Phosphorus nuclear magnetic resonance studies on the effect of duration of contraction in bull‐frog skeletal muscles.

Abstract

1. Contraction and recovery of bull-frog skeletal muscles were studied using 31P nuclear magnetic resonance (NMR), with a time resolution of 16 s. at 4.degree. C. The muscles were stimulated tetanically for various periods (0.2-10 s) at a sarcomere length of 2.4 .mu.m. Changes in the concentrations of inorganic phosphate (Pi), phosphocreatine (PCr) and other metabolites were studied for repeated cycles of contraction and recovery. 2. In resting muscles, bathed in a solution gassed with 95% O2 and 5% CO2, the concentration of Pi was 1.15 .+-. 0.21 mmol kg-1 wet weight (mean .+-. one S.D., n = 12), that of ATP was 3.32 .+-. 0.15 mmol Kg-1 (mean .+-. one S.D., n = 12) and that of sugar phosphates was less than 0.5 mmol kg-1. The intracellular pH (pHi) was 7.22 .+-. 0.01 (mean .+-. one S.D., n = 12). These results are averages for fibres which probably have different values. 3. One stimulating the muscles pHi shifted in the alkaline direction and subsequently recovered. The extent of the alkaline shift was linearly related to the contraction duration (0.2-10 s) with a rate of 0.01 pH unit s-1. 4. The increase in Pi with stimulus duration was biphasic, consisting of an early burst, 0.38 .+-. 0.10 mmol kg-1 (.+-. 1 S.D., N = 5)., complete within about 0.2-0.5s, followed by a slower steady-state increase. The steady-state rate of Pi increase was 0.33 .+-. 0.02 mmol kg-1 s-1 (.+-. 1 S. D., n = 5) in agreement with the results of previous studies involving chemical analyses. 5. The time course of Pi recovery was well described by a single exponential. Intensities of ATP, sugar phosphates and the peaks in the phosphodiester region did not change during a 2 s tetanus. 6. The amount of PCr hydrolysed (-.DELTA.PCr), associated with contractions of various durations, coincided well with Pi increase (.DELTA.Pi). This was found during the whole recovery period except for the initial few minutes following relaxation when -.DELTA.PCr was significantly smaller than .DELTA.Pi. The difference was 0.35 .+-. 0.03 mmol kg-1 (mean .+-. 1 SD.D., n = 3) immmediately after a 2 s tetanus. 7. In contractions of 2 s or longer, the extent of the temporal separation between .DELTA.Pi and -.DELTA.PCr was almost the same, but in contractions of less than 2 s it was significantly reduced. 8. Peaks in the phosphodiester region showed a systematic seasonal variation. The concentrations measured was over 2 mmol kg-1 in December and less than 0.5 mmol kg-1 in June. 9. In conclusion, these results suggest that Pi is released and ADP remains bound to the myosin heads for a long time after relaxation.