Step In Excitation-contraction Coupling Research Articles

Differential effects of tetracaine on charge movements and Ca2+ signals in frog skeletal muscle.

The effects of tetracaine on charge movements and on antipyrylazo III signals monitoring intracellular delta [Ca2+] were compared in cut frog semitendinosus muscle fibers in a single vaseline gap-voltage clamp. Low tetracaine concentrations (25-40 microM) markedly reduced delta [Ca2+] signals and shifted the rheobase. However, they neither influenced charge movement nor that peak delta [Ca2+] value associated with the contractile threshold. Higher tetracaine concentrations (100-200 microM) partly inhibited charge movements in cut fibers. They separated a steeply voltage-sensitive charge, some of whose features resembled 'q gamma' reported in intact fibers, and whose movement preceded delta [Ca2+] signals at threshold. These findings: (a) directly confirm an earlier suggestion that tetracaine acts on steps in excitation-contraction coupling rather than myofilament activation; (b) show that tetracaine at low concentrations can directly interfere with sarcoplasmic reticular calcium release without modifying charge movement; (c) show that the tetracaine-sensitive charge, first found in intact fibers, also exists in cut fibers; and (d) make it unlikely that tetracaine-sensitive charge transfer is a consequence of Ca2+ release as suggested on earlier occasions.

2] Measurement of Ca2+ release in skinned fibers from skeletal muscle

Publisher Summary This chapter describes in detail, the methods for measurement of Ca 2+ release in skinned fibers that are used in laboratory. Ca 2+ release from the sarcoplasmic reticulum (SR) is one of the most important steps in excitation-contraction coupling of skeletal muscle. However, while various kinds of stimuli experimentally applied to the SR can be shown to cause Ca 2+ release, the mechanism of physiological Ca 2+ release from the SR is not yet known. In the experiments on Ca 2+ release from the SR, one must take the following facts into account. (1) There are several Ca 2+ release mechanisms in the SR, which are different at least in their activation mechanisms, but probably also in their Ca 2+ channels. Therefore, determinations regarding one of the release mechanisms cannot be applied automatically to Ca 2+ release in general. (2) SR membrane also has a strong Ca 2+ uptake activity through the calcium pump ATPase. Therefore, unless the Ca 2+ -pump is inhibited by the experimental condition, Ca 2+ released by any means tends to activate the pump and partly be taken up again. (3) Ca 2+ also activates one of the Ca 2+ release mechanisms present in the SR, the so-called Ca 2+ -induced Ca 2+ release mechanism.

The Chemical Transmission Mechanism of Excitation-Contraction Coupling in Skeletal Muscle

There is a temperature-dependent lag between depolarization of transverse tubules by the action potential and onset of calcium release from the sacromplasmic reticulum that reveals the occurrence of a chemical step in excitation-contraction coupling. Recent studies suggest that in vertebrate muscle inositol 1,4,5-trisphosphate may act as a chemical link in this process.

Excitation of skinned muscle fibers by imposed ion gradients. II. Influence of quercetin and ATP removal on the Ca2+-insensitive component of stimulated 45Ca efflux.

Ionic gradients imposed by choline Cl replacement of K methanesulfonate (Mes) at constant [K][Cl] product stimulate 45Ca efflux from skinned muscle fibers; a small, sustained Ca2+-insensitive efflux component, observed in EGTA, appears to grade a much larger Ca2+-dependent component responsible for contractile activation and is likely to reflect intermediate steps in excitation-contraction coupling. The present studies examined ATP-related effects on the Ca2+-insensitive stimulation. 45Ca efflux was measured on segments of frog semitendinosus muscle skinned by microdissection, with isometric force monitored continuously. The Ca2+-insensitive component was potentiated by quercetin, a flavonoid thought to inhibit the sarcoplasmic reticulum (SR) Ca pump by stabilizing a phosphorylated intermediate. Quercetin increased the stimulated net 45Ca release in the absence of EGTA, as expected from inhibition of reaccumulation, but its effectiveness in EGTA indicated potentiation of unidirectional efflux as such. Quercetin also increased unstimulated (control) 45Ca efflux in EGTA, to a smaller extent; potentiation appeared to be a function of efflux, with stimulation above control loss increased approximately 2.6-fold. ATP removal before stimulation, which led to rigor force and increased stiffness, prevented all quercetin effects in EGTA. ATP removal by itself inhibited ionic stimulation of the Ca2+-insensitive component, with little residual increase above the parallel control loss. Addition of the nonhydrolyzable ATP analogue AMP-PCP ([adenylyl-beta,gamma-methylene]diphosphate) (0.8 mM) after ATP removal gave similar results to ATP-free solution, which suggests that adenine nucleotide binding alone does not support stimulation by choline Cl. These results imply a fundamental role for ATP in the excitation of skinned fibers by imposed diffusion potentials; they also suggest that ATP regulates the SR Ca efflux channel, in a manner that could provide the positive feedback in Ca2+-dependent Ca release.

