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

Abstract

1. 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 Ca2+-free solutions had no influence on either the mechanical or the luminescent responses in twitches. In Ca2+-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. 10. In K+-contractures and during the plateaus of tetani the aequorin signal changes slowly enough so that it seems unlikely that substantial gradients of [Ca2+] exist at the sarcomere level. Under such circumstances the amplitude of the aequorin response probably does reflect the [Ca2+] in equilibrium with the myofilaments.