CALIBRATION OF INDO-1 AND RESTING INTRACELLULAR [CA](I) IN INTACT RABBIT CARDIAC MYOCYTES

Fluorescent Ca indicators have been extremely valuable in understanding intracellular [Ca] ([Ca](i)) regulation in many cell types. The calibration of these indicators in the intracellular environment, however, has been a continuous challenge. We performed in vivo calibrations of indo-1 in isolated rabbit ventricular myocytes loaded with the acetoxymethylester form of indo-1 and used the perforated patch variation of whole cell voltage clamp, Voltage, [Na], and [K] gradients were eliminated to approach equilibrium. We also took advantage of the powerful Na/Ca exchange in cardiac myocytes so that [Ca](i) would be equilibrated with [Ca](o) (because there was no [Na] or voltage gradient). The equilibration of [Na] and [Ca] across the membrane was tested by measuring the reversal potential of Na current and poking the cell to test for changes in [Ca](i)-dependent fluorescence ratio. The apparent dissociation constant, K-d for indo-1 in the cellular environment was 844 nM, which is similar to 2-3 times higher than that in aqueous solutions. In a separate series of experiments, a null point approach was used to determine the [Ca](i) in intact cells at rest for very long periods (82 +/- 6 nM). This is lower than that measured 15 s after a train of steady-state twitches ([Ca](i) = 294 +/- 53 nM). These experiments also allowed the direct assessment of the shortening versus [Ca](i) relationship in intact cells.