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Review
, 15 (10), 2683-98

Calcium and Reactive Oxygen Species in Acute Pancreatitis: Friend or Foe?

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Review

Calcium and Reactive Oxygen Species in Acute Pancreatitis: Friend or Foe?

David M Booth et al. Antioxid Redox Signal.

Abstract

Significance: Acute pancreatitis (AP) is a debilitating and, at times, lethal inflammatory disease, the causes and progression of which are incompletely understood. Disruption of Ca(2+) homeostasis in response to precipitants of AP leads to loss of mitochondrial integrity and cellular necrosis.

Recent advances: While oxidative stress has been implicated as a major player in the pathogenesis of this disease, its precise roles remain to be defined. Recent developments are challenging the perception of reactive oxygen species (ROS) as nonspecific cytotoxic agents, suggesting that ROS promote apoptosis that may play a vital protective role in cellular stress since necrosis is avoided.

Critical issues: Fresh clinical findings have indicated that antioxidant treatment does not ameliorate AP and may actually worsen the outcome. This review explores the complex links between cellular Ca(2+) signaling and the intracellular redox environment, with particular relevance to AP.

Future directions: Recent publications have underlined the importance of both Ca(2+) and ROS within the pathogenesis of AP, particularly in the determination of cell fate. Future research should elucidate the subtle interplay between Ca(2+) and redox mechanisms that operate to modulate mitochondrial function, with a view to devising strategies for the preservation of organellar function.

