Exocytosis Of Zymogen Granules Research Articles

Zymogen granule exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity.

The dynamics of the fusion pore that forms between a secretory vesicle and the plasma membrane are important in the regulation of both exocytosis and endocytosis. Here, we describe characteristics of fusion during zymogen granule exocytosis in exocrine pancreatic acinar cells. By using fluorescence recovery after photobleaching techniques, we show that the fusion pore remains open to allow free aqueous exchange with the vesicle lumen. There is no lipid interchange between the plasma and granule membranes during this time, and at the end of its life, the intact granule shrinks in situ, probably by a gradual pinching off of membrane patches. We propose that the protracted fusion pore lifetime is adapted to permit compound exocytosis, whereby the lingering primary granule acts as a conduit through which the contents of a secondary granule can be released. The lack of lipid intermixing may then facilitate selective recycling of granule membrane and preservation of apical membrane integrity.

Calcium-induced calcium release

Why is calcium so important? Intracellular calcium controls a diverse array of cellular processes from fertilisation through gene transcription, muscle contraction to cell death. Changes in calcium levels can occur in microseconds or hours, they can be microscopic or propagate throughout cells and tissues.How are calcium levels increased within cells? Increases in intracellular calcium arise through either calcium influx across the plasma membrane or release from intracellular calcium stores, which is usually either the endoplasmic reticulum (ER) or, in muscle, the sarcoplasmic reticulum (SR). Release of calcium from the ER/SR is activated by a variety of second messengers, such as inositol 1,4,5-trisphosphate (IP3), cyclic ADP ribose (cADPr) or, significantly, by calcium itself.What is calcium-induced calcium release? This is the process – commonly known by the acronym CICR – whereby calcium promotes its own release from intracellular calcium stores. The diffusion of calcium within cells is greatly retarded by buffers. CICR therefore provides a means for amplification of microscopic initiation events into propagating calcium signals. For calcium to control cellular activities it has to reach targets that may be distant from initiation sites. Without CICR, cells would have to rely on the slow dissipative diffusion of calcium.What channels mediate CICR? IP3 receptors and ryanodine receptors are the major calcium release channels that display CICR. These structurally related channels are widely expressed and mediate calcium release in muscle, neurons and non-excitable cells. IP3 dictates the sensitivity of IP3 receptors to calcium. Calcium can directly activate ryanodine receptors, although cADPr may also act as a sensitiser.How does calcium modulate release channel activity? Calcium binding sites have been found in the primary sequences of both ryanodine receptors and IP3 receptors. CICR therefore occurs via direct binding of calcium to channels. In addition to the direct binding, calcium exerts some effects through accessory proteins such as calmodulin. IP3 receptors and ryanodine receptors exist as macromolecular complexes containing proteins such as kinases and phosphatases which serve to regulate the sensitivity of these channels to calcium.Where does CICR occur in biology? During each heartbeat an influx of calcium through L-type voltage-operated channels provides the trigger to induce CICR from juxtaposed ryanodine receptors on the SR, resulting in cardiac muscle contraction. Examples of CICR in non-excitable cells include IP3-induced calcium oscillations and waves following fertilisation of mammalian eggs, and the spread of calcium waves in pancreatic acinar cells that underlie zymogen granule exocytosis and fluid secretion.Are there any other manifestations of CICR? Although IP3 receptors and ryanodine receptors are believed to be the primary CICR channels, a few other forms of CICR have been reported. For example, a protein, polycystin 2, which is mutated in autosomal dominant polycystic kidney disease has been shown to operate as a CICR channel. The permeability transition pore of mitochondria can open in response to elevated matrix calcium levels. This results in the rapid release of calcium from mitochondria, which can affect neighbouring mitochondria leading to a propagating signal. The term CICR is most commonly used to imply the activation of release channels by the direct binding of calcium. Calcium can, however, also modulate the activity of enzymes that produce calcium-mobilising metabolites such as IP3, arachidonic acid and nitric oxide.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Receptor biology and intracellular regulatory mechanisms in pancreatic acinar cells

