Estimation of systolic and diastolic free intracellular Ca2+ by titration of Ca2+ buffering in the ferret heart

Spectroscopic Ca²⁺-indicators are thought to report values of free intracellular Ca²⁺ concentration ([Ca²⁺]_i) that may differ from unperturbed values because they add to the buffering capacity of the tissue. To check this for the heart we have synthesized a new ¹⁹F-labelled NMR Ca²⁺ indicator, 1,2-bis-[2-bis(carboxymethyl)amino-4,5-difluorophenoxy]ethane (‘4,5FBAPTA’), with a low affinity (K_d 2950 nM). The new indicator and four previously described ¹⁹F-NMR Ca²⁺ indicators 1,2-bis-[2-(1 - carboxyethyl)(carboxymethyl)amino - 5 - fluorophenoxy]ethane (‘DiMe-5FBAPTA’), 1,2-bis-[2-(1-carboxyethyl)(carboxymethyl)amino-4-fluorophenoxy]ethane (‘DiMe-4FBAPTA’), 1,2-bis-[2-bis(carboxymethyl)amino-5-fluorophenoxy]ethane (‘5FBAPTA’) and 1,2-bis-[2-bis(carboxymethyl)amino-5-fluoro-4-methylphenoxy]ethane (‘MFBAPTA’), with dissociation constants for Ca²⁺ ranging from 46 to 537 nM, have been used to measure [Ca²⁺]_i, over the range from less than 100 nM to more than 3 μM, in Langendorff-perfused ferret hearts (30 °C, pH 7.4, paced at 1.0 Hz) by ¹⁹F-NMR spectroscopy. Loading hearts with indicators resulted in buffering of the Ca²⁺ transient. The measured end-diastolic and peak-systolic [Ca²⁺]_i were both positively correlated with indicator K_d. The positive correlations between indicator K_d and the measured end-diastolic and peak-systolic [Ca²⁺]_i were used to estimate the unperturbed end-diastolic and peak-systolic [Ca²⁺]_i by extrapolation to K_d = 0 (diastolic) and to K_d = ∞ (systolic) respectively. The extrapolated values in the intact beating heart were 161 nM for end-diastolic [Ca²⁺]_i and 2650 nM for peak-systolic [Ca²⁺]_i, which agree well with values determined from single cells and muscle strips.