Mechanisms underlying phosphate-induced failure of Ca2+ release in single skinned skeletal muscle fibres of the rat

1. Single mechanically skinned fibres from rat extensor digitorum longus (EDL) muscles were used to investigate the mechanisms underlying inorganic phosphate (P-i) movements between the myoplasm and the sarcoplasmic reticulum (SR). Force transients elicited by caffeine/low Mg2+ application were used to assess the rate of P-i-induced inhibition of SR Ca2+ release and the subsequent recovery of Ca2+ release following removal of myoplasmic P-i. 2. Myoplasmic P-i reduced XR Ca2+ release in a concentration- and time-dependent manner. A 10 s exposure to 10, 20 and 50 mM myoplasmic P-i reduced SR Ca2+ release by 12 +/- 9, 29 +/- 5 and 82 +/- 5%, respectively. 3. Removal of myoplasmic ATP at the time of P-i exposure significantly increased the rate and extent of SR Ca2+ release inhibition. For example, Ca2+ release was reduced by 86 +/- 6% (n = 6) after 20 s exposure to 20 mM P-i in the absence of ATP compared with only 47 +/- 5% (n = 5) in the presence of ATP. 4. The half and full recovery times for XR Ca2+ release following washout of myoplasmic P-i were 35 s and similar to 7 min, respectively. Recovery of Ca2+ release was unaffected by the absence of ATP during washout of P-i but was prevented when fibres were washed in the presence of high myoplasmic P-i (30 mM). Neither the P-i transporter blocker phenylphosphonic acid (PHPA) nor the anion channel blockers anthracene-9-carboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) affected the rate of recovery of SR Ca2+ release. 5. These results show that P-i entry and exit from the SR occur primarily through a passive pathway that is insensitive to well-known anion channel blockers. P-i inhibition of XR Ca2+ release appears to be a complicated phenomenon influenced by the rate of P-i movement across the XR as well as by the rate, extent and species of Ca2+-P-i precipitate formation in the SR lumen. The more rapid inhibitory effect of P-i in the absence of myoplasmic ATP suggests that P-i may inhibit XR Ca2+ release more efficiently during the later stages of fatigue.