Alcohol Redirects CCK‐Mediated Apical Exocytosis to the Acinar Basolateral Membrane in Alcoholic Pancreatitis

Abstract

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 lowdose CCK-stimulated secretion by blocking physiologic apical exocytosis and redirecting exocytosis to lessefficient 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 PMMunc18c 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.

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

“…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).…”

“…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.…”

“…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.…”

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

“…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).…”

Direct binding of parotid secretory protein to phosphatidylinositol phosphates.

“…Alternatively, the constitutive pathway, which is common to all eukaryotic cells, secretes protein in the absence of any stimulation. The constitutive secretory pathways allow secretion from both the apical and basolateral (into the circulation) cell surfaces [1, [11][12][13]. The other secretory pathways are the minor regulated secretory pathway originating from maturing secretory granules, and the constitutive-like secretory pathway originating from secretory granules but not requiring any stimulation [1].…”