No 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.

Despite long-continued interest in the subject, uncertainty still exists about some fundamental aspects of the osmotic behaviour of striated muscle cells. One unsettled problem has to do with the extent to which the fibre adjusts its volume in response to changes in the osmotic pressure of the bathing medium. There is abundant evidence, both from indirect observations and from direct measurements of volume, that under conditions in which one might expect the transfer of solutes across the cell membrane to be slight and over a moderate range of osmotic strengths, the striated muscle fibre exhibits ideal or nearly ideal osmotic behaviour (in the sense that it behaves as a freely distensible semipermeable bag containing a fixed amount of solute and a certain amount of solid or osmotically inactive matter) (e.g. Sato, 1954a; Dydynska & Wilkie, 1963;Reuben, Lopez, Brandt & Grundfest, 1963). However, recent studies in which changes in fibre volume were estimated from measurements of the widths of single muscle fibres have suggested departures from ideal behaviour at very high and very low osmotic strengths. Dydynska & Wilkie (1963) found that the widths of fibres in the sternocutaneus muscle of the frog did not decrease as much as would have been expected when the muscle was placed in solutions whose osmotic strength had been made more than twice normal by the addition of sucrose. This finding was in contrast with the results of their chemical estimates of fibre water in the sartorius muscle, which indicated that the muscle fibres exhibited ideal osmotic behaviour up to osmotic strengths at least four times that of the normal bathing solution. Reuben et al. (1963) estimated changes in the volume of isolated single fibres of the semitendinosus muscle of the frog, and found that the fibres appeared to swell less than might have been expected when the osmotic strength of the bathing solution was reduced by more than half. This finding is in qualitative agreement with the results of some chemical estimates of fibre water made on whole muscles by Grieve

Light emission from the calcium-sensitive bioluminescent protein aequorin was measured at calcium ion concentrations of 10(-9) to 10(-2) molar. At very low Ca2+ concentations, light emission is independent of calcium ion concentration. The maximum slope of the log-log plot of light as a function of calcium ion concentration is about 2.5. The complete relation is well described by a two-state model involving three calcium-binding sites.

Aequorin, which luminesces in the presence of calcium, was injected into photoreceptor cells of Limulus ventral eye. A bright light stimulus elicited a large increase in aequorin luminescence, the aequorin response, indicating a rise of intracellular calcium ion concentration, Ca/. The aequorin response reached a maximum after the peak of the electrical response of the photoreceptor, decayed during a prolonged stimulus, and returned to an undetectable level in the dark. Reduction of Ca, reduced the amplitude of the aequorin response by a factor no greater than 3. Raising Cao increased the amplitude of the aequorin response. The aequorin response became smaller when membrane voltage was clamped to successively more positive values. These results indicate that the stimulus-induced rise of Cai may be due in part to a light-induced influx of Ca and in part to release of Ca from an intracellular store. Our findings are consistent with the hypothesis that a rise in Cai is a step in the sequence of events underlying light-adaptation in Limulus ventral photoreceptors. Aequorin was also injected into photoreceptors of Balanus. The aequorin responses were similar to those recorded from Limulus cells in all but two ways: (a) A large sustained aequorin luminescence was measured during a prolonged stimulus, and (b) removal of extracellular calcium reduced the aequorin response to an undetectable level.