Sarcoplasmic reticulum contains adenine nucleotide-activated calcium channels

Rapid calcium efflux from the sarcoplasmic reticulum (SR) is a necessary step in excitation-contraction coupling in skeletal muscle and is thought to be mediated by a calcium channel. Calcium efflux has been studied in fragmented SR vesicles by radioisotope efflux and fluorescence measurements. Several laboratories have reported that adenine nucleotides can stimulate calcium efflux from SR. In recent reports, Ca2+ release with a first-order rate constant as high as 100 s-1 has been observed for nucleotide-stimulated Ca2+ release from SR vesicles. Also, radioisotope efflux was blocked by Mg2+ and micromolar concentrations of the polycationic dye, ruthenium red. These high rates of transport are difficult to reconcile with a mechanism other than passive diffusion through a nucleotide-activated 'calcium release channel'. Using the fusion technique for inserting SR proteins into planar lipid bilayers, we report here single-channel recordings of calcium release channels from purified 'heavy' SR membranes. Channels have been identified on the basis of their activation by adenine nucleotides, blockade by ruthenium red, and selectivity for divalent cations. Surprisingly, the channel studied here exhibits an unusually large conductance of 170 pS in 50 mM Ba2+ while still being capable of discriminating against monovalent cations by a permeability ratio, P(Ba)/P(Cs) = 11.4.

Calcium release and sarcoplasmic reticulum membrane potential in frog skeletal muscle fibres.

Single twitch fibres were dissected from frog muscle, stretched to a sarcomere spacing greater than or equal to 3.9 micron, then mounted for optical recording. The experiments were carried out at 15-17 degrees C. In some cases D2O Ringer solution was used instead of H2O Ringer solution to reduce movement and any related optical artifacts. Following action potential stimulation, both the amplitude and time course of the change in intrinsic retardation (Baylor & Oetliker's second component, 1975) were found to be approximately independent of wavelength between 480 and 750 nm (D2O Ringer solution). Fibres were injected with the Ca2+-sensitive dye Arsenazo III so that changes in myoplasmic free [Ca2+] could be estimated by measuring changes in dye-related absorbance at 660 nm. The time course of free [Ca2+] was compared with the time course of two other optical signals which have been previously suggested to monitor s.r. (sarcoplasmic reticulum) membrane potential, intrinsic retardation and Nile Blue A fluorescence (Bezanilla & Horowicz, 1975). In D2O Ringer solution the retardation time course was closely similar to that of free [Ca2+] whereas the fluorescence time course was considerably slower. Thus, it is possible that either the retardation signal or Nile Blue A fluorescence (or both) monitors free [Ca2+] rather than s.r. potential. If so, the underlying mechanism which senses Ca2+ must do so very rapidly in the case of retardation and with a delay in the case of Nile Blue A. Changes in Nile Blue A fluorescence were measured in a voltage-clamped fibre (H2O Ringer solution). Only small changes were observed during hyperpolarization or small depolarization whereas relatively large changes were observed near mechanical threshold. These increased e-fold in magnitude every 4-5 mV. This steep voltage dependence, similar to that already shown for intrinsic retardation and [Ca2+], provides additional evidence that Nile Blue A fluorescence monitors a step in excitation-contraction coupling. Theoretical waveforms of s.r. membrane potential were computed using a typical waveform of s.r. Ca2+ release (from Baylor, Chandler & Marshall, 1983) under the assumption that Ca2+ crosses the s.r. membrane as electrical current. Voltage waveforms were calculated using several combinations of electrical parameters for the s.r. membrane. Only certain combinations gave theoretical potential changes similar to the intrinsic retardation or Nile Blue A fluorescence signal.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of action of angiotensin II on excitation-contraction coupling in the rat portal vein.

