TIME COURSE OF CHEMICAL CHANGE AND ENERGY PRODUCTION DURING CONTRACTION OF FROG SKELETAL MUSCLE

Inhibition of sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBQ) in frog skeletal muscle fibers at 10 degrees C prolonged the half time of the fall of the Ca2+ transient by 62% and twitch force by 100% and increased peak force by 120% without increasing the amplitude of the Ca2+ signal. In the presence of TBQ the rate of relaxation and the rate of fall of Ca2+ became progressively slower in a series of twitches until relaxation failed. Relaxation rate decreased with a time course (approximately 2 s-1) similar to the Mg2+ off rate from purified parvalbumin (PA; 3.6 s-1). TBQ slowed the rate of fall of Ca2+ (5-fold) and force (8-fold) in a 0.3-s tetanus so that the rate of fall of Ca2+ (approximately 2.5 s-1) was similar to the Mg2+ off rate from PA. TBQ caused a near total failure of both Ca2+ sequestration and relaxation in a 1.1-s tetanus, during which PA would be saturated with Ca2+ and could not contribute to relaxation. Thus, when the SR Ca(2+)-ATPase is inhibited, Mg(2+)-PA can sequester Ca2+ and produce relaxation at a rate that is defined by the Mg2+ off rate from PA.

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Sodium channel gating currents in frog skeletal muscle.

The Journal of General Physiology ◽

10.1085/jgp.82.5.679 ◽

1983 ◽

Vol 82 (5) ◽

pp. 679-701 ◽

Cited By ~ 19

Author(s):

D T Campbell

Keyword(s):

Skeletal Muscle ◽

Sodium Channel ◽

Time Course ◽

Channel Gating ◽

Total Charge ◽

Charge Movement ◽

Frog Skeletal Muscle ◽

Gating Currents ◽

Gating Mechanism ◽

Charge Movements

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.

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Quantitative x-ray elemental imaging in stimulated single intact skeletal muscle fibers: The fate of calcium in the presence of ryanodine

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102535 ◽

1988 ◽

Vol 46 ◽

pp. 90-91

Author(s):

J. Sommer ◽

P. Ingram ◽

A. LeFurgey ◽

R. Nassar ◽

T. High

Keyword(s):

Skeletal Muscle ◽

Electrical Stimulation ◽

Time Course ◽

Imaging System ◽

Quantitative Imaging ◽

Muscle Fibers ◽

Frog Skeletal Muscle ◽

X Ray ◽

Skeletal Muscle Fibers ◽

Freeze Dried

We are involved in a continuing series of experiments aimed at a complete description,in terms of morphology and quantitative topochemistry, of the time course of spatial distributions of physiologically important elements during excitation-contraction coupling (ECC) at different time intervals (fractions of msec) following electrical stimulation of single, intact frog skeletal muscle fibers. In this present study wg report such distributions for Ca after 1,2 and 3 min of electrical stimulation in the presence of 2x10-4 M ryanodine, an alkaloid that, in time, causes irreversible muscle contractures.Single, intact frog skeletal muscle fibers were quick-frozen, cryosectioned, freeze-substituted and in one case freeze-fractured. The freeze-dried cryosections were subjected to electron probe X-ray microanalysis (EPXMA) in a JEOL 1200EX analytical electron microscope equipped with a Tracor Northern X-ray detector and a fully quantitative imaging system. Both, 64/64 pixel images (ambient temp.), and small raster probes (cold stage,-115 °C) for better statistics, were obtained, each from the same section.

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Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra.

The Journal of General Physiology ◽

10.1085/jgp.97.2.271 ◽

1991 ◽

Vol 97 (2) ◽

pp. 271-301 ◽

Cited By ~ 95

Author(s):

M Konishi ◽

S Hollingworth ◽

A B Harkins ◽

S M Baylor

Keyword(s):

