ACETYLCHOLINE ACTIVATES INTRACELLULAR MOVEMENT OF INSULIN GRANULES INPANCREATIC BETA-CELLS VIA INOSITOL TRIPHOSPHATE-DEPENDENT MOBILIZATION OF INTRACELLULAR CA2+

Intracellular movement of secretory granules is a proximal stage in th e secretory cascade that ends in the release product from cells. We in vestigated mechanisms underlying the control of this movement by acety lcholine using an insulinoma cell line, MIN6, in which acetylcholine i ncreases both insulin secretion and granule movement. The peak activat ion of movement was observed 3 min after an acetylcholine challenge. T he effects were nullified by the muscarinic inhibitor atropine, phosph olipase C (PLC) inhibitors (D 609 and compound 48/80), and pretreatmen t with the Ca2+ pump inhibitor, thapsigargin, Inhibitors of Ca2+-depen dent phospholipase A(2) (arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphate) also partially inhibited the movement ca used by acetylcholine, but downregulation of protein kinase C by overn ight incubation with the phorbol ester 12-o-tetradecanoylphorbol-13-ac etate failed to exert any influence. Acetylcholine stimulation of gran ule movement was not reproduced by membrane depolarization with high K +. Phosphorylation of the endogenous myosin light chain in MIN6 cells was increased by addition of acetylcholine and decreased by the Ca2+ c helator BAPTA ,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid) . The calmodulin inhibitor W-7 and the myosin light-chain kinase inhib itor ML-9 decreased the motile events in the beta-cells under both non stimulated and acetylcholine-stimulated conditions. These findings led us to conclude that inositol triphosphate causes Ca2+ mobilization by muscarinic activation of PLC, leading to intracellular translocation of insulin granules to the ready-releasable pool in pancreatic beta-ce lls via Ca2+/calmodulin-dependent phosphorylation of myosin light chai ns.