Continuing progress is being made in understanding the regulation of pancreatic acinar cell function by receptor-activated intracellular signaling mechanisms. Knowledge of how ligands interact at the molecular level with their receptors and activate heterotrimeric G proteins is increasing. In addition to inositol trisphosphate, intracellular messengers include cyclic ADP ribose, nicotinic acid adenine dinucleotide phosphate, arachidonic acid, and diacylglycerol. Ca signaling involves the interaction of inositol trisphosphate, cyclic ADP ribose, and nicotinic acid adenine dinucleotide phosphate with distinct subcellular Ca stores. Ca signals ultimately induce exocytosis of zymogen granules and identification of the proteins involved on the granule and plasma membrane, and understanding of their roles is continuing. Other receptor-activated signaling pathways primarily regulate nonsecretory events. Considerable progress has been made in understanding how the mammalian target of rapamycin pathway regulates protein synthesis through translation factors and ribosomal proteins. Other pathways in acinar cells include the mitogen-activated protein kinases, the tyrosine kinases, and the transforming growth factor-beta-Smad pathways.

Acetylcholine-Induced Zymogen Granule Exocytosis: Comparison Between Acini and Single Pancreatic Acinar Cells

Numerous studies have been carried out on the agonist-evoked calcium responses of single pancreatic acinar cells; however, several reports have shown that dissociation of the exocrine pancreas into predominantly single cells has an adverse effect on agonist-evoked amylase secretion. To determine whether single acinar cells behave in an anomalous manner compared with cells within an intact acinus, we measured exocytosis in both single acinar cells and acini (2-5 cells) present in the same preparation. Exocytosis of individual zymogen granules was quantified in real-time by using the technique of continuous time-differential analysis of brightfield digital images. Basal rates of exocytosis were low in both single cells and intact acini. Application of 10 microM acetylcholine for 6 minutes stimulated a biphasic secretory response in acinar cells. Additionally, we found that exocytotic events occur repetitively in specific locations within the apical domain; i.e., there are exocytotic "hot spots." There were no statistically significant differences between the exocytotic rates, nor were there any differences in the characteristics of the exocytotic hot spots of single cells compared with those of acini. We conclude that time-differential analysis of brightfield images appears to be a useful tool for the investigation of the role of gap junctions in zymogen granule exocytosis and that single acinar cells provide a reasonable model for studies of acinar cell signaling and secretion.

Two Modes of Secretion in Pancreatic Acinar Cells: Involvement of Phosphatidylinositol 3-Kinase and Regulation by Capacitative Ca2+ Entry

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. The part played by Ca2+ released from intracellular stores in the regulation of secretion is well established; however, the role of Ca2+ influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (≥10 μM) elicited an additional component of exocytosis despite reducing Ca2+ influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (∼70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 μM) was potentiated (∼45%) by wortmannin. Exocytosis stimulated by 1 μM acetylcholine in the absence of extracellular Ca2+ was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca2+ entry through store-operated Ca2+ channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd3+ (2 μM), but not by inhibition of noncapacitative Ca2+ entry by SB203580 (10 μM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca2+ influx.

Sequential-replenishment mechanism of exocytosis in pancreatic acini.

Here we report exocytosis of zymogen granules, as examined by multiphoton excitation imaging in intact pancreatic acini. Cholecystokinin induces Ca 2+ oscillations that trigger exocytosis when the cytosolic Ca 2+ concentration exceeds 1 microM. Zymogen granules fused with the plasma membrane maintain their Omega-shaped profile for an average of 220 s and serve as targets for sequential fusion of granules that are located within deeper layers of the cell. This secondary exocytosis occurs as rapidly as the primary exocytosis and accounts for most exocytotic events. Granule-granule fusion does not seem to precede primary exocytosis, indicating that secondary fusion events may require a plasma-membrane factor. This sequential-replenishment mechanism of exocytosis allows the cell to take advantage of a large supply of fusion-ready granules without needing to transport them to the plasma membrane.

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

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 microM). 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 approximately 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 microM 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.

Exocytosis and movement of zymogen granules observed by VEC-DIC microscopy in the pancreatic tissue en bloc.

