Ex vivo human pancreatic slice preparations offer a valuable model for studying pancreatic exocrine biology.

A genuine understanding of human exocrine pancreas biology and pathobiology has been hampered by a lack of suitable preparations and reliance on rodent models employing dispersed acini preparations. We have developed an organotypic slice preparation of the normal portions of human pancreas obtained from cancer resections. The preparation was assessed for physiologic and pathologic responses to the cholinergic agonist carbachol (Cch) and cholecystokinin (CCK-8), including 1) amylase secretion, 2) exocytosis, 3) intracellular Ca2+ responses, 4) cytoplasmic autophagic vacuole formation, and 5) protease activation. Cch and CCK-8 both dose-dependently stimulated secretory responses from human pancreas slices similar to those previously observed in dispersed rodent acini. Confocal microscopy imaging showed that these responses were accounted for by efficient apical exocytosis at physiologic doses of both agonists and by apical blockade and redirection of exocytosis to the basolateral plasma membrane at supramaximal doses. The secretory responses and exocytotic events evoked by CCK-8 were mediated by CCK-A and not CCK-B receptors. Physiologic agonist doses evoked oscillatory Ca2+ increases across the acini. Supraphysiologic doses induced formation of cytoplasmic autophagic vacuoles and activation of proteases (trypsin, chymotrypsin). Maximal atropine pretreatment that completely blocked all the Cch-evoked responses did not affect any of the CCK-8-evoked responses, indicating that rather than acting on the nerves within the pancreas slice, CCK cellular actions directly affected human acinar cells. Human pancreas slices represent excellent preparations to examine pancreatic cell biology and pathobiology and could help screen for potential treatments for human pancreatitis.

Palmitic acid increases invasiveness of pancreatic cancer cells AsPC-1 through TLR4/ROS/NF-κB/MMP-9 signaling pathway.

Pancreatic cancer (PC) is an aggressive malady with proclivity for early metastasis. Overexpression of toll-like receptor 4 (TLR4) in pancreatic ductal adenocarcinoma, the most common type of pancreatic malignancy, correlates to tumor size, lymph node involvement, venous invasion and pathological stage. Lipopolysaccharides (LPS) are natural TLR4 ligands that have been shown to increase the invasive ability of PC cells. However, rapid inactivation of circulating LPS and low systemic absorption of inhaled LPS from the bronchoalveolar compartment make other agonists such as saturated fatty acids more suitable to be considered for TLR4-related cell invasiveness. Interestingly, PC risk was strongly associated to intake of saturated fat from animal food sources, in particular to consumption of saturated palmitic acid (PA). In the present study, we investigated the influence of PA on the invasive capacity of human PC cells AsPC-1. Using specific inhibitors, we found that PA stimulation of these tumor cells induced a TLR4-mediated cell invasion. Our results also indicate that the signaling events downstream of TLR4 involved generation of reactive oxygen species, activation of nuclear factor-kappa beta, and secretion and activation of matrix metalloproteinase 9 (MMP-9). Furthermore, PA stimulation decreased the levels of the micro RNA 29c (miR-29c). Of note, while inhibition of miR-29c increased MMP-9 mRNA levels, MMP-9 secretion and activation, and invasiveness, miR-29c mimic abrogated all these PA-stimulated effects. These results strongly suggest that miR-29c could be an attractive potential pharmacological agent for antitumoral therapy in PC.

ER stress-associated CTRC mutants decrease stimulated pancreatic zymogen secretion through SIRT2-mediated microtubule dysregulation.

