Ca²⁺ Signaling Occurs via Second Messenger Release from Intraorganelle Synthesis Sites

Cyclic ADP-ribose is an important Ca²⁺-mobilizing cytosolic messenger synthesized from β-NAD⁺ by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCβ and ARCγ are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a β-NAD⁺ transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca²⁺ wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.