Action Potentials, Afterpotentials, and Excitation-Contraction Coupling in Frog Sartorius Fibers without Transverse Tubules

In frog sartorius muscle fibers in which the transverse tubular system has been disrupted by treatment with glycerol, action potentials which are unaccompanied by twitches can be recorded. These action potentials appear to be the same as those recorded in normal fibers except that the early afterpotential usually consists of a small hyperpolarization of short duration. After a train of action potentials no late afterpotential is seen even when the membrane potential is changed from the resting level. In fibers without transverse tubules hyperpolarizing currents do not produce a creep in potential. The interruption of excitation-contraction coupling, the changes in the afterpotentials, and the disappearance of creep are all attributed to the lack of a transverse tubular system.     

Radiocalcium Release by Stimulated and Potassium-Treated Sartorius Muscles of the Frog

Stimulation of frog (Rana pipiens) sartorius muscle accelerates release of Ca⁴⁵, but only during the period of stimulation. No appreciable difference is obtained in the calcium released per impulse whether stimulation is at a rate of 20/sec. or 0.5/sec. However, prior stimulation may appreciably increase the loss per impulse. In unfatigued muscles, the minimum amount of calcium liberated during an isotonic twitch is estimated to be about that previously calculated to enter, viz. 0.2 µµmole/cm². The time course of radiocalcium release during potassium depolarization depends on the nature of the contracture. When contracture is isometric, the rate of escape is doubled and declines only slowly; if isotonic, the rate is quadrupled but declines in a few minutes to a level maintained at about double that before potassium. The minimal calcium release during the first 10 minutes of potassium treatment is estimated to be about the same in both cases and about one-half to one-third the uptake. This, and especially the close equality of calcium entry and exit during electrical stimulation, are pointed out as not necessarily inconsistent with a transitory net entry of calcium, comparable to the influx, into restricted regions of the individual fibers.     

Fractionation of Sodium Efflux in Frog Sartorius Muscles by Strophanthidin and Removal of External Sodium

The influence of strophanthidin, ouabain, and the removal of external sodium on the sodium efflux from frog sartorius muscle was measured. In freshly dissected muscles strophanthidin and ouabain in maximally effective concentrations reduced the efflux of sodium by about 50%. Of the sodium efflux which is strophanthidin-insensitive about 75% is inhibited after complete replacement of external sodium by lithium. In the absence of strophanthidin replacement of external sodium by lithium, calcium, or magnesium produces an initial rise in the sodium efflux, followed by a fall in the efflux as the exposure of the muscles to sodium-free media is continued. When the muscles are exposed for prolonged periods in sodium-free media, the fraction of internal sodium lost per minute is higher when returned to normal Ringer fluid than it was initially. The activation of sodium efflux by external sodium after long periods in sodium-free solutions is partly strophanthidin-sensitive and partly strophanthidin-insensitive. The internal sodium concentration is an important factor in these effects. The effects of temperature on the sodium efflux were also measured. Above 7°C the Q₁₀ of both the strophanthidin-sensitive and strophanthidin-insensitive sodium efflux is about 2.0. Below 7°C the strophanthidin-insensitive sodium efflux has a Q₁₀ of about 7.4.     

Activation Heat in Frog Sartorius Muscle

Upon excitation of a muscle with two stimuli and variation of the interval between them up to the end of the period of full mechanical fusion, an increment of the isometric heat over that found in a single twitch is obtained. This is a good approximation to the activation heat, directly at 0°C, or after certain corrections which become important at higher temperature. The activation heat so found is independent of the muscle length and nearly independent of temperature. It is increased by nitrate and caffeine.     

