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1. The effects of acetylcholine upon the output of amylase, Ca²⁺ efflux and membrane potential of pancreatic acinar cells have been measured in segments of mouse pancreas superfused in vitro.

2. Amylase output was measured continuously using an on-line automated fluorimetric method; Ca²⁺ efflux was monitored by measuring the release of ⁴⁵Ca²⁺ from pre-labelled tissue; and intracellular recordings of acinar transmembrane potentials were obtained with glass micro-electrodes. In some experiments membrane potentials, and in others ⁴⁵Ca²⁺ efflux, were measured concomitantly with amylase release.

3. Acetylcholine depolarized the acinar cells, increased tissue ⁴⁵Ca²⁺ efflux and raised amylase output, each with a similar dose-dependence, i.e. a maximal response at 10^-5 M, threshold 10^-8 M, and ED₅₀ values of 0·7 x 10^-7 M, 0·5 x 10^-7 M, and 2 x 10^-7 M for depolarization, amylase release, and ⁴⁵Ca²⁺ efflux, respectively.

4. In response to acetylcholine both depolarization and ⁴⁵Ca²⁺ efflux preceded or coincided with the increase in amylase output.

5. Acetylcholine 10^-5 M and [K]₀ 47 mM were without effect on ⁴⁵Ca²⁺ efflux in the presence of atropine (3 x 10^-6 M) but pancreozymin (0·3 u./ml.) still elicited a marked increase in ⁴⁵Ca²⁺ release.

6. These results suggest that the stimulatory action of acetylcholine on the pancreatic acinar cell involves, sequentially, a specific receptor-activated increase in membrane permeability, depolarization, Ca²⁺ mobilization and amylase release. These events are discussed in relation to the integrated mechanism of stimulus-secretion coupling.