Previous experiments1,2 have shown that inorganic phosphate (Pi) 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, Pi stimulates the reversal of the SR Ca2+-ATPase which reduces the SR Ca2+ content independent of the formation of Ca2+-Pi precipitates3. Therefore, we sought to determine if under our conditions cytoplasmic CP alters the effect of Pi on Ca2+ release observed previously1,2.
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 described1,2. 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 Pi 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 Pi on Ca2+ release confirm our previous conclusions1,2 that Pi 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.