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A minireview about the universal molecular machinery and mechanism of secretion in …

Biology Articles » Cell biology » Molecular Machinery and Mechanism of Cell Secretion » The Regulated Expulsion of Intravesicular Contents During Secretion

The Regulated Expulsion of Intravesicular Contents During Secretion
- Molecular Machinery and Mechanism of Cell Secretion


Once the membrane-bound secretory vesicle fuses at the base of porosomes, establishing continuity between the two compartments, how is the vesicle content expelled? Studies reveal that vesicle swelling is required for the expulsion of intravesicular contents during secretion (46). It has been demonstrated (46) that the extent of vesicle swelling is directly proportional to the amount of intravesicular contents expelled, hence, providing cells with the ability to further regulate the amount of release of secretory products. Direct observations of the requirement of secretory vesicle swelling in secretion (46) provides, for the first time, an understanding of the appearance of empty and partially empty vesicles following secretion (10, 26, 47).

Electron micrograph of pancreatic acinar cells and isolated live pancreatic acinar cells in near physiological buffer, when imaged using AFM at high force (200–300 pN), reveal the profile of the secretory vesicles called zymogen granules (ZGs), lying immediately under the apical plasma membrane of the cells. Within 2.5 mins of exposure to a physiological secretory stimulus (1 µM carbamylcholine), the majority of ZGs within cells swell, followed by a decrease in ZG size, by which time most of the release of secretory products from within ZGs had occurred. These studies (46) reveal, for the first time in live cells, intracellular swelling of secretory vesicles following stimulation of secretion and their deflation following partial discharge of vesicular contents. Measurements of intra-cellular ZG size further revealed that different vesicles swell differently following a secretory stimulus. This differential swelling among ZGs within the same cell may explain why, following stimulation of secretion, some intracellular ZGs demonstrate the presence of less vesicular content than others following secretion, because they have discharged more of their contents due to greater swelling (10).

To determine precisely the role of swelling in the expulsion of intravesicular contents, the electrophysiological ZG-reconstituted lipid bilayer fusion assay (17), as described earlier, has been employed (46). The ZGs used in the bilayer fusion assays were characterized for their purity and their ability to respond to a swelling stimulus. As previously reported (1921), exposure of isolated ZGs (Fig. 9A and BGo) to GTP resulted in ZG swelling (Fig. 9CGo; Ref. 46). Once again, similar to what is observed in live acinar cells, each isolated ZG responded differently to the same swelling stimulus. For example, the red arrowhead points to a ZG of which the diameter increased by 29% as opposed to the green arrowhead pointing to a ZG that increased only by a modest 8%. The differential response of isolated ZGs to GTP was further assessed by measuring changes in the volume of isolated ZGs of different sizes (Fig. 9DGo). The ZGs in the exocrine pancreas range in size from 0.2 to 1.3 µm in diameter (19). Not all ZGs swell following a GTP challenge. The volume of most ZGs increases between 5% and 20%. However, larger increases, up to 45%, are observed only in vesicles ranging from 250 to 750 nm in diameter (Fig. 9DGo). These studies demonstrate that, following stimulation of secretion, ZGs within pancreatic acinar cells swell, followed by a release of intravesicular contents through porosomes (7, 8) at the cell plasma membrane and a return to resting size on completion of secretion. On the contrary, isolated ZGs stay swollen following exposure to GTP because there is no outlet for release of the intravesicular contents. In acinar cells, little or no secretion is detected 2.5 mins following stimulation of secretion, although the ZGs within them have swelled. However, 5 mins following stimulation, ZGs deflated and the intravesicular {alpha}-amylase released from the acinar cell is detectable, suggesting the involvement of ZG swelling in secretion.

In the electrophysiological bilayer fusion assay, immunoisolated fusion pores or porosomes from the exocrine pancreas were isolated and functionally reconstituted (8) into the lipid membrane of the bilayer apparatus and membrane conductance and capacitance continually monitored (Fig. 10AGo). Reconstitution of the porosome into the lipid membrane results in a small increase in capacitance (Fig. 10BGo), possibly due to the increase in membrane surface area contributed by incorporation of porosomes, ranging in size from 100 to 150 nm in diameter (8). Isolated ZGs, when added to the cis compartment of the bilayer chamber, fuse at the porosome-reconstituted lipid membrane (Fig. 10AGo), which is detected as a step increase in membrane capacitance (Fig. 10BGo). However, even after 15 mins of ZG addition to the cis compartment of the bilayer chamber, little or no release of the intravesicular enzyme {alpha}-amylase is detected in the trans compartment of the chamber (Fig. 10C and DGo). On the contrary, exposure of ZGs to 20 µM GTP induces swelling (1921) and results both in the potentiation of fusion as well as a robust expulsion of {alpha}-amylase into the trans compartment of the bilayer chamber (Fig. 10C and DGo). These studies demonstrated that, during secretion, secretory vesicle swelling is required for the efficient expulsion of intravesicular contents.

