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 Ca²-mobilizing messenger that releases Ca² 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 Ca² 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 [Ca²]i, which was independent of extracellular Ca² and blocked by the NAADP antagonist Ned-19 or the vacuolar H⁺-ATPase inhibitor bafilomycin A1, indicating Ca² release from acidic endolysosomal Ca² stores. The Ca² response was unaffected by xestospongin C but was partially blocked by ryanodine or thapsigargin. NAADP triggered heterogeneous local Ca² signals, including a diffuse increase in cytosolic [Ca²], Ca² sparks, Ca² bursts, and regenerative Ca² release. The diffuse Ca² increase and Ca² bursts were ryanodine-insensitive, presumably arising from different endolysosomal sources. Ca² sparks and regenerative Ca² 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 Ca² signals interact with both ryanodine and inositol 1,4,5-trisphosphate receptors during agonist stimulation. Our results show thatNAADPmediates complex global and local Ca² signals. Depending on the physiological stimuli, these diverse Ca² signals may serve to regulate different cellular functions in PASMCs.