The Sodium-Potassium Exchange Pump: II. Analysis of Na+-Loaded Frog Sartorius Muscle

A model for the Na-K exchange pump was applied to data on Na⁺-loaded frog sartorius muscle, and was used to relate the rate of adenosine triphosphate (ATP) hydrolysis to the electrical properties of the cell membrane. Membrane hyperpolarization was considered to arise from an electrical current which was produced by the hydrolysis reaction coupled to ion movements, and which flowed across the membrane. The reaction rate, as calculated from hyperpolarization, agreed with direct measurements of ATP hydrolysis and with the rate estimated from Na⁺ tracer efflux studies. Although Na⁺ is actively extruded, the model showed that K⁺ is inwardly transported if the potassium permeability of the membrane is less than about 6.6 × 10^-6 cm/sec, as is suggested by resistance data. Calculations indicated that the reaction conductance L_rr was relatively constant when compared with the reaction rate and reaction free energy for large changes in internal and external ionic concentrations. Its value agreed with the value obtained from the dependence of Na⁺ tracer efflux on external K⁺. A set of experiments was suggested which would provide a more complete interpretation of the data.     

Swelling of the Transverse Tubular System in Frog Sartorius

Electron microscopy shows that the transverse tubular system of frog sartorius swells in Ringer fluid in which NaCl is partially replaced by sucrose (sucrose isotonic solutions). At constant tonicity, the degree of swelling is roughly proportional to the decrease in ionic strength and to the sucrose concentration of the bathing solution. Swelling is time-dependent and reversible within 2 hr. The late after potential which follows a train of impulses is prolonged with swelling, but not to the extent expected from the model of Adrian and Freygang. This discrepancy remains unexplained, as does the mechanism of swelling of the transverse tubular system, although some suggestions are offered. One is that the transverse tubular system contains fixed charges and swells like a fixed charge gel.     

The Pattern of Activation in the Sartorius Muscle of the Frog

The development of isometric twitch tension has been compared with the redevelopment of isometric tension in the fully active frog sartorius muscle following release. At 0°C the rate of rise of isometric twitch tension is the same as that for the muscle in the fully active state at the same tension but not until about 40 msec. after the stimulus and then only for a few milliseconds. The rates of rise of tension in the twitch and in the redevelopment of tension in the fully active muscle appear to be nearly the same at low tensions. Substitution of nitrate for chloride in the Ringer's solution bathing the muscle retards the development of tension during the early part of the contraction phase of the twitch and the effect reaches a maximum within 3 minutes after changing the solutions. These observations have been discussed in connection with some possible patterns of activation and the hypothesis has been advanced that the rate of activation of a sarcomere is determined mainly by the rate at which the transverse component of the link between excitation and contraction is propagated inwards from the periphery to the center of the fiber. This hypothesis has been discussed in relation to others concerning the nature of excitation-contraction coupling.     

Potassium Fluxes in Desheathed Frog Sciatic Nerve

Desheathed frog (R. pipiens) sciatic nerves were soaked in Na-deficient solutions, and measurements were made of their Na and K contents and of the movements of K⁴². When a nerve is in Ringer's solution, the Na fluxes are equal to the K fluxes, and about 75 per cent of the K influx is due to active transport. The Na content and the Na efflux are linearly related to the Na concentration of the bathing solution, while the K content and the K fluxes are not so related. When a nerve is in a solution in which 75 per cent of the NaCl has been replaced by choline chloride or sucrose, the active K influx exceeds the active Na efflux, and the K content is maintained. When a nerve is soaked in a solution that contains Li, the K⁴² uptake is inhibited, and the nerve loses K and gains Li. When a Li-loaded nerve recovers in a Li-free solution, K is taken up in exchange for Li. This uptake of K requires Na in the external solution. It is concluded that the active transports of K and of Na may be due to different processes, that an accumulation of K occurs only in exchange for an intracellular cation, which need not be Na, and that Na plays a specific, but unknown, role in K transport.     

Contraction and Action Potentials of Frog Heart Muscles Soaked in Sucrose Solution

Isolated auricles or ventricles from the frog continue to contract, either spontaneously or when stimulated, for from 2 to 4 hours after they are placed in isotonic sucrose solution. After the muscles stop contracting in sucrose solution, contractility is partially restored when the muscles are placed in chloride Ringer's. However, contractility is usually not restored if the muscles are placed in sulfate Ringer's. Ventricles soaked in sucrose solution at 4–7°C continue to contract for 12 to 24 hours and during the first few hours in sucrose solution the contractions often are enhanced. Several types of experiment indicate that the sucrose solution does replace the Ringer's in the extracellular space. Auricles and ventricles also continue to conduct action potentials, with an overshoot, for from 30 to 360 minutes after being placed in sucrose solution. Muscles soaked in sucrose until they are inexcitable rapidly recover in chloride Ringer's but often fail to recover in sulfate Ringer's. The results are discussed in relation to theories about the generation of the action potential in cardiac muscle, and the role of the extracellular fluid in contraction.     

