Voltage dependence of the Fura-2 transient in rabbit left atrial myocytes at 37 degrees C.
We used the whole-cell patch-clamp technique and monitoring of Fura-2 fluorescence to investigate the voltage dependence of the L-type Ca current (ICa,L) and intracellular Ca (Cai) transient in rabbit atrial myocytes at 37 degrees C. Imaging the atrial cell membrane with Di-4-ANNEPS showed (in contrast to ventricular cells) that atrial cells had very few transverse tubules. We measured ICa,L using a Cs-based internal dialysis solution to eliminate interfering K currents. The voltage dependence of peak ICa,L amplitude was bell-shaped: ICa,L was maximal at +10 mV and declined at more negative and positive potentials. For measuring the Fura-2 (Cai) transient, we used a K-based internal dialysis solution to preserve normal excitation-contraction coupling. Ryanodine (20 microM) plus thapsigargin (2 microM) (blockers of the sarcoplasmic reticulum, SR) abolished the phasic component of the Fura-2 transient (n = 5), demonstrating that the phasic Fura-2 transient provided an index of the magnitude of SR release. The Fura-2 transient also showed bell-shaped voltage dependence, but this was different from that for ICa,L. The Fura-2 transient peaked at +30 mV and partially declined at more positive potentials; but at potentials where inward ICa,L was small (if not absent), the phasic Fura-2 transient still attained a significant amplitude. We used a rapid application of nifedipine (32 microM), and of nifedipine plus 5 mM Ni, to assess the ability of ICa,L and reverse-mode Na-Ca exchange to trigger SR Ca release. With test pulses to +10 mV and +60 mV, a rapid switch to nifedipine (which blocked ICa,L) produced no significant reduction in phasic Fura-2 transient amplitude. This suggests that in the absence of ICa,L, another mechanism was able to trigger SR release. With pulses to +10 and +60 mV, a single beat switch to nifedipine plus 5 mM Ni almost completely abolished the phasic transient. Since 5 mM Ni inhibits Na-Ca exchange, this suggests that, in the absence of ICa,L, trigger Ca entry via reverse Na-Ca exchange was able to activate SR Ca release in atrial cells at 37 degrees C. The mechanisms underlying the Fura-2 transient in atrial cells, and differences with pre-existing data from rabbit ventricular cells, are discussed.
Mitcheson
J S
JS
Department of Physiology University of Bristol, School of Medical Sciences, UK.
Hancox
J C
JC
Levi
A J
AJ
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D004558:Electric Stimulation; D004594:Electrophysiology; D005456:Fluorescent Dyes; D016257:Fura-2; D006321:Heart / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018613:Microscopy, Confocal; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger
D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium; D016257:Fura-2