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) Ca2+ regulation was studied in mechanically skinned skeletal muscle fibres from rat extensor digitorium longus (EDL). Preparations were perfused with solutions 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 marked prolongation of the descending phase of the caffeine-induced fluorescence 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 following application of the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA). This suggests that (i) the descending phase of the caffeine-induced Ca2+ transient is dependent on the rate of Ca2+ uptake by the SR and (ii) prolongation 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 the [PCr] may influence SR Ca2+ regulation by limiting local ATP regeneration by endogenous CK. After treatment with DNFB, PCr withdrawal had no effect on the Ca2+ transient, confirming that PCr does not have an additional direct effect on the SR. 5. The Ca2+ efflux associated with PCr withdrawal was insensitive to ryanodine or Ruthenium Red, but was effectively abolished by pretreatment with the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA). This suggests that the Ca2+ efflux associated with PCr withdrawal is independent of the SR Ca2+ channel, 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.
Duke
A M
AM
School of Biology, University of Leeds, Leeds LS2 9JT, UK.
Steele
D S
DS
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D004139:Dinitrofluorobenzene / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D009994:Osmolar Concentration; D010539:Permeability; D010725:Phosphocreatine / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism*
D010725:Phosphocreatine; D002110:Caffeine; D004139:Dinitrofluorobenzene; D000252:Calcium-Transporting ATPases; D002118:Calcium