Mechanism of latency relaxation in frog skeletal muscle

The latency relaxation is a small drop of tension before skeletal muscle begins to develop active tension. This phenomenon was found nearly one century ago but its origin has not been clarified. In this review, the hypotheses for its mechanism are discussed in terms of the recent experimental results using X-ray diffraction. The latency relaxation takes place almost simultaneously as the structural change of the regulatory protein troponin, an unspecified structural change of the thick filament, and increase in stiffness. It seems difficult to associate all of these with the latency relaxation by assuming a simple mechanism.     

Determinants of relaxation rate in skinned frog skeletal muscle fibers

The influences of sarcomere uniformity andCa²⁺ concentration on the kineticsof relaxation were examined in skinned frog skeletal muscle fibersinduced to relax by rapid sequestration ofCa²⁺ by the photolysis of theCa²⁺ chelator, diazo-2, at10°C. Compared with an intact fiber, diazo-2-induced relaxationexhibited a faster and shorter initial slow phase and a fast phase witha longer tail. Stabilization of the sarcomeres by repeated releases andrestretches during force development increased the duration of the slowphase and slowed its kinetics. When force of contraction was decreasedby lowering the Ca²⁺concentration, the overall kinetics of relaxation was accelerated, withthe slow phase being the most sensitive toCa²⁺ concentration. Twitchlikecontractions were induced by photorelease ofCa²⁺ from a cagedCa²⁺ (DM-Nitrophen), withsubsequent Ca²⁺ sequestration byintact sarcoplasmic reticulum orCa²⁺ rebinding to cagedCa²⁺. These twitchlike responsesexhibited relaxation kinetics that were about twofold slower than thoseobserved in intact fibers. Results suggest that the slow phase ofrelaxation is influenced by the degree of sarcomere homogeneity andrate of Ca²⁺ dissociation fromthin filaments. The fast phase of relaxation is in part determined bythe level of Ca²⁺ activation.

     

Activation heat and latency relaxation in relation to calcium movement in skeletal and cardiac muscle

Measurements of activation heat, initial heat, twitch tension, and latency relaxation were made using thin-layer, vacuum-deposited thermopiles and isometric force transducers, respectively. Experiments were performed on frog skeletal muscle fiber bundles and on rabbit right ventricular papillary muscles at 0, 15, ans 21 degrees C in normal and 1.75X to 2.5X mannitol hyperosmotic bathing solutions. In skeletal muscle, activation heat, obtained by stretching to zero overlap, was only slightly affected by 1.75X hyperosmotic solution and consisted of a fast and a slow component. Both components have a refractory period and a relatively refractory period which can be demonstrated by double pulse stimulation. The twitch potentiators Zn2+ and caffeine increase the total activation heat and the magnitude and rate of the fast component. The temporal relation between the latency relaxation and activation heat is demonstrated. The latency relaxation is independent of the number of sarcomeres in series in a muscle. Activation heat and latency relaxation records from heart muscle are obtained in 2.5X hyperosmotic bathing solution. A model of excitation--contraction coupling is presented which indicates that (1) the downstroke of the latency relaxation monitors the functioning of the Ca2+-permeability or debinding mechanism in the terminal cisternae, (2) the fast component of activation heat monitors the amount of Ca2+ bound to troponin C, and (3) the total amplitude of activation heat is a measure of the total quantity of Ca2+ cycled in a twitch.     

Minimal latency of calcium release in frog twitch muscle fibres

Intracellular release of calcium in frog skeletal muscle fibres was monitored by the use of arsenazo III, in response to voltage clamped depolarizing pulses. A latency of a few milliseconds was evident between the onset of depolarization and the first detectable rise in the arsenazo-calcium signal, and this decreased logarithmically as the depolarization was increased. The minimal latency with strong depolarization (to +20 to +100 mV) was about 2 ms at 5 degrees C. This delay appears to be sufficiently long to be compatible with a chemically mediated coupling mechanism between depolarization and calcium release from the sarcoplasmic reticulum.     