Pancreatitis has been suspected for a long time to have an autodigestive genesis. The main events occurring in the pancreatic acinar cell that initiate acute pancreatitis include inhibition of zymogen secretion and intracellular activation of proteases. Chymotrypsin C (CTRC) is a protective protease that limits trypsin and trypsinogen proteolytic activity. Hereditary pancreatitis-associated CTRC mutants such as p.A73T and p.G61R precipitate within the endoplasmic reticulum (ER) causing ER stress. We found that expression of these mutants reduces amylase secretion from carbachol-stimulated rat pancreatic acinar cells AR42J and isolated mice pancreatic acini. Furthermore, this expression also reduces the levels of acetylated tubulin by increasing both the levels and phosphorylation of the deacetylase SIRT2. Remarkably, inhibition of SIRT2 not only greatly recovers tubulin acetylation, but also amylase secretion in pancreatic acinar cells and isolated acini. However, SIRT2 inhibition does not rescue secretion of the CTRC mutants. These results strongly suggest that CTRC variants associated to ER stress inhibit secretagogue-stimulated pancreatic zymogen secretion by altering microtubule stability. Of note, the extent of this inhibition correlates with the degree of ER stress exhibited by the particular CTRC variant.

Omega-3 PUFA docosahexaenoic acid decreases LPS-stimulated MUC5AC production by altering EGFR-related signaling in NCI-H292 cells.

Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Lipopolysaccharides (LPS) are known to stimulate the production of mucin 5AC (MUC5AC) via epidermal growth factor receptor (EGFR) in human airway cells. Noteworthy, we have previously demonstrated that EGFR/Rac1/reactive oxygen species (ROS)/matrix metalloproteinase 9 (MMP-9) is a key signaling cascade regulating MUC5AC production in airway cells challenged with LPS. Various reports have shown an inverse association between the intake of polyunsaturated fatty acids (PUFA) of the n-3 (omega-3) family or fish consumption and COPD. In the present study, we investigated the influence of docosahexaenoic acid (DHA), one of the most important omega-3 PUFA contained in fish oil, on the production of MUC5AC in LPS-challenged human airway cells NCI--H292. Our results indicate that DHA is capable of counteracting MUC5AC overproduction in LPS-stimulated cells by abrogating both EGFR phosphorylation and its downstream signaling pathway. This signaling pathway not only includes Rac1, ROS and MMP-9, but also NF-κB, since we have found that ROS require NF-κB activity to induce MMP-9 secretion and activation.

Microenvironmental factors and extracellular matrix degradation in pancreatic cancer.

Pancreatic cancer is a devastating malady with proclivity for early metastasis, accounting for its poor prognosis. Pancreatic ductal adenocarcinoma, the most common type of pancreatic malignancy, exhibits an over-expression of several growth factors such as epidermal growth factor and transforming growth factor beta, which correlate with a decrease in patient survival. These growth factors as well as hypoxia-reoxygenation conditions have been shown to increase pancreatic tumor cell invasiveness. This review will focus on the signaling pathways used by these distinct microenvironmental factors to promote extracellular matrix degradation and invasion by pancreatic tumor cells.

VAMP8 is the v-SNARE that mediates basolateral exocytosis in a mouse model of alcoholic pancreatitis.

In rodents and humans, alcohol exposure has been shown to predispose the pancreas to cholinergic or viral induction of pancreatitis. We previously developed a rodent model in which exposure to an ethanol (EtOH) diet, followed by carbachol (Cch) stimulation, redirects exocytosis from the apical to the basolateral plasma membrane of acinar cells, resulting in ectopic zymogen enzyme activation and pancreatitis. This redirection of exocytosis involves a soluble NSF attachment receptor (SNARE) complex consisting of syntaxin-4 and synapse-associated protein of 23 kDa (SNAP-23). Here, we investigated the role of the zymogen granule (ZG) SNARE vesicle-associated membrane protein 8 (VAMP8) in mediating basolateral exocytosis. In WT mice, in vitro EtOH exposure or EtOH diet reduced Cch-stimulated amylase release by redirecting apical exocytosis to the basolateral membrane, leading to alcoholic pancreatitis. Further reduction of zymogen secretion, caused by blockade of both apical and basolateral exocytosis and resulting in a more mild induction of alcoholic pancreatitis, was observed in Vamp8(-/-) mice in response to these treatments. In addition, although ZGs accumulated in Vamp8(-/-) acinar cells, ZG-ZG fusions were reduced compared with those in WT acinar cells, as visualized by electron microscopy. This reduction in ZG fusion may account for reduced efficiency of apical exocytosis in Vamp8(-/-) acini. These findings indicate that VAMP8 is the ZG-SNARE that mediates basolateral exocytosis in alcoholic pancreatitis and that VAMP8 is critical for ZG-ZG homotypic fusion.

