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

Abstract

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.

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).…”

“…It could therefore be potentially advantageous to combine CRAC blockade with inhibition of Ca 2+ release from the internal stores using a synthetic membrane permeable calmodulin activator (29). Overall, our data provide a unique proof of principle that pharmacological CRAC channel inhibition could become an effective tool for reducing the cellular Ca 2+ overload that is such an important feature of the changes occurring in the initial phase of acute pancreatitis.…”

“…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.…”

“…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.…”