The effects of the NMR shift-reagents Dy(PPP)(2), Dy(TTHA) and Tm(DOTP) ondeveloped pressure in isolated perfused rat hearts. The role of shift-reagent calcium complexes

The Na-23 NMR shift-reagent complexes (Dy(PPP)(2), Dy(TTHA), and Tm(DOTP)) bind stoichiometric amounts of Ca2+. Thus, in perfused rat heart systems, a supplementation of Ca2+ is required to maintain the requisite extracellula r free calcium concentration ([Ca-0](f)) and to approximate a physiological level of contractile function. The amount of reagent-bound Ca2+ in a heart perfusate that contains a shift-reagent depends on: (1) Ca2+ binding by ex cess ligand used during the preparation of the shift-reagent; and (2) the C a2+ binding affinity of the shift-reagent. To address point 1), we introduc ed a H-1 and P-31 NMR spectroscopic titration method to quantify directly t he concentration of the excess ligand. We also used this method to minimize the amount of excess Ligand (L) and thus the amount of Ca-L complex. To ad dress point (2), we determined the stepwise K-d (muM) values of the Ca comp lexes of the three shift-reagents.: Dy(PPP)2, K-d1 = 0.09, K-d2 = 7.9; Dy(T THA), K-d1 = 10.66, K-d2 = 10.12; and Tm(DOTP), K-d1 = 0.502, K-d2 = 4.98. The K-d values of the Ca complexes of the phosphonate and triphosphate base d shift-reagents, Tm(DOTP) and Dy(PPP)(2), respectively, are lower than tho se of the polyaminocarboxylate-based Dy(TTHA), indicating stronger Ca. bind ing affinities for the former two types of complexes, We have also shown a positive correlation between [Ca-0](f) and left ventricular developed press ure (LVDP) in perfused rat hearts. Dy(TTHA) has shown no effect on LVDP v [ Ca-0](f). The LVDP values in the presence of the phosphonate and triphospha te based shift-reagents, however, were significantly higher than expected f rom the [Ca-0](f) levels alone. Thus a positive inotropic effect, independe nt of [Ca-0](f), is evident in the presence of Tm(DOTP) or Dy(PPP)(2). (C) 2001 Academic Press.