Autoradiographic Studies of Intracellular Calcium in Frog Skeletal Muscle

Abstract

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 Ca 45 . The muscles had been fixed with an oxalate-containing osmium solution at rest at room temperature, at rest at 4 0°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 /A 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 /j) 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.A considerable body of evidence has been accumulated to suggest that calcium plays a critical role in excitation-contraction coupling in muscle. Small amounts of calcium are necessary for maximum contraction and maximum adenosine-triphosphatase activity in muscle models (1, 31) Associated with contraction of intact muscle fibers there is an accelerated exchange of calcium probably involving both membrane and intracellular calcium (2, 3). Hill has shown on theoretical grounds that insufficient time exists between membrane depolarization and muscle activation for a substance to diffuse from the cell surface to the cell center (4). Therefore, the "activator" which directly initiates the contraction must either be transmitted by a process faster than diffusion or must be stored inside the cell at a site near the contractile proteins. Huxley and Taylor have shown that the membrane excitation may be con-455

Intracellular calcium translocation during the contraction–relaxation cycle in scorpionfish swimbladder muscle

“…Intracellular calcium translocation during the contraction-relaxation cycle in scorpionfish swimbladder muscle is taken up by the LT and the FC of the SR, thus supporting the observations of Winegrad (1965Winegrad ( , 1968 in frog skeletal muscle.…”

“…during relaxation In the present study, we have clearly answered the question of whether the myoplasmic Ca 2+ is taken up by the LT (Winegrad, 1965(Winegrad, , 1968 or by the TC (Somlyo et al, 1981) during relaxation of vertebrate striated muscle fibres. The answer we have obtained is intermediate between the above two cases; after onset of relaxation in the SBM fibres, the myoplasmic Ca 2+ is taken up not only by the LC and FC but also by the TC (Fig.·4, Table·2), which is consistent with the uniform distribution of Ca pump proteins all over the SR membrane except for the triadic junctional region (Jorgensen et al, 1979;Saito et al, 1984).…”

“…In vertebrate skeletal muscle, the myoplasmic Ca 2+ concentration is regulated by the release of Ca 2+ from, and its uptake by, the sarcoplasmic reticulum (SR) (Ebashi and Endo, 1968). Using 45 Ca autoradiography and quick-freezing techniques, Winegrad (1965Winegrad ( , 1968 investigated intracellular Ca translocation during the contraction-relaxation cycle in frog skeletal muscle, and reported that the Ca 2+ released from the terminal cisternae (TC) of the SR into the myoplasm is taken up at the longitudinal tubules of the SR during relaxation, and then gradually returns to the TC. Subsequent development of electron probe X-ray microanalysis technique has made it possible to study the intracellular Ca distribution with much higher spatial resolution.…”

The measurement and dynamic implications of thin filament lengths in heart muscle.

“…One of the rat atrial trabeculae that had been embedded for transverse sectioning was cut from its block and remounted to allow subsequent sectioning with the filaments parallel to the knife edge. The sarcomere length in these longitudinal sections, after a 6 % shrinkage correction (Page, 1974) (Winegrad, 1965) and the loss is likely to be even less and more uniform with the harder embedding material used in these experiments. Any loss in material during sectioning will, however, be compensated for by the use of the thick filament and the sarcomere length for calibration.…”

Ultrastructure of heart muscle.

“…The structural complexes of junctional SR, junctional processes and plasmalemma in both cardiac and skeletal muscle are called "couplings" (10) and their consistent occurrence in most muscles has led to the tempting assumption that they are the anatomical sites at which the action potential is translated into calcium release for contraction. Indeed, calcium movements toward the Z line (where most couplings are located during relaxation), and toward the A band during contraction have been intimated by several kinds of experiments (34,35). Nevertheless, the direct demonstration of Ca2+ release from junctional SR has not been accomplished as yet, and although the free and junctional SR are clearly Ca2+ sinks, it does not necessarily follow that Ca2+ is released for contraction from either or both.…”