APPS November 2002 Meeting Abstract 1106

William A. Macdonald, D. George Stephenson, Department of Zoology, Faculty of Science and Technology, La Trobe University, Bundoora, Victoria, 3083.

ADP accumulates during fatigue and it has previously been shown that physiological levels of ADP (10-40µmol/l) affect sarcoplasmic reticulum (SR) Ca²⁺ content ¹. With this in mind, it was of interest to investigate the effect of physiologically relevant concentrations of ADP on action potential induced twitch responses in skinned muscle.

Male Long-Evans Hooded rats were killed by halothane overdose. Single extensor digitorum longus fibres were isolated, mechanically skinned under paraffin oil and mounted on a force transducer. The skinned fibre preparation permits access to the myoplasm by removal of the sarcolemma and therefore allows for manipulation of the myoplasmic content. Fibres were bathed in a standard solution, mimicking the intracellular environment and contained (mmol/l) HEPES, 90; HDTA, 50; Na⁺, 36; K⁺, 126; ATP, 8; CP, 10; Mg²⁺ free, 1; pH 7.10). The creatine kinase reaction ADP + CP ←→ ATP + Cr, K_app = 200², together with endogenous creatine kinase activity was used to buffer ADP concentrations. Varied amounts of Cr were added to buffer ADP to required levels. Fibres were electrically stimulated via two parallel platinum wire electrodes with single 2ms, 50 V/cm pulses every 15s³.

After 3 min equilibration at 40µmol/l ADP (12mmol/l Creatine), the twitch response was reversibly decreased by 35 ± 2 % compared with controls in the same fibre. The duration at half peak height was also reduced by 19 ± 3 % of control values. The results demonstrate that SR Ca²⁺ release, during a twitch, is reduced by ADP in µM range. The reduction in Ca²⁺ release could be the consequence of ADP-induced reduction in SR Ca²⁺ content alone, or in combination with an ADP-dependent reduction in Na⁺/K⁺ pump activity. Either way, these results give clear evidence that increasing [ADP], in the µmol/l range, is an important factor in muscle fatigue.