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 (Kd 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 Kd. The positive correlations between indicator Kd 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 Kd = 0 (diastolic) and to Kd = ∞ (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.

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