Interdependent effects of inorganic phosphate and creatine phosphate on sarcoplasmic reticulum Ca2+ regulation in mechanically skinned rat skeletal muscle

1. The effects of creatine phosphate (CP) and inorganic phosphate (P-i) on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in mechanically skinned muscle fibres from rat extensor digitorum longus (EDL) muscles. Changes in [Ca2+] were detected using fura-2 fluorescence, during continuous perfusion or when the solution surrounding the preparation was restricted to approximately 6 mul by stopping perfusion. 2. In solutions with 5 mM ATP and 10 mM CP, stopping the flow for 2-3 min had no effect on [Ca2+] within the bath. This suggests that SR Ca2+ uptake is balanced by an efflux under these conditions. 3. In solutions with CP, the introduction of P-i induced a small transient rise in [Ca2+], due to Ca2+ loss from the SR. Following equilibration with solutions containing P-i (greater than or equal to5 mM), a maintained decrease in [Ca2+] occurred when the flow was stopped. This is consistent with calcium phosphate (Ca-P-i) precipitation within the SR, resulting in maintained Ca2+ uptake. 4. In the absence of CP, the [Ca2+] within the bath increased progressively when the flow was stopped. This rise in [Ca2+] was inhibited by an alternative ATP regenerating system comprising phosphoendolpyruvate (PEP) and pyruvate kinase (PR). Therefore, the loss of Ca2+ from the SR may result from local ADP accumulation and the consequent reversal of the SR Ca2+ pump. 5. In the absence of CP, the initial Ca2+ release associated with the introduction of P-i increased markedly. Following prolonged equilibration with solutions containing P-i, a rise in [Ca2+] occurred within the bath when the flow was stopped. Maintained Ca2+ uptake associated with Ca-P-i precipitation was not apparent at any level of P-i tested (1-60 mM), when CP was absent. 6. These results suggest that withdrawal of CP is associated with activation of a SR Ca2+ efflux pathway. This may involve reversal of the SR Ca2+ pump, due to local ADP accumulation. In the absence of CP, the dominant influence of P-i appears to involve further Ca2+ efflux via the SR Ca2+ pump. The possible relevance of these effects to skeletal muscle fatigue is considered.