Within minutes or even seconds following stimulation of secretion, empty and partially empty secretory vesicles accumulate within cells. There may be two possible explanations for such accumulation of partially empty vesicles. Following fusion at the porosome, secretory vesicles may either remain fused for a brief period and therefore time would be the limiting factor for partial expulsion. An alternate scenario would be that secretory vesicles may not swell enough and therefore are unable to generate the required intravesicular pressure for complete discharge. Results from published studies (Fig. 10Go) suggest that it would be highly unlikely that generation of partially empty vesicles would result from brief periods of vesicle fusion at porosomes. Because, after addition of ZGs to the cis compartment of the bilayer apparatus, membrane capacitance continues to increase, although little or no detectable secretion occurred even after 15 mins (Fig. 10Go), it is suggested that either variable degrees of vesicle swelling or repetitive cycles of fusion and swelling of the same vesicle, or both, may operate during the secretory process. Under these circumstances, empty and partially empty vesicles could be generated within cells following secretion. To test this hypothesis, two key parameters have been examined (46). One is whether the extent of swelling is the same for all ZGs exposed to a certain concentration of GTP. The second is whether ZG is capable of swelling to different degrees. And if so, whether there is a correlation between extent of swelling and the quantity of intravesicular contents expelled. The answer to the first question is clear, that different ZGs respond to the same stimulus differently. Studies (46) reveal that different ZGs within cells, or in isolation, undergo different degrees of swelling, even though they are exposed to the same stimulus (carbamylcholine for live pancreatic acinar cells or GTP for isolated ZGs). The requirement of ZG swelling for expulsion of vesicular contents is further confirmed, when GTP dose dependently increased ZG swelling is translated into increased secretion of {alpha}-amylase (46). Although higher GTP concentrations elicited an increased ZG swelling, the extent of swelling between ZGs once again varied.

To determine if a similar or an alternate mechanism is responsible for the release of secretory products in a fast secretory cell, such as neurons, synaptosomes and synaptic vesicle preparation from rat brain has been studied (46). Because synaptic vesicle membrane is known to possess both Gi and Go proteins, it was hypothesized that GTP and Gi-agonist (mastoparan) would mediate vesicle swelling. To test this hypothesis, isolated synaptosomes were lysed to obtain synaptic vesicles and synaptosomal membrane. Isolated synaptosomal membrane, when placed on mica and imaged by the AFM in near-physiological buffer, reveal on the cytosolic side the presence of 40–50-nm diameter synaptic vesicles still docked to the presynaptic membrane. Similar to the ZGs, exposure of synaptic vesicles to 20 µM GTP results in an increase in synaptic vesicle swelling. However, exposure to Ca2+ results in the transient fusion of synaptic vesicles at the presynaptic membrane, expulsion of intravesicular contents, and a consequent decrease in size of the synaptic vesicle. Additionally, as observed in ZGs of the exocrine pancreas, not all synaptic vesicles swell and, if they do, they swell to different degrees even though they had been exposed to the same stimulus. This differential response of synaptic vesicles within the same nerve terminal may dictate and regulate the potency and efficacy of neurotransmitter release. To further confirm synaptic vesicle swelling and determine the swelling rate, light-scattering experiments to monitor vesicle size have also been performed (46). Light-scattering studies demonstrate a mastoparan-dose-dependent increase in synaptic vesicle swelling. Mastoparan (20 µM) induced a time-dependent (in seconds) increase of synaptic vesicle swelling, as opposed to the control peptide (Mast-17).

These studies (46) demonstrate that, following stimulation of secretion, ZGs, the membrane-bound secretory vesicles, in exocrine pancreas swell. Different ZGs swell differently, and the extent of their swelling dictates the amount of intravesicular contents to be expelled. ZG swelling is therefore a requirement for the expulsion of vesicular contents in the exocrine pancreas. Similar to ZGs, synaptic vesicle swelling enables the expulsion of neurotransmitters at the nerve terminal. This mechanism of vesicular expulsion during cell secretion may explain why partially empty vesicles are observed in secretory cells following secretion. The presence of empty secretory vesicles could result from multiple rounds of fusion-swelling-expulsion a vesicle may undergo during the secretory process. These results reflect the precise and regulated nature of cell secretion from the exocrine pancreas to neurons.

What is the molecular mechanism of secretory vesicle swelling? This has been addressed in studies using isolated ZGs (1921). Isolated ZGs from exocrine pancreas swell rapidly in response to GTP (19). These studies suggested the involvement of rapid water gating into ZGs following exposure to GTP. Therefore, when the possible involvement of water channels, or aquaporins, in ZG swelling was explored (20), results from the study demonstrate the presence of aquaporin-1 (AQP1) at the ZG membrane and its participation in GTP-mediated vesicle water entry and swelling (20). To further understand the molecular mechanism of secretory vesicle swelling, the regulation of AQP1 in ZGs has been determined (21). Detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, coisolates AQP1, PLA2, Gαi3, potassium channel IRK-8, and the chloride channel ClC-2 (21). Exposure of ZGs to either the potassium channel blocker glyburide or the PLA2 inhibitor ONO-RS-082 blocked GTP-induced ZG swelling. Red blood cells, known to possess AQP1 at the plasma membrane, also swell on exposure to the Gαi-agonist mastoparan and respond similarly to ONO-RS-082 and glyburide as do ZGs (21). Additionally, liposomes reconstituted with the AQP-1 immunoisolated complex from solubilized ZGs were found to swell in response to GTP. Glyburide or ONO-RS-082 abolished the GTP effect in reconstituted liposomes. Furthermore, immunoisolate-reconstituted planar lipid membrane demonstrate conductance, which is sensitive to glyburide and an AQP1 specific antibody. These results demonstrate a Gαi3-PLA2-mediated pathway and potassium channel involvement in AQP-1 regulation (21), contributing to our understanding of the molecular mechanism of ZG swelling.

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