Diffusible magnesium in frog skeletal muscle cells

Total diffusible magnesium concentration in frog skeletal muscle is 5.2 mM as determined by electron probe microanalysis of 0.2 nl liquid samples. The calculated free Mg concentration, 0.2 mM, is at the lower end of the range of values reported by others as calculated by methods using nuclear magnetic resonance, Mg-selective microelectrodes, and metallochromic indicator dyes. Magnesium is but one of many elements of physiological importance in muscle that can be analyzed using this novel liquid-sampling and x-ray spectroscopic method.     

A new method for excitation-contraction uncoupling in frog skeletal muscle

The mechanical activity of frog sartorius muscle fibers can be uncoupled from the electrical activity of their surface membranes by immersing the preparation in Ringer solution containing either 1.5 or 2.0 M of formamide for 15--20 min. This uncoupling is not reversed when the muscle is transferred to normal frog Ringer solution. Formamide does not affect the electrical activity of the sciatic nerve branch, and both endplate potentials and miniature endplate potentials may be recorded from the uncoupled muscles. Prolonged exposure to formamide, beyond the time needed to paralyze, causes neuromuscular block.     

Delayed rectification in the transverse tubules: origin of the late after-potential in frog skeletal muscle

Tetanic stimulation of skeletal muscle fibers elicits a train of spikes followed by a long-lasting depolarization called the late after- potential (LAP). We have conducted experiments to determine the origin of the LAP. Isolated single muscle fibers were treated with a high potassium solution (5 mM or 10 mM K) followed by a sudden reduction of potassium concentration to 2.5 mM. This procedure produced a slow repolarization (K repolarization), which reflects a diffusional outflow of potassium from inside the lumen of the transverse tubular system (T system). Tetanic stimulation was then applied to the same fiber and the LAP was recorded. The time courses of K repolarization and LAP decay were compared and found to be roughly the same. This approximate equality held under various conditions that changed the time courses of both events over a wide range. Both K repolarization and the LAP became slower as fiber radius increased. These results suggest that LAP decay and K repolarization represent the same process. Thus, we conclude that the LAP is caused by potassium accumulation in the T system. A consequence of this conclusion is that delayed rectification channels exist in the T system. A rough estimation suggests that the density of delayed rectification channels is less in the T system than in the surface membrane.     

Calcium in excitation--contraction coupling of frog skeletal muscle

A principal step in the process leading to muscle contraction is the intracellular release of Ca2+. We have detected and compared some physical and chemical events that reflect Ca2+ release in contracting frog skeletal muscle cells, described the effects of some agents that are believed to alter intracellular Ca2+ release during contraction, and speculated about the role of Ca2+ release in influencing some of the mechanical properties of frog muscle. The specific physical features recorded were changes in striation spacing, myofibrillar orientation, and force development. The chemical feature was the relative change in intracellular [Ca2+] recorded as light emission from cells microinjected with the Ca2+-sensitive protein aequorin. The presence or absence of a correlation among these variables has been used (i) to evaluate the action of some agents thought to change intracellular Ca2+ release in excitation--contraction (E--C) coupling, (ii) to further substantiate the effects of cell length on Ca2+ release, and (iii) to examine some details of models for E--C coupling. The results showed that potentiating agents enhance and prolong intracellular Ca2+ release without changing the rate of Ca2+ removal during E--C coupling. This extra Ca2+ does not produce the same effect on contractions at all lengths. Contractility is inversely related to cell length, and Ca2+-induced activation is normally less than maximum not only at short lengths but also at optimal striation spacings.     

