Real‐time studies of zymogen granule exocytosis in intact rat pancreatic acinar cells

Abstract

An adequate understanding of secretion requires the measurement of exocytosis on the same time scale as that used for second messenger dynamics. To investigate the kinetics of ACh‐evoked secretion in pancreatic acinar cells, exocytosis of zymogen granules was quantified by continuous, time‐differential analysis of digital images. The validity of this method was confirmed by simultaneous fluorescence imaging of quinacrine‐loaded zymogen granules. Basal rates of exocytosis were low (0.2 events min−1). ACh stimulated a biphasic increase in secretory activity, maximal rates exceeding 20 events min−1 after 10 s of ACh application (10 μm). Over the next 15 s the rate of exocytosis fell to less than 4 events min−1; then began a second phase of secretion that peaked 15 s later at ∼11 events min−1, but subsequently declined in the continued presence of agonist. Measurements of fura‐2 fluorescence demonstrated a biphasic increase in intracellular [Ca2+] ([Ca2+]i). Comparison of the [Ca2+]i records and time‐differential analysis revealed that the fall in exocytotic rate following the initial burst occurred despite the fact that [Ca2+]i remained high. The second phase of secretion depended on both [Ca2+]i and [ACh]. At 10 μm ACh there was a decrease in the steepness of the relationship between [Ca2+]i and exocytosis that led to an enhancement of the slow secretory phase. We propose that acinar cells contain two pools of secretory vesicles: a small pool of granules that is exocytosed rapidly, but is quickly depleted; and a reserve pool of granules that can be recruited by ACh in a process that is modulated by second messengers other than calcium.

Spatiotemporal analysis of exocytosis in mouse parotid acinar cells

“…Time-differential images were generated by subtraction of each brightfield frame by its preceding frame. Timedifferential imaging has been used and validated previously in pancreatic acinar cells and other cell types as a method of visualizing individual secretory granule fusions (2). Parotid acinar cells were amenable for this approach, likely because of the large size and phase-dark core of their ZSGs.…”

Water Channels and Zymogen Granules in Salivary Glands

“…Norepinephrine also acts at β 2 -ADRs in salivary glands and activates AC via the activation of Gsα protein, thereby increasing the amount of cAMP (Fig. 4) 2+ release with an initial rapid rise followed by a gradual decline to the plateau level observed under unstimulated conditions (94). Cevimeline-and MCh-induced exocytosis in rat parotid glands are completely inhibited by BAPTA-AM, KN-93 (CaM kinase II inhibitor), ML-9 (MLCK inhibitor), L-NAME (nNOS inhibitor), ODQ (GC-s inhibitor), or KT5823 (PKG inhibitor), but not KT5720 (PKA inhibitor) (21,54).…”

The Plasma Membrane Q-SNARE Syntaxin 2 Enters the Zymogen Granule Membrane during Exocytosis in the Pancreatic Acinar Cell

“…Because a finite time will be required for syntaxin 2 to diffuse around the open fusion pore into the granule membrane, these features may represent cases where fixation has "frozen" a granule after fusion pore opening but before significant syntaxin 2 migration. After 5 min of stimulation, the peak of the exocytotic burst is virtually complete (17). To study an earlier phase of the exocytotic response, we imaged cells that had been fixed after a 1-min stimulation, when exocytosis is occurring robustly (17).…”

“…After 5 min of stimulation, the peak of the exocytotic burst is virtually complete (17). To study an earlier phase of the exocytotic response, we imaged cells that had been fixed after a 1-min stimulation, when exocytosis is occurring robustly (17). Analysis of 10 images taken at this time showed that there were 5.1 Ϯ 0.5 (n ϭ 33 cells) LF Texas Red/dextran-positive exocytotic events per acinar cell, of which 3.7 Ϯ 0.5 (i.e.…”