Figures

FIG. 1.
FIG. 1.
Interplay between reactive oxygen species (ROS) and Ca2+ signaling. Physiological stimulation of pancreatic acinar cells by acetylcholine (ACh) and cholecystokinin (CCK), acting at muscarinic receptors (M3) and cholecystokinin (CCK1) receptors, respectively, raises intracellular Ca2+ levels. Bile acids, such as taurolithocholic acid 3-sulfate (TLC-S), act at Gpbar1 receptors. In response to receptor stimulation, the second messengers inositol 1, 4, 5 trisphosphate (IP3), nicotinic acid adenine dinucleotide phosphate (NAADP), and cyclic ADP ribose (cADPR) are generated releasing Ca2+ from intracellular stores via the IP3 receptor (IP3R) and the ryanodine receptor (RyR); Ca2+ release may further precipitate Ca2+-induced Ca2+-release (CICR) contributing to depletion of the endoplasmic reticulum (ER) store. The fall in ER Ca2+ is sensed by stromal interaction molecule 1 (STIM1) protein that translocates to the plasma membrane and couples with Orai units, allowing store-operated Ca2+ entry to ensue and refilling of internal stores. Sustained rises of cytosolic Ca2+ are normally limited by clearance of Ca2+ to the cell exterior via the plasma membrane Ca2+-ATPase (PMCA) or taken up into the ER store by the sarcoendoplasmic Ca2+-ATPase (SERCA). ROS produced by the mitochondrial electron transport chain (ETC) may interact with targets involved in cellular Ca2+ homeostasis, thereby modifying their activity (stars: numbers represent reference material; see text for details).
FIG. 2.
FIG. 2.
Bile acid-induced mitochondrial metabolic inhibition: the importance of sustained Ca2+ elevations. Confocal images showing that the bile acid TLC-S induces sustained increases of cytosolic Ca2+ ([Ca2+]c) in a triplet of pancreatic acinar cells loaded with the Ca2+ indicator Fluo4 (upper panel). The result of sustained [Ca2+]c increases is uptake of Ca2+ by mitochondria. Simultaneous measurements of NAD(P)H autofluorescence (lower panel) and mitochondrial Ca2+ ([Ca2+]m) using the mitochondrial Ca2+ probe Rhod2 (center panel) show that application of TLC-S (500 μM) to a doublet of pancreatic acinar cells induces a large sustained rise of [Ca2+]m and marked loss of NAD(P)H autofluorescence intensity as mitochondrial function is inhibited. Note the predominantly perigranular distribution of both NAD(P)H and Rhod2 localized to mitochondria [adapted from Ref. (14)].
FIG. 3.
FIG. 3.
ROS induce pancreatic acinar cell apoptosis. (A) Confocal images of a cluster of pancreatic acinar cells (left) showing intracellular ROS detected by 5-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester (CM-DCFDA; lower panel) and NAD(P)H autofluorescence (upper panel), and normalized fluorescence measurements (right) demonstrating that application of menadione (30 μM) markedly increased intracellular ROS, whereas NAD(P)H was concomitantly reduced as redox cycling of the quinone occurred. (B) Menadione induced apoptosis (mid gray; measured with rhodamine 110-aspartic acid amide, a general caspase substrate) but had no effect on cellular necrosis (dark gray; measured with propidium iodide uptake). Clearance of menadione-induced ROS with the antioxidant N-acetyl-L-cysteine abolished the increase in apoptosis demonstrating the specific involvement of ROS in pancreatic acinar apoptosis. Necrosis and apoptosis shown as a proportion of total cells counted [adapted from Refs. (25, 26)].
FIG. 4.
FIG. 4.
Bile acid-induced mitochondrial ROS generation in human and murine pancreatic acinar cells. (A) Intracellular ROS generation by 500 μM TLC-S measured with 5-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester (CM-DCFDA) in a human pancreatic acinar cell. (B) Thin confocal sections (<2 μm) of a small cluster of murine pancreatic acinar cells reveal a typical mitochondrial distribution NAD(P)H autofluorescence (upper panel) at the beginning of the experiment. Application of TLC-S caused an increase of intracellular ROS, detected with DCFDA (lower panel), in the mitochondrial region, whereas NAD(P)H was concomitantly decreased [adapted from Ref. (14)].
FIG. 5.
FIG. 5.
Schematic representation of the proposed mechanism of bile-induced apoptosis within the pancreatic acinar cell. Bile acids stimulate the bile receptor Gpbar1, which elicits concentration-dependent increases in cytosolic Ca2+ ([Ca2+]c). Free [Ca2+]c enters the mitochondrial matrix increasing the mitochondrial Ca2+ concentration ([Ca2+]m), resulting in mitochondrial ROS production and subsequent caspase activation. Clearance of ROS with the antioxidant N-acetyl-L-cysteine (NAC) or inhibition of mitochondrial ROS production with antimycin-A and rotenone, agents that block the ETC, abolishes apoptosis. Preincubation with the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), loaded in membrane-permeable-AM form, significantly dampens [Ca2+]c elevations, abolishing both ROS production and apoptosis [adapted from Ref. (14)].
FIG. 6.
FIG. 6.
Effects of intracellular ROS and cytosolic Ca2+ ([Ca2+]c) on cell death induced by bile acids. A schematic representation shows how manipulation of ROS and Ca2+ may influence cell fate. When mitochondrial Ca2+ concentration ([Ca2+]m) is elevated following bile acid-induced [Ca2+]c rises, mitochondrial ROS generation is triggered, which promotes apoptosis. As [Ca2+]m becomes further increased/sustained mitochondrial function is compromised, probably via opening of the mitochondrial permeability transition pore (MPTP), and ATP production falls leading to necrosis. The bar chart below shows the results of an in vitro cell death assay evaluating the effects of agents that manipulate either [Ca2+]c or ROS on TLC-S induced toxicity. The antioxidant NAC markedly reduced TLC-S-induced ROS and apoptosis, whereas the NADPH quinone oxidoreductase 1 (NQO1) inhibitor 2,4-dimethoxy-2-methylnaphthalene (DMN) potentiated both ROS and apoptosis. Conversely, NAC augmented necrosis, whereas DMN reduced this form of cell death. A combination (B+C) of the Ca2+ chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and caffeine, an IP3R blocker, completely prevented cell death caused by the bile acid, returning both apoptosis and necrosis to control levels. Patch pipette administration of intracellular ATP was able to prevent TLC-S-induced necrosis (27) [adapted from Ref. (14)].
FIG. 7.
FIG. 7.
Schematic model of the complex interplay between [ROS]I, [Ca2+]C, and [Ca2+]M in bile acid-induced pancreatitis. Sustained elevations of [Ca2+]C in pancreatic acinar cells acutely affect mitochondrial function; the outcome would depend on the level and duration of these pathological signals. Mitochondria uptake Ca2+ from the cytosol raising [Ca2+]m levels, an action that stimulates mitochondrial [ROS]I generation and promotion of apoptosis. This may constitute a protective mechanism since stressed cells may be successfully disposed of by macrophages, thereby removing the risk of releasing deleterious digestive enzymes. However, greater and/or more sustained stress by high [Ca2+]C and [Ca2+]m levels elicits depolarization of mitochondria with loss of ATP production; this fall in energy is the crucial trigger for necrosis. Leakage of intracellular contents, cytokine release, and inflammation may ensue that result in activation, proliferation, and infiltration of neutrophils, initiating a cycle that ultimately may lead to a systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). Antioxidant (AOx) treatment may affect the development of acute pancreatitis at several places but which may have differing outcomes. First, inhibition of ROS released by neutrophils is likely to reduce the damage associated with the inflammatory response; however, a reduction of ROS at the local level might conversely prevent apoptosis of pancreatic acinar cells thereby increasing necrosis and organ damage [adapted from Refs. (14, 25)].

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