Ultraslow contractile inactivation in frog skeletal muscle fibers

After a contracture response, skeletal muscle fibers enter into a state of contractile refractoriness or inactivation. Contractile inactivation starts soon after membrane depolarization, and causes spontaneous relaxation from the contracture response. Here we demonstrate that contractile inactivation continues to develop for tens of seconds if the membrane remains in a depolarized state. We have studied this phenomenon using short (1.5 mm) frog muscle fibers dissected from the Lumbricalis brevis muscles of the frog, with a two-microelectrode voltage-clamp technique. After a contracture caused by membrane depolarization to 0 mV, from a holding potential of -100 mV, a second contracture can be developed only if the membrane is repolarized beyond a determined potential value for a certain period of time. We have used a repriming protocol of 1 or 2 s at -100 mV. After this repriming period a fiber, if depolarized again to 0 mV, may develop a second contracture, whose magnitude and time course will depend on the duration of the period during which the fiber was maintained at 0 mV before the repriming process. With this procedure it is possible to demonstrate that the inactivation process builds up with a very slow time course, with a half time of approximately 35 s and completion in greater than 100 s. After prolonged depolarizations (greater than 100 s), the repriming time course is slower and the inactivation curve (obtained by plotting the extent of repriming against the repriming membrane potential) is shifted toward more negative potentials by greater than 30 mV when compared with similar curves obtained after shorter depolarizing periods (10-30 s). These results indicate that important changes occur in the physical state of the molecular moiety that is responsible for the inactivation phenomenon. The shift of the inactivation curve can be partially reversed by a low concentration (50 microM) of lanthanum ions. In the presence of 0.5 mM caffeine, larger responses can be obtained even after prolonged depolarization periods, indicating that the fibers maintain their capacity to liberate calcium.     

Furosemide-sensitive cation transport in frog skeletal muscle fibers

Furosemide-inhibitable components in unidirectional cation fluxes have been identified in frog skeletal muscle. In sodium loaded muscles, placed in sodium-free rubidium lithium media, furosemide (1 mM) inhibits partially rubidium and lithium influxes as well as potassium and sodium outfluxes. The furosemide-inhibitable components were found to depend on the presence of ouabain. They were greatly diminished in sodium-free magnesium media and were present in chloride-free nitrate containing media. The dependence of furosemide-inhibitable sodium efflux on internal sodium content was also described.     

Sodium channel gating currents in frog skeletal muscle

Charge movements similar to those attributed to the sodium channel gating mechanism in nerve have been measured in frog skeletal muscle using the vaseline-gap voltage-clamp technique. The time course of gating currents elicited by moderate to strong depolarizations could be well fitted by the sum of two exponentials. The gating charge exhibits immobilization: at a holding potential of -90 mV the proportion of charge that returns after a depolarizing prepulse (OFF charge) decreases with the duration of the prepulse with a time course similar to inactivation of sodium currents measured in the same fiber at the same potential. OFF charge movements elicited by a return to more negative holding potentials of -120 or -150 mV show distinct fast and slow phases. At these holding potentials the total charge moved during both phases of the gating current is equal to the ON charge moved during the preceding prepulse. It is suggested that the slow component of OFF charge movement represents the slower return of charge "immobilized" during the prepulse. A slow mechanism of charge immobilization is also evident: the maximum charge moved for a strong depolarization is approximately doubled by changing the holding potential from -90 to -150 mV. Although they are larger in magnitude for a -150-mV holding potential, the gating currents elicited by steps to a given potential have similar kinetics whether the holding potential is -90 or -150 mV.     

Acoustic signals from frog skeletal muscle.

Acoustic, force, and compound muscle action-potential signals were recorded simultaneously during maximal isometric twitches of frog gastrocnemius muscles. The onset of sound production occurred after the onset of muscle depolarization but before the onset of external force production. Acoustic waveforms consisted of oscillations that initially increased in amplitude, followed by decaying oscillations. The peak-to-peak acoustic amplitude increased with increasing temperature with a Q10 of 2.6 +/- 0.2 over a range of 7.0-25.0 degrees C. The acoustic amplitude increased with increasing muscle length up to approximately 90% of the optimal length for force generation. As length was increased further, the acoustic amplitude decreased. Microphones positioned on opposite sides of the muscle recorded acoustic signals that were 180 degrees out of phase. These results provided evidence that sound production is produced by lateral oscillations of muscle. The oscillation frequency may provide a measure of mechanical properties of muscle.     

