Effects of creatine phosphate on Ca2+ regulation by the sarcoplasmic reticulum in mechanically skinned rat skeletal muscle fibres

1. The effect of creatine phosphate (PCr) on sarcoplasmic reticulum (SR) Ca 2+ regulation was studied in mechanically skinned skeletal muscle fibres fr om rat extensor digitorium longus (EDL). Preparations were perfused with so lutions mimicking the intracellular milieu and the [Ca2+] within the muscle was monitored continuously using fura-2. 2. Brief application of 40 mM caffeine caused a transient increase in [Ca2] due to SR Ca2+ release, and an associated tension response. Withdrawal of PCr resulted in (i) a slow transient release of Ca2+ from the SR (ii) a ma rked prolongation of the descending phase of the caffeine-induced fluoresce nce ratio transient and (iii) a decrease in the Ca2+ transient amplitude to 69.2 +/- 2.7% (n = 16) of control responses. 3. Prolongation of the caffeine-induced Ca2+ transient also occurred follow ing application of the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA). Thi s suggests that (i) the descending phase of the caffeine-induced Ca2+ trans ient is dependent on the rate of Ca2+ uptake by the SR and (ii) prolongatio n associated with PCr withdrawal may also reflect a decrease in the net Ca2 + uptake rate. 4. The effects of PCr withdrawal were mimicked by addition of the creatine kinase (CK) inhibitor 2,4-dinitro-1-fluorobenzene (DNFB). Hence, reducing t he [PCr] may influence SR Ca2+ regulation by limiting local ATP regeneratio n by endogenous CK. After treatment with DNFB, PCr withdrawal had no effect on the Ca2+ transient, confirming that PCr does not have an additional dir ect effect on the SR. 5. The Ca2+ efflux associated with PCr withdrawal was insensitive to ryanod ine or Ruthenium Red, but was effectively abolished by pretreatment with th e SR Ca2+ pump inhibitor cyclopiazonic acid (CPA). This suggests that the C a2+ efflux associated with PCr withdrawal is independent of the SR Ca2+ cha nnel, but may involve reversal or inhibition of the Ca2+ ATPase. 6. These data suggest that Ca2+ regulation by the SR is strongly dependent on the supply of ATP via endogenous CK. Depletion of PCr may contribute to impaired SR Ca2+ regulation known to occur in intact skeletal muscle under conditions of fatigue.