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 (Pi) 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 microl 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 Pi induced a small transient rise in [Ca2+], due to Ca2+ loss from the SR. Following equilibration with solutions containing Pi (> or = 5 mM), a maintained decrease in [Ca2+] occurred when the flow was stopped. This is consistent with calcium phosphate (Ca-Pi) 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 phosphoenolpyruvate (PEP) and pyruvate kinase (PK). 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 Pi increased markedly. Following prolonged equilibration with solutions containing Pi, a rise in [Ca2+] occurred within the bath when the flow was stopped. Maintained Ca2+ uptake associated with Ca-Pi precipitation was not apparent at any level of Pi 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 Pi appears to involve further Ca2+ efflux via the SR Ca2+ pump. The possible relevance of these effects to skeletal muscle fatigue is considered.
Duke
A M
AM
School of Biology, University of Leeds, Leeds LS2 9JT, UK.
Steele
D S
DS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000172:deficiency / Q000378:metabolism* / Q000493:pharmacokinetics; D011232:Chemical Precipitation; D003402:Creatine Kinase / Q000037:antagonists & inhibitors; D004056:Differential Threshold; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006652:Histological Techniques; D008297:Male; D018482:Muscle, Skeletal / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism / Q000494:pharmacology*; D010725:Phosphocreatine / Q000494:pharmacology*; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D004791:Enzyme Inhibitors; D010710:Phosphates; D010725:Phosphocreatine; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D003402:Creatine Kinase; D002118:Calcium