Identification of ATP-sensitive Potassium Channel in Frog Ventricular Myocytes

Nuclear magnetic resonance (NMR) microimaging and proton relaxation times were used to monitor differences between the hydration state of the nucleus and cytoplasm in the Rana pipiens oocyte. Individual isolated ovarian oocytes were imaged in a drop of Ringer's solution with an in-plane resolution of 80 μm. Proton spin echo images of oocytes arrested in prophase I indicated a marked difference in contrast between nucleoplasm and cytoplasm with additional intensity gradations between the yolk platelet-rich region of the cytoplasm and regions with little yolk. Neither shortening τ_e (spin echo time) to 9 msec (from 18 msec) nor lengthening τ_r (spin recovery time) to 2 sec (from 0.5 sec) reduced the observed contrast between nucleus and cytoplasm. Water proton T₁ (spin-lattice) relaxation times of oocyte suspensions indicated three water compartments that corresponded to extracellular medium (T₁= 3.0 sec), cytoplasm (T₁= 0.8 sec) and nucleoplasm (T₁= 1.6 sec). The 1.6 sec compartment disappeared at the time of nuclear breakdown. Measurements of plasma and nuclear membrane potentials with KCl-filled glass microelectrodes demonstrated that the prophase I oocyte nucleus was about 25 mV inside positive relative to the extracellular medium. A model for the prophase-arrested oocyte is proposed in which a high concentration of large impermeant ions together with small counter ions set up a Donnan-type equilibrium that results in an increased distribution of water within the nucleus in comparison with the cytosol. This study indicates: (i) a slow exchange between two or more intracellular water compartments on the NMR time-scale, (ii) an increased rotational correlation time for water molecules in both the cytoplasmic and nuclear compartments compared to bulk water, and (iii) a higher water content (per unit dry mass) of the nucleus compared to the cytoplasm, and (iv) the existence of a large (about 75 mV positive) electropotential difference between the nuclear and cytoplasmic compartments. Received: 18 January 1996/Revised: 29 April 1996     

电磁场对青蛙骨骼肌单根肌纤维效应的研究

电磁场对完整和去膜青蛙肌纤维作用的比较研究表明,交变电场通过改变膜电位引起肌肉收缩,在此过程中收缩蛋白质的空间位置而非自身构象发生变化,横桥尤其是S－2片段,在伴随横桥从弱耦合状态向强耦合状态过渡时远离粗肌丝而向细肌丝运动,使其与粗肌丝骨架的平均取向比松弛状态或静息状态时相对增大．一般强度恒定磁场对肌纤维膜电位状态及肌纤维内部蛋白质分子的运动及其相互作用影响极其微弱．     

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.     

Afterpotentials and transduction properties in different types of central neurones

In mammalian central neurones, the soma-dendritic spike is generally followed by afterpotentials, a brief depolarizing potential (delayed depolarization) and a more longlasting afterhyperpolarization (AHP). These afterpotentials, and in particular the AHP, have long been considered as important factors in the control of excitation-to-frequency transduction. Analysis of the afterpotential properties has been performed on various types of central neurones; spinal alpha-motoneurones, dorsal spinocerebellar tract cells, rubrospinal neurones and hippocampal CA1 pyramidal cells. These investigations have shown the afterpotentials to differ considerably in their characteristics among these types of neurones. Studies of the firing behaviour of the neurones have also shown great variations in their firing properties, the observed differences being well in accord with those expected on the basis of their different afterpotential characteristics. The results suggest that the afterpotentials play a major role in the control of excitation-to-frequency transduction in several types of central neurones.     

