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 calcium indicator, 1,2- bis-[2-bis(carboxymethyl)amino-4,5-difluorophenoxy] ethane (4,5FBAPTA) with a low affinity (Kd: 2950nM). The new indicator and four previously described 19F-NMR Ca2+ indicators, 1,2-bis-[2- bis(carboxymethyl)amino-5-fluorophenoxy]ethane (5-FBAPTA), 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) and 1,2-bis-[2-bis(carboxymethyl)amino-5-fluoro-4-methylphenoxy]ethane (MFBAPTA), with dissociation constants for Ca2+ ranging from 46 to 537nM have been used to measure [Ca2+]i, over the range <100nM to >3µM, in Langendorff-perfused ferret hearts (30°C, pH 7.4, paced at 1.0Hz) by 19F-NMR spectroscopy. Loading hearts within dicators 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 enddiastolic 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 161nM for end-diastolic [Ca2+]i and 2650nM for peak systolic [Ca2+]i which agree well with values determined from single cells and muscle strips.