The efflux of calcium from single crab and barnacle muscle fibres

Abstract

1. The efflux of calcium, as the isotope (45)Ca, has been investigated from single muscle fibres from the barnacle Balanus nubilus and from the crab Maia squinado.2. If the isotope was initially injected with sufficient calcium (5-65 mM) to cause a contraction, the efflux did not follow first order kinetics. There was an early rapid phase which reached a peak after 5-10 min and then declined slowly over a period of 50-150 min to a low residual value.3. Injection of the isotope with the calcium-binding agent EGTA, so that the injected free calcium concentration was ca. 2 x 10(-8)M, abolished the initial rapid loss of calcium. The efflux rose to give a steady value after 10-15 min and its magnitude was similar to the value of the residual efflux.4. The rate constant for the low residual loss was ca. 7 x 10(-4) min(-1) for Maia and ca. 17 x 10(-4) min(-1) for Balanus. The rate constant predicted a calcium efflux of 0.4 p-mole/cm(2).sec for Maia and 1-2 p-mole/cm(2).sec for Balanus at 16-25 degrees C based on the total fibre calcium concentration.5. The residual calcium efflux was not affected by 0.5 mM ouabain or 0 potassium salines applied externally. It was stimulated, some 10-15 times in Maia and to a lesser extent in Balanus, by salines containing 600 mM potassium or 2-5 mM caffeine. The increased efflux was associated with a brisk contraction.6. External application of salines containing 20, 40 or 60 mM potassium or 0.5 mM caffeine in Maia produced some stimulation of the residual efflux but no visible contraction.7. Pre-treatment of Maia fibres with 40 mM potassium or 0.5 mM caffeine salines abolished the ability of the fibres to respond to higher concentrations of these agents. A depletion of a releasable calcium fraction by these subthreshold stimuli could explain this phenomenon.8. Electrical stimulation, the injection of 50 mM calcium chloride or 50 mM caffeine produced an elevated calcium efflux which was associated with a contraction.9. Intracellular injections of EGTA only lowered the residual efflux by up to half its initial value. This suggests that calcium can be released rapidly within these muscle fibres and that the sarcoplasmic calcium concentration is not much altered from its normal value by the injection.10. The experiments suggest that in Maia, changes in the calcium efflux reflect in magnitude, but not in time course, the internal calcium changes which can be observed with the calcium-sensitive protein, aequorin.