Ca2+ release triggered by NAADP in hepatocyte microsomes

Abstract

NAADP (nicotinic acid-adenine dinucleotide phosphate) is fast emerging as a new intracellular Ca2+-mobilizing messenger. NAADP induces Ca2+ release by a mechanism that is distinct from IP3 (inositol 1,4,5-trisphosphate)- and cADPR (cADP-ribose)-induced Ca2+ release. In the present study, we demonstrated that micromolar concentrations of NAADP trigger Ca2+ release from rat hepatocyte microsomes. Cross-desensitization to IP3 and cADPR by NAADP did not occur in liver microsomes. We report that non-activating concentrations of NAADP can fully inactivate the NAADP-sensitive Ca2+-release mechanism in hepatocyte microsomes. The ability of thapsigargin to block the NAADP-sensitive Ca2+ release is not observed in sea-urchin eggs or in intact mammalian cells. In contrast with the Ca2+ release induced by IP3 and cADPR, the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ concentration and pH (in the range 6.4-7.8). The NAADP-elicited Ca2+ release cannot be blocked by the inhibitors of the IP3 receptors and the ryanodine receptor. On the other hand, verapamil and diltiazem do inhibit the NAADP- (but not IP3- or cADPR-) induced Ca2+ release.

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).

Figure 2. Dose-dependence of the NAADP-induced Ca²⁺ release in rat liver microsomes

Microsomes were actively loaded with Ca²⁺ in the presence of 1 mM ATP and were assayed for Ca²⁺ release using different concentrations of NAADP in the range 0.01–10 μM. Results are means±S.E.M. for five independent experiments.

Figure 3. Unique homologous desensitization pattern of the NAADP receptors

(A) Homologous desensitization of NAADP receptors by subthreshold concentrations of NAADP. Actively loaded microsomes (●) were pre-treated with 0.1 μM NAADP for 2 min (starting 3 min after uptake was initiated, indicated by the arrow) and were then challenged to a supramaximal concentration of NAADP (10 μM, ○). NAADP-induced Ca²⁺ release from non-pre-treated microsomes (▼). The inset shows the Ca²⁺ efflux at 5 min of Ca²⁺ loading from microsomes incubated with non-activating concentrations of NAADP and non-pre-treated microsomes. (B) Dose–response curve of NAADP (●) and the residual Ca²⁺ release by a supramaximal concentration of NAADP (10 μM) after 2 min of pre-incubation with concentrations of NAADP between 0.1 nM and 10 μM (○).

Figure 4. Effects of thapsigargin and bafilomycin A1 on the cADPR- and NAADP-elicited Ca²⁺ release in rat liver microsomes

The actively loaded vesicles were pre-incubated with thapsigargin (1 μM) for at least 2 min and with bafilomycin A1 (1 μM) for at least 5 min before Ca²⁺ release was induced with supramaximal concentrations of cADPR and NAADP (both 10 μM). Closed bars represent the Ca²⁺ release from non-pre-treated microsomes, while open bars show the Ca²⁺ efflux from microsomes treated with thapsigargin (1 μM) and hatched bars represent the effect of bafilomycin A1 (1 μM).

Figure 5. Ca²⁺- and pH-dependence of the NAADP-induced Ca²⁺ release

(A) Extravesicular free Ca²⁺ concentration-dependence of the IP₃-, cADPR- and NAADP-mediated system in passively loaded liver microsomes. Extravesicular pCa (4–8) was set by EGTA (200–750 μM), NAADP (■), IP₃ (◆) and cADPR (□) were applied at supramaximal concentrations (10 μM). (B) Differential effect of pH on the cADPR- and NAADP-sensitive Ca²⁺-releasing system. The pH of the Ca²⁺-release medium was changed from 6.4 to 7.8, and the amount of ⁴⁵Ca²⁺ released by 10 μM cADPR (□) and 10 μM NAADP (■) was determined.

Figure 6. Pharmacological properties of the intracellular Ca²⁺ channels mediated by IP₃, cADPR and NAADP

The ⁴⁵Ca²⁺ release by supramaximal concentrations of IP₃, cADPR and NAADP (all 10 μM) was challenged in the presence of heparin (100 μg/ml), ryanodine (5 μM), Ruthenium Red (5 μM), verapamil (100 μM) and diltiazem (100 μM).