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

Abstract

An intracellular glass scintillator (Caldwell et Lea, 1973) was used to obtain a continuous record of the influx of 45Ca into single muscle fibers of the barnacle, Balanus nubilus. In the presence of intracellular EGTA [ethylene glycol bis (.beta.-aminoethyl ether) tetraacetic acid] (final concentration > 2 mM/kg), the scintillator detected an initial fast phase to the influx (half-time = 18.3 min, compartment size = 4.1% fiber volume) followed by a slow, linear phase which gave a value for the Ca influx of 1.2 pmol .cntdot. cm-2 .cntdot. s-1. The 45Ca efflux was measured with the scintillator by transferring a 45Ca-loaded fiber into 45Ca-free saline. Two exponential phases of efflux were detected with half-times of 16.2 and 500 min. The characteristics of the fast phases of influx and efflux were similar to those of the influx of the impermeant sucrose and inulin, suggesting that the fast phase represented exchange with the extracellular cleft spece. This phase was insensitive to external La3+ (2 mM). The slow phase probably represented the Ca flux across the surface membrane. It was inhibited by external La3+ (2 mM) and stimulated by replacing external Na+ with Li+. When EGTA-injected fibers 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 pmol .cntdot. cm-2. External D600 [.alpha. isopropyl-.alpha.[N-methyl-N-homoveratryl-.alpha.-aminopropyl]-3,4,5-trimethoxyphenyl acetonitrile] (5 .times. 10-4 M) inhibited Ca spike and the extra Ca influx. At lower intracellular EGTA concentrations (3.6-11 mM/kg), a 50 mV depolarization of 250 ms duration gave a mean extra Ca influx of 80 pmol .cntdot. cm-2. The upper value was 145 pmol .cntdot. cm-2 and this would increase the total internal Ca by 4.1 .mu.M/kg. 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 fiber with no intracellular EGTA.