Calcium mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) in rat astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to release intracellular Ca2+ in several types of cells. We have used Ca2+-sensitive fluorescent dyes (Fura-2, Fluo-4) to measure intracellular Ca2+ in astrocytes in culture and in situ. Bath-applied NAADP elicited a reversible and concentration-dependent Ca2+ rise in up to 90% of astrocytes in culture (EC50 = 7 μM). The NAADP-evoked Ca2+ rise was maintained in the absence of extracellular Ca2+, but was suppressed after depleting the Ca2+ stores of the ER with ATP (20 μM), with cyclopiazonic acid (10 μM) or with ionomycin (5 μM). P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′4′-disulfonic acid (PPADS, 100 μM), IP3 receptor blocker 2-aminoethoxydiphenyl borate (2-APB, 100 μM) and PLC inhibitor U73122 (10 μM) also reduced or suppressed the NAADP-evoked Ca2+ rise. NAADP still evoked a Ca2+ response after application of glycyl-l-phenylalanine-β-naphthylamide (GPN, 200 μM), which permeabilizes lysosomes, or preincubation with H+-ATPase inhibitor bafilomycin A1 (4 μM) and of p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, 2 μM), that impairs mitochondrial Ca2+ handling. In acute brain slices, NAADP (10 μM) evoked Ca2+ transients in cerebellar Bergmann glial cells and in hippocampal astrocytes. Our results suggest that NAADP recruits Ca2+ from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in mammalian astrocytes, at least partly by activating metabotropic P2Y receptors.