Free [ADP] and aerobic muscle work follow at least second order kinetics in rat gastrocnemius in vivo

The relationship between free cytosolic [ADP] land [P-i]) and steady-state aerobic muscle work in rat gastrocnemius muscle in vivo using P-31 NMR was investigated. Anesthetized rats were ventilated and placed in a custom-buil t cradle fitted with a force transducer that could be placed into a 7-tesla NMR magnet. Muscle work was induced by supramaximal sciatic nerve stimulat ion that activated all fibers, Muscles were stimulated at 0.1, 0.2, 0.3, 0. 4, 0.5, 0.8, 1.0, and 2.0 Hz until twitch force, phosphocreatine, and P-i w ere unchanged between two consecutive spectra acquired in 4-min blocks (8-1 2 min), Parallel bench experiments were performed to measure total tissue g lycogen, lactate, total creatine, and pyruvate in freeze-clamped muscles af ter 10 min of stimulation at each frequency. Up to 0.5 Hz, there was no sig nificant change in muscle glycogen, lactate, and the lactate/pyruvate ratio s between 8-12 min. At 0.8 Hz, there was a 17% fall in glycogen and a 65% r ise in the muscle lactate with a concomitant fall in pH, Above this frequen cy, glycogen fell rapidly, lactate continued to rise, and ATP and pH declin ed. On the basis of these force and metabolic measurements, we estimated th e maximal mitochondrial capacity (V-max) to be 0.8 Hz. Free [ADP] was then calculated at each submaximal workload from measuring all the reactants of the creatine kinase equilibrium after adjusting the K'(CK) to the muscle te mp (30 degrees C), pH, and pMg, We show that ADP (and P-i) and tension-time integral follow a Pill relationship with at least a second order function. The K-0.5 values for free [ADP] and [P-i] were 48 mu M and 9 mM, respectiv ely. Our data did not fit any form of the Michaelis-Menten equation. We the refore conclude that free cytosolic [ADP] and [P-i] could potentially contr ol steady-state oxidative phosphorylation in skeletal muscle in vitro.