CALCIUM TRANSIENTS AND CALCIUM RELEASE IN RAT FAST-TWITCH SKELETAL-MUSCLE FIBERS

1. Calcium transients were recorded from cut segments of fast-twitch rat skeletal muscle fibres stretched to 3.7-4.0 mum per sarcomere and voltage clamped at a holding potential of - 80 mV using the double Vaseline-gap technique. Calcium transients were monitored simultaneously with the two calcium indicators antipyrylazo III (AP III) and fura-2. AP Ill was used to record the calcium changes in response to 10-200 ms depolarizing pulses to different membrane potentials while fura-2 monitored the slow decay of the transient (during 16-20 s) and the resting calcium concentration. Experiments were performed at 14-17-degrees-C. 2. For 50-100 ms depolarizing pulses calcium transients were first detected between - 30 and - 20 mV in a total of twenty-one fibres. The transients recorded with AP III showed a plateau for small pulses (- 20 mV) and a steady increase during stronger pulses (- 10 mV and more positive). Upon repolarization the transients decayed towards the baseline. The signal recorded simultaneously with fura-2 showed a continuous increase of the transient during the pulses at all membrane potentials. The amplitude of the calcium transients for the large pulses could not be followed with fura-2 due to saturation of the dye. 3. The signals obtained with both dyes were used to determine the kinetics of the calcium-fura-2 reaction inside the fibres. The mean values of the kinetic parameters were: the on rate constant (k(on)) = 5.1 x 10(8) m-1 s-1, the off rate constant (k(off)) 26 s-1, and k(off)/k(on) (K(D)) = 69.7 nm. 4. The fast phase of decay of the calcium transients after the pulses was studied from the records obtained with AP III. For depolarizing pulses of the same duration, the rate of decay of the transients after the pulse was slower the stronger the depolarization. For pulses to the same membrane potential, the rate of decay was slower the longer the pulse duration. Both stimulating patterns indicated saturation of the removal system in the muscle fibres due to occupancy of slowly equilibrating myoplasmic calcium binding sites by released calcium. 5. The fast phase of decay of the signals obtained with AP III was well fitted with a model of the system for removing calcium from the myofilament space. 6. The rate of calcium release (R(rel)) from the sareoplasmic reticulum was calculated once the removal system was characterized in the same fibre. The R(rel) waveform during a pulse consisted of a fast initial peak and a steady level of smaller amplitude. The peak appeared for depolarizations about 10 mV more positive than needed to see the steady level in some fibres. The average peak release for pulses to 0 or + 10 mV was 7.5 mum ms-1 with a range of variation from 3.0 to 17.5 mum ms-1. 7. In addition to the fast decay of [Ca2+] studied with AP III, we examined a slower decay of [Ca2+] after the pulse using fura-2 signals sampled for up to 20 s after a pulse. The time course of the slow decay was analysed and quantified by fitting the records with two exponential functions plus a constant. The mean values of the time constants (tau) and their amplitudes (A) were: tau(s1) = 0.81 s, A(s1) = 33.3 nm; T(s2) = 15.8 s, A(s2) = 12.4 nm. The value of the constant was set as the resting calcium concentration measured at the baseline level before the depolarizing pulse for each of the eleven fibres studied.