Skeletal Muscle ◽

Time Course ◽

Dissociation Constants ◽

Diffusion Constant ◽

Preceding Paper ◽

Excitation Wavelength ◽

Frog Skeletal Muscle ◽

Fluorescent Indicator ◽

Single Action

Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations indicate that furaptra reacts with Ca2+ and Mg2+ with 1:1 stoichiometry, with dissociation constants of 44 microM and 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in a frog skeletal muscle fiber stimulated electrically, the indicator is expected to respond to the change in myoplasmic free [Ca2+] (delta[Ca2+]) with little interference from changes in myoplasmic free [Mg2+]. The apparent longitudinal diffusion constant of furaptra in myoplasm was found to be 0.68 (+/- 0.02, SEM) x 10(-6) cm2 s-1 (16-16.5 degrees C), a value which suggests that about half of the indicator was bound to myoplasmic constituents of large molecular weight. Muscle membranes (surface and/or transverse-tubular) appear to have some permeability to furaptra, as the total quantity of indicator contained within a fiber decreased after injection; the average time constant of the loss was 302 (+/- 145, SEM) min. In fibers containing less than 0.5 mM furaptra and stimulated by a single action potential, the calibrated peak value of delta[Ca2+] averaged 5.1 (+/- 0.3, SEM) microM. This value is about half that reported in the preceding paper (9.4 microM; Konishi, M., and S. M. Baylor. 1991. J. Gen. Physiol. 97:245-270) for fibers injected with purpurate-diacetic acid (PDAA). The latter difference may be explained, at least in part, by the likelihood that the effective dissociation constant of furaptra for Ca2+ is larger in vivo than in vitro, owing to the binding of the indicator to myoplasmic constituents. The time course of furaptra's delta[Ca2+], with average values (+/- SEM) for time to peak and half-width of 6.3 (+/- 0.1) and 9.5 (+/- 0.4) ms, respectively, is very similar to that of delta[Ca2+] recorded with PDAA. Since furaptra's delta[Ca2+] can be recorded at a single excitation wavelength (e.g., 420 nm) with little interference from fiber intrinsic changes, movement artifacts, or delta[Mg2+], furaptra represents a useful myoplasmic Ca2+ indicator, with properties complementary to those of other available indicators.

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Effects of previous activity on the energetics of activation in frog skeletal muscle.

The Journal of General Physiology ◽

10.1085/jgp.75.6.617 ◽

1980 ◽

Vol 75 (6) ◽

pp. 617-631 ◽

Cited By ~ 12

Author(s):

J A Rall

Keyword(s):

Skeletal Muscle ◽

Time Delay ◽

Time Course ◽

Frog Skeletal Muscle ◽

Active Force ◽

Force Development ◽

Uptake Sites ◽

Contractile Activation ◽

Terminal Cisternae ◽

Activation Heat

Effects of previous activity on the ability of frog skeletal muscle at 0 degrees C to liberate energy associated with contractile activation, i.e., activation heat (AH), have been examined. Earlier work suggests that activation heat amplitude (as measured from muscles stretched to lengths where active force development is nearly abolished) is related to the amount of Ca2+ released upon stimulation. After a twitch, greater than 2 s is required before a second stimulus (AHt) can liberate the same activation heat as a first stimulus (AH infinity), i.e., (AHt)/(AH infinity) = 1 -0.83 e-1.40t, where t is time in seconds. Caffeine introduces a time delay in the recovery of the ability to generate activation heat after a twitch. After a tetanus, the activation heat is depressed to a greater extent at any time than after a twitch. The activation heat elicited by a stimulus 1 s after a tetanus is depressed progressively with respect to tetanus duration up to 3 s. For tetani of 3, 40, and 80 s duration the postetanus activation heat is comparably depressed. The time-course of the recovery of the ability of the muscle to produce activation heat after a tetanus can be described as (AHt)/(AH infinity) = 1 -0.80 e-0.95t -0.20 e-0.02t. Greater than 90 s is required before the posttetanus activation heat is equal to the pretetanus value. The faster phase of recovery is similar to recovery after the twitch and the slower phase may be associated with the return of calcium to the terminal cisternae from uptake sites in the longitudinal sarcoplasmic reticulum.