TGF-ß1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2.

Human pancreatic cancer invasion and metastasis have been found to correlate with increased levels of active matrix metalloproteinase 2 (MMP-2). The multifunctional cytokine transforming growth factor beta 1 (TGF-ß1) has been shown to increase both secretion of MMP-2 and invasion by several pancreatic cancer cell types. In the present study, we investigated the signaling pathway involved in TGF-ß1-promoted MMP-2 secretion and invasion by human pancreatic cancer cells SW1990. Using specific inhibitors, we found that stimulation of these tumor cells with TGF-ß1 induced secretion and activation of the collagenase MMP-2, which was required for TGF-ß1-stimulated invasion. Our results also indicate that signaling events involved in TGF-ß1-enhanced SW1990 invasiveness comprehend activation of Rac1 followed by generation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate-oxidase, activation of nuclear factor-kappa beta, release of interleukin-6, and secretion and activation of MMP-2.

Hypoxia-reoxygenation increase invasiveness of PANC-1 cells through Rac1/MMP-2.

Pancreatic cancer is an aggressive malignancy with proclivity to early metastasis. High expression and activation of the collagenase matrix metalloproteinase-2 (MMP-2) have been found in human pancreatic cancer tissues, being these increased levels of active MMP-2 correlated to tumor invasion and metastasis. Hypoxia and reoxygenation (H-R) are critical pathophysiological conditions during ischemia-reperfusion injury, which has been shown to enhance both invasion and metastasis. In the present study, we investigated the effects of H-R on MMP-2 levels and the invasiveness properties of human pancreatic cancer cells PANC-1. Using specific inhibitors, we found that H-R treatment of these tumor cells induced secretion and activation of MMP-2, which was required for H-R-stimulated basement membrane degradation and cell invasion. Our results also indicate that signaling events involved in H-R-enhanced PANC-1 invasiveness comprehend PI3K-dependent activation of Rac1, which mediated the formation of NADPH-generated reactive oxygen species responsible for MMP-2 secretion and activation.

EGF promotes invasion by PANC-1 cells through Rac1/ROS-dependent secretion and activation of MMP-2.

Cancer metastasis involves tumor cells invading the surrounding tissue. Remodeling of tissue barriers depends on the ability of tumor cells to degrade the surrounding collagen matrix and then migrate through the matrix defects. Epidermal growth factor (EGF) has been shown to regulate tumor cell invasion through activation of matrix metalloproteinase-2 (MMP-2) in various tumor cell types. In the present study, we investigated the role of MMP-2 and the signaling pathway involved in EGF-promoted invasion by human pancreatic cancer cells PANC-1. Using specific inhibitors, we found that EGF stimulation of these tumor cells induced secretion and activation of the collagenase MMP-2, which was required for EGF-stimulated basement membrane degradation and cell invasion. Our results also indicate that signaling events downstream of EGF receptor involved PI3K- and Src-dependent activation of Rac1, which mediated the NADPH-generated reactive oxygen species responsible for MMP-2 secretion and activation.

LPS-stimulated MUC5AC production involves Rac1-dependent MMP-9 secretion and activation in NCI-H292 cells.

Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Previous studies have reported that lipopolysaccharides (LPS) stimulate mucin 5AC (MUC5AC) production via epidermal growth factor receptor (EGFR) in human airway cells. Moreover, this production was shown to depend on the expression and activity of matrix metalloproteinase 9 (MMP-9), which is increased in COPD patients' serum. In the present study we investigated the signaling pathway mediating LPS-stimulated secretion and activation of MMP-9, and the regulatory effects of this pathway on the production of MUC5AC in the human airway cells NCI-H292. Using specific inhibitors, we found that LPS-stimulated cells secreted and activated MMP-9 via EGFR. Our results also indicate that signaling events downstream of EGFR involved PI3K-dependent activation of Rac1, which mediated the NADPH-generated reactive oxygen species responsible for MMP-9 secretion and activation. Finally, we observed that EGFR/PI3K/Rac1/NADPH/ROS/MMP-9 regulate MUC5AC production in LPS-challenged NCI-H292 cells.

Munc18/SNARE proteins' regulation of exocytosis in guinea pig duodenal Brunner's gland acini.

AIM: To examine the molecular mechanism of exocytosis in the Brunner's gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner's gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy. RESULTS: One and 6 micromol/L carbachol evoked a dose-dependent dilation of Brunner's gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact. CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner's gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.

Recent insights into the cellular mechanisms of acute pancreatitis.

In acute pancreatitis, initiating cellular events causing acinar cell injury includes co-localization of zymogens with lysosomal hydrolases, leading to premature enzyme activation and pathological exocytosis of zymogens into the interstitial space. This is followed by processes that accentuate cell injury; triggering acute inflammatory mediators, intensifying oxidative stress, compromising the microcirculation and activating a neurogenic feedback. Such localized events then progress to a systemic inflammatory response leading to multiorgan dysfunction syndrome with resulting high morbidity and mortality. The present review discusses some of the most recent insights into each of these cellular processes postulated to cause or propagate the process of acute pancreatitis, and also the role of alcohol and genetics.

Acute pancreatitis: the stress factor.

Acute pancreatitis is an inflammatory disorder of the pancreas that may cause life-threatening complications. Etiologies of pancreatitis vary, with gallstones accounting for the majority of all cases, followed by alcohol. Other causes of pancreatitis include trauma, ischemia, mechanical obstruction, infections, autoimmune, hereditary, and drugs. The main events occurring in the pancreatic acinar cell that initiate and propagate acute pancreatitis include inhibition of secretion, intracellular activation of proteases, and generation of inflammatory mediators. Small cytokines known as chemokines are released from damaged pancreatic cells and attract inflammatory cells, whose systemic action ultimately determined the severity of the disease. Indeed, severe forms of pancreatitis may result in systemic inflammatory response syndrome and multiorgan dysfunction syndrome, characterized by a progressive physiologic failure of several interdependent organ systems. Stress occurs when homeostasis is threatened, and stressors can include physical or mental forces, or combinations of both. Depending on the timing and duration, stress can result in beneficial or harmful consequences. While it is well established that a previous acute-short-term stress decreases the severity of experimentally-induced pancreatitis, the worsening effects of chronic stress on the exocrine pancreas have received relatively little attention. This review will focus on the influence of both prior acute-short-term and chronic stress in acute pancreatitis.

Chronic stress sensitizes rats to pancreatitis induced by cerulein: role of TNF-α.