The dynamic aspects of exocytosis, especially in the normal acinar tissue en bloc, have remained unclear. We visualized exocytosis directly in the tissue of the exocrine pancreas of rodents by video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy to investigate various exocytosis-related rates and the relationship between the movement of granules and exocytotic responses. Stimulation of the tissue with bethanechol or cholecystokinin caused many of the zymogen granules in the apical pole to disappear abruptly. The exocytotic transients of individual granules were completed in 0.48-0.65 s. Granules destined to participate in the exocytotic response moved randomly at velocities of approximately 0.06 microm/s or less during stimulation. In the tissue preparation, granules located far from the apical pole frequently moved back and forth for 1-7 microm without showing exocytosis. Colchicine suppressed this movement and the late phase of the secretory response. Real-time (VEC-DIC) observation of granule dynamics revealed that the initial step of exocytosis was not coupled directly with the microtubule-dependent translocation but with a continuous, slow Brownian fluctuation of granules.

Mast cell: pivotal player in lethal acute pancreatitis.

The fearful aura of acute pancreatitis stems from the imagery of an organ and organism under threat of cannibalization by pancreatic enzymes that have become active prematurely. This century‐old autodigestion theory1 is all too plausible when one is confronted with the necrohaemorrhagic remains of the gland in a patient who succumbs from multisystem organ failure, as do 10–20% of cases. It was challenged2 when the average interval from onset of symptoms to death was found to be shorter (under 48 h) and the degree of initial shock greater with interstitial pancreatitis—which accounts for a quarter of the toll3—than with wholesale coagulative necrosis, an infarct‐like lesion associated with hyaline occlusion of venules and capillaries.2 The literature is colossal—4844 titles in a Medline search from 1966, and 260 in 1999—but the current position can be gleaned from a recent monograph,4 an assortment of reviews5–,11 and supplementary papers. Of necessity, much reliance has been placed on studies of experimental pancreatitis to unravel the earliest aberrations. From these and clinical studies, it is now believed that the ‘attack’ begins in the acinar cell, with a functional blockade in the secretory pathway towards exocytosis of zymogen granules. It is also agreed that multiorgan collapse is primarily due to hyperinflammation, abetted by activation of coagulation, fibrinolytic, kallikrein‐kinin and complement enzyme cascades with consumption of α2 macroglobulin (α2M). The debate concerns the nature of the trigger mechanism and why it should evoke an exaggerated inflammatory reaction and cause (subclinical) disturbances in distant organs even when the disease seems to be mild. In the latter context, the injured acinar cell is now known to step up its production of platelet‐activating factor (PAF)—a physiological secretagogue but which at high doses inhibits secretion—and to synthesize heat‐shock and acute‐phase proteins, …

Selective exocytosis of zymogen granules induces non-parallel secretion in short-term cholecystokinin-stimulated rats.

Parallel studies on pancreatic enzyme secretion and zymogen granule enzyme composition have been carried out in rats subjected to infusion of cholecystokinin (CCK) (1.25 microgram/kg per h) over 30 min. Flow cytometric analysis showed a significant decrease in the mean value of granule size after CCK stimulation. The amount of trypsinogen stored in each individual zymogen granule was significantly lower at 30 min of CCK infusion, but no variation in intragranular amylase content was observed. As a result, the amylase/trypsinogen ratio was significantly increased in the zymogen granules that remained in the pancreas of rats stimulated with CCK for 30 min. A significantly greater proportion of trypsin than amylase was secreted after 30 min CCK infusion. Our results support the existence of different types of granules loaded with different proportions of enzymes. We conclude that short-term CCK stimulation induces the selective release of large granules containing a high proportion of trypsinogen, which leads to a non-parallelism of enzyme secretion.

