The Suppression of the Late After-Potential in Rubidium-Containing Frog Muscle Fibers

The late after-potential that follows trains of impulses in frog muscle fibers is virtually absent when most of the intracellular potassium is replaced by rubidium and the muscle is immersed in rubidium-containing Ringer's fluid. Its amplitude is also reduced in freshly dissected, potassium-containing muscle fibers that are immersed directly in Rb-Ringer's fluid. These findings are discussed in terms of the model for muscle membrane of Adrian and Freygang (1962 a, b) and in relation to the report of Adrian (1964) that Rb-containing muscle fibers do not exhibit the variations in potassium permeability as a function of membrane potential that are found in fibers with normal intracellular potassium concentration immersed in Ringer's fluid.     

Graded Activation in Frog Muscle Fibers

The membrane potential of frog single muscle fibers in solutions containing tetrodotoxin was controlled with a two-electrode voltage clamp. Local contractions elicited by 100-ms square steps of depolarization were observed microscopically and recorded on cinefilm. The absence of myofibrillar folding with shortening to striation spacings below 1.95 µm served as a criterion for activation of the entire fiber cross section. With depolarizing steps of increasing magnitude, shortening occurred first in the most superficial myofibrils and spread inward to involve axial myofibrils as the depolarization was increased. In contractions in which the entire fiber cross section shortened actively, both the extent of shortening and the velocity of shortening at a given striation spacing could be graded by varying the magnitude of the depolarization step. The results provide evidence that the degree of activation of individual myofibrils can be graded with membrane depolarization.     

Longitudinal Impedance of Skinned Frog Muscle Fibers

Longitudinal impedance of skinned muscle fibers was measured with extracellular electrodes and an oil gap method in which a central longitudinal section of fiber is insulated by oil while the ends of the fiber are bathed in conducting pools of relaxing solution. Intact single fibers were isolated from frog semitendinosus muscle and the sarcolemma removed either by mechanical or chemical methods. Stray capacitance across the oil gap was measured after each experiment and its admittance subtracted from the admittance of the fiber and oil gap. Effects of impedance at the ends of the fiber were eliminated by measuring the impedance with two lengths of fiber in the oil gap and subtracting the impedance at the shorter length from that at the longer length. Longitudinal impedance so determined for mechanically and chemically skinned fibers exhibited zero phase shift from 1 to 10,000 Hz, i.e., the longitudinal impedance of skinned fibers is purely resistive. If we assume that our skinned fibers are a model of the sarcoplasm of muscle, we conclude that the equivalent circuit of the sarcoplasm is a resistor.     

Transverse Impedance of Single Frog Skeletal Muscle Fibers

The transverse electrical impedance of single frog skeletal muscle fibers was measured at 31 frequencies that ranged from 1 to 100,000 Hz. Each fiber was bathed entirely in Ringer's solution, but it was positioned so that a central length of 5 mm was in a hollow plastic disk and was electrically isolated from the ends of the fiber. The diameter of the segment of the fiber in the disk was measured and then the segment was pressed from opposite sides by two insulating wedges. Electrical current was passed transversely through the segment between two platinum-platinum black electrodes that were located in the pools of Ringer's solution within the disk. The results were corrected for stray parallel capacitance, series resistance of the Ringer's solution between the fiber and the electrodes, parallel shunt resistance around the fiber, and the phase shift of the measuring apparatus. A nonlinear least-squares routine was used to fit a lumped equivalent circuit to the data from six fibers. The equivalent circuit that was chosen for the fibers contained three parallel branches; each branch was composed of a resistor and a capacitor in series. The model also included a seventh adjustable parameter that was designed to account for the degree of compression of the fibers by the insulating wedges. The branches of the equivalent circuit were assumed to represent the electrical properties of: (a) the myoplasm in series with the membrane capacitance that was exposed directly to the pools of Ringer's solution; (b) the capacitance and series resistance of the transverse tubules that were exposed directly to the pools of Ringer's solution; (c) the membrane capacitance in series with the shunt resistance between the fibers and the insulating wedges. The results gave no indication that current entered the sarcoplasmic reticulum.     