AIM: To investigate chronic stress as a susceptibility factor for developing pancreatitis, as well as tumor necrosis factor-α (TNF-α) as a putative sensitizer. METHODS: Rat pancreatic acini were used to analyze the influence of TNF-α on submaximal (50 pmol/L) cholecystokinin (CCK) stimulation. Chronic restraint (4 h every day for 21 d) was used to evaluate the effects of submaximal (0.2 µg/kg per hour) cerulein stimulation on chronically stressed rats. RESULTS: In vitro exposure of pancreatic acini to TNF-α disorganized the actin cytoskeleton. This was further increased by TNF-α/CCK treatment, which additionally reduced amylase secretion, and increased trypsin and nuclear factor-κB activities in a protein-kinase-C δ and ε-dependent manner. TNF-α/CCK also enhanced caspases' activity and lactate dehydrogenase release, induced ATP loss, and augmented the ADP/ATP ratio. In vivo, rats under chronic restraint exhibited elevated serum and pancreatic TNF-α levels. Serum, pancreatic, and lung inflammatory parameters, as well as caspases'activity in pancreatic and lung tissue, were substantially enhanced in stressed/cerulein-treated rats, which also experienced tissues' ATP loss and greater ADP/ATP ratios. Histological examination revealed that stressed/cerulein-treated animals developed abundant pancreatic and lung edema, hemorrhage and leukocyte infiltrate, and pancreatic necrosis. Pancreatitis severity was greatly decreased by treating animals with an anti-TNF-α-antibody, which diminished all inflammatory parameters, histopathological scores, and apoptotic/necrotic markers in stressed/cerulein-treated rats. CONCLUSION: In rats, chronic stress increases susceptibility for developing pancreatitis, which involves TNF-α sensitization of pancreatic acinar cells to undergo injury by physiological cerulein stimulation.

Inhibition of Rac1 decreases the severity of pancreatitis and pancreatitis-associated lung injury in mice.

Pancreatitis is a disease with high morbidity and mortality. In vitro experiments on pancreatic acini showed that supramaximal but not submaximal cholecystokinin (CCK) stimulation induces effects in the acinar cell that can be correlated with acinar morphological changes observed in the in vivo experimental model of cerulein-induced pancreatitis. The GTPase Rac1 was previously reported to be involved in CCK-evoked amylase release from pancreatic acinar cells. Here, we demonstrate that pretreatment with the Rac1 inhibitor NSC23766 (100 microM, 2 h) effectively blocked Rac1 translocation and activation in CCK-stimulated pancreatic acini, without affecting activation of its closely related GTPase, RhoA. This specific Rac1 inhibition decreased supramaximal (10 nM) CCK-stimulated acinar amylase release (27.% reduction), which seems to be connected to the reduction observed in serum amylase (46.6% reduction) and lipase levels (46.1% reduction) from cerulein-treated mice receiving NSC23766 (100 nmol h(-1)). The lack of Rac1 activation also reduced formation of reactive oxygen species (ROS; 20.8% reduction) and lactate dehydrogenase release (LDH; 24.3% reduction), but did not alter calcium signaling or trypsinogen activation in 10 nM CCK-stimulated acini. In the in vivo model, the cerulein-treated mice receiving NSC23766 also presented a decrease in both pancreatic and lung histopathological scores (reduction in oedema, 32.4 and 66.4%; haemorrhage, 48.3 and 60.2%; and leukocyte infiltrate, 53.5 and 43.6%, respectively; reduction in pancreatic necrosis, 65.6%) and inflammatory parameters [reduction in myeloperoxidase, 52.2 and 38.9%; nuclear factor kappaB (p65), 61.3 and 48.6%; and nuclear factor kappaB (p50), 46.9 and 44.9%, respectively], together with lower serum levels for inflammatory (TNF-alpha, 40.4% reduction) and cellular damage metabolites (LDH, 52.7% reduction). Collectively, these results suggest that pharmacological Rac1 inhibition ameliorates the severity of pancreatitis and pancreatitis-associated lung injury through the reduction of pancreatic acinar damage induced by pathological digestive enzyme secretion and overproduction of ROS.

Modulation of the K(v)4.3 channel by syntaxin 1A.

