The effect of phosphate on the relaxation of frog skeletal muscle

The effect of phosphate on the relaxation of isometrically contracting sing le skinned fibres from the semitendinosus muscle of the frog Rana temporari a has been investigated using laser pulse photolysis of the photolabile cag ed calcium-chelator diazo-2 to rapidly reduce the Ca2+ (<2 ms) within the f ibre and produce >90% relaxation of force. Relaxation occurred in two phase s - an initial linear shoulder which lasted approximately 20 ms followed by a double-exponential phase which gave two rate constants, k(1) (43.4+/-1.8 s(-1), mean +/-SEM, n=14) and k(2) (15.6+/-0.3 s(-1), mean +/-SEM, n=14) a t 12 degrees C. Increased phosphate concentrations did not affect the linea r phase, but slowed the double-exponential phase following photolysis of di azo-2 in a dose-dependent fashion (k(50)=0.9 mM for k(1), 1.15 mM for k(2)) . Reducing the concentration of contaminating phosphate (from 640 mu M to 1 00 mu M) led to an increase in the rate of the double-exponential phase (k( 1)=67.1+/-4.4 s(-1), k(2)=19.7+/-0.6 s(-1), mean+/-SEM, n=12). Time-resolve d measurements of sarcomere length during relaxation, both in control fibre s and in the presence of a raised phosphate concentration, reveal a <2% cha nge throughout the whole relaxation transient, and less than 0.1% at the en d of the linear phase. This finding implies that gross changes in sarcomere length do not contribute to the decay of the relaxation transient seen upo n diazo-2 photolysis. Our results suggest that cross-bridges in states prio r to phosphate release are already committed to force generation and must r elax by releasing phosphate, rather than by a reversal of the force-generat ing step to a weakly bound, low-force phosphate-bound state. These findings also indicate that an increase in the phosphate concentration within muscl e fibres plays an important part in the slowing of relaxation observed in s keletal muscle fatigue and that the relaxation transients observed upon dia zo-2 photolysis represent a disengagement of the cross-bridges.