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Creatine kinase (CK) release in response to excessive electrically stimulated contractile activity has been studied in isolated rat soleus muscles. The exacerbation of CK release induced by contractile activity was found to be directly related to the length of time for which the muscle was stimulated and indirectly related to the recovery of force following the end of stimulation. 31P-NMR studies were undertaken using a recirculating superfused muscle preparation and demonstrated that muscles subjected to two different stimulation protocols (stimulation for 0.5 s every 2 s in oxygenated medium or for 1.5 s every 2 s in anoxic medium) had similar falls in ATP content and pH despite a Substantially greater release of CK from the muscles stimulated under anoxia. However, stimulated muscles under anoxia showed a more rapid fall and reduced recovery of phosphocreatine and a greater sustained elevation of inorganic phosphate than muscles in oxygenated medium. It is concluded that only part of the increased loss of CK from muscles stimulated in anoxic medium can be explained by release from cells which have lost energy supplies and therefore that other mechanisms must exist which allow release of CK and other cytosolic enzymes from muscle cells.

(1) A recirculating isolated superfused skeletal muscle preparation has been developed for the study of rat soleus muscles at physiological temperature using 31P Nuclear Magnetic Resonance (NMR). (2) This system has been used to study intracellular muscle high energy phosphate content and pH during experimental damage to the muscle induced by 2,4-dinitrophenol, deoxycholate and the calcium ionophore, A23187. (3) Results indicate that release of intracellular cytosolic enzymes from damaged skeletal muscle may be induced by phosphocreatine (PCr) and adenosine trisphosphate (ATP) depletion, but under certain circumstances intracellular enzymes can be released from skeletal muscle without any fall in muscle PCr or ATP content.

The 31P-NMR technique has been used to assess the intracellular ratios and concentrations of mobile ATP and ADP and the intracellular pH in an insulin-secreting cell line, RINm5F. The single-channel current-recording technique has been used to investigate the effects of changes in the concentrations of ATP and ADP on the gating of nucleotide-dependent K+ channels. Adding ATP to the membrane inside closes these channels. However, in the continued presence of ATP adding ADP invariably leads to the reactivation of ATP-inhibited K+ channels, even at ATP4-/ADP3- concentration ratios greater than 7:1. Interactions between ATP4- and ADP3- seem competitive. An increase in the concentration ratio ATP4-/ADP3- consistently evoked a decrease in the open-state probability of K+ channels; conversely, a decrease in ATP4-/ADP3- increased the frequency of K+ channel opening events. Channel gating was also influenced by changes in the absolute concentrations of ATP4- and ADP3-, at constant free concentration ratios. ADP-evoked stimulation of ATP-inhibited channels did not result from phosphorylation of the channel, as ADP-beta-S, a nonhydrolyzable analog of ADP, not only stimulated but enhanced ADP-induced activation of K+ channels, in the presence of ATP. Similarly, ADP was able to activate K+ channels in the presence of two nonhydrolyzable derivatives of ATP, AMP-PNP and beta gamma methylene ATP.