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

Abstract

Ca2+ signaling in vascular smooth muscle cells involves global and local interactions between multiple Ca2+ influx and release pathways. Ca2+ release pathways include IP3 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 Ca2+ 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 [Ca2+]i. It was blocked by the NAADP antagonist NED‐19 or the acidic vacuolar H+‐ATPase bafilomycin A, suggesting Ca2+ release from the lysosomal Ca2+ stores. The Ca2+ response was independent of extracellular Ca2+ influx, and was unaffected by the IP3 receptor blocker xestospongin C; but was partially inhibited by ryanodine or thapsigargin. Moreover, NAADP caused dramatic increase in local Ca2+ 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 Ca2+ sparks, the elementary Ca2+ events mediated via the ryanodine receptors. Our results, hence, show that the NAADP‐sensitive release channels are functionally expressed and Ca2+ signals from the NAADP channel‐gated lysosomal stores cross‐activate ryanodine receptors to amplify Ca2+ release in PASMCs.