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

Abstract

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. ©2013 by The American Society for Biochemistry and Molecular Biology, Inc.