Ca2+ Signaling in the Pancreatic Acinus

Change in cytosolic free calcium ion concentration [Ca2+]c, resulting from receptor activation by an appropriate agonist, functions as a cardinal intracellular signaling in the stimulus-secretion coupling in a wide variety of secretory cells including the acini of the pancreas. Ratiometric imaging of [Ca2+]c dynamics by UV-laser scanning confocal microscopy led us to conclude that in the cholecystokinin (CCK)-8-induced recurrent [Ca2+]c, spiking increases initially in the basolateral margin of the acinus and propagates to the luminal margin. [Ca2+]c in this initial cell increased rapidly and uniformly to the maximum level. The decrease in [Ca2+]c in the initial cell coincided with a small increase in [Ca2+]c in the luminal regions of the bilateral neighboring cells followed by uniform maximal increase in [Ca2+]c in these neighboring cells. A series of [Ca2+]c dynamics was repeated to form recurrent Ca2+ spiking. The temporal sequences of [Ca2+]c dynamics recorded during continuous stimulation with CCK-8 at a physiologic concentration in individual acinar cells forming the acinus were displayed on the identical time scale. The figure indicates that the signaling is not synchronous in cells forming an acinus. From these and other results, we proposed a model in which CCK-8 at a low physiologic concentration binds to highly sensitive CCK receptor interacting with heterotrimeric guanosine 5'-triphosphate-binding proteins of the Gq class, generate Ins 1,4,5-P3, and recurrent [Ca2+]c, spiking. The recurrent Ca2+ spiking maintains a sustained secretory response, recurrent exocytosis of zymogen granules, and concomitant secretion of isotonic NaCl.

Mechanisms of Protein Secretion in Endocrine and Exocrine Cells

Publisher Summary This chapter describes that dense core vesicles (DCVs) exocytosis in exocrine cells proceeds by mechanisms that are less clearly understood and involves proteins or protein isoforms different from those employed in neural/endocrine secretion. Biochemical and molecular biological studies have resulted in the characterization of at least 25 types of synaptic vesicle (SV) membrane proteins that may mediate aspects of SV function including calcium-dependent exocytosis, endocytosis, and neurotransmitter loading. In neuronal cells, there are important physiological differences between the exocytosis of SVs that contain neurotransmitters such as glutamate and the exocytosis of larger DCVs that contain neuropeptides, including calcium sensitivity and speed. Microinjection and genetic studies have documented the importance of SNARE proteins and synaptotagmin in neurosecretion without revealing their sites of action and mechanisms. Of the other isoforms of synaptotagmin characterized, synaptotagmin III has the most appealing characteristics as a calcium sensor for DCV exocytosis. Expression of synaptotagmin isoforms in the acinar pancreas has not been reported, so it is uncertain whether this protein family plays a role in calcium sensing for zymogen granule exocytosis.

Myosin I is associated with zymogen granule membranes in the rat pancreatic acinar cell

BACKGROUND & AIMS: The mechanisms whereby intracellular messengers mediate zymogen granule transport and exocytosis in the pancreatic acinar cell are not well defined. Electron microscopy has shown a periluminal network of actin in the acinar cell, suggesting a role for actin and myosin in the transport process. The possible involvement of two types of myosin in the secretory process was investigated, and their distribution in acinar cells was determined.METHODS: Antibodies specific to myosin I or to myosin II were used for immunocytochemistry and Western blot analysis. Ultrastructural studies were also performed.RESULTS: Western blot analysis showed that myosin I and myosin II were present in total pancreatic homogenate but that only myosin I was present on isolated zymogen granules and their membranes. By immunocytochemistry, myosin I was shown in the apical aspect of acinar cells colocalized with glycoprotein 2, a marker for zymogen granules, and actin. By immunocytochemistry, myosin I was also localized on isolated zymogen granules.CONCLUSIONS: The immunolocalization of myosin I to zymogen granule membranes and its close association with periluminal actin suggest that myosin I plays a direct role in the process of transport and exocytosis of zymogen granules in the pancreatic acinar cell.(Gastroenterology 1997 Aug;113(2):649-58)

Evidence That Zymogen Granules Are Not a Physiologically Relevant Calcium Pool: DEFINING THE DISTRIBUTION OF INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS IN PANCREATIC ACINAR CELLS

