ABT-200, a norepinephrine uptake inhibitor, blocks Ca2+ signals in chromaffin cells

We previously described inhibition by racemic (+/-)-(1'R*,3R*)-3-phenyl-1- [1',2',3',4'-tetrahydro-5',6'-methylenedioxy-1'- napthalenyl-methyl]-pyrrolidine methanesulfonate (ABT-200), and its two constituent enantiomers, SS,ABT-200 and RR,ABT-200, of nicotine-stimulated but not histamine-stimulated catecholamine release from bovine adrenal chromaffin cells. To test the hypothesis that this inhibition reflects a blockade of Ca2+ influx, we used fura-2 loaded chromaffin cells to investigate cytosolic Ca2+ signals. We found that SS,ABT-200 inhibited nicotine- and K(+)-stimulated Ca2+ signals, both of which depend on Ca2+ influx. However, the early phase of the histamine-stimulated Ca2+ signals, which depends on Ca2+ mobilization from intracellular stores, was unaffected. We also examined ion flux through the nicotinic receptor by measuring 86rubidium+ (86Rb+) efflux from preloaded mouse midbrain synaptosomes. We found that SS,ABT-200 partially inhibited nicotine-stimulated 86Rb+ efflux, suggesting that it blocks ion flux through the nicotinic receptor directly. These data support a model in which ABT-200 blocks nicotine-stimulated catecholamine release by inhibiting cation flux through multiple channels.