Enhanced susceptibility to pancreatitis in severe hypertriglyceridaemic lipoprotein lipase-deficient mice and agonist-like function of pancreatic lipase in pancreatic cells

Gryshchenko

Ferdek

et al. 2013

171

225

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca 2+ concentration inside pancreatic acinar cells ([Ca 2+ ] i ), due to excessive release of Ca 2+ stored inside the cells followed by Ca 2+ entry from the interstitial fluid. The sustained [Ca 2+ ] i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca 2+ release-activated Ca 2+ channels (CRAC) would prevent sustained elevation of [Ca 2+ ] i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca 2+ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca 2+ . Ca 2+ entry then occurred upon admission of Ca 2+ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca 2+ entry in a concentrationdependent manner within the range of 1 to 50 μM (IC 50 = 3.4 μM), but had little or no effect on the physiological Ca 2+ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca 2+ ] i , which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca 2+ ] i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.capacitative Ca 2+ entry | alcohol metabolite | pancreas | hepatocyte Ca 2+ entry | AR42JA cute pancreatitis is a human disease mostly caused by alcohol abuse or complications from biliary disease. In this disease, against which there is currently no effective therapy, digestive proenzymes are prematurely activated inside the acinar cells leading to autodigestion and necrosis (1-3). Intracellular Ca 2+ plays a critical role in the initiation of this disease process (2-4), but intracellular Ca 2+ also plays a critical role in the physiological regulation of the normal exocytotic secretion of the digestive proenzymes (5).The pancreatic acinar cells are capable of generating multiple patterns of cytosolic Ca 2+ signals depending on the type and concentration of the stimulating agent (5). The physiological Ca 2+ signals regulating secretion-evoked by the neurotransmitter acetylcholine (ACh) or the hormone cholecystokinin (CCK)-consist of repetitive short-lasting rises in the cytosolic Ca 2+ concentration ([Ca 2+ ] i ). These are mostly confined to the apical area, in which the secretory (zymogen) granules (ZGs) are concentrated, by a belt of perigranular mitochondria operating as a firewall against...

Section: Discussionmentioning

confidence: 99%

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

Section: Concentration Dependence Of the Acute Effects Of Gsk-7975a Omentioning

confidence: 99%

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

Gryshchenko

Ferdek

et al. 2013

171

225

“…Recent work has shown that palmitoleic acid ethyl ester (POAEE) releases Ca 2+ from both the endoplasmic reticulum (ER) and an acid store in the apical part of pancreatic acinar cells and indicates that it is the release from the acid store, predominantly through IP 3 receptors (IP 3 Rs) of types 2 and 3, that is principally responsible for the intracellular activation of trypsinogen (12). Long-chain fatty acids, for example palmitoleic acid, can induce a more slowly developing Ca 2+ release also causing intracellular trypsinogen activation (10,13,14), which to a large extent depends on inhibition of mitochondrial function and subsequent reduction in the capacity to remove excess Ca 2+ from the cytosol through ATP-dependent Ca 2+ pumps (10,15).…”

mentioning

confidence: 99%

Calmodulin protects against alcohol-induced pancreatic trypsinogen activation elicited via Ca ²⁺ release through IP ₃ receptors

Lür

Ferdek

et al. 2011

Alcohol abuse is a major global health problem, but there is still much uncertainty about the mechanisms of action. So far, the effects of ethanol on ion channels in the plasma membrane have received the most attention. We have now investigated actions on intracellular calcium channels in pancreatic acinar cells. Our aim was to discover the mechanism by which alcohol influences calcium homeostasis and thereby understand how alcohol can trigger premature intracellular trypsinogen activation, which is the initiating step for alcohol-induced pancreatitis. We used intact or twophoton permeabilized acinar cells isolated from wild-type mice or mice in which inositol trisphosphate receptors of type 2 or types 2 and 3 were knocked out. In permeabilized pancreatic acinar cells even a relatively low ethanol concentration elicited calcium release from intracellular stores and intracellular trypsinogen activation. The calcium sensor calmodulin (at a normal intracellular concentration) markedly reduced ethanol-induced calcium release and trypsinogen activation in permeabilized cells, effects prevented by the calmodulin inhibitor peptide. A calmodulin activator virtually abolished the modest ethanol effects in intact cells. Both ethanol-elicited calcium liberation and trypsinogen activation were significantly reduced in cells from type 2 inositol trisphosphate receptor knockout mice. More profound reductions were seen in cells from double inositol trisphosphate receptor (types 2 and 3) knockout mice. The inositol trisphosphate receptors, required for normal pancreatic stimulus-secretion coupling, are also responsible for the toxic ethanol action. Calmodulin protects by reducing calcium release sensitivity.

“…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 emptying of intracellular Ca 2ϩ stores, causing Ca 2ϩ -dependent necrosis (9,13,14).…”

mentioning

confidence: 99%

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

Lür

Sherwood

et al. 2009

100

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...