Figure 1. NAADP-induced ⁴⁵Ca²⁺ release from active loaded hepatocyte microsomes.

(A) The time course of the Ca²⁺ uptake by liver microsomes was determined using ⁴⁵Ca²⁺, as described in the Materials and methods section. Accumulation (■) of Ca²⁺ was started by addition of 1 mM ATP. The amount of mobilizable Ca²⁺ was determined by adding 5 μM ionomycin (arrow) to the medium. The effect on ⁴⁵Ca²⁺ uptake of 1 μM thapsigargin (△) and 1 μM bafilomycin A1 (□) was also tested. Bafilomycin A1 was added to the microsomes 5 min before ⁴⁵Ca²⁺ uptake was initiated. (B) Comparison of the Ca²⁺-mobilizing characteristics of IP₃ (○), cADPR (●) and NAADP (▼) (10 μM each). CICR (▽) was determined by adjusting extravesicular free Ca²⁺ levels to pCa 6 using EGTA (100 μM). Results are means±S.E.M. for six to twelve determinations on at least four different experimental days. The inset shows the total amount of Ca²⁺ efflux triggered by IP₃, cADPR and NAADP after 5 s of Ca²⁺ release. (C) Microsomes sequestered Ca²⁺ in the presence of an ATP-regenerating system (2 units/ml creatine-kinase and 4 mM phosphocreatine) and released calcium in response to subsequent addition of cADPR (●, 10 μM), IP₃ (○, 10 μM) and NAADP (▼, 10 μM).