PHARMACOLOGICAL PROPERTIES OF THE CA2-RELEASE MECHANISM SENSITIVE TO NAADP IN THE SEA-URCHIN EGG()

1 The sea urchin egg homogenate is an ideal model to characterize Ca2-release mechanisms because of its reliability and high signal-to-nois e-ratio. Apart from the InsP(3)- and ryanodine-sensitive Ca2+-release mechanisms, it has been recently demonstrated that this model is respo nsive to a third independent mechanism, that has the pyridine nucleoti de, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endog enous agonist. 2 The sea urchin egg homogenate was used to characteriz e 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 ryano dine 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-i nduced Ca2+-release were insensitive to these agents. L-type Ca2+ chan nel blockers did not displace [P-32]-NAADP binding, suggesting that th eir 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 Ca 2+-release selectively over InsP(3)- and cyclic ADPR-induced Ca2+-rele ase. 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 intracellula r Ca2+-release mechanisms. 6 The presence of K+ was not essential for NAADP-induced Ca2+-release, since substitution of K+ with other monova lent 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 d epending 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 alkal ine 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 biochemicall y the novel Ca2+-release mechanism sensitive to NAADP. Such characteri zation will help future research aimed at understanding the role of NA ADP in mammalian systems.