Sodium Ions in the Electromechanical Coupling System of Myocardium and Skeletal Muscles of the Frog Rana temporaria

The role of the transsarcolemmal Na⁺ gradient in providing phasic contraction of myocardium and skeletal muscles of the common frog Rana temporaria was studied. It was shown that a decrease of the gradient by means of total or partial substitution of Na⁺ for Ch⁺ or Li⁺ produced a tissue-specific reduction of the isometric tension developed by the muscles as well as of the maximum rate of its development. The functional mechanisms of use of the Na⁺-gradient for intracellular mobilization of activator of the muscle contraction, Ca²⁺ ions, are considered, and the experimental evidences for significance of this gradient increase to provide intensification of the process of activation of the discrete (single, rhythmic) contraction of vertebrate phasic muscles are presented. The revealed differences between the studied muscles reflect adaptive rearrangement levels of ionic mechanisms of the electromechanical coupling system in these muscles, which are achieved in the course of evolution of their contractile function.     

Activation of Na/H Exchange in Erythrocytes of Frog Rana ridibunda

Transport of Na⁺ in isolated erythrocytes of the frog Rana ridibunda was studied using radioactive isotope ^{22 22}Na. Treatment of erythrocytes with -adrenergic agonist isoproterenol (ISP) or with a combination of ISP and phosphodiesterase blocker 3-isobutyl-methyl-xanthine (IBMX) did not affect the Na⁺ transport into the cells. These data indicated that cAMP-dependent protein kinase A did not participate in regulation of the Na⁺ transport into the frog erythrocytes. Incubation of erythrocytes with protein kinase C activator phorbol ester (PMA, 0.15 µM) led to a pronounced increase of ^{22 22}Na accumulation and intracellular Na⁺ concentration. These changes of the Na⁺ transport into the cells were completely blocked in the presence of 50 µM ethyl-isopropyl-amiloride (EIPA), a selective blocker of the NHE1-isoform of Na⁺/H⁺ exchanger. Hence, PMA produced activation of Na⁺/H⁺ exchange in frog erythrocytes. The unidirectional Na⁺ influx into erythrocytes amounted, on average, to 0.99 ± 0.12 and 147 ± 9 mmol/l cells/h for control and PMA-treated cells, respectively. The EIPA concentration producing a 50% inhibition of the PMA-induced Na⁺ influx (IC₅₀) was 0.28 µM. A high sensitivity of the frog Na/H exchanger to EIPA indicates its similarity with the mammalian NHE1 isoform. The obtained data for the first time clearly indicate an important role of PKC in Na/H exchange regulation in the frog red blood cells.     

The Roles of Calcium in Excitation-Contraction Coupling in Various Muscles of the Frog, Mouse, and Barnacle

In rectus abdominis muscles of the frog the active shorteningprovoked by 15–40 mM K was supported by Ca, Sr, and Ba,but not by Ni, Co, Mn, Cd, or Zn ions. Addition of the lattercations to a solution containing Ca decreased the responsesin a manner suggesting competitive inhibition. The shorteningof the rectus muscle found in divalent cation-free, low K solutionsis abolished by Ni, Co, Mn, and Mg. In rat muscles a transientincrease in the contractural responses to elevated potassiumwas observed when Ni was applied following partial washout ofCa. In single muscle fibers of the barnacle, development oftension was supported by Ca and Sr, and the other divalent cationswere without effect. Retention of Ca⁴⁵ in barnacle resting musclefibers soaked in solutions containing 10 mM Ca for 2 min andsubsequently washed for 10 min was 60 ± 3.1 mµMCa/g, whereas retention of Ca⁴⁵ in contracting muscles similarlyexposed to Ca⁴⁵ was 156 + 17 mµM Ca/g of fresh muscle.The results are compatible with the idea that activation ofcontraction in some types of muscles is due to entry of extracellularCa.     

