Contraction and recovery of living muscles studied by 31p nuclear magnetic resonance

Abstract

31P NMR was used to measure the concentrations of P-containing metabolites within living tissue. Methods for maintaining muscles in physiological condition were developed, stimulating them and recording tension while simultaneously accumulating their 31P NMR spectra. Experiments were performed on frog sartorii and frog and toad gastrocnemii at 4.degree. C. The NMR signals from 31P (the naturally occurring phosphorus) was weak and signal averaging were required. In order to follow the time course of reactions it was necessary to maintain the muscles in a steady state for many hours while they were undergoing repeated contractions. Signals were accumulated in separate computer bins according to time after initiation of contraction. By these means spectra were obtained which corresponded to the different intervals during the contraction and recovery cycle. In the absence of stimulation, the spectra of frog sartorius muscles and of their extracts indicated concentrations of ATP, phosphoryl creatine (PCr), Pi and sugar phosphates (sugar P) which were in reasonable agreement with the values obtained by chemical analysis. Unidentified resonances representing unknown compounds appeared in the spectra of both frog and toad muscle; 1 of these was much larger in spectra from toad than from frog. An additional small, unidentified resonance which was specific to toad muscle was found. Spectra accumulated during actual contractions (1 s tetani every 2 min) did not differ dramatically from those accumulated throughout the 2 min cycle of contraction and partial recovery. Following 25 s tetanii, approximately 20% of the PCr was hydrolyzed; it was then rebuilt exponentially with a half-time of about 10 min. The increase in [Pi] immediately after contraction and the time course of its disappearance corresponded to the changes in [PCr]. During the later half of the recovery period the concentration of Pi was reduced to below that in resting muscle. The [sugar P] remained very high (.simeq. 4 mmol kg-1) throughout the 56 min interval between contractions. When frog sartorii were tetanized for 1 s every 2 min, the changes in [PCr] and [Pi] between contractions were not observed because too little signal was obtained from these small muscles. When toad gastrocnemii were similarly stimulated, the changes in these compounds were readily detected and were even greater than expected. The position of the Pi resonance was used to monitor intracellular pH and pH changes. The average intracellular pH in unstimulated frog sartorius muscles was 7.5. After a 25 s tetanus this moved in the acid direction by a few 10th of a pH unit and returned to its pre-stimulation value before the end of the recovery period. After a 1 s contraction of toad gastrocnemius the environment of Pi became slightly more alkaline for the 1st few seconds.