Impedance of Frog Skeletal Muscle Fibers in Various Solutions

The linear circuit parameters of 140 muscle fibers in nine solutions are determined from phase measurements fitted with three circuit models: the disk model, in which the resistance to radial current flow is in the lumen of the tubules; the lumped model, in which the resistance is at the mouth of the tubules; and the hybrid model, in which it is in both places. The lumped model fails to fit the data. The disk and hybrid model fit the data, but the optimal circuit values of the hybrid model seem more reasonable. The circuit values depend on sarcomere length. The conductivity of the lumen of the tubules is less than, and varies in a nonlinear manner with, the conductivity of the bathing solution, suggesting that the tubules are partially occluded by some material like basement membrane which restricts the mobility of ions and has fixed charge. The x2.5 hypertonic sucrose solution used in many voltage clamp experiments produces a large increase in the radial resistance, suggesting that control of the potential across the tubular membranes would be difficult to achieve. Glycerol-treated fibers have 90% of their tubular system insulated from the extracellular solution and 10% connected to the extracellular solution through a high resistance. We discuss the implications of our results for calculations of the nonlinear properties of muscle fibers, including the action potential and the radial spread of contraction.     

The Relation Between the Late After-Potential and the Size of the Transverse Tubular System of Frog Muscle

This is an investigation of the effects on the late after-potential of immersing frog sartorius muscles in three kinds of modified Ringer's fluid; hypertonic, low chloride, and potassium-free. The late after-potential has been attributed to the depolarizing effect of an accumulation of potassium, during a preceding train of impulses, in the intermediary space of the model of a muscle fiber proposed by Adrian and Freygang. Both the hypertonic and low chloride solutions prolonged the late after-potential reversibly and the potassium-free solution shortened it. The effect of the low potassium solution fitted those data calculated from the model, but the effect of the hypertonic and low chloride solutions required an increase in size of the intermediary space of the model in order to fit the calculated data. An electron microscopic study of the muscles showed that the size of the transverse tubular system changed reversibly in the hypertonic and low chloride solutions in almost the amount necessary to fit the experimental data to the calculated data. This agreement between the change in size of the transverse tubular system and that of the intermediary space indicates that the intermediary space may be the transverse tubular system.     

Measurement of the Impedance of Frog Skeletal Muscle Fibers

Impedance measurements are necessary to determine the passive electrical properties of cells including the equivalent circuits of the several pathways for current flow. Such measurements are usually made with microelectrodes of high impedance (some 15 MΩ) over a wide frequency range (1-10,000 Hz) and so are subject to many errors. An input amplifier has been developed which has negligible phase shift in this frequency range because it uses negative feedback to keep tiny the voltage on top of the microelectrode. An important source of artifact is the extracellular potential produced by capacitive current flow through the wall of the microelectrodes and the effective resistance of the bathing solution. This artifact is reduced some 10 times by shielding the current microelectrode with a conductive paint. The residual artifact is analyzed, measured, and subtracted from our results. The interelectrode coupling capacitance is reduced below 2 × 10^-17 F and can be neglected. Phase and amplitude measurements are made with phase-sensitive detectors insensitive to noise. The entire apparatus is calibrated at different signal to noise ratios and the nature of the extracellular potential is investigated. The phase shift in the last 5-20 μm of the microelectrode tip is shown to be small and quite independent of frequency under several conditions. Experimental measurements of the phase characteristic of muscle fibers in normal Ringer are presented. The improvements in apparatus and the physiological significance of impedance measurements are discussed. It is suggested that the interpretation of impedance measurements is sensitive to small errors and so it is necessary to present objective evidence of the reliability of one's apparatus and measurements.     

The Effect of Bathing Solution Tonicity on Resting Tension in Frog Muscle Fibers

Resting tension and short-range elastic properties of isolated twitch muscle fibers of the frog have been studied while bathed by solutions of different tonicities. Resting tension in isotonic solution at 2.3-µm sarcomere spacing averaged 0.46 mN·mm^-2 and was proportional to the fiber cross-section area. Hypertonic solutions, containing 0.1–0.5 mM tetracaine to block contracture tension, caused a small sustained tension increase, which was proportional to the fiber cross-section area and which reached 0.9 mN·mm^-2 at two times normal tonicity (2T). Further increases in tonicity caused little increase in tension. Hypotonic solutions decreased tension. Thus, tension at 2.3 µm is a continuous, direct function of tonicity. The dependence of tension on tonicity lessened at greater sarcomere lengths. At 3.2 µm either a very small rise or, in some fibers, a fall in tension resulted from an increase in tonicity. Hypertonic solutions also decreased the tension of extended sarcolemma preparations. In constant-speed stretch experiments the elastic modulus, calculated from the initial part of the stretch response, rose steeply with tonicity over the whole range investigated (1–2.5T). The results show that tension and stiffness of the short-range elastic component do not increase in parallel in hypertonic solutions.     

