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Inorganic phosphate reduces sarcoplasmic reticulum Ca²⁺ release in the presence and absence of cytoplasmic creatine phosphate in mammalian skeletal muscle

Previous experiments^1,2 have shown that inorganic phosphate (P_i) enters the sarcoplasmic reticulum (SR) of skeletal muscle and precipitates with Ca²⁺ to reduce the free [Ca²⁺] available for release. This may underlie the reduction in Ca²⁺ release seen in the late stages of fatigue. These previous experiments were conducted in the absence of creatine phosphate (CP) to mimic fatigued muscle. However, a recent study has suggested that in the absence of CP, P_i stimulates the reversal of the SR Ca²⁺-ATPase which reduces the SR Ca²⁺ content independent of the formation of Ca²⁺-P_i precipitates³. Therefore, we sought to determine if under our conditions cytoplasmic CP alters the effect of P_i on Ca²⁺ release observed previously^1,2.

Male Hooded Wistar rats were rapidly killed with CO₂ in accordance with the University of Adelaide's Animal Ethics guidelines. Mechanically skinned fibres were used here as previously described^1,2. Skinned fibres were immersed in cytoplasmic-like solutions (K-HDTA)^1,2; in some the [Pi] and [CP] were varied appropriately. Full SR Ca²⁺ release was elicited by using a K-HDTA solution containing 30 mM caffeine and low free [Mg²⁺] (0.05 mM). The initial endogenous SR Ca²⁺ content was first determined and then fibres were Ca²⁺ loaded to the same level before exposure to P_i solutions with or without CP (30s).

Ca²⁺ release from skinned fast-twitch fibres was equally reduced following a brief exposure to 50 mM Pi (30s) in the presence or absence of 10 mM CP. The absence of any difference in the effects of P_i on Ca²⁺ release confirm our previous conclusions^1,2 that P_i enters the SR where it precipitates with Ca²⁺ thereby reducing the amount of Ca²⁺ available for release.

(1) Posterino, G. S. & Fryer, M. W. (1998). Journal of Physiology. 512.1, pp97-108.

(2) Duke, A.M. & Steele, D.S. (2001). Journal of Physiology. 531.3, pp729-742.

(3) Fryer, M. W., et. al. (1995). Journal of Physiology. 482, pp123-140.