Pancreatic protease activation by alcohol metabolite depends on Ca ²⁺ release via acid store IP ₃ receptors

Abstract

Toxic alcohol effects on pancreatic acinar cells, causing the often fatal human disease acute pancreatitis, are principally mediated by fatty acid ethyl esters (non-oxidative products of alcohol and fatty acids), emptying internal stores of Ca 2؉ . This excessive Ca 2؉ liberation induces Ca 2؉ -dependent necrosis due to intracellular trypsin activation. Our aim was to identify the specific source of the Ca 2؉ release linked to the fatal intracellular protease activation. In 2-photon permeabilized mouse pancreatic acinar cells, we monitored changes in the Ca 2؉ concentration in the thapsigarginsensitive endoplasmic reticulum (ER) as well as in a bafilomycinsensitive acid compartment, localized exclusively in the apical granular pole. We also assessed trypsin activity in the apical granular region. Palmitoleic acid ethyl ester (POAEE) elicited Ca 2؉ release from both the ER as well as the acid pool, but trypsin activation depended predominantly on Ca 2؉ release from the acid pool, that was mainly mediated by functional inositol 1,4,5-trisphosphate receptors (IP3Rs) of types 2 and 3. POAEE evoked very little Ca 2؉ release and trypsin activation when IP3Rs of both types 2 and 3 were knocked out. Antibodies against IP3Rs of types 2 and 3, but not type 1, markedly inhibited POAEE-elicited Ca 2؉ release and trypsin activation. We conclude that Ca 2؉ release through IP3Rs of types 2 and 3 in the acid granular Ca 2؉ store induces intracellular protease activation, and propose that this is a critical process in the initiation of alcohol-related acute pancreatitis.calcium ͉ inositol trisphopshate receptors ͉ pancreatitis T he pancreatic acinar cell is potentially dangerous because it produces a range of precursor digestive enzymes (zymogens) that, if inappropriately activated inside the cells, cause autodigestion resulting in the often fatal human disease acute pancreatitis (1, 2).Exocytotic secretion of zymogens is controlled by local cytosolic Ca 2ϩ spikes in the apical granular region, generated by small quantities of Ca 2ϩ released from internal stores (3, 4). In contrast, prolonged global cytosolic [Ca 2ϩ ] elevationsassociated with emptying the Ca 2ϩ stores-cause intracellular trypsin activation and transform the normally electron dense zymogen granules (ZGs) into empty looking vacuoles (2, 5-7). The vacuoles are post-exocytotic, endocytic structures, and it is in these vacuoles that trypsin activation occurs (8).The association between alcohol abuse and acute pancreatitis is well known (1, 2, 9), but the exact mechanism by which alcohol initiates the disease is unclear. Hypertriglyceridemia is also a recognized cause of pancreatitis (10) and the presence of high concentrations of fatty acid ethyl esters [FAEEs, non-oxidative products of alcohol and fatty acids (FAs)], particularly in the pancreas, was reported in a postmortem study of subjects intoxicated by alcohol at the time of death (11). FAEEs induce trypsin activation and vacuole formation (12) and also elicit global sustained [Ca 2ϩ ] i elevations, due to e...

Ca 2+ release-activated Ca 2+ channel blockade as a potential tool in antipancreatitis therapy

“…4C). Similar levels of inhibition by GSK-7975A were observed in experiments assessing trypsin activity, using the specific trypsin substrate BZIPAR (10,28,29). Preincubation of pancreatic acinar cells with POAEE (100 μM for 1 h) induced substantial increase of trypsin activity (from 3.4 ± 0.5% of cells in control to 15 ± 1.1% in the POAEE-treated groups).…”

“…Although it has been known for 40 years that cytosolic Ca 2+ signals in pancreatic acinar cells are initiated by release from internal stores (5), it has also been recognized for a long time that following the initial release from the intracellular stores there is an important Ca 2+ entry phase that is essential for refilling the stores and indeed for stimulant-evoked enzyme secretion (48)(49)(50). Previous investigations have demonstrated that the most important mediators of acinar cell damage, namely alcohol, fatty acids, fatty acid ethyl esters, and bile acids all primarily release Ca 2+ from various internal stores (28,29,37,42,43,51), but that this initial phase is followed by store-operated Ca 2+ entry, which plays a crucial role in the destruction of the cells (2,4,5,10). Our electrophysiological data (Fig.…”

“…3D). (42,43), but in combination-as fatty acid ethyl esters-they are much more powerful Ca 2+ releasers (37) (28). Fig.…”

“…Endocytic Ca 2+ uptake (24) could also be important and does occur in pancreatic acinar cells (25). It is also the case that there are important intracellular Ca 2+ stores (acid) outside the ER (4,(26)(27)(28)(29). Ca 2+ release from acid storeslocated in ZGs, endosomes, and lysosomes-plays a crucial role in intracellular trypsinogen activation elicited by alcohol and fatty acid ethyl esters in experiments on permeabilized pancreatic acinar cells (28,29), and trypsinogen activation induced by hyperstimulation occurs in postexocytotic endocytic (and acid) structures (25).…”

“…It is also the case that there are important intracellular Ca 2+ stores (acid) outside the ER (4,(26)(27)(28)(29). Ca 2+ release from acid storeslocated in ZGs, endosomes, and lysosomes-plays a crucial role in intracellular trypsinogen activation elicited by alcohol and fatty acid ethyl esters in experiments on permeabilized pancreatic acinar cells (28,29), and trypsinogen activation induced by hyperstimulation occurs in postexocytotic endocytic (and acid) structures (25). In view of the complexities of pancreatic acinar cell Ca 2+ signaling in physiology and pathology and, in particular, the uncertainty about the nature of the Ca 2+ entry pathways, it is unknown whether blockade of Ca 2+ -selective CRAC channels could in principle be effective as therapy against acute pancreatitis.…”