Alcohol-Induced Protein Kinase Cα Phosphorylation of Munc18c in Carbachol-Stimulated Acini Causes Basolateral Exocytosis

Cosen-Binker, Laura Iris; Lam, Patrick; Binker, Marcelo Gustavo; Gaisano, Herbert Y.

doi:10.1053/j.gastro.2007.01.042

Cited by 43 publications

(58 citation statements)

References 36 publications

(57 reference statements)

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“…Using a rat model of a 6-week ethanol (EtOH) diet (ED) that resulted in blood alcohol levels equivalent to those seen in clinical intoxication (5), followed by i.p. injections of submaximal concentrations of Cch or CCK simulating postprandial stimulation, we observed apical blockade and a redirection of exocytosis to the basolateral PM as well as pancreatitis (6)(7)(8). In those studies we began to reveal the putative molecules that mediate basolateral exocytosis, which we found to be consistent with the paradigm of the "SNARE hypothesis" (9,10).…”

Section: Introductionsupporting

confidence: 64%

“…Instead, Vamp8 deletion resulted in inhibition of agonist-stimulated secretion, not only in pancreatic acinar cells (15) but also in other exocrine tissues (20). While these reports collectively suggest the role of Vamp8 in apical exocytosis, Vamp8 deletion also abrogated supramaximal CCK-induced pancreatitis (15), which when taken in the context of our recent work (4,(6)(7)(8) would suggest a possible role for Vamp8 in basolateral exocytosis as well. Nonetheless, the precise role of Vamp8 in each of these exocytotic compartments has not been unequivocally demonstrated.…”

Section: Introductionmentioning

confidence: 60%

“…Specifically, Munc18c bound syntaxin-4 (Syn-4) on the basolateral PM, which prevented Syn-4 binding to cognate soluble NSF attachment receptor (SNARE) proteins (4,(6)(7)(8). ED or EtOH pretreatment of acini followed by submaximal Cch (6) or CCK stimulation (7,8) induced PKCα-mediated threonine phosphorylation of Munc18c, which caused its displacement from Syn-4, and activated Syn-4 assembly with synapse-associated protein of 23 kDa (SNAP-23) and vesicle-associated membrane protein (VAMP) proteins to form the SNARE complex that we postulated to mediate basolateral exocytosis.…”

Section: Introductionmentioning

confidence: 99%

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

Cosen-Binker¹,

Binker²,

Wang³

et al. 2008

J. Clin. Invest.

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117

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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 synapseassociated 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.

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Section: Introductionsupporting

confidence: 64%

Section: Introductionmentioning

confidence: 60%

Section: Introductionmentioning

confidence: 99%

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

Cosen-Binker¹,

Binker²,

Wang³

et al. 2008

J. Clin. Invest.

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117

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“…We previously reported that pancreatic acini employed Munc18c-Syn-4-VAMP8-SNAP23 as the putative SM-SNARE complex mediating basolateral exocytosis that causes pancreatitis [25][26][27]. We showed that all beta cell Syns displayed promiscuous binding to VAMP2 and VAMP8 and to SNAP25 and SNAP23, although each Syn has a preference for a particular VAMP [16,17].…”

