Modulation of store-operated ca²⁺ entry by cyclic-ADP-ribose

Store-operated Ca²⁺ entry plays an important role in Ca²⁺ homeostasis in cells but the mechanisms of control of these channels are not completely understood. We describe an investigation of the role of the CD38-cyclic-ADP-ribose (cADPR)-ryanodine-channel (RyR) signaling pathway in store-operated Ca²⁺ entry in human smooth muscle. We observed that human myometrial cells have a functional store-operated Ca²⁺ entry mechanism. Furthermore, we observed the presence of transient receptor potential 1, 3, 4, 5, and 6 ion channels in human myometrial cells. Store-operated Ca²⁺ transient was inhibited by at least 50-70% by several inhibitors of the RyR, including ryanodine (10 μM), dantrolene (10 μM), and ruthenium red (10 μM). Furthermore, the cell permeable inhibitor of the cADPR-system, 8Br-cADPR (100 μM), is a potent inhibitor of the store-operated entry, decreasing the store operated entry by 80%. Pre-incubation of cells with 100 μM cADPR and the hydrolysis-resistant cADPR analog 3-deaza-cADPR (50 μM), but not with ADP-ribose (ADPR) leads to a 1.6-fold increase in the store-operated Ca²⁺ transient. In addition, we observed that nicotinamide (1-10 mM), an inhibitor of cADPR synthesis, also leads to inhibition of the store-operated Ca²⁺ transient by 50-80%. Finally, we observed that the transient receptor potential channels, RyR, and CD38 can be co-immunoprecipitated, indicating that they interact in vivo. Our observations clearly implicate the CD38-cADPR-ryanodine signaling pathway in the regulation of store-operated Ca²⁺ entry in human smooth muscle cells.