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

Abstract

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...

Calcium and ATP control multiple vital functions

“…B 371: 20150418 in figure 2, but see next section). The initial release of Ca 2þ from intracellular stores, particularly from the ER, also triggers opening of store-operated Ca 2þ channels (not included in figure 2, but see later section), which enable store refilling [29,30]. In the neighbouring insulin-secreting beta-cells, a very different relationship exists between regulation of Ca 2þ and ATP (figure 2b).…”

“…By monitoring simultaneously the emptying of the ER Ca 2þ store (after inhibition of the ER Ca 2þ pumps by thapsigargin) and the CRAC current, it can be seen that the rise of the current follows closely the decrease in the intra-store [Ca 2þ ] [29]. Excessive primary Ca 2þ release from intracellular stores is generally the initiating event in the actions of toxic agents or excessive concentrations of neurotransmitters and hormones [27,29], but cytosolic Ca 2þ overload rstb.royalsocietypublishing.org Phil. Trans.…”

“…This seems to be the case in pancreatitis. In the quantitatively dominant acinar cells, toxic agents, such as fatty acid ethyl esters or bile acids, cause excessive intracellular Ca 2þ release followed by excessive CRAC-mediated Ca 2þ entry [27,29,34]. If sustained, this would activate trypsin inside the cells and lead to necrosis with leakage of activated proteases into the interstitial fluid.…”

CFTR: A new horizon in the pathomechanism and treatment of pancreatitis

“…Cytosolic Ca 2+ -overload and the associated mitochondrial dysfunction during ethanolinduced pancreatitis could be attenuated by inhibiting the PM Ca 2+ entry channels (such as the store operated Ca 2+ channel Orai1 [104]). Intriguingly, inhibiting PM Ca 2+ -channels also up-regulates several molecular chaperones, which could be an added advantage in stabilizing CFTR.…”

“…These events, jointly, elicit the conformational destabilization of newly synthesized CFTR, which leads to the channel accelerated metabolic turnover at the ER and PM [53]. Accordingly, we envision multifaceted therapeutic approaches to alleviate the exocrine pancreas damage in pancreatitis.Cytosolic Ca 2+ -overload and the associated mitochondrial dysfunction during ethanolinduced pancreatitis could be attenuated by inhibiting the PM Ca 2+ entry channels (such as the store operated Ca 2+ channel Orai1 [104]). Intriguingly, inhibiting PM Ca 2+ -channels also up-regulates several molecular chaperones, which could be an added advantage in stabilizing CFTR.…”