Autoradiographic Studies of Intracellular Calcium in Frog Skeletal Muscle

Autoradiographs consisting of a 1000 A thick tissue section and a 1400 A thick emulsion film have been prepared from frog toe muscles labeled with Ca⁴⁵. The muscles had been fixed with an oxalate-containing osmium solution at rest at room temperature, at rest at 4°C, during relaxation following K⁺ depolarization or after prolonged depolarization. From 6 to 39 per cent of K⁺ contracture tension was produced during fixation. The grains in the autoradiographs were always concentrated in the center 0.2 to 0.3 µ of the I band and the region of the overlapping of the thick and thin filaments. The greater the tension produced during fixation, the greater was the concentration in the A band and the smaller the concentration in the I band. Autoradiographs of two muscles fixed by freeze-substitution resembled those of muscles which produced little tension during osmium fixation. Muscles which shortened during fixation produced fewer grains. In the narrow (<2.0 µ) sarcomeres of the shortened muscles, grain density decreased with decreasing sarcomere width. A theoretical analysis of the significance of these grain distributions is proposed and discussed.     

Intracellular Calcium Movements of Frog Skeletal Muscle during Recovery from Tetanus

Radioautographs of ⁴⁵Ca-labeled frog skeletal muscles have been prepared using freeze-dry and vapor fixation techniques to avoid displacement of the isotope during the preparation of the radioautographs. ⁴⁵Ca has been localized in resting muscles exposed to ⁴⁵Ca Ringer's for 5 min or 5 hr and in isotopically labeled muscles recovering from tetanic stimulation at room temperature or at 4°C. In muscles soaked at rest for 5 min ⁴⁵Ca was present almost exclusively in the terminal cisternae. In all other muscles there were three sites at which the isotope was concentrated: (a) the terminal cisternae, (b) the intermediate cisternae and the longitudinal tubules, and (c) the A band portion of the myofibrils. The terminal cisternae were labeled more rapidly than the myofibrils, but both exchanges were accelerated by electrical stimulation. The amount of ⁴⁵Ca in the longitudinal tubules and the intermediate cisternae decreased with time after a tetanus as the amount in the terminal cisternae increased. It is proposed that electrical stimulation releases calcium from the terminal cisternae and that relaxation occurs from the binding of the released calcium by the longitudinal tubules and the intermediate cisternae. Complete recovery from mechanical activity involves the transport of this bound calcium into the reticulum and its subsequent binding by the terminal cisternae. Resting exchange of calcium occurs primarily between the terminal cisternae and the transverse tubules.     

Contractile Activation in Frog Skeletal Muscle

Contractile activation was studied in frog single muscle fibers treated with tetrodotoxin to block action potentials. The membrane potential in a short segment of the fiber was controlled with a two-electrode voltage clamp, and the contractile response of superficial myofibrils in this segment was observed microscopically. The strength-duration relation for contractile activation was similar to that reported by Adrian, Chandler, and Hodgkin (1969); at 3.9°C, the contraction threshold was –44 mV for long depolarizing pulses (100-ms) and increased to +64 mV for 2-ms depolarizations. Hyperpolarizing postpulses shifted the threshold for 2-ms pulses to more positive values, and a similar, but smaller, effect was produced by hyperpolarizing prepulses. The decay of excitability following subthreshold pulses showed two apparently distinct components; at 3.9°C, excitability fell to 50% of its initial value within 4 ms, while the subsequent decline of excitability proceeded with a half-time of about 20 ms.     

The Site of Calcium Binding in Relation to the Activation of Myofibrillar Contraction

Skeletal muscle myofibrils, in the presence of 2 mM MgCl₂ at pH 7.0, were found to have two classes of calcium-binding sites with apparent affinity constants of 2.1 x 10⁶ M ^-1 (class 1) and ∼3 x 10⁴ M ^-1 (class 2), respectively. At free calcium concentrations essential for the activation of myofibrillar contraction (∼10^-6 M) there would be significant calcium binding only to the class 1 sites. These sites could bind about 1.3 µmoles of calcium per g protein. Extraction of myosin from the myofibrils did not alter their calcium-binding parameters. Myosin A, under identical experimental conditions, had little affinity for calcium. The class 1 sites are, therefore, presumed to be located in the I filaments. The class 1 sites could only be detected in F actin and myosin B preparations which were contaminated with the tropomyosin-troponin complex. Tropomyosin bound very little calcium. Troponin, which in conjunction with tropomyosin confers calcium sensitivity on actomyosin systems, could bind 22 µmoles of calcium per g protein with an apparent affinity constant of 2.4 x 10⁶ M ^-1. In view of the identical affinity constants of the myofibrils and troponin and the much greater number of calcium-binding sites on troponin it is suggested that calcium activates myofibrillar contraction by binding to the troponin molecule.     

