracellular effects of insulin might be mediated by such an effect.     

Changes in frog skeletal muscle action potentials following hypophysectomy and adrenalectomy

Summary Hypophysectomy and adrenalectomy caused changes in skeletal muscle action potentials. Stimulation for 3–5 minutes, or immersion for 60 minutes in Ringer, caused a reduction in the action potential amplitude.Presented by Academician L. A. Orbeli