1 The action of angiotensin II (At II) has been studied on the electrical and mechanical activity of the vascular smooth muscle of the rat portal vein.2 At low concentrations (between 5 x 10(-10) and 10(-9) M) At II induces an acceleration of spontaneous action potential (AP) discharge without change in the resting membrane potential. The frequency and size of the associated contractions are simultaneously augmented. Under these conditions the size of the spikes is not affected, thus suggesting that At II triggers the release of Ca(2+) from internal stores.3 The increase in AP discharge rate produced by low concentrations of At II results from an acceleration of the pacemaker potential. Furthermore, in the presence of 10 mM tetraethylammonium (TEA), there is an acceleration of the repolarizing phase of AP.4 Ouabain (10(-3) M) inhibits the increase in rhythmic activity induced by low concentrations of At II (in the presence of 10 mM TEA), thus suggesting that the Na-K pump is directly or indirectly involved in this action of the peptide.5 At higher concentrations, At II produces a concentration-dependent depolarization with an EC(50) of 1.2 x 10(-8) M and a maximum of 10(-7) M. The associated contraction has an EC(50) of 3.3 x 10(-8) M and a maximum of 3 x 10(-7) M.6 Ouabain (3 x 10(-3) M) depolarizes the cell membrane. Under these conditions, At II (10(-7) M) has a slight depolarizing effect, but it still produces a large tonic contraction.7 It is concluded, that At II acts on different steps of excitation-contraction coupling, depending on the concentration. At low levels, the peptide mainly accelerates spike discharge, through a mechanism involving the Na-K pump. At higher concentrations, At II depolarizes the cell membrane. The contraction is then activated by the influx of Ca(2+) due to secondary AP discharge and the release of Ca(2+) from intracellular stores. Pharmacomechanical coupling has an important role in the triggering of contractions both at high and at low concentrations of At II.

The T-SR junction in contracting single skeletal muscle fibers.

The junction between the T system and sarcoplasmic reticulum (SR) of frog skeletal muscle was examined in resting and contracting muscles. Pillars, defined as pairs of electron-opaque lines bounding an electron-lucent interior, were seen spanning the gap between T membrane and SR. Feet, defined previously in images of heavily stained preparations, appear with electron-opaque interiors and as such are distinct from the pillars studied here. Amorphous material was often present in the gap between T membrane and SR. Sometimes the amorphous material appeared as a thin line parallel to the membranes; sometimes it seemed loosely organized at the sites where feet have been reported. Resting single fibers contained 39 +/- 14.3 (mean +/- SD; n = 9 fibers) pillars/micrometer2 of tubule membrane. Single fibers, activated by a potassium-rich solution at 4 degrees C, contained 66 +/- 12.9 pillars/micrometer2 (n = 8) but fibers contracting in response to 2 mM caffeine contained 33 +/- 8.6/micrometer2 (n = 5). Pillar formation occurs when fibers are activated electrically, but not when calcium is released directly from the SR; and so we postulate that pillar formation is a step in excitation-contraction coupling.

Activation of fast skeletal muscle: contributions of studies on skinned fibers.

The membrane potential of vertebrate twitch fibers closely controls Ca fluxes between intracellular compartments, which in turn control contraction. Recent work on intracellular Ca movement is reviewed in the general context of current efforts to synthesize physiological, biochemical, and structural observations on the contractile mechanism and its regulation, emphasizing the increasing role of functionally skinned fibers in this synthesis. Skinned fiber preparations, with removed or disrupted sarcolemma, bridge the gap between properties of isolated subsystems and their constrained operation in the intact fiber. Recent studies indicate that the surface action potential propagates along the transverse tubules, but not the sarcoplasmic reticulum (SR), which appears to be a distinct intracellular compartment. Voltage-dependent charge movements in the transverse tubules probably control Ca flux across the SR membranes. Current questions concern the mechanism of the signal that bridges the junctional gap between the two membrane systems, the mechanism and properties of the activated Ca efflux to the myofilament space, and the operation of the Ca pump of the SR during activation. New methods applied to intact fibers, cut fibers, skinned fibers, and subcellular systems are yielding the kind of information needed for a complete description of these central steps in excitation-contraction coupling and of Ca regulation of the myofilaments.