Tension in Isolated Frog Muscle Fibers Induced by Hypertonic Solutions

The effect of hypertonic solutions on the tension of isolated twitch muscle fibers of the frog has been investigated. Increased tonicity up to about 1.7 times normal (1.7 T) caused a very small, graded, maintained tension increase. Above about 1.7 T a large, transient contracture response was superimposed on the small tension. The contracture response was graded with tonicity and reached a maximum at 2.5 T of 108 ± 25 mN·mm² a third of the maximum tetanic tension in isotonic solution. Contracture tension developed with a delay which decreased with increased tonicity. The contracture threshold was lower and the delay shorter in small fibers than in large. Contractures were obtained equally well in depolarized as in polarized fibers. They were completely suppressed by 0.1–0.5 mM tetracaine. The possible mechanism responsible for the tension-inducing effect of hypertonic solutions is discussed in terms of the close similarity between the properties of these contractures and those caused by caffeine, and it is suggested that the effect is due to a release of calcium from internal stores.     

Inotropic Effects of Trains of Impulses Applied during the Contraction of Cardiac Muscle

The application of a train of supramaximal stimuli during the absolute refractory period of a cardiac muscle preparation has two effects: a depression of the contraction during which it is applied, and a large potentiation of subsequent contractions. The former is ascribed to a direct effect upon the cell membrane, and is an indication of the continued control of the contractile event by this membrane. The latter is explained as a sudden liberation of norepinephrine by a stimulation of embedded nerve elements, which norepinephrine then distributes itself through the tissue and finally diffuses away.     

The Influence of Sodium-Free Solutions on the Membrane Potential of Frog Muscle Fibers

The membrane potential of frog sartorius muscle fibers in a Cl- and Na-free Ringer's solution when sucrose replaces NaCl is about the same as that in normal Ringer's solution. The K⁺ efflux is also about the same in the two solutions but muscles lose K and PO₄ in sucrose Ringer's solutions. The membrane potential in sucrose Ringer's solution is equal to that given by the Nernst equation for a K⁺ electrode, when corrections are made for the activity coefficients for K⁺ inside and outside the fiber. For a muscle in normal Ringer's solution, the measured membrane potential is within a few millivolts of E_K. This finding is incompatible with a 1:1 coupled Na-K pump. It is consistent with either no coupling of Na efflux to K influx, or a coupling ratio of 3 or greater.     

The Role of Sodium Current in the Radial Spread of Contraction in Frog Muscle Fibers

The membrane potential of isolated muscle fibers was controlled with a two-electrode voltage clamp, and the radial extent of contraction elicited by depolarizing pulses of increasing magnitude was observed microscopically. Depolarizations of the fiber surface only 1–2 mv greater than the contraction threshold produced shortening throughout the entire cross-section of the muscle fiber. The radial spread of contraction was less effective in fibers exposed to tetrodotoxin or to a bathing medium with a greatly reduced sodium concentration. The results provide evidence that depolarization of a muscle fiber produces an increase in sodium conductance in the T tubule membrane and that the resultant sodium current contributes to the spread of depolarization along the T system.     

Caffeine- and Potassium-Induced Contractures of Frog Striated Muscle Fibers in Hypertonic Solutions

The effect of hypertonic solutions on the caffeine- and KCl-induced contractures of isolated fibers of frog skeletal muscle was tested. Hypertonic solutions, twice the normal osmotic strength, prepared by adding NaCl or sucrose, potentiate the caffeine-induced contractures. The fibers may develop tensions of 3.6 kg/cm² of fiber transverse section. The same hypertonic medium reduced the peak tension of KCl-induced contractures. Thus the hypertonic condition does not affect the contractile mechanism itself. These findings give further support to the view that the differential effect of hypertonic solution is on the excitation-contraction coupling mechanism. Extracellular calcium is not essentially required for the first few of a series of caffeine-induced contractures either in hypertonic or in isotonic solutions.     