Three Types of Single Voltage-Dependent Potassium Channels in the Sarcolemma of Frog Skeletal Muscle

Patch-clamp experiments in the sarcolemma of frog skeletal muscle evidenced the presence of three types of voltage-dependent single-channel K⁺ currents. According to their unitary conductance at a membrane voltage of +40 mV, we classified them as 16-, 13-, and 7-pS K⁺ channels. The 16-pS K⁺ channels are active close to a membrane voltage of −80 mV and they do not become inactivated during voltage pulses of 100 ms. Within 10 min after beginning the recording, these channels developed rundown with an exponential time course. The 13-pS K⁺ channels are active near −60 mV; upon a 100-ms depolarization, they exhibited inactivation with an approximate exponential time course. The 7-pS K⁺ channels were recorded at voltages positive to 0 mV. In patches containing all three types of K⁺ channels, the ensemble average currents resemble the kinetic properties of the macroscopic delayed rectifier K⁺ currents recorded in skeletal muscle and other tissues. In conclusion, the biophysical properties of unitary K⁺ currents suggest that these single-channel K⁺ currents may underlie the macroscopic delayed K⁺ currents in frog skeletal muscle fibers. In addition, since the 16- and 13-pS channels were more frequently recorded, both are the main contributors to the delayed K⁺ currents.     

Free and Esterified Coenzyme A in the Liver and Muscles of Chronically Hyperammonemic Mice Treated with Sodium Benzoate

Ammonia toxicity and relative sodium benzoate toxicity alters the energy metabolism, leading to a decrease of adenosine triphosphate and free coenzyme A levels. The object of the present study was to analyze the hepatic and muscular acyl-coenzyme A profiles in chronically hyperammonemic mice treated with varying doses of the sodium benzoate. An enzymatic method was used for the measurement of free coenzyme A, acetyl-coenzyme A, and medium and long chain acyl-coenzyme A. Untreated chronic hyperammonemia resulted in a decrease in acetylcoenzyme A and an increase in the long chain acyl coenzyme A in the liver, accompanied by an increase in total coenzyme A in the muscular tissues. Treatment with sodium benzoate at moderate doses, caused a decrease in the hepatic free and esterified coenzyme A while these were increased at higher doses. We conclude that chronic hyperammonemia is responsible for qualitative changes in the free and esterified coenzyme A profile in the liver, while causing qualitative and quantitative changes in the muscular tissue, probably due to an inhibition of mitochondrial oxidation. The sodium benzoate had a biphasic effect on the hepatic content of free and esterified coenzyme A, suggesting a degradation of coenzyme A at moderate doses. However, at a higher dose of benzoate, the possibility of glycine mobilization and/or a significant formation of acylcarnitines is proposed as an important factor in an increase of the hepatic total coenzyme A.     

Photosynthetic Electron Transport in Plastids in Detached Cucumber Cotyledons Treated with 6-Benzylaminopurine and Potassium

Etiolated cotyledons of cucumber (Cucumis sativus L.) were excisedand incubated for 16 days on agar in the absence or in the presenceof 6-benzylaminopurine (BA) (3 µM) or KCl (10 mM) undercontinuous light. Treatment with BA or KCl increased growthand greening of cotyledons, KCl being particularly active duringthe last few days of the incubation. The rate of photosynthetic electron transport reached a maximumafter 3 days, as did the chlorophyll content, and then it declinedby approximately 95%. An uncoupling from phosphorylation wasobserved in older cotyledons. BA and KCl increased the rateof electron flow per cotyledon, and KCl delayed the onset ofthe decline in this rate. PS I and PS II activities per chlorophyllunit were lower in BA-treated cotyledons, but KCl-treated cotyledonsbehaved like the controls. Fluorescence analysis indicated that the level of active chlorophyllsinvolved in the transfer of energy in PS II decreased between3 and 7 days under all conditions tested. In BA-treated cotyledons,the proportion of active chlorophylls was consistently lowerthan that in the control. Thus, it appears that, in the absenceof exogenous K⁺, cytokinin has only a low antisenescence effecton photosynthetic activity, and that K⁺ may enhance the photosyntheticelectron flow between plastoquinone and plastocyanin via thecytochrome b₆-f complex. (Received August 2, 1989; Accepted January 23, 1990)