Most assays of secretion in exocrine tissues involve the collection and measurement of secretory products, such as amylase. The methods are slow, have poor time resolution and give no insight into the exocytotic process of single-vesicle events. We now describe initial results obtained using 2-photon microscopy to identify and follow single vesicle events in submandibular acinar cells.

Mice were humanely killed and the submandibular gland removed. The gland was then minced and incubated in collagenase (Worthington CLSPA) for 5-10 min at 37 °C. The tissue was then resuspended in extracellular solution (containing [mM] NaCl 135, KCL 5, MgCl₂ 1, CaCl₂ 2, Glucose 10, Hepes 10 -pH 7.4 NaOH) and gently triturated to produce a preparation of large clusters of acinar cells. The clusters were then placed on Poly-l-ornithine coated coverslips and Oregon Green (100 ÁM, Molecular Probes) added to the extracellular solution. The cell clusters were imaged using 2-photon microscopy (Leica) with light excitation at 890 nm and emitted lighted, longer than 505 nm, collected. Images were then processed using Metamorph software (Universal Imaging).

In unstimulated cells the fluorescent dye in the extracellular media labels the ductal system of the acinus but is excluded from cell interiors. We stimulated cells with 1 ÁM isoproterenol (b adrenergic agonist) and with a latency of 64.85 s ( ± 31.1 s mean ± S.E.M., n = 6 records) observed the appearance of small spots of fluorescence exclusively in the apical pole of individual acinar cells. These spots had a diameter of 1.04 Ám ( ± 0.07 mean ± S.E.M., n = 15 events). The time-course, size and location of these spots are consistent with the entry of extracellular dye through the fusion pore of single secretory vesicles undergoing exocytosis. In parallel experiments we measured the intracellular calcium response of submandibular cells to stimulation with the same concentration of isoproterenol. Resting calcium was found to be 43 nM ( ± 4.6 mean ± S.E.M., n = 6 records), calcium measured 65 s after application of agonist (same time as latency to secretory response) was 45 nM ( ± 5.7 mean ± S.E.M.). Subsequent application of 500 nM Acetylcholine raised calcium to 390 nM ( ± 225 mean ± S.E.M.) demonstrating our ability to detect a calcium response.

Using our new assay we are able to visualize single-vesicle events in response to isoproterenol in submandibular acinar cells. Our evidence indicates that secretion is relatively slow and that there is no dependence on a rise in intracellular calcium. Future experiments will aim to describe the response more fully.

This work was supported by The Wellcome Trust