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

1991 ◽  
Vol 97 (2) ◽  
pp. 271-301 ◽  
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.

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

1998 ◽  
Vol 112 (3) ◽  
pp. 297-316 ◽  
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+].

Effect of diabetes and insulin treatment of diabetic rats on total RNA, poly(A)+ RNA, and mRNA in skeletal muscle

1991 ◽  
Vol 260 (3) ◽  
pp. C409-C416 ◽  
Author(s):  
J. D. Kent ◽  
S. R. Kimball ◽  
L. S. Jefferson
Keyword(s):  
Skeletal Muscle ◽  
Ribosomal Rna ◽  
Time Course ◽  
Diabetic Rats ◽  
In Vitro Translation ◽  
Specific Gene ◽  
Insulin Administration ◽  
Tissue Mass ◽  
Total Rna

We have assessed the time course of alterations in several biochemical parameters and expression of specific mRNAs in gastrocnemius muscle following both the induction of diabetes and the administration of insulin to diabetic rats. Muscle mass, total RNA, and total protein were reduced, whereas poly(A)+ RNA relative to total RNA was increased following the induction of diabetes. All the above parameters, with the exception of poly(A)+ RNA, were reciprocally and rapidly altered following administration of insulin to 3-day diabetic animals. These changes suggest that during the induction of diabetes 1) total cellular protein is reduced at a rate that is less than the reduction in gastrocnemius mass, whereas RNA is reduced at a rate 1.5 times the reduction in tissue mass, and 2) poly(A)+ RNA is elevated relative to total RNA. After insulin administration, there appears to be coordinate synthesis of both poly(A)+ RNA and ribosomal RNA, assuming 85% of total RNA is ribosomal. Therefore, we conclude that poly(A)+ RNA is more stable than ribosomal RNA during diabetes, whereas the amounts of poly(A)+ RNA and ribosomal RNA are increased at the same rates following insulin administration to diabetic animals. Analysis of expression of specific gene products over the same time course, as assessed by in vitro translation of total RNA followed by two-dimensional gel analysis, suggests that there are a few mRNAs that are very rapidly altered in response to insulin administration. The mRNAs that are altered demonstrate variable temporal patterns of either repression or full or transient expression. These rapid, but limited, alterations in gene expression may prove important in the development of the defects that occur in skeletal muscle in response to diabetes.

scholarly journals Simultaneous measurement of Ca2+ in muscle with Ca electrodes and aequorin. Diffusible cytoplasmic constituent reduces Ca(2+)-independent luminescence of aequorin.

1991 ◽  
Vol 98 (6) ◽  
pp. 1141-1160 ◽  
Author(s):  
L A Blatter ◽  
J R Blinks
Keyword(s):  
Skeletal Muscle ◽  
Simultaneous Measurement ◽  
Light Emission ◽  
Ringer Solution ◽  
Light Signal ◽  
Frog Skeletal Muscle ◽  
Stable Level ◽  
Total Absence ◽  
Normal Ringer

Estimates of cytoplasmic Ca2+ concentration ([Ca2+]i) were made essentially simultaneously in the same intact frog skeletal muscle fibers with aequorin and with Ca-selective microelectrodes. In healthy fibers under truly resting conditions [Ca2+]i was too low to be measured reliably with either technique. The calibration curves for both indicators were essentially flat in this range of [Ca2+], and the aequorin light signal was uniformly below the level to be expected in the total absence of Ca2+. When [Ca2+]i had been raised to a stable level below the threshold for contracture by increasing [K+]o to 12.5 mM, [Ca2+]i was 38 nM according to aequorin and 59 nM according to the Ca-selective microelectrodes. These values are not significantly different. Our estimates of [Ca2+]i are lower than most others obtained with microelectrodes, probably because the presence of aequorin in the cells allowed us to detect damaging microelectrode impalements that otherwise we would have had no reason to reject. The observation that the light emission from aequorin-injected fibers in normal Ringer solution was below the level expected from the Ca(2+)-independent luminescence of aequorin in vitro was investigated further, with the conclusion that the myoplasm contains a diffusible macromolecule (between 10 and 30 kD) that interacts with aequorin to reduce light emission in the absence of Ca2+.

Parvalbumin relaxes frog skeletal muscle when sarcoplasmic reticulum Ca(2+)-ATPase is inhibited

1996 ◽  
Vol 270 (2) ◽  
pp. C411-C417 ◽  
Author(s):  
Y. Jiang ◽  
J. D. Johnson ◽  
J. A. Rall
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Relaxation Rate ◽  
Time Course ◽  
Muscle Fibers ◽  
Peak Force ◽  
Half Time ◽  
Frog Skeletal Muscle ◽  
Twitch Force ◽  
Total Failure

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.

scholarly journals In Vitro Study of Mechanical and Kinetic Properties of Myosin II from Frog Skeletal Muscle

2009 ◽  
Vol 96 (3) ◽  
pp. 614a
Author(s):  
Ravikrishnan Elangovan ◽  
Marco Capitanio ◽  
Francesco S. Pavone ◽  
Vincenzo Lombardi
Keyword(s):  
Skeletal Muscle ◽  
Myosin Ii ◽  
In Vitro Study ◽  
Vitro Study ◽  
Kinetic Properties ◽  
Frog Skeletal Muscle

In vivo expression patterns of MyoD, p21, and Rb proteins in myonuclei and satellite cells of denervated rat skeletal muscle

2004 ◽  
Vol 287 (2) ◽  
pp. C484-C493 ◽  
Author(s):  
Minenori Ishido ◽  
Katsuya Kami ◽  
Mitsuhiko Masuhara
Keyword(s):  
Skeletal Muscle ◽  
Satellite Cell ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Expression Patterns ◽  
Cell Types ◽  
Cell Nuclei ◽  
Myogenic Regulatory Factor

MyoD, a myogenic regulatory factor, is rapidly expressed in adult skeletal muscles in response to denervation. However, the function(s) of MyoD expressed in denervated muscle has not been adequately elucidated. In vitro, it directly transactivates cyclin-dependent kinase inhibitor p21 (p21) and retinoblastoma protein (Rb), a downstream target of p21. These factors then act to regulate cell cycle withdrawal and antiapoptotic cell death. Using immunohistochemical approaches, we characterized cell types expressing MyoD, p21, and Rb and the relationship among these factors in the myonucleus of denervated muscles. In addition, we quantitatively examined the time course changes and expression patterns among distinct myofiber types of MyoD, p21, and Rb during denervation. Denervation induced MyoD expression in myonuclei and satellite cell nuclei, whereas p21 and Rb were found only in myonuclei. Furthermore, coexpression of MyoD, p21, and Rb was induced in the myonucleus, and quantitative analysis of these factors determined that there was no difference among the three myofiber types. These observations suggest that MyoD may function in myonuclei in response to denervation to protect against denervation-induced apoptosis via perhaps the activation of p21 and Rb, and function of MyoD expressed in satellite cell nuclei may be negatively regulated. The present study provides a molecular basis to further understand the function of MyoD expressed in the myonuclei and satellite cell nuclei of denervated skeletal muscle.

scholarly journals Sodium channel gating currents in frog skeletal muscle.

1983 ◽  
Vol 82 (5) ◽  
pp. 679-701 ◽  
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.

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

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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