Length-dependent electromechanical coupling in single muscle fibers.

In single muscle fibers from the giant barnacle, a small decrease in muscle length decreases both the calcium activation and the peak isometric tension produced by a constant current stimulus. The effect is most pronounced if the length change immediately precedes the stimulation. In some cases, the decrease in tension with shortening can be accounted for almost entirely by a decrease in calcium release rather than changes in mechanical factors such as filament geometry. During the constant current stimulation the muscle membrane becomes more depolarized at longer muscle lengths than at the shorter muscle lengths. Under voltage clamp conditions, when the membrane potential is kept constant during stimulation, there is little length dependence of calcium release. Thus, the effect of length on calcium release is mediated through a change in membrane properties, rather than an effect on a subsequent step in excitation-contraction coupling. Stretch causes the unstimulated fiber membrane to depolarize by about l mV while release causes the fiber membrane to hyperpolarize by about the same amount. The process causing this change in potential has an equilibrium potential nearly 10 mV hyperpolarized from the resting level. This change in resting membrane potential with length may account for the length dependence of calcium release.

Charge movement and mechanical repriming in skeletal muscle.

1. Muscles were placed in a solution which depolarized the membrane to -30 to -20 mV so that mechanical activation was made refractory. Mechanical repriming and the recovery of voltage dependent charge movement were studied using a voltage clamp technique. 2. Mechanical repriming was investigated by determining the duration of a hyperpolarizing pulse required to elicit a just-visible contraction for various post-pulse potentials. As the post-pulse potential was made more positive shorter repriming times were required to produce a threshold contraction. The relationship approached a minimum repriming time for very positive post-pulse potentials. 3. These results suggest that hyperpolarization gradually removes some component of the activation mechanism from a refractory state and that the effectiveness of the amount which has recovered depends on the post-pulse potential. A quantitative explanation is given using a simple model in which the essential component is assumed to be the charge movement process. 4. The rate of repriming contraction is voltage dependent; at -160 mV the rate is about twice that at -120 mV. Between 4 and 10 degrees C the rate has a Q10 of about 9. 5. Recovery of charge movement was studied using a repriming duration less than that required to produce a threshold contraction. The observed charge movement increased linearly with repriming time, consistent with the approximately linear initial segment of a slow exponential recovery process. Extrapolation of the recovery curve indicated that 2-5 n/CmuF of charge is reprimed in the time necessary to reprime a threshold contraction. 6. The charge which recovers during a subthreshold repriming pulse is distributed according to membrane potential in the same way as a fully reprimed charge. 7. These results are consistent with the hypothesis that voltage dependent charge movement is an intermediate step in excitation-contraction coupling. 8. The characteristics of a second type of charge movement are also described.

Inhibition of membrane calcium activation by neomycin and streptomycin in crab muscle fibers.

Experiments were performed on single, isolated muscle fibers of the crabCallinectes danae to investigate the effects of neomycin and streptomycin on membrane Ca++ conductance in relation to excitation-contraction coupling (ECC). Neomycin (2–8 mg/ml) hyperpolarizes the fiber and increases the effective membrane resistance. The antibiotic increases the threshold for, and reduces the amplitude of the graded membrane responses which result from activation of Ca++ conductance on membrane depolarization. Treatment of the fiber with either procaine or tetraethylammonium (TEA) converts the graded responses into regenerative Ca-spikes. Neomycin (2–4 mg/ml) increases spike threshold, reduces the rate of rise of the upstroke and shortens the duration of the action potentials. With higher neomycin concentrations (up to 8 mg/ml) the spikes are abolished. Streptomycin (8 mg/ml) is also capable of blocking Ca-spikes. As a consequence of their effects on membrane Ca++ conductance the antibiotics reduce the mechanical output associated with both graded responses and Ca-spikes. The data support the postulate that membrane Ca++ activation is an essential step in ECC in skeletal muscle.