Section: Syn-4 Depletion Diminishes Biphasic Gsis By Reducing Exocytomentioning

confidence: 88%

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells

et al. 2015

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Aims/hypothesis Of the four exocytotic syntaxins (Syns), much is now known about the role of Syn-1A (pre-docked secretory granules [SGs]) and Syn-3 (newcomer SGs) in insulin exocytosis. Some work was reported on Syn-4's role in biphasic glucose-stimulated insulin secretion (GSIS), but its precise role in insulin SG exocytosis remains unclear. In this paper we examine this role in human beta cells. Methods Endogenous function of Syn-4 in human islets was assessed by knocking down its expression with lentiviral single hairpin RNA (lenti-shRNA)-RFP. Biphasic GSIS was determined by islet perifusion assay. Single-cell analysis of exocytosis of red fluorescent protein (RFP)-positive beta cells (exhibiting near-total depletion of Syn-4) was by patch clamp capacitance measurements (Cm) and total internal reflection fluorescence microscopy (TIRFM), the latter to further assess single SG behaviour. Co-immunoprecipitations were conducted on INS-1 cells to assess exocytotic complexes. Results Syn-4 knockdown (KD) of 77% in human islets caused a concomitant reduction in cognate Munc18c expression (46%) without affecting expression of other exocytotic proteins; this resulted in reduction of GSIS in the first phase (by 42%) and the second phase (by 40%). Cm of RFP-tagged Syn-4-KD beta cells showed severe inhibition in the readily releasable pool (by 71%) and mobilisation from reserve pools (by 63%). TIRFM showed that Syn-4-KD-induced inhibition of first-phase GSIS was attributed to reduction in exocytosis of both pre-docked and newcomer SGs (which undergo minimal residence or docking time at the plasma membrane before fusion). Second-phase inhibition was attributed to reduction in newcomer SGs. Stx-4 co-immunoprecipitated Munc18c, VAMP2 and VAMP8, suggesting that these exocytotic complexes may be involved in exocytosis of pre-docked and newcomer SGs. Conclusions/interpretation Syn-4 is involved in distinct molecular machineries that influence exocytosis of both predocked and newcomer SGs in a manner functionally redundant to Syn-1A and Syn-3, respectively; this underlies Syn-4's role in mediating portions of first-phase and second-phase GSIS.

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“…It would be of particular interest to know this because ACh stimulation of acinar cells may be a more physiologically relevant situation than CCK stimulation because 1) ACh is the primary stimulant for pancreatic secretion in human acinar cells and 2) even in rodents, neurogenic pathways that locally secrete ACh likely contribute to a major component of CCK-induced secretion as well as pancreatitis (20,21). In addition, ethanol was recently shown to disrupt ACh-induced acinar cell events such as secretion by redirecting exocytosis from the apical to the basolateral membrane (22). For these reasons, we examined whether ethanol can modulate intracellular events initiated by stimulation with the ACh analog carbachol.…”

Section: Ethanol Enhances Carbachol-induced Protease Activation-mentioning

confidence: 99%

Ethanol Enhances Carbachol-induced Protease Activation and Accelerates Ca2+ Waves in Isolated Rat Pancreatic Acini

Orabi¹,

Shah²,

Muili

et al. 2011

Journal of Biological Chemistry

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Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70 -90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca 2؉ within the pancreatic acinar cell. An important conductor of this Ca 2؉ is the basolaterally localized, intracellular Ca 2؉ channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca 2؉ signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca 2؉ . Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 M). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca 2؉ wave from 9 to 18 m/s (p < 0.0005; n ‫؍‬ 18 -22 cells/group); an increase in Ca 2؉ wave speed was also observed with a change from physiologic concentrations of carbachol (1 M) to a supraphysiologic concentration (1 mM) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n ‫؍‬ 10 -16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca 2؉ waves.Pancreatitis is a life-threatening inflammatory disorder of the pancreas that leads to more than 30,000 deaths per year (1). Alcohol-associated pancreatitis accounts for 30% of acute cases and 70 -90% of chronic cases. Further, alcoholic pancreatitis carries the highest mortality rate among all etiologies (2). However, the mechanisms by which ethanol mediates pathology are largely unknown.Alcohol can exert diverse effects on the pancreas. Ethanol exposure has been linked to abnormal blood flow, leading to ischemic changes, and increased sphincter of Oddi dysfunction, resulting in pancreatic duct hypertension (2).In addition, ethanol appears to directly predispose the acinar cell to pathological changes including oxidant stress (3), membrane fragility (4), mitochondrial uncoupling (5, 6), and basolateral exocytosis (7). Several lines of evidence also link ethanol to aberrant Ca 2ϩ 3 signaling (6). High amplitude, aberrant, intracellular Ca 2ϩ waves that propagate from the apical to basolateral region of the acinar cell predispose to early features of pancreatitis, particularly intra-acinar protease activation (8, 9). We know that intracellular Ca 2ϩ release is responsible for the onset of this aberrant Ca 2ϩ signal (10). In addition, we have previously shown that the ryanodine receptor (RYR), 4 a major intracel...

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Alcohol-Induced Protein Kinase Cα Phosphorylation of Munc18c in Carbachol-Stimulated Acini Causes Basolateral Exocytosis

Cited by 43 publications

References 36 publications

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

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

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells

Ethanol Enhances Carbachol-induced Protease Activation and Accelerates Ca2+ Waves in Isolated Rat Pancreatic Acini

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