Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Activates Global and Heterogeneous Local Ca2+ Signals from NAADP- and Ryanodine Receptor-gated Ca2+ Stores in Pulmonary Arterial Myocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-mobilizing messenger that releases Ca(2+) from endolysosomal organelles. Recent studies showed that NAADP-induced Ca(2+) release is mediated by the two-pore channels (TPCs) TPC1 and TPC2. However, the expression of TPCs and the NAADP-induced local Ca(2+) signals have not been examined in vascular smooth muscle. Here, we found that both TPC1 and TPC2 are expressed in rat pulmonary arterial smooth muscle cells (PASMCs), with TPC1 being the major subtype. Application of membrane-permeant NAADP acetoxymethyl ester to PASMCs elicited a biphasic increase in global [Ca(2+)]i, which was independent of extracellular Ca(2+) and blocked by the NAADP antagonist Ned-19 or the vacuolar H(+)-ATPase inhibitor bafilomycin A1, indicating Ca(2+) release from acidic endolysosomal Ca(2+) stores. The Ca(2+) response was unaffected by xestospongin C but was partially blocked by ryanodine or thapsigargin. NAADP triggered heterogeneous local Ca(2+) signals, including a diffuse increase in cytosolic [Ca(2+)], Ca(2+) sparks, Ca(2+) bursts, and regenerative Ca(2+) release. The diffuse Ca(2+) increase and Ca(2+) bursts were ryanodine-insensitive, presumably arising from different endolysosomal sources. Ca(2+) sparks and regenerative Ca(2+) release were inhibited by ryanodine, consistent with cross-activation of loosely coupled ryanodine receptors. Moreover, Ca(2+) release stimulated by endothelin-1 was inhibited by Ned-19, ryanodine, or xestospongin C, suggesting that NAADP-mediated Ca(2+) signals interact with both ryanodine and inositol 1,4,5-trisphosphate receptors during agonist stimulation. Our results show that NAADP mediates complex global and local Ca(2+) signals. Depending on the physiological stimuli, these diverse Ca(2+) signals may serve to regulate different cellular functions in PASMCs.