Effects of cytosolic ATP on spontaneous and triggered Ca2+-induced Ca2+ release in permeabilised rat ventricular myocytes

1. The effects of cytosolic ATP on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in saponin-permeabilised rat ventricular myocytes. [Ca2+] within the cells was monitored using Fura-2 or Fluo-3 fluorescence. Spontaneous cyclic Ca2+ release from the SR was induced by increasing the bathing [Ca2+] to 200-300 nM, in solutions weakly Ca2+ buffered with 0.05 mM EGTA. Alternatively, Ca2+-induced Ca2+ release (CICR) was triggered by a rapid increase in [Ca2+] induced by flash photolysis of Nitr-5 (0.08 mM), replacing EGTA in the solution. 2. Stepwise reductions in [ATP] were associated with corresponding decreases in the frequency and increases in the amplitude of spontaneous Ca2+ transients. A decrease from 5 mM to 0. 1 mM ATP, reduced the release frequency by 48.6 +/- 7 % (n = 7) and almost doubled the amplitude of the Ca2+ transient. Marked prolongation of the spontaneous Ca2+ transient occurred when [ATP] was further reduced to 10 microM, consistent with inhibition of the SR Ca2+ pump. 3. These effects of ATP were compared with other interventions that inhibit Ca2+ uptake or reduce the sensitivity of the SR Ca2+ release mechanism. Inhibition of the SR Ca2+ pump with cyclopiazonic acid (CPA) markedly reduced the spontaneous Ca2+ release frequency, without changing the amplitude. The descending phase of the Ca2+ transient was prolonged in the presence of CPA, while the rising phase was unaffected. In contrast, desensitisation of the SR Ca2+ release mechanism with tetracaine decreased the frequency of spontaneous release, but markedly increased the amplitude. 4. CICR triggered by flash photolysis of Nitr-5 appeared to be more sensitive to cytosolic [ATP] than spontaneous release and was generally delayed by a decrease to 2.5 mM ATP. In the presence of 0.1-0.2 mM ATP, release often failed completely or was not consistently triggered. Some preparations exhibited Ca2+ release 'alternans', whereby every alternate trigger induced a response. 5. These results suggest that the increase in spontaneous Ca2+ release amplitude and the decrease in frequency that occurs as [ATP] is reduced from 1 mM to 100 microM, is mainly due to desensitisation of the SR Ca2+ release mechanism, which allows the SR Ca2+ content to reach a higher level before release occurs. At very low [ATP], a reduction in the SR Ca2+ uptake rate may also contribute to the decrease in release frequency. CICR triggered by photolysis of Nitr-5 appeared to be more sensitive to cytosolic [ATP]. The possible underlying mechanisms and the relevance of these results to myocardial ischaemia or hypoxia is considered.