The effects of Ca 2+-free perfusion and the calcium paradox on [ 125I] endothelin-1 binding to rat cardiac membranes

Abstract

The binding characteristics of [ 125I]endothelin-1 (ET-1) to cardiac membranes isolated from rat hearts subjected to Ca 2+-free perfusion or the Ca 2+ paradox were examined. The effect of treatment with 2, 3 butanedione monoxime (BDM), which inhibits the tissue damage associated with the calcium paradox, was also investigated. Membranes from rat hearts perfused under control conditions bound [ 125I]ET-1 to a single population of sites with a B max of 107·7 ± 3.7 fmol/mg protein and an affinity ( K D) of 153 ± 12 pM. Ten minutes of Ca 2+-free perfusion resulted in a significant ( P < 0.01) increase in B max to 167.5 ± 8.3 fmol/mg protein without change in K D. Ca 2+ repletion following Ca 2+-free perfusion tended to increase further the B max (180.6 ± 10.4 fmol/mg protein) without change in K D. Treatment with BDM attenuated but did not prevent the rise in B max following Ca 2+-free perfusion. Following Ca 2+ repletion, however, B max returned to control levels in the BDM treated group. These changes were not associated with changes in the ability of ET-1 and ET-3 to inhibit [ 125I]ET-1 binding. The results demonstrate that Ca 2+-free perfusion is associated with an increase in the binding site density of [ 125I]ET-1 which is maintained or further increased upon Ca 2+ repletion. If, however, the tissue damage associated with the Ca 2+ paradox is prevented with BDM, Ca 2+ repletion is associated with a reversal of the increase due to Ca 2+-free perfusion.