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.
Levi
A J
AJ
Department of Physiology, School of Medical Sciences, University of Bristol, UK. allan.levi@bristol.ac.uk
Issberner
J
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D009532:Nickel / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D011817:Rabbits; D012433:Ryanodine / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D013327:Strophanthidin / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D019831:Sodium-Calcium Exchanger; D012433:Ryanodine; D013327:Strophanthidin; D019284:Thapsigargin; D009532:Nickel; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D009543:Nifedipine; D002118:Calcium; D016257:Fura-2