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.

ACh (250 μM) 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 (τ) 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 μM) for much longer periods in the presence than in the absence of the agonist.

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 τ for the refilling of the RRP from 31 to 19 s.

Collectively, the effects of ACh on the RRP and τ result in a > 10‐fold stimulation of the rate at which granules are supplied for release.