A key event leading to exocytosis of pancreatic acinar cell zymogen granules is the inositol 1,4,5-trisphosphate (InsP3)-mediated release of Ca2+ from intracellular stores. Studies using digital imaging microscopy and laser-scanning confocal microscopy have indicated that the initial release of Ca2+ is localized to the apical region of the acinar cell, an area of the cell dominated by secretory granules. Moreover, a recent study has shown that InsP3 is capable of releasing Ca2+ from a preparation enriched in secretory granules (Gerasimenko, O., Gerasimenko, J., Belan, P., and Petersen, O. H., (1996) Cell 84, 473-480). In the present study, we have investigated the possibility that zymogen granules express InsP3 receptors and are thus Ca2+ release sites. Immunofluorescence staining, obtained with antisera specific to types I, II, or III InsP3 receptors and analyzed by confocal fluorescence microscopy revealed that all InsP3 receptor types were present in acinar cells. The type II receptor localized exclusively to an area close to or at the luminal plasma membrane. While types I and III InsP3 receptors displayed a similar luminal distribution, these receptors were also present at low levels in nuclei. The localization of InsP3 receptor was in marked contrast to the distribution of amylase, a zymogen granule content protein. In a zymogen granule fraction prepared in an identical manner to the aforementioned report demonstrating InsP3-induced Ca2+ release, immunoblotting demonstrated the presence of types I, II, and III InsP3 receptors. Ca2+ release from this preparation in response to InsP3, but not thapsigargin, could also be demonstrated. In contrast, when the zymogen granules were further purified on a Percoll gradient, InsP3 receptors were undetectable, and InsP3 failed to release Ca2+. Transmission electron microscopy performed on both preparations showed that the Percoll-purified granule preparation consisted of essentially pure zymogen granules, whereas the granules prepared without this step were enriched in granules but also contained significant contamination by mitochondria, endoplasmic reticulum, and nuclei. It is concluded that zymogen granules do not express InsP3 receptors and thus are not a site of Ca2+ release relevant to the secretory process in the pancreatic acinar cell.

Micromolar and submicromolar Ca2+ spikes regulating distinct cellular functions in pancreatic acinar cells.

Agonists induce Ca2+ spikes, waves and oscillations initiating at a trigger zone in exocrine acinar cells via Ca2+ release from intracellular Ca2+ stores. Using a low affinity ratiometric Ca2+ indicator dye, benzothiazole coumarin (BTC), we found that high concentrations of agonists transiently increased Ca2+ concentrations to the micromolar range (>10 microM) in the trigger zone. Comparison with results obtained with a high affinity Ca2+ indicator dye, fura-2, indicated that fura-2 was in fact saturated with Ca2+ during the agonist-induced Ca2+ spikes in the trigger zone. We further revealed that the micromolar Ca2+ spikes were necessary for inducing exocytosis of zymogen granules investigated using capacitance measurements. In contrast, submicromolar Ca2+ spikes selectively gave rise to sequential activation of luminal and basal ion channels. These results suggest new functional diversity in Ca2+ spikes and a critical role for the micromolar Ca2+ spikes in exocytotic secretion from exocrine acinar cells. Our data also emphasize the value of investigating the Ca2+ signalling using low affinity Ca2+ indicators.

Secretin potentiates cholecystokinin-stimulated amylase release by AR4-2J cells via a stimulation of phospholipase C.

Alterations in the activity of phospholipase C (PLC) are thought to be the primary intracellular events leading to pancreatic acinar cell exocytosis of zymogen granules. When multiple hormones, each of which may stimulate different signal transduction pathways, bind to cell surface receptors, the cell must integrate these signals into a common response through communication (cross-talk) among intracellular second messengers. We show that cholecystokinin (CCK) induces amylase secretion from AR4-2J pancreatic acinar cells via stimulation of PLC activity. Secretin indirectly stimulated the PLC pathway through cross-talk of the activated cAMP pathway to potentiate the CCK-stimulated amylase secretion. Therefore, secretin potentiated the acinar cell secretory response to CCK by cAMP-mediated cross-talk with the PLC signal transduction pathway.

Evidence for an apical, nonregulated protein secretion in pig exocrine pancreas.