Swelling of Skinned Muscle Fibers of the Frog: Experimental Observations

Frog skeletal muscle fibers, mechanically skinned under water-saturated silicone oil, swell upon transfer to aqueous relaxing medium (60 mM KCl; 3 mM MgCl₂; 3 mM ATP; 4 mM EGTA; 20 mM Tris maleate; pH = 7.0; ionic strength 0.15 M). Their cross-sectional areas, estimated with an elliptical approximation, increase 2.32-fold (±0.54 SD). Sarcomere spacing is unaffected by this swelling. Addition of 200 mM sucrose to relaxing medium had no effect on fiber dimensions, whereas decreasing pH to 5.0 caused fibers to shrink nearly to their original (oil) size. Decreasing MgCl₂ to 0.3 mM caused fibers to swell 10%, and increasing MgCl₂ to 9 mM led to an 8% shrinkage. Increasing ionic strength to 0.29 M with KCl caused a 26% increase in cross-sectional area; decreasing ionic strength to 0.09 M had no effect. Swelling pressure was estimated with long-chain polymers, which are probably excluded from the myofilament lattice. Shrinkage in dextran T10 (number average mol wt 6,200) was transient, indicating that this polymer may penetrate into the fibers. Shrinkage in dextran T40 (number average mol wt 28,000), polyvinylpyrrolidone (PVP) K30 (number average mol wt 40,000) and dextran T70 (number average mol wt 40,300) was not transient, indicating exclusion. Maximal calcium-activated tension is decreased by 21% in PVP solutions and by 31% in dextran T40 solutions. Fibers were shrunk to their original size with 8 × 10^-2 g/cm³ PVP K30, a concentration which, from osmometric data, corresponds to an osmotic pressure (II/RT) of 10.5 mM. As discussed in the text, we consider this our best estimate of the swelling pressure. We find that increasing ionic strength to 0.39 M with KCl decreases swelling pressure slightly, whereas decreasing ionic strength to 0.09 M has no effect. We feel these data are consistent with the idea that swelling arises from the negatively charged nature of the myofilaments, from either mutual filamentary repulsion or a Donnan-osmotic mechanism.     

The Mode of Transverse Spread of Contraction Initiated by Local Activation in Single Frog Muscle Fibers

Isolated single frog muscle fibers were locally activated by applying negative current pulses to a pipette whose tip was in contact with the fiber surface. In contrast to the graded inward spread of contraction initiated by a moderate depolarization, the contraction in response to a strong negative current was observed to spread transversely around the whole perimeter but not through the center of the fiber. This response was elicited only with pipettes of more than 6 µ diameter. The response was still present if the sodium of the Ringer solution was replaced by choline, or the chloride was replaced by nitrate or propionate. The duration of the response appeared to be independent of the duration of stimulating current in fresh fibers, while the contraction lasted as long as the current went on in deteriorated fibers. The contraction was first initiated at the area of fiber surface covered by the pipette, and spread around the perimeter of the fiber with a velocity of 0.8–6 cm/sec. Possible mechanisms of the response are discussed in connection with the properties of the transverse tubular system, the possibility of some self-propagating process along the walls of the tubules being suggested.     

Tension in Skinned Frog Muscle Fibers in Solutions of Varying Ionic Strength and Neutral Salt Composition

The maximal calcium-activated isometric tension produced by a skinned frog single muscle fiber falls off as the ionic strength of the solution bathing this fiber is elevated declining to zero near 0.5 M as the ionic strength is varied using KCl. When other neutral salts are used, the tension always declines at high ionic strength, but there is some difference between the various neutral salts used. The anions and cations can be ordered in terms of their ability to inhibit the maximal calcium-activated tension. The order of increasing inhibition of tension (decreasing tension) at high ionic strength for anions is propionate^- SO₄^-- < Cl^- < Br^-. The order of increasing inhibition of calcium-activated tension for cations is K⁺ Na⁺ TMA⁺ < TEA⁺ < TPrA⁺ < TBuA⁺. The decline of maximal calcium-activated isometric tension with elevated salt concentration (ionic strength) can quantitatively explain the decline of isometric tetanic tension of a frog muscle fiber bathed in a hypertonic solution if one assumes that the internal ionic strength of a muscle fiber in normal Ringer's solution is 0.14–0.17 M. There is an increase in the base-line tension of a skinned muscle fiber bathed in a relaxing solution (no added calcium and 3 mM EGTA) of low ionic strength. This tension, which has no correlate in the intact fiber in hypotonic solutions, appears to be a noncalcium-activated tension and correlates more with a declining ionic strength than with small changes in [MgATP], [Mg], pH buffer, or [EGTA]. It is dependent upon the specific neutral salts used with cations being ordered in increasing inhibition of this noncalcium-activated tension (decreasing tension) as TPrA⁺ < TMA⁺ < K⁺ Na⁺. Measurements of potentials inside these skinned muscle fibers bathed in relaxing solutions produced occasional small positive values (<6 mV) which were not significantly different from zero.     

