1 The sea urchin egg homogenate is an ideal model to characterize Ca2+-release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP(3)- and ryanodine-sensitive Ca2+-release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist. 2 The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca2+-releasing agent, NAADP, compared to inositol trisphosphate (InsP(3)) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors. 3 NAADP-induced Ca2+-release was blocked by L-type Ca2+-channel blockers and by Bay K 8644, while InsP(3)- and cyclic ADPR-induced Ca2+-release were insensitive to these agents. L-type Ca2+ channel blockers did not displace [P-32]-NAADP binding, suggesting that their binding site was different. Moreover, stopped-flow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion. 4 Similarly, a number of K+-channel antagonists blocked NAADP-induced Ca2+-release selectively over InsP(3)- and cyclic ADPR-induced Ca2+-release. Radioligand studies showed that these agents were not competitive antagonists. 5 As has been shown for InsP(3) and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca2+-release mechanisms. 6 The presence of K+ was not essential for NAADP-induced Ca2+-release, since substitution of K+ with other monovalent cations in the experimental media did not significantly alter Ca2+ release by NAADP. On the contrary, cyclic ADPR and InsP(3)-sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K+. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP-induced Ca2+-release. While the alkaline condition permitted InsP(3) and cyclic ADPR-induced Ca2+ release, the acidic condition completely hampered both Ca2+-release mechanisms. 7 The present results characterize pharmacologically and biochemically the novel Ca2+-release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.
