The Pancreas Misled: Signals to Pancreatitis

Review Published online: September 25, 2007

Pancreatology 2007;7:436–446 DOI: 10.1159/000108960

The Pancreas Misled: Signals to Pancreatitis David N. Criddle a Euan McLaughlin a, b John A. Murphy a, b Ole H. Petersen a Robert Sutton b MRC Group, a Physiological Laboratory, University of Liverpool, and b Division of Surgery and Oncology, Royal Liverpool University Hospital, Liverpool, UK

Key Words Pancreas ⴢ Pancreatitis ⴢ Calcium signalling ⴢ Signal pathway ⴢ Cell death

suggest prophylaxis or treatment targets, more work is required to define the mechanisms and interactions of cell signalling pathways in the pathogenesis of pancreatitis. Copyright © 2007 S. Karger AG, Basel and IAP

Abstract Acute pancreatitis is an increasingly common and sometimes severe disease for which there is little specific therapy. Chronic pancreatitis is a common and grossly debilitating sequel that is largely irreversible, whatever treatment is adopted. In the face of these burdens, the absence of specific treatments is a spur to research. The acinar cell is the primary target of injury from alcohol metabolites, bile, hyperlipidaemia, hyperstimulation and other causes. These induce abnormal, prolonged, global, cytosolic calcium signals, the prevention of which also prevents premature digestive enzyme activation, cytokine expression, vacuole formation and acinar cell necrosis. Such agents increase calcium entry through the plasma membrane and/or increase calcium release from intracellular stores, shown to result from effects on calcium channels and calcium pumps, or their energy supply. A multitude of signalling mechanisms are activated, diverted or disrupted, including secretory mechanisms, lysosomal regulators, inflammatory mediators, cell survival and cell death pathways, together with or separately from calcium. While recent discoveries have increased insight and

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The Burden of Pancreatitis

Acute pancreatitis is a major and sometimes lethal disease with a steadily increasing incidence; currently between 20 and 100 per 100,000 per year are accurately recorded [1, 2]. This increase reflects increasing alcohol consumption that is continuing [3], although there are various other precipitants. One fifth of cases are severe, featuring pancreatic necrosis and other local complications, and/or organ failure and sepsis that necessitate prolonged intensive care and hospital stay, leading to death in 20–30% of these severe cases. Survivors frequently suffer prolonged debility that may include pancreatic exocrine and endocrine insufficiency. At present acute pancreatitis lacks any specific therapy that is generally agreed to prevent or ameliorate its course, although measures that correct precipitants (e.g. cholecystectomy for gallstones) prevent recurrence [3, 4]. Chronic pancreatitis causes hospital admission rates approaching one half of those for acute pancreatitis, rates that are also increasing Robert Sutton, DPhil, FRCS, Prof. Surg. Division of Surgery and Oncology, University of Liverpool Royal Liverpool University Hospital, Daulby Street Liverpool L69 3GA (UK) Tel. +44 151 706 4170, Fax +44 151 706 5826, E-Mail [email protected]

[4]. Pancreatic exocrine and/or endocrine insufficiency develops, accompanied by chronic, severe and at times crippling pain. Again, little if any treatment has any effect on the course of the disease, although removal of a precipitant (e.g. alcohol abuse) can slow progression, while other measures including pain relief, endoscopic and surgical procedures are directed at complications of the disease [5]. Acute pancreatitis may precede chronic pancreatitis from alcohol excess, hyperlipidaemia, trypsinogen (PRSS1) or cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and autoimmunity [6, 7], while parenchymal necrosis is a feature of both forms of the disease [2, 8]. Recurrent experimental acute pancreatitis may progress to become chronic pancreatitis, further evidence of a continuum of organ injury [9, 10]. All pancreatitis features acinar cell injury and destruction, brought about by the same precipitants. It is likely that the same or similar mechanisms of injury occur in all pancreatitis, even though there is marked variation of inflammatory responses and in the recruitment of other cell types. How precipitants of pancreatitis alter normal signalling mechanisms to mislead the pancreas, injure acinar cells, disrupt exocrine secretion, induce inflammation and cause parenchymal necrosis is addressed in this review.

Calcium and Stimulus-Secretion Coupling

Calcium signalling pathways in pancreatic acinar cells have been extensively investigated and elucidated [11], although our understanding remains incomplete. As in numerous physiological systems [12] it is clear that precise spatio-temporal calcium signals are generated in the pancreas by hormones and neurotransmitters, notably cholecystokinin and acetylcholine [11]. The calcium signals are tightly linked to the physiological release of inactive zymogens from the apex of the acinar cell [13] into the pancreatic duct and duodenum, where the zymogens are activated. The vast majority of studies have been carried out using cholecystokinin-8, widely thought to be the physiological secretory hormone; however, recent evidence calls this view into question, suggesting that other forms of this peptide, such as cholecystokinin-58, may in fact be more relevant [14, 15]. For example, cholecystokinin-58 is the only detectable form of cholecystokinin in the rat [14], where water and enzyme secretion are tightly coupled in pancreatic secretion stimulated by cholecystokinin-58 but not cholecystokinin-8 [15]. To resolve conSignals to Pancreatitis

