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

Abstract

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70 -90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca 2؉ within the pancreatic acinar cell. An important conductor of this Ca 2؉ is the basolaterally localized, intracellular Ca 2؉ channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca 2؉ signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca 2؉ . Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 M). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca 2؉ wave from 9 to 18 m/s (p < 0.0005; n ‫؍‬ 18 -22 cells/group); an increase in Ca 2؉ wave speed was also observed with a change from physiologic concentrations of carbachol (1 M) to a supraphysiologic concentration (1 mM) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n ‫؍‬ 10 -16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca 2؉ waves.Pancreatitis is a life-threatening inflammatory disorder of the pancreas that leads to more than 30,000 deaths per year (1). Alcohol-associated pancreatitis accounts for 30% of acute cases and 70 -90% of chronic cases. Further, alcoholic pancreatitis carries the highest mortality rate among all etiologies (2). However, the mechanisms by which ethanol mediates pathology are largely unknown.Alcohol can exert diverse effects on the pancreas. Ethanol exposure has been linked to abnormal blood flow, leading to ischemic changes, and increased sphincter of Oddi dysfunction, resulting in pancreatic duct hypertension (2).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). High amplitude, aberrant, intracellular Ca 2ϩ waves that propagate from the apical to basolateral region of the acinar cell predispose to early features of pancreatitis, particularly intra-acinar protease activation (8, 9). We know that intracellular Ca 2ϩ release is responsible for the onset of this aberrant Ca 2ϩ signal (10). In addition, we have previously shown that the ryanodine receptor (RYR), 4 a major intracel...

Bile Acids Induce Pancreatic Acinar Cell Injury and Pancreatitis by Activating Calcineurin

“…Our findings that blockade of intracellular Ca 2ϩ reduced calcineurin activation are in general agreement with previous reports that sustained Ca 2ϩ signals are required to activate calcineurin (22,23 (12,25,46), Ca 2ϩ store depletion, and the subsequent opening of store operated Ca 2ϩ entry (SOCE) channels (47)(48)(49).…”

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

“…In vivo, administration of dantrolene blocked pancreatic injury when given before bile acid infusion, and it also ameliorated pancreatitis when given as therapy after bile infusion. In previous work, we and others have shown that basolateral Ca 2ϩ amplitude and Ca 2ϩ wave speed with supraphysiological secretagogue stimulation were dependent on the RyR (22,29,32,36,45). In our current work, we find that RyR inhibition reversed the pattern of the acinar cell Ca 2ϩ signals (in most acinar cells) induced by pathological concentrations of bile acids from a peak-plateau to that of Ca 2ϩ oscillations.…”

Raf-1 Kinase Inhibitory Protein (RKIP) Mediates Ethanol-induced Sensitization of Secretagogue Signaling in Pancreatic Acinar Cells

“…Exposing either experimental animals or isolated pancreatic acinar cells to EtOH sensitizes the cells to physiologic secretagogue stimulation (22)(23)(24)(25)(26) and deregulates normal agonist-in-duced Ca 2ϩ responses and zymogen activation (27,28). Specifically, it has been shown that after treatment of isolated pancreatic acinar cells either with EtOH or its non-oxidative metabolites, physiological concentrations of secretagogue induces sustained increases in [Ca 2ϩ ] cyto that are no longer limited to the apical pole but spread throughout the acinar cell (28,29).…”

“…Specifically, it has been shown that after treatment of isolated pancreatic acinar cells either with EtOH or its non-oxidative metabolites, physiological concentrations of secretagogue induces sustained increases in [Ca 2ϩ ] cyto that are no longer limited to the apical pole but spread throughout the acinar cell (28,29). Associated with the prolonged aberrant Ca 2ϩ signal is an inhibition of apical zymogen secretion (30), impaired mitochondrial function (i.e.…”

“…Associated with the prolonged aberrant Ca 2ϩ signal is an inhibition of apical zymogen secretion (30), impaired mitochondrial function (i.e. reduced ATP generation) (31), the generation of reactive oxygen species (27, 32) Ca 2ϩ -dependent intracellular zymogen activation (28,(33)(34)(35), and Ca 2ϩ -dependent necrotic cell death (36 -38).…”