The SNARE protein syntaxin 1A (Syn1A) is known to inhibit delayed rectifier K(+) channels of the K(v)1 and K(v)2 families with heterogeneous effects on their gating properties. In this study, we explored whether Syn1A could directly modulate K(v)4.3, a rapidly inactivating K(v) channel with important roles in neuroendocrine cells and cardiac myocytes. Immunoprecipitation studies in HEK293 cells coexpressing Syn1A and K(v)4.3 revealed a direct interaction with increased trafficking to the plasma membrane without a change in channel synthesis. Paradoxically, Syn1A inhibited K(v)4.3 current density. In particular, Syn1A produced a left-shift in steady-state inactivation of K(v)4.3 without affecting either voltage dependence of activation or gating kinetics, a pattern distinct from other K(v) channels. Combined with our previous reports, our results further verify the notion that the mechanisms involved in Syn1A-K(v) interactions vary significantly between K(v) channels, thus providing a wide scope for Syn1A modulation of exocytosis and membrane excitability.

Alcohol-induced protein kinase Calpha phosphorylation of Munc18c in carbachol-stimulated acini causes basolateral exocytosis.

BACKGROUND & AIMS: Acute or chronic alcohol treatment does little to the exocrine pancreas but predisposes the pancreas to postprandial cholinergic stimulation that triggers cellular events leading to pancreatitis. This alcohol-induced susceptibility mechanism of pancreatitis is unknown. METHODS: We employed alcohol-treated dispersed rat pancreatic acini and alcohol diet-fed rats to examine the effects of submaximal carbachol-induced changes in exocytosis (FM1-43 epifluorescence imaging and electron microscopy), Munc18c cellular translocation (confocal microscopy and subcellular fractionation), and protein kinase C (PKC) alpha-induced phosphorylation in relation to pancreatitis. RESULTS: Acute low-dose alcohol (20 mmol/L) in vitro exposure or chronic alcohol diet reduces postprandial cholinergic-stimulated amylase secretion from rat pancreatic acinar cells by blocking apical exocytosis and redirecting exocytosis to less efficient basolateral plasma membrane sites. This ectopic exocytosis is mediated by PKCalpha-induced phosphorylation of Munc18c, causing Munc18c displacement from the basolateral plasma membrane into the cytosol in which it undergoes proteolytic degradation; these processes can be blocked by PKCalpha inhibition. CONCLUSIONS: We conclude that sequential low-dose alcohol and postprandial cholinergic stimulation can induce PKCalpha-mediated Munc18c plasma membrane displacement. This relieves cognate SNARE proteins on zymogen granules and basolateral membrane to complex and consummate pathologic ectopic exocytosis at the basolateral surface. This change in vesicle trafficking may be related to the pathogenesis of pancreatitis.

Alcohol/cholecystokinin-evoked pancreatic acinar basolateral exocytosis is mediated by protein kinase C alpha phosphorylation of Munc18c.

The pancreatic acinus is the functional unit of the exocrine pancreas whose role is to secrete zymogens into the gut lumen for food digestion via apical exocytosis. We previously reported that supramaximal CCK induced apical blockade and redirected exocytosis to ectopic sites on the basolateral plasma membrane (BPM) of this polarized cell, leading to pancreatitis. Basolateral exocytosis was mediated by protein kinase C phosphorylation of BPM Munc18c, causing its displacement into the cytosol and activation of BPM-bound Syntaxin-4 to form a SNARE complex. To mimic the conditions of alcoholic pancreatitis, we now examined whether 20 mm alcohol followed by submaximal CCK might mimic supramaximal CCK in inducing these pathologic exocytotic events. We show that a non-secretory but clinically relevant alcohol concentration (20 mm) inhibited submaximal CCK (50 pM)-stimulated amylase secretion by blocking apical exocytosis and redirecting exocytosis to less efficient BPM, indeed mimicking supramaximal CCK (10 nM) stimulation. We further demonstrate that basolateral exocytosis caused by both stimulation protocols is mediated by PKC alpha-induced phosphorylation of Munc18c: 1) PKC alpha is activated, which binds and induces phosphorylation of PM-Munc18c at a Thr site, and these events can be inhibited by PKC alpha blockade; 2) PKC alpha inhibition blocks Munc18c displacement from the BPM; 3) PKC alpha inhibition prevents basolateral exocytosis but does not rescue apical exocytosis. We conclude that 20 mm alcohol/submaximal CCK as well supramaximal CCK stimulation can trigger pathologic basolateral exocytosis in pancreatic acinar cells via PKC alpha-mediated activation of Munc18c, which enables Syntaxin-4 to become receptive in forming a SNARE complex in the BPM; and we further postulate this to be an underlying mechanism contributing to alcoholic pancreatitis.

