title: Estimation of systolic and diastolic free intracellular Ca2+ by titration of Ca2+ buffering in the ferret heart.
abstract: Spectroscopic Ca(2+)-indicators are thought to report values of free intracellular Ca(2+) concentration ([Ca(2+)](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 (19)F-labelled NMR Ca(2+) 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 (19)F-NMR Ca(2+) 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 Ca(2+) ranging from 46 to 537 nM, have been used to measure [Ca(2+)](i), over the range from less than 100 nM to more than 3 microM, in Langendorff-perfused ferret hearts (30 degrees C, pH 7.4, paced at 1.0 Hz) by (19)F-NMR spectroscopy. Loading hearts with indicators resulted in buffering of the Ca(2+) transient. The measured end-diastolic and peak-systolic [Ca(2+)](i) were both positively correlated with indicator K(d). The positive correlations between indicator K(d) and the measured end-diastolic and peak-systolic [Ca(2+)](i) were used to estimate the unperturbed end-diastolic and peak-systolic [Ca(2+)](i) by extrapolation to K(d)=0 (diastolic) and to K(d)=infinity (systolic) respectively. The extrapolated values in the intact beating heart were 161 nM for end-diastolic [Ca(2+)](i) and 2650 nM for peak-systolic [Ca(2+)](i), which agree well with values determined from single cells and muscle strips.
authors.affiliation: Section of Cardiovascular Biology, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.
authors.forename: H L
authors.initials: HL
authors.lastname: Kirschenlohr
authors.forename: A A
authors.initials: AA
authors.lastname: Grace
authors.forename: J I
authors.initials: JI
authors.lastname: Vandenberg
authors.forename: J C
authors.initials: JC
authors.lastname: Metcalfe
authors.forename: G A
authors.initials: GA
authors.lastname: Smith
mesh_terms: D000818:Animals; D001794:Blood Pressure; D002118:Calcium / Q000032:analysis / Q000502:physiology*; D005289:Ferrets; D006321:Heart / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D009200:Myocardial Contraction
chemical_list: D002118:Calcium