Introduction 1 3 4 1–3 1 4 7 1 8 1 − 1 9 10 1 − 2+ 2+ 2+ 10 2+ We found that hypotonic shock markedly increased ATP, adenosine diphosphate (ADP), uridine triphosphate (UTP), and uridine diphosphate (UDP) concentrations in perfusates, which peaked at approximately 2.5 min. Nucleotide release was almost completely abolished from cells loaded with BAPTA and, under isotonic conditions, could be evoked by elevation of intracellular calcium with the calcium ionophore ionomycin. High nucleotide diphosphate (NDP) concentrations in perfusates of stimulated cells suggested that an NDP-rich compartment, e.g., the secretory pathway, contributed to this release. Together with real-time FM1-43 fluorescence experiments, our results strongly indicate that calcium-dependent exocytosis is a major mechanism of adenosine and uridine nucleotide release from A549 cells. Materials and methods Cells Human lung carcinoma A549 cells were grown in Dulbecco’s modified eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 56 U/ml penicillin-G and 56 μg/ml streptomycin sulfate. All constituents of the culture media were from GIBCO-BRL (Burlington, ON, Canada). ATP efflux was measured from cell monolayers grown to confluency on 24 × 60-mm glass coverslips. Fura-2 calcium imaging and FM1-43 microscopy experiments were performed on cells grown on circular 15-mm diameter no. 1 glass coverslips. Nucleotide efflux assay 10 2 3 2 2 2 4 2 4 2 4 2 2 HPLC quantification of adenine and uridine nucleotides 11 2 2 14 14 14 32 32 2 2 32 32 12 2 N 4 FM1-43 studies 13 14 2 2 xz xz Fura-2 calcium measurements 2 2 2 Chemicals For calcium-imaging experiments, Fura-2-AM was obtained from Molecular Probes, Invitrogen Corp. (Burlington, ON, Canada). Probenicid, Pluronic F127 and all other reagents were from Sigma Aldrich (Oakville, ON, Canada). Results Kinetics of nucleotide release 1 1 Fig. 1 a b c a b  +2 i +2 i 2+ 2 2+ 2 +2 i +2 i 2 +2 i Fig. 2 2+ i a 2+ b c n 2+ i d 340 380 2+ i  FM1-43 fluorescence changes implicate vesicular exocytosis 3 4 Fig. 3 a xz b  Fig. 4 a xz Δ CON ▪ ION • ION 0 Ca b  Discussion 1 +2 i 2 +2 i − 10 +2 i +2 i 2+ 2+ +2 i 2 +2 i 50 2+ 13 3 xz 15 10 16 17 18 19 20 21 22 25 1 26 2+ 2+