Arrested maturation of Neisseria-containing phagosomes in the absence of the lysosome-associated membrane proteins, LAMP-1 and LAMP-2.

Mature, microbicidal phagosomes are rich in the lysosome-associated membrane proteins, LAMP-1 and LAMP-2, two highly glycosylated proteins presumed to form a protective barrier lining the phagosomal membrane. Pathogenic Neisseria secrete a protease that selectively cleaves LAMP-1, suggesting a critical role for LAMP proteins in the microbicidal competence of phagosomes. To determine the requirement for LAMP proteins in bacterial phagocytosis, we employed embryonic fibroblasts isolated from knockout mice lacking lamp-1, lamp-2 or both genes, as well as small interfering RNA (siRNA)-mediated knockdown of LAMP expression in a human epithelial cell line. Like wild-type cells, those lacking either LAMP-1 or LAMP-2 alone formed phagosomes that gradually acquired microbicidal activity and curtailed bacterial growth. In contrast, LAMP-1 and LAMP-2 double-deficient fibroblasts failed to kill engulfed Neisseria gonorrhoeae. In these cells, maturation was arrested prior to the acquisition of Rab7. As a result, the Rab7-interacting lysosomal protein (RILP, a Rab7 effector) was not recruited to the phagosomes, which were consequently unable to undergo dynein/dynactin-mediated centripetal displacement along microtubules and remained in a predominantly peripheral location. The inability of such phagosomes to migrate towards lysosomes likely contributed to their incomplete maturation and inability to eliminate bacteria. These findings suggest that neisserial degradation of LAMP-1 is not sufficient to affect its survival within the phagosome, and establish LAMP proteins as critical components in the process whereby phagosomes acquire microbicidal capabilities.

Alcohol redirects CCK-mediated apical exocytosis to the acinar basolateral membrane in alcoholic pancreatitis.

The molecular mechanism of clinical alcohol-induced pancreatitis remains vague. We had reported that experimental high-dose cholecystokinin (CCK)-induced pancreatitis is in part because of excessive aberrant basolateral exocytosis. High-dose CCK caused Munc18c on basolateral plasma membrane (BPM) to dissociate from syntaxin (Syn)-4, activating Syn-4 to complex with plasma membrane (PM)-SNAP-23 and granule-VAMP to mediate basolateral exocytosis. We now hypothesize that alcohol could render the acinar cell BPM conducive to exocytosis by a similar mechanism. Weakly stimulating postprandial doses of alcohol (20-50 mM) inhibited postprandial low-dose CCK-stimulated secretion by blocking physiologic apical exocytosis and redirecting exocytosis to less-efficient basal PM (visualized by FM1-43 fluorescence imaging) and lateral PM sites (electron microscopy). Alcohol or low-dose CCK had no effect on PM-Munc18c, but alcohol preincubation enabled low-dose CCK to displace Munc18c from BPM, leading to SNARE complex assembly in the BPM. Similarly, alcohol diet-fed rats did not exhibit morphologic defects in the pancreas nor affected PM-Munc18c behavior, but subsequent intraperitoneal injections of low-dose CCK analog cerulein caused Munc18c displacement from BPM and cytosolic degradation, which contributed to pancreatitis. We conclude that alcohol induces BPM-Munc18c to become receptive to postprandial CCK-induced displacement into the cytosol, a process which facilitates SNARE complex assembly that in turn activates restricted BPM sites to become available for aberrant exocytosis into the interstitial space, where zymogen activation would take place and cause pancreatitis.