Inconstant association of aequorin luminescence with tension during calcium release in skinned muscle fibres.

IT is now generally accepted that the release of calcium ions from intracellular stores (presumably the sarcoplasmic reticulum (SR)) is a key step in excitation-contraction coupling in skeletal muscle1–4. One of the most useful preparations for studying the mechanism of release is the mechanically skinned muscle fibre preparation, originally devised by Natori5, and subsequently modified for use in aqueous solutions 6–9. The skinned fibre has the advantage that the cell membrane is removed, allowing free access of substances placed in the bathing medium to intracellular structures, but at the same time causing minimal disruption of these structures. The partially skinned (split) fibre has the further advantage that the patency of the T tubules is maintained13.

Membrane calcium activation in excitation-contraction coupling.

Depolarization thresholds for eliciting tension and Ca electrogenesis have been compared in isolated crayfish muscle fibers. Just-detectable tensions and Ca spikes induced after treatment with procaine were elicited with intracellularly applied depolarizing currents of fixed duration. Both thresholds were found to increase in a similar manner in fibers exposed to increased concentrations of Ca in the bathing solution or addition of other divalent cations (Mg, Mn, Ni). However, antagonistic effects between divalent cations were also demonstrated. Substitution of increasing amounts of NaSCN for NaCl in the standard saline produced a progressive decrease in both thresholds. The correlation in the change in thresholds for the two processes supports the hypothesis that a change in membrane Ca conductance is an integral step in excitation-contraction coupling.

The steps between depolarization and the increase in the respiration of frog skeletal muscle.

1. For many years it has been known that when muscles are depolarized by raising [K(+)](out) there is an increase in respiration, even at levels of depolarization below the threshold for a detectable contracture.2. K(+)-stimulated respiration occurs in muscles in which protein synthesis is blocked with puromycin. Stimulation does not depend upon activation of phosphorylase kinase. In muscle poisoned with IIA and kept in N(2), depolarizations below the threshold for contracture cause a fall in creatine phosphate. Apparently an ATPase is activated by depolarization; the resulting ADP is probably the trigger for the increase in oxygen uptake.3. When the T-tubules are destroyed by the glycerol-osmotic shock method depolarization does not produce an increase in respiration.4. Caffeine is known to stimulate respiration at concentrations below the threshold for producing a contracture. Muscles that have been made refractory to stimulation by potassium are still stimulated by caffeine: the action of caffeine is not antagonized by an increase in extracellular Mg(2+). Caffeine must act on a later step in excitation-contraction coupling.5. K(+)-stimulated respiration ultimately depends on the presence of Ca(2+) in the Ringer. However, the Ca(2+) can be replaced by Ni(2+). It is known that Ni(2+) does not activate actomyosin. Ni(2+) is not sequestered by isolated fragments of the sarcoplasmic reticulum. It seems that the Ni(2+) or Ca(2+) in the extracellular solution is required for a superficial step in excitation-contraction coupling.6. Respiration is also often stimulated when muscles are placed in an isotonic sucrose solution, even though the fibres are hyperpolarized. A trace amount of Ca(2+) in the sucrose solution is probably necessary for the response.7. An interaction between Ca(2+) and a superficial membrane receptor appears to be an essential, early step in excitation-contraction coupling.

The relation between membrane potential and contraction in single crayfish muscle fibres.

The series of events following the action of the nerve transmitter and leading to contraction is called excitation-contraction coupling (Sandow, 1952). Most investigators consider the first step in excitation-contraction coupling in striated muscle to be a reduction of the membrane potential (Kuffler, 1946; Katz, 1950; Sten-Knudsen, 1954, 1960; Watanabe, 1958; Huxley & Taylor, 1958; for contrary views see Csapo & Suzuki, 1958; Hoyle & Wiersma, 1958). The purpose of the present experiments, in a crustacean striated muscle, was twofold: first, to determine the relation between membrane potential and the development of tension; secondly, to determine how electrical stimulation of the muscle leads to contraction. With the aid of techniques for intracellular electrical stimulation and intracellular potential recording it was possible simultaneously to study the interrelations between membrane potential, transmembrane current and contraction in single muscle fibres.