CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells.
1. Measurements of cell capacitance were used to investigate the mechanisms by which acetylcholine (ACh) stimulates Ca2+-induced exocytosis in single insulin-secreting mouse pancreatic B-cells. 2. ACh (250 microM) increased exocytotic responses elicited by voltage-clamp depolarizations 2.3-fold. This effect was mediated by activation of muscarinic receptors and dependent on elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) attributable to mobilization of Ca2+ from intracellular stores. The latter action involved interference with the buffering of [Ca2+]i and the time constant (tau) for the recovery of [Ca2+]i following a voltage-clamp depolarization increased 5-fold. As a result, Ca2+ was present at concentrations sufficient to promote the replenishment of the readily releasable pool of granules (RRP; > 0.2 microM) for much longer periods in the presence than in the absence of the agonist. 3. The effect of Ca2+ on exocytosis was mediated by activation of CaM kinase II, but not protein kinase C, and involved both an increased size of the RRP from 40 to 140 granules and a decrease in tau for the refilling of the RRP from 31 to 19 s. 4. Collectively, the effects of ACh on the RRP and tau result in a > 10-fold stimulation of the rate at which granules are supplied for release.
Department of Islet Cell Physiology, Islet Discovery Research, Novo Nordisk A/S, Novo Allé, DK-2880 Bagsvaerd, Denmark. jlg@novo.dk
J
J
Gromada
M
M
Høy
E
E
Renström
K
K
Bokvist
L
L
Eliasson
S
S
Göpel
P
P
Rorsman
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000201:enzymology*; D004553:Electric Conductivity; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects*; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018721:Muscarinic Agonists / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013268:Stimulation, Chemical
D004791:Enzyme Inhibitors; D018721:Muscarinic Agonists; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000109:Acetylcholine; D002118:Calcium