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Model of Sarcomeric Ca2+ Movements, Including ATP Ca2+ Binding and Diffusion, during Activation of Frog Skeletal Muscle

The Journal of General Physiology ◽

10.1085/jgp.112.3.297 ◽

1998 ◽

Vol 112 (3) ◽

pp. 297-316 ◽

Cited By ~ 119

Author(s):

S.M. Baylor ◽

S. Hollingworth

Keyword(s):

Skeletal Muscle ◽

Action Potential ◽

Metal Binding ◽

Time Course ◽

Current Knowledge ◽

Diffusion Constant ◽

Compartment Model ◽

Half Width ◽

Frog Skeletal Muscle ◽

And Diffusion

Cannell and Allen (1984. Biophys. J. 45:913–925) introduced the use of a multi-compartment model to estimate the time course of spread of calcium ions (Ca2+) within a half sarcomere of a frog skeletal muscle fiber activated by an action potential. Under the assumption that the sites of sarcoplasmic reticulum (SR) Ca2+ release are located radially around each myofibril at the Z line, their model calculated the spread of released Ca2+ both along and into the half sarcomere. During diffusion, Ca2+ was assumed to react with metal-binding sites on parvalbumin (a diffusible Ca2+- and Mg2+-binding protein) as well as with fixed sites on troponin. We have developed a similar model, but with several modifications that reflect current knowledge of the myoplasmic environment and SR Ca2+ release. We use a myoplasmic diffusion constant for free Ca2+ that is twofold smaller and an SR Ca2+ release function in response to an action potential that is threefold briefer than used previously. Additionally, our model includes the effects of Ca2+ and Mg2+ binding by adenosine 5′-triphosphate (ATP) and the diffusion of Ca2+-bound ATP (CaATP). Under the assumption that the total myoplasmic concentration of ATP is 8 mM and that the amplitude of SR Ca2+ release is sufficient to drive the peak change in free [Ca2+] (Δ[Ca2+]) to 18 μM (the approximate spatially averaged value that is observed experimentally), our model calculates that (a) the spatially averaged peak increase in [CaATP] is 64 μM; (b) the peak saturation of troponin with Ca2+ is high along the entire thin filament; and (c) the half-width of Δ[Ca2+] is consistent with that observed experimentally. Without ATP, the calculated half-width of spatially averaged Δ[Ca2+] is abnormally brief, and troponin saturation away from the release sites is markedly reduced. We conclude that Ca2+ binding by ATP and diffusion of CaATP make important contributions to the determination of the amplitude and the time course of Δ[Ca2+].

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Side bridge geometry after quick-freezing of stimulated and unstimulated frog skeletal muscle fibers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076056 ◽

1983 ◽

Vol 41 ◽

pp. 464-465

Author(s):

J.R. Sommer ◽

R. Nassar ◽

S. Walker

Keyword(s):

Skeletal Muscle ◽

Time Course ◽

Muscle Fibers ◽

Freeze Fracture ◽

Freeze Substitution ◽

Frog Skeletal Muscle ◽

Specimen Holder ◽

Skeletal Muscle Fibers ◽

Quick Freezing ◽

The Impact

Quick-freezing allows the structural analysis of timed perturbations of morphology. We are presenting preliminary results concerning the feasibility of studying directly the side bridge geometry of actin-myosin interactions within the time course of a twitch in single intact frog skeletal muscle fibers, both by freeze-substitution and freeze-fracture after quick-freezing, and following various time intervals between stimulation and impact of the fibers on a liquid He-cooled copper block.Materials and Methods. The quick-freezing device was a "Slammer"(Polaron) for which the electronics had been redesigned; they are capable, in combination with a Grass S48 stimulator, of any stimulation interval between 0 and 1 sec prior to freezing, including tetanus. The actual elapsed time between stimulation and freezing is recorded with a digital clock. Single intact tendonto- tendon frog skeletal muscle fibers (semitendinosus of r. temporaria) or toe muscle bundles (r.pipiens) were isolated by sharp dissection and placed between coextensive Pt stimulation wires on blackened 2% agarose, the height of which on the specimen holder was adjusted appropriately with respect to a spacer ring both, to calibrate the impact time and to prevent smashing of the fibers.

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Repriming of delayed potassium conductance in frog skeletal muscle

Journal of Experimental Biology ◽

10.1242/jeb.87.1.217 ◽

1980 ◽

Vol 87 (1) ◽

pp. 217-228

Author(s):

E. W. Ballou

Keyword(s):

