A comparison of the effects of ATP and tetracaine on spontaneous Ca2+ release from rat permeabilised cardiac myocytes

Abstract

1. Fluo-3 fluorescence measurements were made in isolated beta-escin permeablised rat cardiac myocytes using confocal microscopy. Perfusion of a mock intracellular solution containing 0.22-0.23 microM Ca(2+) and 5 mM ATP elicited regular waves of Ca(2+) (approximately every 5 s) due to spontaneous release of Ca(2+) from the sarcoplasmic reticulum (SR). 2. An approximately linear relationship was noted between Ca(2+) wave velocity (v) and amplitude (sigma). Under the control conditions the ratio of velocity to amplitude (v/sigma) varied little and was 99.8 +/- 2.5 m s(-1) microM(-1) (n = 78). 3. Reduction of [ATP] in the bathing solution to 0.5 and 0.2 mM ATP progressively decreased Ca(2+) wave frequency and propagation velocity while increasing the amplitude. The changes in Ca(2+) wave characteristics in 0.5 mM ATP were similar to those observed during perfusion with 50 microM tetracaine. In 0.2 mM ATP the decline of [Ca(2+)] during a Ca(2+) wave was slowed suggesting a lowered rate of Ca(2+) re-uptake by the SR Ca(2+) pump. 4. Reduction of [ATP] to 0.1 mM abolished Ca(2+) waves after 15-20 s. Returning the [ATP] to 5 mM caused a burst of high frequency and large amplitude waves. Mean velocity of the first wave on returning to 5 mM ATP was larger than normal but the v/sigma value was 32 +/- 6 % of control (n = 6). In the similar burst on removal of 100 microM tetracaine v/sigma was higher than control (166 +/- 9 %, n = 6). 5. Rapid application of caffeine (10 mM) was used to assess the SR Ca(2+) content. This showed that SR Ca(2+) increased as [ATP] was reduced or [tetracaine] was increased. The highest SR Ca(2+) content was observed after perfusion with 0.1 mM ATP, which was 245 +/- 15 % of control values. 6. Returning [ATP] from 0.1 mM to 5 mM caused a burst of high frequency, large amplitude Ca(2+) waves. But recovery after incubation with 300 microM tetracaine resulted in SR Ca(2+) release with no coherent wave pattern. The reason for this discrepancy is discussed.