EFFECTS OF CREATINE-PHOSPHATE AND INORGANIC-PHOSPHATE ON THE SARCOPLASMIC-RETICULUM OF SAPONIN-TREATED RAT-HEART

1. Ventricular trabeculae from rat heart were permeabilized by treatment with saponin. In the presence of 150 nM Ca2+, application of 20 mM caffeine released Ca2+ from the sarcoplasmic reticulum (SR), resulting in a transient contracture. Ca2+ released from the SR was detected using fura-2 fluorescence. The amplitudes of the caffeine-induced Ca2+ transients were used to assess SR Ca2+ content. 2. In the absence of creatine phosphate (CP), introduction of 5-30 mM inorganic phosphate (P-i) caused a net release of Ca2+ from the SR. Subsequent caffeine-induced Ca2+ and tension transients were smaller in the presence of P-i. Under these conditions, 30 mM P-i decreased the caffeine-induced Ca2+ transients by 45 +/- 3.1% (mean +/- S.D., n = 14). On removal of P,(i) the [Ca2+] transiently decreased and the caffeine-induced Ca2+ transients returned to control levels over 4-6 min. 3. In the presence of CP (5-15 mM), the Ca2+ transients were unaffected by the introduction of P-i (5-30 mM) or slightly increased in amplitude. P-i (30 mM) significantly increased the caffeine-induced Ca2+ transients by 7 +/- 8.8% (mean +/- S.D., n = 19, P < 0.05) in the presence of 15 mM CP. The release of Ca2+ on addition of P-i and decrease in [Ca2+] on P-i withdrawal was less pronounced or absent completely in the presence of CP. The inhibitory effects of P-i on caffeine-induced Ca2+ release became apparent as the [CP] was decreased from 5 to 0 mM 4. In the presence of the creatine phosphokinase inhibitor dinitro-fluorobenzene (DNFB) the effects of P-i (in the presence of CP) were qualitatively similar to the results obtained in the absence of CP, although the decrease in caffeine-induced Ca2+ release was less pronounced. 5. These results suggest that the rise in [P-i](i) during ischaemia or anoxia will have little effect on the regulation of Ca2+ by the SR while the [CP](i) remains above 5 mM. However, as the [CP] decreases below 5 mM, the accumulation of P-i within the cytosol will progressively reduce the SR Ca2+ content. CP may act in conjunction with endogenous creatine phosphokinase to modify the response of the SR to P-i, and possible mechanisms are considered.