Effects of Cannabinoids on Caffeine Contractures in Slow and Fast Skeletal Muscle Fibers of the Frog

The effect of cannabinoids on caffeine contractures was investigated in slow and fast skeletal muscle fibers using isometric tension recording. In slow muscle fibers, WIN 55,212-2 (10 and 5 μM) caused a decrease in tension. These doses reduced maximum tension to 67.43 ± 8.07% (P = 0.02, n = 5) and 79.4 ± 14.11% (P = 0.007, n = 5) compared to control, respectively. Tension-time integral was reduced to 58.37 ± 7.17% and 75.10 ± 3.60% (P = 0.002, n = 5), respectively. Using the CB₁ cannabinoid receptor agonist ACPA (1 μM) reduced the maximum tension of caffeine contractures by 68.70 ± 11.63% (P = 0.01, n = 5); tension-time integral was reduced by 66.82 ± 6.89% (P = 0.02, n = 5) compared to controls. When the CB₁ receptor antagonist AM281 was coapplied with ACPA, it reversed the effect of ACPA on caffeine-evoked tension. In slow and fast muscle fibers incubated with the pertussis toxin, ACPA had no effect on tension evoked by caffeine. In fast muscle fibers, ACPA (1 μM) also decreased tension; the maximum tension was reduced by 56.48 ± 3.4% (P = 0.001, n = 4), and tension-time integral was reduced by 57.81 ± 2.6% (P = 0.006, n = 4). This ACPA effect was not statistically significant with respect to the reduction in tension in slow muscle fibers. Moreover, we detected the presence of mRNA for the cannabinoid CB₁ receptor on fast and slow skeletal muscle fibers, which was significantly higher in fast compared to slow muscle fiber expression. In conclusion, our results suggest that in the slow and fast muscle fibers of the frog cannabinoids diminish caffeine-evoked tension through a receptor-mediated mechanism.     

Effects of Verapamil and Gadolinium on Caffeine-Induced Contractures and Calcium Fluxes in Frog Slow Skeletal Muscle Fibers

In this work, we tested whether L-type Ca²⁺ channels are involved in the increase of caffeine-evoked tension in frog slow muscle fibers. Simultaneous net Ca²⁺ fluxes and changes in muscle tension were measured in the presence of caffeine. Isometric tension was recorded by a mechanoelectrical transducer, and net fluxes of Ca²⁺ were measured noninvasively using ion-selective vibrating microelectrodes. We show that the timing of changes in net fluxes and muscle tension coincided, suggesting interdependence of the two processes. The effects of Ca²⁺ channel blockers (verapamil and gadolinium) were explored using 6 mm caffeine; both significantly reduced the action of caffeine on tension and on calcium fluxes. Both caffeine-evoked Ca²⁺ leak and muscle tension were reduced by 75% in the presence of 100 μm GdCl₃, which also caused a 92% inhibition of net Ca²⁺ fluxes in the steady-state condition. Application of 10 μm verapamil to the bath led to 30% and 52% reductions in the Ca²⁺ leak caused by the presence of caffeine for the peak and steady-state values of net Ca²⁺ fluxes, respectively. Verapamil (10 μm) caused a 30% reduction in the maximum values of caffeine-evoked muscle tension. Gd³⁺ was a more potent inhibitor than verapamil. In conclusion, L-type Ca²⁺ channels appear to play the initial role of trigger in the rather complex mechanism of slow fiber contraction, the latter process being mediated by both positive Ca²⁺-induced Ca²⁺ release and negative (Ca²⁺ removal from cytosol) feedback loops. Lana Shabala and Xóchitl Trujillo contributed equally to this study.