title: Calcium measurements with a new high-affinity n.m.r. indicator in the isolated perfused heart.
abstract: A new n.m.r. indicator, 1,2-bis-(2-[1-(hydroxycarbony)ethyl- (hydroxycarbonylmethyl)]amino-5-fluorophenoxy)ethane (DiMe-5FBAPTA), with a higher affinity for calcium (apparent Kd 46 nM, pH 7.2, 30 degrees C) than the parent 5FBAPTA chelator (Kd 537 nM, pH 7.1, 30 degrees C) has been used to measure the cardiac intracellular free Ca2+ ([Ca2+]i). DiMe-5FBAPTA was loaded into Langendorff-perfused ferret hearts maintained at 30 degrees C using the acetoxymethyl ester (AM) derivative. The intracellular concentration required to achieve an adequate signal-to-noise (S/N) ratio (> 10:1) for the n.m.r. spectra caused a similar reduction in developed pressure to that obtained using 5FBAPTA-AM. The DiMe-5FBAPTA was used to estimate [Ca2+]i in diastole, through the calcium transient and at rest in the presence of the slow calcium channel blocker diltiazem. At a pacing frequency of 1.0 Hz, end-diastolic [Ca2+]i was 198 +/- 30 nM (n = 9), and reducing the pacing frequency to 0.2 Hz lowered [Ca2+]i to 89 +/- 13 nM (n = 5). Perfusion with diltiazem (100 microM) for 60 min lowered [Ca2+]i to 10 +/- 1 nM (n = 4) in unpaced hearts and to 94 +/- 24 nM (n = 4) in hearts paced at 1.0 Hz. The [Ca2+]i transient measured with DiMe-5FBAPTA was sharper and delayed compared with the transient measured previously with 5FBAPTA. Co-loading the two indicators provided evidence that the indicator with the higher Kd had a dominant effect on the end-diastolic [Ca2+]i. The lower values for end-diastolic [Ca2+]i obtained with DiMe-5FBAPTA are consistent with fluorescent indicator measurements. These observations suggest that perturbations of [Ca2+]i caused by the new indicator are less than those induced by 5FBAPTA. DiMe-5FBAPTA therefore represents a useful step in the development of 19F-n.m.r. calcium indicators.
authors.affiliation: Department of Biochemistry, University of Cambridge, U.K.
authors.forename: H L
authors.initials: HL
authors.lastname: Kirschenlohr
authors.forename: A A
authors.initials: AA
authors.lastname: Grace
authors.forename: S D
authors.initials: SD
authors.lastname: Clarke
authors.forename: Y
authors.initials: Y
authors.lastname: Shachar-Hill
authors.forename: J C
authors.initials: JC
authors.lastname: Metcalfe
authors.forename: P G
authors.initials: PG
authors.lastname: Morris
authors.forename: G A
authors.initials: GA
authors.lastname: Smith
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis*; D002614:Chelating Agents; D004110:Diltiazem / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D005289:Ferrets; D006321:Heart / Q000187:drug effects / Q000502:physiology; D006324:Heart Arrest, Induced; D006339:Heart Rate; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium / Q000737:chemistry*; D010477:Perfusion
chemical_list: D002614:Chelating Agents; D007202:Indicators and Reagents; C082393:1,2-bis(2-(1-hydroxycarbonyl)ethyl-(hydoxycarbonylmethyl)amino-5-fluorophenoxy)ethane; D004533:Egtazic Acid; C039877:5,5'-difluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D004110:Diltiazem; D002118:Calcium