Effect on the fura‐2 transient of rapidly blocking the Ca2+ channel in electrically stimulated rabbit heart cells.

1. We used a rapid solution switcher technique to investigate mechanisms that might trigger intracellular Ca2+ release in rabbit ventricular myocytes. The study was carried out at 36 degrees C, intracellular Ca2+ (Ca2+i) was monitored with fura‐2, and myocytes were electrically stimulated. 2. In patch‐clamped cells, using the switcher to apply 20 microM nifedipine (an L‐type Ca2+ current (ICa,L) blocker) 4 s before a depolarization to +10 mV reduced the amplitude of ICa,L to 10.25 +/‐ 2.25% of control (mean +/‐ S.E.M., n = 7 cells). 3. In externally stimulated cells, a rapid switch to 20 microM nifedipine 4 s before a stimulus reduced the amplitude of the fura‐2 transient to 64.01 +/‐ 2.09% of control (mean +/‐ S.E.M., n = 19 cells). Using an in vivo calibration curve for fura‐2, this was equivalent to a reduction in the Ca2+ transient to 50% during nifedipine application. Since an identical nifedipine switch reduced ICa,L to 10.25%, it would seem that blocking a large fraction of ICa,L inhibited only half the Ca2+ transient. 4. The Na(+)‐Ca2+ exchanger is inhibited by 5 mM nickel. Switching to 20 microM nifedipine +5 mM nickel 4 s before a stimulus abolished the fura‐2 transient completely, consistent with the hypothesis that Ca2+ entry via reverse Na(+)‐Ca2+ exchange might trigger a fraction of the fura‐2 transient that remained during nifedipine. 5. After the Na(+)‐K+ pump was inhibited by strophanthidin to increase intracellular Na+ (Na+i), a switch to 20 microM nifedipine became progressively less effective in reducing the fura‐2 transient. This suggests that as Na+i rose, other mechanisms (perhaps reverse Na(+)‐Ca2+ exchange) appeared able to substitute for ICa,L in triggering the Ca2+ transient. 6. In cells depleted of Nai+ to inhibit the triggering of sarcoplasmic reticulum (SR) Ca2+ release by reverse Na(+)‐Ca2+ exchange, a nifedipine switch reduced the fura‐2 transient to 10.9 +/‐ 4.19% (mean +/‐ S.E.M., n = 7; equivalent to 6.5% of the Ca2+ transient). 7. A switch to Na(+)‐free (Li+) solution 100 ms before an electrical stimulus caused an increase in the fura‐2 transient of 12.2 +/‐ 1.5% (mean +/‐ S.E.M., n = 7; equivalent to a 22% increase in the Ca2+ transient). 8. The results confirm that ICa,L is an important trigger for SR Ca2+ release and the resulting Ca2+ transient. However, since 50% of the Ca2+ transient remained when ICa,L was largely inhibited, it would seem likely that other SR trigger mechanisms might exist in addition. These data are consistent with the idea that Ca2+ entry via reverse Na(+)‐Ca2+ exchange during the upstroke of the normal cardiac action potential might trigger a fraction of SR Ca2+ release and the resulting Ca2+ transient.