Determination of ionic calcium in frog skeletal muscle fibers

Ionic calcium concentrations were measured in frog skeletal muscle fibers using Ca-selective microelectrodes. In fibers with resting membrane potentials more negative than -85 mV, the mean pCa value was 6.94 (0.12 microM). In fibers depolarized to -73 mV with 10-mM K the mean pCa was 6.43 (0.37 microM). This increase in the intracellular [Ca2+] could be related to the higher oxygen consumption and heat production (Solandt effect) reported to occur under these conditions. Caffeine, 3 mM, also produced an increase in the free ionic calcium to a pCa of 6.52 (0.31 microM) without changes in the membrane potential. Lower caffeine concentrations, 1 and 2 mM, did not change the fiber pCa. Lower Ca concentrations in the external medium effectively reduced the internal ionic calcium to an estimated pCa of 7.43 (0.03 microM).     

Localization of acid organelles in frog skeletal muscle fibers

By means of fluorescent and phase-contrast microscopy the distribution of acid membrane organelles in normal and vacuolated frog skeletal muscle fibers has been studied. The vacuolation of the T-system was produced by loading and subsequent removal of glycerol (80-110 mM), or it appeared as a result of Zenker's necrosis. Acridine orange (AO) was used as a marker for acid intracellular compartments. AO accumulated in granules localized near the nuclear poles (more seldom around the nucleus)' and in the intermyofibrillar spaces. Typically the AO granules make up short longitudinal chains or regular pairs, where the distances between neighboring granules are short-dated to sarcomere lengths. Almost all granules emit in red, but about one third of them simultaneously emit in green, which is characteristic of AO monomers. In the vicinity of necrotic boundary or under the influence of brefeldin A, a green component of fluorescence appears in most granules. Treatment with monensin leads to granule disappearance. Vacuoles accompanying the glycerol treatment or developing of necrosis do not accumulate AO and exert no effect on the localization of AO-granules. The nature of cellular organelles accumulating AO in skeletal muscle fibers is discussed.     

K-current fluctuations in inward-rectifying channels of frog skeletal muscle

Summary K currents and K-current fluctuations were recorded in inwardly rectifying K channels of frog skeletal muscle under voltage-clamp conditions. External application of 0.2 to 10mm Cs reduces the inward mean K current but produces a distinct increase of the spectral density of K-current fluctuations. The additional fluctuations arise from the random blocking by Cs ions. From the variance of current fluctuations, the steady-state current and the probability of the open unblocked channel an effective single-channel conductance ^* was calculated. ^* strongly depends on the external Cs concentration (7.8 pS at 0.2mm Cs, 2.1 pS at 10mm Cs). This dependence is interpreted in terms of a two-step blocking process: (1) a fast exchange of Cs ions between the external solution and a first binding site inside the channel which leads to the Cs-modulated effective single-channel conductance, and (2) a slow Cs binding to a second site deeper in the channel which produces the observed current fluctuations. With this hypothesis we obtained a real single-channel conductance of 10 pS and a real density ofn4 inwardly rectifying channels per m² of muscle surface area.     

A novel frog skin peptide containing function to induce muscle relaxation

A novel bioactive peptide (polypedarelaxin 1) was identified from the skin secretions of the tree frog, Polypedates pingbianensis. Polypedarelaxin 1 is composed of 21 amino acid residues with a sequence of QGGLLGKVSNLANDALGILPI. Its primary structure was further confirmed by cDNA cloning and mass spectrometry analysis. Polypedarelaxin 1 was found to elicit concentration-dependent relaxation effects on isolated rat ileum. It has no antimicrobial and serine protease inhibitory activities. BLAST search revealed that polypedarelaxin 1 did not show similarity to known proteins or peptides. Especially, polypedarelaxin 1 do not contain conserved structural motifs of other amphibian myotropic peptides, such as bradykinins, bombesins, cholecystokinin (CCK), and tachykinins, indicating that polypedarelaxin 1 belongs to a novel family of amphibian myotropic peptide.