Activation in a Skeletal Muscle Contraction Model with a Modification for Insect Fibrillar Muscle

A sliding filament model for muscle contraction is extended by including an activation mechanism based on the hypothesis that the binding of calcium by a regulating protein in the myofibrils must occur before the rate constant governing the making of interactions between cross-bridges and thin filament sites can take on nonzero values. The magnitude of the rate constant is proportional to the amount of bound calcium. The model's isometric twitch and rise of force in an isometric tetanus are similar to the curves produced by real muscles. It redevelops force after a quick release in an isometric tetanus faster than the initial rise. Quick release experiments on the model during an isometric twitch show that the “active state” curve produced is different from the postulated calcium binding curve. The force developed by the model can be increased by a small quick stretch delivered soon after activation to values near the maximum generated in an isometric tetanus. Following the quick stretch, the force remains near the tetanic maximum for a long time even though the calcium binding curve rises to a peak and subsequently decays by about 50%. The model satisfies the constraint of shortening with a constant velocity under a constant load. Modifications can be made in the model so that it produces the delayed force changes following step length changes characteristic of insect fibrillar muscle.     

Depolarization of the Internal Membrane System in the Activation of Frog Skeletal Muscle

"Skinned" muscle fibers, single fibers from the frog semitendinosus muscle in which the sarcolemma had been removed, could be reversibly activated by electrical stimulation. Electrical responsiveness was abolished when the skinned fiber was prepared from a muscle exposed to a cardiac glycoside, and the development of responsiveness was delayed when the muscle was bathed in high potassium solution. The findings were taken as evidence that active sodium-potassium exchange across the internal membranes restored electrical excitability, after the sarcolemma had been removed, by establishing a potential gradient across the internal membranes. In general, the contractions were graded with the strength of the applied current. On occasion, however, "all-or-none" type responses were seen, raising the possibility that the internal membranes were capable of an electrically regenerative response. Activation could also be produced by an elevation of the intracellular chloride ion concentration or a decrease in the intracellular potassium, ion concentration, suggesting that depolarization of some element of the internal membrane system, that is, a decrease in the potential of the lumen of the internal membrane system relative to the potential of the myofibrillar space, was responsible for activation in these experiments. The distribution of both the electrically induced contractions and those produced by changes in the intracellular ion concentrations indicated that the responsive element of the internal membrane system was electrically continuous over many sarcomeres.     

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.     

Anion Permeability of Frog Skeletal Muscle

Unidirectional chloride effluxes from small bundles of muscle fibers were measured under equilibrium conditions. It was found that chloride effluxes are described by the constant field theory with a chloride permeability constant, P_cl, which is independent of the chloride concentration and the membrane potential. The value of P_cl at neutral pH was found to be 5 x 10^-6 cm/sec. Chloride movements were markedly depressed at low pH and increased at high pH. It is concluded that chloride fluxes are independent of each other over a wide pH range. The effect of nitrate on the chloride effluxes was measured. It was found that both external and internal nitrate alone reduced the chloride efflux with the external nitrate appearing more effective than internal nitrate due to the nonequilibrium nature of the experimental conditions. Under equilibrium conditions the reduction of the chloride efflux by nitrate was greater than the external nitrate effect, both of which were dependent on the relative proportion of nitrate in the bathing solution. These results are consistent with the hypothesis that the inhibition of the chloride movements by nitrate is essentially symmetrical with regard to the inside and outside surfaces of the muscle membranes. The relative action of nitrate on the chloride efflux was independent of the external pH despite marked changes in the absolute values of the fluxes measured.     

Acetylcholine Synthesizing Enzymes in Frog Skeletal Muscle

Acetylcholine synthesis in homogenates of frog sartorius muscle was measured by a radiometric method with a low blank. Choline acetyltransferase activity was very low (V_max, 2 nmol g¹ h^?1, K_mfor choline, approx. 50 μ, m ). The enzyme was found only in the endplate area and disappeared after denervation; it was inactivated by 4-(1-naphthylvinyl)pyridine. At high substrate concentrations its activity was overshadowed by the acetylcholine-synthesizing activity of a different enzyme not saturated by 10 mm -choline. The non-specific enzyme was present at and away from the endplate area, and it was not affected by denervation.     

