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 intracel lular Ca2+ concentration ([Ca2+](i)) that may differ from unperturbed value s because they add to the buffering capacity of the tissue. To check this f or the heart we have synthesized a new F-19-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 de scribed F-19-NMR Ca2+ indicators 1,2-bis-[2-(1-carboxyethyl)(caraboxymethyl )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 dissoci ation constants for Ca2+ ranging from 46 to 537 nM, have been used to measu re [Ca2+](i), over the range from less than 100 nM to more than 3 mu M, in Langendorff-perfused ferret hearts (30 degrees C, pH 7.4, paced at 1.0 Hz) by F-19-NMR spectroscopy. Loading hearts with indicators resulted in buffer ing of the Ca2+ transient. The measured end-diastolic and peak-systolic [Ca 2+](i) were both positively correlated with indicator K-d. The positive cor relations between indicator K-d and the measured end-diastolic and peak-sys tolic [Ca2+](i) were used to estimate the unperturbed end-diastolic and pea k-systolic [Ca2+](i) by extrapolation to K-d=0 (diastolic) and to K-d = inf inity (systolic) respectively. The extrapolated values in the intact beatin g heart were 161 nM for end-diastolic [Ca2+](i) and 2650 nM for peak-systol ic [Ca2+](i), which agree well with values determined from single cells and muscle strips.