VAMP8 is the v-SNARE that mediates basolateral exocytosis in a mouse model of alcoholic pancreatitis
Laura I. Cosen-Binker, Marcelo G. Binker, Cheng-Chun Wang, Wanjin Hong, Herbert Y. Gaisano
Laura I. Cosen-Binker, Marcelo G. Binker, Cheng-Chun Wang, Wanjin Hong, Herbert Y. Gaisano
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Research Article Inflammation

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

Abstract

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.

Authors

Laura I. Cosen-Binker, Marcelo G. Binker, Cheng-Chun Wang, Wanjin Hong, Herbert Y. Gaisano

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Figure 3

Regulated enzyme secretion is reduced but not ablated in Vamp8–/– acini and exhibits further reduction with alcohol pretreatment.

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Regulated enzyme secretion is reduced but not ablated in Vamp8–/– acini ...
(A) Western blot showing that VAMP8 is absent in Vamp8–/– mouse pancreatic tissue, while VAMP2 levels are increased. Protein (10 μg) of each fraction was separated on SDS-PAGE and immunoblotted with antibodies to VAMP8, VAMP2, and actin. Representative of 3 independent experiments, n = 3 (analysis shown in Supplemental Figure 1). (B) Accumulation of amylase in Vamp8–/– pancreases. The amylase activity of adult WT and Vamp8–/– pancreases were determined and presented as arbitrary units. Each value is the mean ± SD of triplicate samples per experiment from 5 independent experiments (n = 15). *P < 0.05 compared with WT mice. (C) Effect of 20 mM EtOH (1 h preincubation) on submaximal (3 μM, 1 h), maximal (80 μM, 1 h), and supramaximal (2 mM, 1 h) Cch stimulated amylase release in WT and Vamp8–/– acini. The amylase secreted into the media was determined and expressed as a percentage of the total cellular amylase of the respective sample. Each value is the mean ± SD of triplicate samples per experiment from 5 independent experiments (n = 15). *P < 0.05 compared with KRH; #P < 0.05 compared with WT mice. (D) Effect of 6-wk ED on 3 μM Cch–stimulated amylase release. Here, dispersed acini were obtained from CD- or ED-fed WT and Vamp8–/– mice and then stimulated with 3 μM Cch (1 h), 1 μM Atp (1 h) followed by 3 μM Cch (1 h), or KRH (control). The amylase secreted into the media was determined and expressed as a percentage of the total cellular amylase of the respective sample. Each value is the mean ± SD of triplicate samples per experiment from 5 independent experiments (n = 15). *P < 0.05 compared with CD; #P < 0.05 compared with WT mice.