The Fura-2 transient can show two types of voltage dependence at 36°C in ventricular myocytes isolated from the rat heart

Abstract

We used the whole-cell patch-clamp method to investigate the voltage dependence of the L-type Ca current (ICa,L) and intracellular Ca (Cai) transient in ventricular myocytes isolated from the rat heart. Intracellular Ca was monitored using Fura-2 and the experiments were carried out at 36 degrees C. We measured ICa,L by using a caesium-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 positive potentials. When ICa,L was integrated over the first 25 ms to estimate the magnitude of Ca entry, this had a very similar voltage dependence to peak ICa,L. In all cells, phasic Fura-2 transients were abolished by 5 microM ryanodine (a blocker of the sarcoplasmic reticulum, SR) showing that the Fura-2 transient provided an index of the magnitude of SR Ca release. For experiments measuring the Cai transient, we used a K-based internal dialysis solution to preserve normal excitation-contraction coupling. In 30-40% of cells, we found that the Fura-2 transient had a bell-shaped voltage dependence. This suggests that, in these cells, the primary trigger mechanism for Ca-induced Ca-release might have been Ca entry via ICa,L. In the remaining 60-70% of cells, the voltage dependence of the Fura-2 transient was not bell-shaped. The Fura-2 transient reached a maximum with a pulse to +10 mV, and the amplitude of the transient did not decline significantly at more positive potentials to this. In cells with a non-bell-shaped voltage dependence of the Fura-2 transient, pulses to potentials as far positive as +140 mV elicited phasic Fura-2 transients. Since this potential exceeded the Nernst potential for Ca, it was unlikely there was any trigger Ca entry via ICa,L at this potential. This would suggest that, in these cells, another trigger for SR Ca release (in addition to ICa,L) might be present. We conclude that rat ventricular myocytes, produced using a standard isolation technique and under standard recording conditions, can show either a bell-shaped or a sigmoidal voltage dependence of the Fura-2 transient.