Secretory proteins are segregated into two pathways out of the trans-Golgi network of regulated secretory cells. To identify proteins specifically secreted by pathways other than the one leading to zymogen granule exocytosis in the exocrine pancreas, conscious permanently cannulated pigs were perfused with atropine to inhibit the regulated fusion of granules. Atropine almost totally inhibited the protein secretion after 1 h of perfusion. The secretion of GP-2, a glycosyl phosphatidylinositol-anchored protein of the zymogen granule membrane, was partially inhibited but was never totally abolished by atropine perfusion. The pattern of proteins secreted under atropine was almost totally different. Soluble GP-2 was the major secretory product. Its specific activity increased 60 times over its normal level in all other conditions. This secretion clearly originated from nonregulated pathways. Results suggest that during the atropine block the apical plasmalemma could be the source of the released GP-2 and that the sustained nature of this release is compatible with a replenishment of the plasmalemma with GP-2 by the continuous exocytosis of vesicles from the nonregulated pathways.

High-Resolution 1H NMR Study of Membrane Phospholipids in Rat Pancreas Stimulated by Caerulein

In this study, the peak observed at 3.2 +/- 0.2 ppm in the 1H nuclear magnetic resonance (NMR) spectra recorded at 60 MHz of rat pancreas stimulated by caerulein was characterized at higher resolution (100 and 300 MHz). Whole pancreas from rats stimulated by caerulein was analyzed ex vivo by 1H NMR spectroscopy at 100 MHz. After suppression of the tissue water peak (OSIRIS method), a peak at 3.2 +/- 0.2 ppm appeared distinctly, along with two other weaker signals (2.8 and 3.5 ppm). No signals were observed in these regions in the spectra recorded from pancreas of 48-h fasted rats. The signal at 3.2 +/- 0.2 ppm was characterized by analysis of the high-resolution 1H NMR (300 MHz) spectra of lipid extracts of rat pancreas. Addition of various pure membrane phospholipids in extracts showed that the peak was due to the N(CH3)3 groups of choline-containing lipids. The weaker signals (2.8 and 3.5 ppm) were attributed to the methylene protons of fatty acids and the glycerol of glycerophosphorylcholine (GPC). A small decrease in phosphatidylcholine (PC) concentration was observed on analysis of these lipid extracts by high-performance liquid chromatography, indicating that the increase in intensity of the 3.2 +/- 0.2 ppm peak was not due to any increase in PC concentration, but rather to a change in conformation of PC, allowing higher mobility of the trimethylamino protons. Lorglumide, a specific inhibitor of caerulein, markedly reduced the intensity of the NMR peak, and pentagastrin, which also stimulates exocytosis of zymogen granules in the pancreas, had a similar but somewhat smaller effect than caerulein.(ABSTRACT TRUNCATED AT 250 WORDS)

Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

Early acinar cell changes in caerulein-induced interstitial acute pancreatitis in the rat

Early ultrastructural and immunohistochemical changes caused by supramaximal secretory stimulation with caerulein were studied in the rat pancreas. The morphological basis for the earlier reported decrease of pancreatic juice secretion after supramaximal caerulein was the appearance of swollen and irregular zymogen-like material containing structures with short segments of budding bristle-coated membranes in the apical parts of acinar cells. Images of exocytosis of zymogen granules were only few. Later, marked vacuolization and signs of autophagocytosis are seen in the basal cytoplasm. Immunohistochemistry showed that the large zymogen containing structures were intensively labelled for trypsin at the early stages of the experiment (4-30 min). Later (1-2 h), the vacuoles were empty or contained occasional, small-labelled granules only. The pancreozymin-receptor antagonist proglumide as well as cycloleucine that inhibits protein synthesis by inhibiting the synthesis of S-adenosylmethionine, effectively prevented the caerulein induced acinar cell changes. The irregular zymogen containing structures with coated pits on their surface indicate disturbed zymogen granule formation leading to the accumulation of large lakes of zymogen material and finally to marked autophagocytosis in acinar cells. The effects of caerulein are receptor-mediated and depend on the process of methylation in the formation of zymogen granules.