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.     

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.     

Motor Force Homeostasis in Skeletal Muscle Contraction

In active biological contractile processes such as skeletal muscle contraction, cellular mitosis, and neuronal growth, an interesting common observation is that multiple motors can perform coordinated and synchronous actions, whereas individual myosin motors appear to randomly attach to and detach from actin filaments. Recent experiment has demonstrated that, during skeletal muscle shortening at a wide range of velocities, individual myosin motors maintain a force of ∼6 pN during a working stroke. To understand how such force-homeostasis can be so precisely regulated in an apparently chaotic system, here we develop a molecular model within a coupled stochastic-elastic theoretical framework. The model reveals that the unique force-stretch relation of myosin motor and the stochastic behavior of actin-myosin binding cause the average number of working motors to increase in linear proportion to the filament load, so that the force on each working motor is regulated at ∼6 pN, in excellent agreement with experiment. This study suggests that it might be a general principle to use catch bonds together with a force-stretch relation similar to that of myosin motors to regulate force homeostasis in many biological processes.     

Intracellular Calcium Binding and Release in Frog Heart

The capacities and affinities of intracellular calcium-binding sites have been studied in frog ventricles, in which the concentration of Ca⁺⁺ in the sarcoplasm can be controlled as a result of treatment with EDTA. The total calcium content of calcium-depleted and nondepleted muscles at rest and muscles generating considerable tension was 0.8, 1.4, and 5.4 µmol/g of muscle, respectively. Net movement of calcium into or out of the cells occurred without change in tension when the sarcoplasmic concentration of Ca⁺⁺ was either of two values, less than 10^-7 M or approximately 5 x 10^-7 M. These data can be explained by the presence of two groups of intracellular calcium sinks which compete with the contractile proteins, one with a capacity of about 0.6 µmol/g and an affinity constant greater than 10⁷ M^-1 and a second with a capacity of 4.0 µmol/g and an affinity constant of about 2 x 10⁶ M^-1. The higher affinity calcium is released by anoxia, oligomycin, or abrupt changes in sarcoplasmic Ca⁺⁺. Muscles soaked in Sr-Ringer's contain electron densities in the sarcoplasmic reticulum and to a lesser extent in the mitochondria.     

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.     

Permeability of Frog Skeletal Muscle Cells to Choline

Using choline-methyl-C¹⁴ as a tracer, it has been shown that choline⁺ penetrates into the cells of resting frog skeletal muscle at a rate similar to that of Na⁺, and that it escapes from these cells much more slowly than does Na⁺. Some implications of these findings are discussed.     

The Temperature Dependence of Intracellular pH in Isolated Frog Skeletal Muscle: Lessons Concerning the Na+-H+ Exchanger

We used ³¹P NMR to investigate the temperature-dependence of intracellular pH (pH _i ) in isolated frog skeletal muscles. We found that ln[H⁺ _i ] is a linear function of 1/T _abs paralleling those of neutral water (i.e., H⁺= OH⁻) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was unaffected by inhibition of glycolysis and was not dependent upon the pH or [Na⁺] in the bathing solution. Insulin stimulation of Na⁺-H⁺ exchange shifted the intercept in the alkaline direction but had no effect on the slope. Acid loading followed by washout resulted in an amiloride-sensitive return to the (temperature dependent) basal pH _i . These results show that the temperature dependence of activation of Na⁺-H⁺ exchange is similar to that of the intracellular buffers, and suggest that constancy of [H⁺]/[OH⁻] with changing temperature is achieved in the short term by intracellular buffering and in the long term by the set-point of the Na⁺-H⁺ exchanger. Proton activation of the exchanger has an apparent standard enthalpy change (ΔH°) under both control and insulin-stimulated conditions that is similar to the ΔH° of the intracellular buffers and approximately half of the ΔH° for the dissociation of water. Thus, the temperature-dependent component of the standard free-energy change (ΔF°) is unaffected by insulin stimulation, suggesting that changes in Arrhenius activation energy (E _a ) may not be a part of the mechanism of hormone stimulation. Received: 12 February 1997/Revised: 1 October 1997     

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.     

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.