Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin.

Abstract

SUMMARY1. Single twitch muscle fibres isolated from frogs and toads were microinjected with the Ca2+-sensitive bioluminescent protein aequorin. The fibres contracted normally and emitted flashes of light (aequorin responses) in response to stimulation for many hours thereafter.2. No luminescence was detected from healthy fibres at rest. 3. The aequorin diffused from the site of injection at a rate consistent with a diffusion coefficient of 5 x 10-8 cm2/sec.4. During trains of isometric contractions there was a progressive reduction in both the amplitude and the rate of decline of the aequorin response, an observation consistent with the theory that Ca is redistributed from sites of release to sites of sequestration under such circumstances.5. In isometric tetani light emission continued to rise long after the plateau of force had been achieved. This and the fact that the amplitude of the tetanic aequorin response increased steeply with increasing stimulus frequency suggest that in tetani the sarcoplasmic [Ca2+] may normally be above the level required to saturate the contractile apparatus.6. Both in twitches and in tetani the amplitude of the aequorin response increased slightly and then decreased substantially as the fibre was stretched progressively beyond slack length.7. In potassium contractures the luminescent and mechanical responses first became detectable at about the same [K+], but for equivalent force luminescence was less intense than in twitches. The aequorin response was biphasic in solutions of high [K+].8. Exposure of the fibre to Ca 2+-free solutions had no influence on either the mechanical or the luminescent responses in twitches. In Ca 2+-free solutions tetanic aequorin responses tended not to be maintained as well as normally, suggesting that intracellular Ca stores do become somewhat depleted.9. In twitches the amplitude of the aequorin response probably reflects the amount of Ca2+ liberated into the cytoplasm rather than a [Ca2+] in equilibrium with the myofilaments. Changes in the rate of decay of the aequorin response may reflect changes in the rate of Ca sequestration by the sarcoplasmic reticulum.

Effects of twitch train on the tetanic contractility of the frog skeletal muscle.

“…The calcium transients during repetitive stimulation and tetanus were also investigated by MILEDI et al (1982) in frog's twitch muscle by using arsenzao III. In agreement with BLINKS et al (1978) they observed a negative staircase of the amplitude of light response together with slowing of its decay (summation). In this respect, the recent observation by HOMSHER and KEAN (1977) that the activation heat per stimulus declined with decrease in the stimulus interval would be interesting.…”

“…The effect may not interfere with the progressing positive staircase per se, within a certain limit of number of stimuli, but could well influence the following tetani by decreasing the supply of activator calcium to the releasing sites. Applying calcium-sensitive bioluminescent aequorin, BLINKS et al (1978) measured the calcium transients in the single twitch muscle fiber of frog and observed a descending staircase of the peak-light intensity when repetitive stimulation was applied. This was interpreted as reflecting a gradual decline of sequestration of calcium by the sarcoplasmic reticulum.…”

Rapid relaxation of single frog skeletal muscle fibres following laser flash photolysis of the caged calcium chelator, diazo‐2

“…In addition the relation between the fall of free Ca 2+ and rate of relaxation has been studied in intact muscle fibres [13,[16][17][18] but these studies on relaxation are limited by the rate at which free calcium can be removed from the sarcoplasm, presumably mainly by the activity of the sarcoplasmic reticulum calcium pump (14 s -1 at 10°C [14] or 25 s -1 following a single twitch decreasing to 10 s -1 after a tetanus of at least 2 s duration [13]). There is also an elevation or 'tail' to the free calcium fall which extends over 5 s in frog at 10°C [13] or 1-2 s in arthropod fibres at 20°C [18].…”

Contractile basis of ameboid movement. VII. Aequorin luminescence during ameboid movement, endocytosis, and capping.

“…The micropipette (arrow) and the tail of the cell are shown by raising the background illumination (A) . The damage response involves an intensive but localized rise in the free calcium ion concentration (8), which causes cortical contraction. Recorded in 1/60 s. luminescence in the tail (I-5-s duration) (Fig.…”

“…The high signal to noise ratio, relative specificity for Ca", lack of toxicity, and ease of signal detection have made this technique attractive for the study of cells containing low free calcium ion concentrations . Aequorin luminescence has already been applied successfully to other motile processes, including the contraction of muscle (3,4,8,23), shuttle streaming in Physarum polycephalum (58), fertilization of Medaka eggs (59), and the contraction of Spirostomum (26) .…”