Characterization of NAADP‐Induced Global and Local Calcium Signals in Rat Pulmonary Arterial Smooth Muscle Cells

Ca 2+ signaling in vascular smooth muscle cells involves global and local interactions between multiple Ca 2+ influx and release pathways. Ca 2+ release pathways include IP 3 receptor‐, ryanodine receptor‐, and nicotinic acid adenine dinucleotide phosphate (NAADP) receptor‐mediated mechanisms. Recent studies demonstrated that the two‐pore channels (TPC1 and TPC2) are NAADP receptors located in the endo‐/lysosomes. Here we characterized the NAADP‐mediated Ca 2+ signals in rat pulmonary arterial smooth muscle cells (PASMCs). Application of the membrane permeable NAADP‐AM (0.25–1 μM) to PASMCs elicited concentration‐dependent increase in global [Ca 2+ ] i . It was blocked by the NAADP antagonist NED‐19 or the acidic vacuolar H + ‐ATPase bafilomycin A, suggesting Ca 2+ release from the lysosomal Ca 2+ stores. The Ca 2+ response was independent of extracellular Ca 2+ influx, and was unaffected by the IP 3 receptor blocker xestospongin C; but was partially inhibited by ryanodine or thapsigargin. Moreover, NAADP caused dramatic increase in local Ca 2+ release events, which was inhibited by ryanodine or NED‐19. The temporal and spatial properties of the NAADP‐induced local release events were comparable to the spontaneous Ca 2+ sparks, the elementary Ca 2+ events mediated via the ryanodine receptors. Our results, hence, show that the NAADP‐sensitive release channels are functionally expressed and Ca 2+ signals from the NAADP channel‐gated lysosomal stores cross‐activate ryanodine receptors to amplify Ca 2+ release in PASMCs.