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 °C, intracellular Ca²⁺ (Ca₁²⁺) was monitored with fura-2, and myocytes were electrically stimulated. 2. In patch-clamped cells, using the switcher to apply 20 μM nifedipine (an L-type Ca²⁺ current (I_Ca.L) blocker) 4 s before a depolarization to +10 mV reduced the amplitude of I_Ca.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 μM nifedipine 4s 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 I_Ca.L to 10-25%, it would seem that blocking a large fraction of I_Ca.L inhibited only half the Ca²⁺ transient. 4. The Na⁺-Ca²⁺ exchanger is inhibited by 5 mM nickel. Switching to 20 μM 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 μM 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 I_Ca.L in triggering the Ca²⁺ transient. 6. In cells depleted of Na_i⁺ 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 I_Ca.L is an important trigger for SR Ca²⁺ release and the resulting Ca²⁺ transient. However, since 50% of the Ca²⁺ transient remained when I_Ca.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.