Calcium fluxes in single muscle fibres measured with a glass scintillator probe

Abstract

1. An intracellular glass scintillator (Caldwell & Lea, 1973) has been used to obtain a continuous record of the influx of 45Ca into single muscle fibres of the barnacle, Balanus nubilus. 2. In the presence of intracellular EGTA (final concentration greater than 3 mM/kg), the scintillator detected an initial fast phase to the influx (half-time = 18.3 min, compartment size = 4.1% fibre volume) followed by a slow, linear phase which gave a value for the Ca influx of 1.2 p-mole . cm-2 . sec-1. The efflux of 45Ca was also measured with the scintillator by transferring a 45Ca-loaded fibre into 45Ca-free saline. Two exponential phases of efflux were detected with half-times of 16.2 and 500 min. 3. The characterisitics of the fast phase of the influx and efflux are similar to those of the influx of the impermeant sucrose and inulin, suggesting that the fast phase represents exchange with the extracellular 'cleft space'. This phase was insensitive to external La3+ (2 mM). 4. The slow phase is considered to represent the flux of Ca across the surface membrane. It was inhibited by external La3+ (2 mM) and stimulated by replacing external Na+ with Li+. 5. When EGTA-injected fibres were depolarized using an axial, intracellular electrode the Ca influx, measured from the slow phase, was increased. At higher concentrations of intracellular EGTA (6--22 mM/kg), the extra Ca influx due to a rectangular, depolarizing current pulse was proportional to the number of Ca spikes it produced. A single Ca spike gave an extra Ca influx of 19--48 p-mole . cm-2. External D600 (5 x 10(-4)M) inhibited both Ca spike and the extra Ca influx. 6. At lower intracellular EGTA concentrations (3.6--11 mM/kg), a 50 mV depolarization of 250 msec duration gave a mean extra Ca influx of 80 p-mole . cm-2. The upper value was 145 p-mole . cm-2 and this would increase the total internal Ca by 4.1 micrometer/kg. It is calculated that if all this extra Ca was bound to the myofibrillar sites for tension, it would only produce 6.2% of the force expected for a similar depolarization in a fibre with no intracellular EGTA.