Contraction and recovery of living muscles studies by 31P nuclear magnetic resonance.
1. Phosphorus nuclear magnetic resonance ((31)P NMR) can be used to measure the concentrations of phosphorus-containing metabolites within living tissue. We have developed methods for maintaining muscles in physiological condition, stimulating them and recording tension while at the same time accumulating their (31)P NMR spectra. Experiments were performed on frog sartorii and frog and toad gastrocnemii at 4 degrees C.2. The NMR signals from (31)P (the naturally occurring phosphorus) is weak, and signal averaging is required. In order to follow the time course of reactions it is necessary to maintain the muscles in a steady state for many hours while they are 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.3. In the absence of stimulation, the spectra of frog sartorius muscles and of their extracts indicated concentrations of adenosine triphosphate (ATP), phosphoryl creatine (PCr), inorganic orthophosphate (P(i)) and sugar phosphates (sugar P) which are in reasonable agreement with the values obtained by chemical analysis.4. We have confirmed that unidentified resonances representing unknown compounds appear in the spectra of both frog and toad muscle; one of these is much larger in spectra from toad than from frog. We have found an additional small, unidentified resonance which appears to be specific to toad muscle.5. 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.6. Following 25 s tetanii, approximately 20% of the PCr had been hydrolysed; it was then rebuilt exponentially with a half-time of about 10 min. The increase in [P(i)] 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 P(i) was reduced to below that in resting muscle. The [sugar P] remained very high ( approximately 4 mmol kg(-1)) throughout the 56 min interval between contractions.7. When frog sartorii were tetanized for 1 s every 2 min, the changes in [PCr] and [P(i)] between contractions could not be observed because too little signal was obtained from these small muscles. However, when toad gastrocnemii were similarly stimulated, the changes in these compounds could be readily detected and were even greater than expected.8. The position of the P(i) resonance can be used to monitor intracellular pH and changes in pH. Under the conditions of our experiments the average intracellular pH in unstimulated frog sartorius muscles was 7.5. After a 25 s tetanus this was observed to move in the acid direction by a few tenths of a pH unit and to return to its pre-stimulation value before the end of the recovery period. After a 1 s contraction of toad gastrocnemius the environment of P(i) became slightly more alkaline for the first few seconds.
Contraction and recovery of living muscles studies by 31P nuclear magnetic resonance. 1. Phosphorus nuclear magnetic resonance ((31)P NMR) can be used to measure the concentrations of phosphorus-containing metabolites within living tissue. We have developed methods for maintaining muscles in physiological condition, stimulating them and recording tension while at the same time accumulating their (31)P NMR spectra. Experiments were performed on frog sartorii and frog and toad gastrocnemii at 4 degrees C.2. The NMR signals from (31)P (the naturally occurring phosphorus) is weak, and signal averaging is required. In order to follow the time course of reactions it is necessary to maintain the muscles in a steady state for many hours while they are 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.3. In the absence of stimulation, the spectra of frog sartorius muscles and of their extracts indicated concentrations of adenosine triphosphate (ATP), phosphoryl creatine (PCr), inorganic orthophosphate (P(i)) and sugar phosphates (sugar P) which are in reasonable agreement with the values obtained by chemical analysis.4. We have confirmed that unidentified resonances representing unknown compounds appear in the spectra of both frog and toad muscle; one of these is much larger in spectra from toad than from frog. We have found an additional small, unidentified resonance which appears to be specific to toad muscle.5. 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.6. Following 25 s tetanii, approximately 20% of the PCr had been hydrolysed; it was then rebuilt exponentially with a half-time of about 10 min. The increase in [P(i)] 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 P(i) was reduced to below that in resting muscle. The [sugar P] remained very high ( approximately 4 mmol kg(-1)) throughout the 56 min interval between contractions.7. When frog sartorii were tetanized for 1 s every 2 min, the changes in [PCr] and [P(i)] between contractions could not be observed because too little signal was obtained from these small muscles. However, when toad gastrocnemii were similarly stimulated, the changes in these compounds could be readily detected and were even greater than expected.8. The position of the P(i) resonance can be used to monitor intracellular pH and changes in pH. Under the conditions of our experiments the average intracellular pH in unstimulated frog sartorius muscles was 7.5. After a 25 s tetanus this was observed to move in the acid direction by a few tenths of a pH unit and to return to its pre-stimulation value before the end of the recovery period. After a 1 s contraction of toad gastrocnemius the environment of P(i) became slightly more alkaline for the first few seconds.