Skeletal Muscle ◽

Time Course ◽

Rana Pipiens ◽

Voltage Dependence ◽

Potassium Conductance ◽

Frog Skeletal Muscle ◽

Semitendinosus Muscle ◽

Sequential Model ◽

Temperature Dependences ◽

High Concentrations

A dissection of the semitendinosus muscle from Rana pipiens was developed for three-microelectrode voltage-clamp studies of the delayed potassium-selective conductance system. The delayed conductance inactivates in muscles bathed in high concentrations of potassium or rubidium, but can be reprimed by hyperpolarizing voltage pulses to membrane potentials beyond −80 mV. The repriming time-course was studied by measuring the delayed conductance that coulde be activated following hyperpolarizing pulses of varying duration. Responses following 20–100 s pulses to potentials between −90 and −140 mV could not be reconciled with an exponential approach to the conductance present in normally polarized fibres. The sigmoid appearance of the early (< 25 s) time course was exaggerated by cooling from 20 to 10 degrees C. This effect was described by a sequential model invoking two inactivated states with different temperature dependences. An explanation is suggested for differences in the kinetics and voltage dependence of repriming between briefly and chronically depolarized muscle cells.

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Energy coupling to sodium transport in frog skeletal muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1978.235.1.c25 ◽

1978 ◽

Vol 235 (1) ◽

pp. C25-C34 ◽

Cited By ~ 5

Author(s):

R. J. Connett ◽

E. T. Hays

Keyword(s):

Skeletal Muscle ◽

Energy Production ◽

Atp Hydrolysis ◽

Energy Coupling ◽

Creatine Phosphate ◽

Coupling Factor ◽

Metabolic Energy ◽

Frog Skeletal Muscle ◽

Energy Turnover ◽

Sodium Efflux

In addition to a strophanthidin-sensitive (SS) sodium efflux, a large component of the sodium efflux in freshly isolated frog skeletal muscle is sodium-activated and strophanthidin-insensitive (SASI). The amount of metabolic energy associated with sodium movement by each of these components was measured and the coupling between sodium movement and adenosine 5'-triphosphate (ATP) hydrolysis in muscle was calculated. Energy production was blocked by iodoacetate and cyanide. Energy turnover was estimated from the change in creatine phosphate (CrP) and ATP contents and expressed as potential energy (PE = CrP + 2ATP). After metabolic poisoning a linear fall of PE occurred (6.3 mumol/g.h). Metabolic poisoning had no effect on the magnitude of the SS or SASI components of sodium efflux. In 2 h the sodium moved, and PE change due to the SS component was 4.35 and 1.66 mumol/g.h, respectively, which gave a coupling factor of 2.6. The amount of sodium moved by the SASI component was similar to that moved by the SS component in 2 h whereas no energy change was observed. It was, therefore, concluded that sodium movement by the SASI component requires no energy input.

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Altered sodium and gating current kinetics in frog skeletal muscle caused by low external pH.

The Journal of General Physiology ◽

10.1085/jgp.84.5.771 ◽

1984 ◽

Vol 84 (5) ◽

pp. 771-788 ◽

Cited By ~ 19

Author(s):

D T Campbell ◽

R Hahin

Keyword(s):

Skeletal Muscle ◽

Time Constant ◽

Time Course ◽

Voltage Dependence ◽

Low Ph ◽

Na Channel ◽

Frog Skeletal Muscle ◽

Gating Current ◽

Gating Charge ◽

External Ph

The effect of low pH on the kinetics of Na channel ionic and gating currents was studied in frog skeletal muscle fibers. Lowering external pH from 7.4 to 5.0 slows the time course of Na current consistent with about a +25-mV shift in the voltage dependence of activation and inactivation time constants. Similar shifts in voltage dependence adequately describe the effects of low pH on the tail current time constant (+23.3 mV) and the gating charge vs. voltage relationship (+22.1 mV). A significantly smaller shift of +13.3 mV described the effect of pH 5.0 solution on the voltage dependence of steady state inactivation. Changes in the time course of gating current at low pH were complex and could not be described as a shift in voltage dependence. tau g, the time constant that describes the time course of the major component of gating charge movement, was slowed in pH 5.0 solution by a factor of approximately 3.5 for potentials from -60 to +45 mV. We conclude that the effects of low pH on Na channel gating cannot be attributed simply to a change in surface potential. Therefore, although it may be appropriate to describe the effect of low pH on some Na channel kinetic properties as a "shift" in voltage dependence, it is not appropriate to interpret such shifts as a measure of changes in surface potential. The maximum gating charge elicited from a holding potential of -150 mV was little affected by low pH.(ABSTRACT TRUNCATED AT 250 WORDS)

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