Adenophostin A, the most potent known agonist of inositol 1,4,5-trisphosphate (InsP3) receptors, stimulated 45Ca2+ release from the intracellular stores of permeabilized hepatocytes. The concentration of adenophostin A causing the half-maximal effect (EC50) was 7.1±0.5nM, whereas the EC50 for InsP3 was 177±26nM; both responses were positively co-operative. In rapid superfusion analyses of 45Ca2+ release from the intracellular stores of immobilized hepatocytes, maximal concentrations of adenophostin A or InsP3 evoked indistinguishable patterns of Ca2+ release. The Ca2+ release evoked by both agonists peaked at the same maximal rate after about 375ms and the activity of the receptors then decayed to a stable, partially (60%) inactivated state with a half-time (t1/2) of 318±29ms for adenophostin A and 321±22ms for InsP3. Dissociation rates were measured by recording rates of InsP3-receptor channel closure after rapid removal of agonist. The rate of adenophostin A dissociation (t1/2, 840±195ms) was only 2-fold slower than that of InsP3 (t1/2, 436±48ms). We conclude that slow dissociation of adenophostin A from InsP3 receptors does not underlie either its high-affinity binding or the reported differences in the Ca2+ signals evoked by InsP3 and adenophostin A in intact cells.

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