Cross-signaling between L-type Ca2+ channels and ryanodine receptors in rat ventricular myocytes.
Calcium-mediated cross-signaling between the dihydropyridine (DHP) receptor, ryanodine receptor, and Na(+)-Ca2+ exchanger was examined in single rat ventricular myocytes where the diffusion distance of Ca2+ was limited to < 50 nm by dialysis with high concentrations of Ca2+ buffers. Dialysis of the cell with 2 mM Ca(2+)- indicator dye, Fura-2, or 2 mM Fura-2 plus 14 mM EGTA decreased the magnitude of ICa-triggered intracellular Ca2+ transients (Cai-transients) from 500 to 20-100 nM and completely abolished contraction, even though the amount of Ca2+ released from the sarcoplasmic reticulum remained constant (approximately 140 microM). Inactivation kinetics of ICa in highly Ca(2+)-buffered cells was retarded when Ca2+ stores of the sarcoplasmic reticulum (SR) were depleted by caffeine applied 500 ms before activation of ICa, while inactivation was accelerated if caffeine-induced release coincided with the activation of ICa. Quantitative analysis of these data indicate that the rate of inactivation of ICa was linearly related to SR Ca(2+)-release and reduced by > 67% when release was absent. Thapsigargin, abolishing SR release, suppressed the effect of caffeine on the inactivation kinetics of ICa. Caffeine-triggered Ca(2+)-release, in the absence of Ca2+ entry through the Ca2+ channel (using Ba2+ as a charge carrier), caused rapid inactivation of the slowly decaying Ba2+ current. Since Ba2+ does not release Ca2+ but binds to Fura-2, it was possible to calibrate the fluorescence signals in terms of equivalent cation charge. Using this procedure, the amplification factor of ICa-induced Ca2+ release was found to be 17.6 +/- 1.1 (n = 4). The Na(+)-Ca2+ exchange current, activated by caffeine-induced Ca2+ release, was measured consistently in myocytes dialyzed with 0.2 but not with 2 mM Fura-2. Our results quantify Ca2+ signaling in cardiomyocytes and suggest the existence of a Ca2+ microdomain which includes the DHP/ ryanodine receptors complex, but excludes the Na(+)-Ca2+ exchanger. This microdomain appears to be fairly inaccessible to high concentrations of Ca2+ buffers.
Adachi-Akahane
S
S
Institute for Cardiovascular Sciences, Georgetown University Medical Center, Washington, DC 20007, USA.
Cleemann
L
L
Morad
M
M
D000818:Animals; D001464:Barium / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002021:Buffers; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000502:physiology; D002352:Carrier Proteins / Q000502:physiology*; D003956:Dialysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016257:Fura-2; D006352:Heart Ventricles / Q000737:chemistry / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D009124:Muscle Proteins / Q000502:physiology*; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011673:Pulsatile Flow; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D019284:Thapsigargin / Q000494:pharmacology
D002021:Buffers; D015220:Calcium Channels; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D009124:Muscle Proteins; D010726:Phosphodiesterase Inhibitors; D019837:Ryanodine Receptor Calcium Release Channel; D019831:Sodium-Calcium Exchanger; D001464:Barium; D002110:Caffeine; D019284:Thapsigargin; D012964:Sodium; D002118:Calcium; D016257:Fura-2