CALCIUM MEASUREMENTS WITH A NEW HIGH-AFFINITY NMR INDICATOR IN THE ISOLATED-PERFUSED HEART

A new n.m.r. indicator, ydroxycarbonylmethyl)!amino-5-fluorophenoxy}et hane (DiMe-5FBAPTA), with a higher affinity for calcium (apparent K(d) 46 nM, pH 7.2, 30-degrees-C) than the parent 5FBAPTA chelator (K(d) 5 37 nM, pH 7.1, 30-degrees-C) has been used to measure the cardiac intr acellular free Ca2+ (Ca2+!i). DiMe-5FBAPTA was loaded into Langendorf f-perfused ferret hearts maintained at 30-degrees-C using the acetoxym ethyl 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 prese nce of the slow calcium channel blocker diltiazem. At a pacing frequen cy of 1.0 Hz, end-diastolic Ca2+!i was 198 +/- 30 nM (n = 9), and red ucing the pacing frequency to 0.2 Hz lowered Ca2+!i to 89 +/- 13 nM ( n = 5). Perfusion with diltiazem (100 muM) for 60 min lowered Ca2+!i to 10 +/- 1 nM (n = 4) in unpaced hearts and to 94 +/- 24 nM (n = 4) i n hearts paced at 1. 0 Hz. The Ca2+)i transient measured with DiMe-5F BAPTA was sharper and delayed compared with the transient measured pre viously with 5FBAPTA. Co-loading the two indicators provided evidence that the indicator with the higher K(d) had a dominant effect on the e nd-diastolic Ca2+!i. The lower values for end-diastolic Ca2+!i obtai ned with DiMe-5FBAPTA are consistent with fluorescent indicator measur ements. These observations suggest that perturbations of Ca2+!i cause d by the new indicator are less than those induced by 5FBAPTA. DiMe-5F BAPTA therefore represents a useful step in the development of F-19-n. m.r. calcium indicators.