INORGANIC-PHOSPHATE DECREASES THE CA2+ CONTENT OF THE SARCOPLASMIC-RETICULUM IN SAPONIN-TREATED RAT CARDIAC TRABECULAE

1. Measurements of [Ca2+] were made in saponin-permeabilized rat ventricular trabeculae using the fluorescent indicator Indo-1. Application of caffeine (20 mm) caused a transient rise in [Ca2+] within the preparation as a result of Ca2+ release from the sarcoplasmic reticulum (SR). The size of the caffeine-induced Ca2+ transient was related to the amount of Ca2+ accumulated by the SR prior to addition of caffeine. Caffeine-induced Ca2+ release was abolished by ryanodine (10 muM), an inhibitor of SR Ca2+ release. 2. At a bathing [Ca2+] of 0.2 muM, the amount of Ca2+ released from the SR on addition of caffeine was sufficient to generate a tension transient. Ca2+ and tension responses were stabilized by application of caffeine at regular intervals (2 min). Addition of 10 mm inorganic phosphate (P(i)) induced a transient increase in [Ca2+] within the preparation due to a net release of Ca2+ from the SR. The amplitude of subsequent caffeine-induced Ca2+ transients were reduced to 65 +/- 7.5% (mean +/- S.D., n = 13) of control. In addition, the accompanying tension transient fell to 45 +/- 6.9% of control. Removal of P(i) caused a transient decrease in the [Ca2+] within the preparation consistent with a net increase in Ca2+ uptake by the SR. Subsequent caffeine-induced Ca2+ and tension transients returned to control levels. 3. Inclusion of P(i) (2-30 mm) in the perfusing solution decreased the size of caffeine-induced Ca2+ and tension transients in a dose-dependent manner. 4. Addition of 10 mm ADP caused a transient increase in [Ca2+] and depressed subsequent caffeine-induced Ca2+ transients to a greater extent than 10 mM P(i). Despite the reduction in Ca2+ release from the SR, tension responses were larger in the presence of 10 mm ADP than under control conditions. This is a consequence of an increase in Ca2+-activated force by ADP. 5. A decrease in the amplitude of caffeine-induced Ca2+ transients also occurred on changing from a solution containing 1 mM ADP and 10 mM P(i) to a solution with 10 mm ADP and 1 mm P(i). This confirms the previous observation that ADP is more effective than P(i) at reducing caffeine-induced Ca2+ released from the SR. 6. Spontaneous oscillations of [Ca2+] and tension occurred in the presence of 0.5 muM Ca2+. These oscillations result from the cyclic release and uptake of Ca2+ from the SR. Under these conditions, introduction of P(i) (10 mM) caused a slow transient release of Ca2+ from the SR followed by complete abolition of spontaneous activity. 7. The results suggest that P(i) (2-30 mm) decreases the Ca2+ content of the SR. This could occur either by stimulating SR Ca2+ release or by partially inhibiting Ca2+ uptake. Based on these results, the rise in intracellular [P(i)] that occurs during ischaemia and anoxia in cardiac muscle would be expected to lower the SR Ca2+ content and hence reduce the amount of Ca2+ released into the cytosol during systole.