Cyclic ADP-ribose-induced calcium release in sea urchin egg homogenates is a cooperative process

Cyclic ADP-ribose (cADPR), an enzymatic product formed from NAD+ in sea urchin eggs and in several mammalian cell types, has been proposed as a second messenger for the regulation of cytosolic free Ca2+ concentration. Although cADPR induces Ca2+ release from an intracellular Ca2+ pool by a mechanism that differs from that used by inositol 1,4,5-trisphosphate, the precise action is unknown. We have analyzed the kinetics of cADPR-induced Ca2+ release at 15 degrees C in sea urchin egg homogenates using the calcium indicator fluo-3. In this system, Ca2+ release induced by cADPR was a positively cooperative process with a Hill coefficient of 1.8 +/- 0.32 (mean +/- SE, n = 15). We also examined the effect of caffeine, which potentiates the action of cADPR, on the kinetics of cADPR-induced Ca2+ release. Caffeine (3 mM) did not affect the degree of cooperativity induced by cADPR, but it did cause an increase in the apparent affinity of cADPR for its cellular target; the apparent Kd, derived from the kinetic analysis, decreased from 176 +/- 50 to 68 +/- 13 nM. We conclude that cADPR-induced Ca2+ release in sea urchin eggs is a cooperative process. A straightforward interpretation of the analysis is that opening of the Ca(2+)-release channel requires the binding of two molecules of cADPR (Hill coefficient of about 2). However, it is not yet known whether cADPR binds directly to the release channel. It is possible that potentiation of the action of cADPR by newly released Ca2+ could also contribute to the observed cooperative effect.