Production of NAADP and its role in Ca 2ϩ mobilization associated with lysosomes in coronary arterial myocytes. The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic

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... analysis of intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca 2ϩ ]i by 711 Ϯ 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca 2ϩ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca 2ϩ release and vacuolar proton pump function, NAADP-induced [Ca 2ϩ ]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca 2ϩ ]i in these cells with a large increase in global Ca 2ϩ level of 1,815 Ϯ 84 nM. Interestingly, before this large Ca 2ϩ increase, a small Ca 2ϩ spike with an increase in [Ca 2ϩ ]i of 529 Ϯ 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca 2ϩ ]i response was completely abolished, whereas Rya (50 M) only markedly blocked the ET-1-induced large global Ca 2ϩ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 Ϯ 4.42% to 51.80 Ϯ 4.36%. Our results suggest that a lysosome-mediated Ca 2ϩ regulatory mechanism via NAADP contributes to ET-1-induced Ca 2ϩ mobilization in CASMCs and consequent vasoconstriction of coronary arteries. ␤-nicotinamide adenine dinucleotide phosphate; adenosine 5Ј-diphosphate-ribosyl cyclase; coronary artery; vascular smooth muscle NICOTINIC ACID (NA) adenine dinucleotide phosphate (NAADP) is produced from NADP ϩ by a replacement of its nicotinamide (Nicot) group with NA via ADP-ribosyl cyclase or its mammalian homologue CD38 (1). NAADP was first identified to mobilize Ca 2ϩ in sea urchin eggs (40), and recently this signaling nucleotide has been shown to act as an endogenous regulator of intracellular Ca 2ϩ in a wide variety of cell types from plants to animals, thereby participating in the regulation of cell functions, such as fertilization, cell proliferation and differentiation, insulin secretion, and nitric oxide signaling, as well as muscle constriction (33, 37, 42, 50) . In contrast to the well-characterized D-myo-inositol 1,4,5-trisphosphate