troversy arising from these recent in vivo observations, comparative studies of the actions of cholecystokinin-58 and cholecystokinin-8 on isolated pancreatic cells are required. The hormone- and neurotransmitter-induced responses of the highly polarised pancreatic acinar cell require the second messengers inositol trisphosphate (IP3), cyclic adenosine dinucleotide phosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) [16–18], as shown in figure 1. These second messengers generate oscillatory rises of cytosolic calcium concentrations through initiation and/or amplification of calcium release from the endoplasmic reticulum via the IP3 receptor (IP3R) and ryanodine receptor (RyR), calcium channels situated within the endoplasmic reticulum membrane [11, 16]. Physiological concentrations of chlolecystokinin-8 induce cytosolic calcium signals primarily via the production of NAADP [17] which may act on its own specific receptor or modulate RyRs [18]; such signals are specifically amplified by IP3 [11, 18]. In contrast, acetylcholine-mediated responses occur principally via the activation of IP3Rs [11]. The transient oscillatory calcium signals induced by physiological concentrations of cholecystokinin or acetylcholine are always generated in the apical pole of the acinar cell [19]. The spread of these signals into the basolateral area is limited by a perigranular firewall of mitochondria [20], which take up local cytosolic calcium [21] via a calcium uniporter [22] for subsequent release and reuptake into the endoplasmic reticulum [23]. Basal cytoplasmic calcium concentrations are restored by SERCA (sacroplasmic/endoplasmic reticulum calcium-ATPase) and PMCA (plasma membrane calcium-ATPase) pumps that transport calcium into the endoplasmic reticulum and out of the cell, respectively [11–13]. Each of the transient rises of the cytosolic calcium concentration elicits a concomitant increase in mitochondrial NADH [24]. This increase occurs ostensibly via the activation of calcium-dependent dehydrogenase enzymes [25] that feeds into the electron respiratory chain to generate a large electrochemical gradient linked to the production of ATP. Thus, tightly controlled cytosolic calcium signals are a feature of normal, energy-dependent acinar cell secretory function.

Toxic Calcium Signals

Sustained high levels of cytosolic calcium are toxic to most if not all cell types, with a significant role for example in ischaemia-reperfusion damage and neurotoxicity Pancreatology 2007;7:436–446

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[26–28]. In the pancreas, a variety of toxins convert apical, cytosolic calcium signals into global signals that spread and persist throughout the acinar cell, and although shortlasting global signals (several seconds) occur from time to time following physiological stimulation, sustained globalisation is toxic [29–37]. It is the prolonged, global elevation of cytosolic calcium concentrations that is toxic rather than any associated change, because when prevented by prior treatment with BAPTA, a calcium chelator, cell injury is prevented [29]. Without BAPTA, toxic effects of abnormal cytosolic calcium concentrations include premature intracellular digestive enzyme activation, cytokine expression, vacuolisation, mitochondrial depolarisation, loss of plasma membrane integrity and subsequent necrosis [29–37]. Many causes of acute pancreatitis induce pancreatic acinar cell injury through such elevations of cytosolic calcium concentrations, including hyperstimulation [29, 31], bile acids [33, 34], fatty acid ethyl esters (non-oxidative ethanol metabolites, see fig. 2) [35, 36], fatty acids that are produced intracellularly from fatty acid ethyl ester metabolism via the action of hydrolase enzymes [36], and pancreatic duct obstruction [37]. Although these findings are from rodent data, the toxins tested are broadly representative of commoner causes of acute and/or chronic pancreatitis in man. Toxic cytosolic calcium signals are generated mainly if not entirely by discharge of the endoplasmic reticulum calcium store [29, 34–36], and maintained by sustained calcium entry [29, 33, 35], predominantly across the basolateral area of the acinar cell via an as yet undefined pathway. Under these conditions physiological calcium signalling is disrupted, with spread of the elevation of cytosolic calcium concentrations throughout the cell [29, 35, 36], as displayed in figure 3, in responses to palmitoleic acid (one of several long chain fatty acids that are toxic to the pancreatic acinar cell, also see fig. 2). Bile acids [33, 34] and fatty acid ethyl esters [35, 36] induce pathological rises of cytosolic calcium concentrations in part through opening of IP3Rs, an effect that can be blocked by caffeine [33, 36]. Recent data point to a role for RyRs, since inhibition of RyR calcium channel release in vitro and in vivo reduces premature intracellular digestive enzyme activation, considered a key marker of and contributor to the development of pancreatitis [38]. Subsequent depletion of endoplasmic reticulum calcium concentrations prompts extracellular calcium entry and uptake into the endoplasmic reticulum via SERCA pumps, followed by further release into the cytosol, which if sustained leads to cellular necrosis [34–36].

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Fig. 1. Normal calcium signalling mechanisms in the pancreatic acinar cell: (1) Cholecystokinin (CCK) binds to its receptor, which then initiates formation of nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP ribose (cADPR). (2) Acetylcholine (ACh) binds to muscarinic receptors, which then initiates formation of inositol trisphosphate (IP3). (3) The second messengers IP3R, NAADP and cADPR bind to their receptors (IP3R and RyR respectively, but no specific receptor for NAADP has been identified) on the endoplasmic reticulum membrane. (4) IP3R is a calcium channel distributed in the apical region, that then releases calcium into the cytosol. (5) RyR is a calcium channel distributed predominantly in the basolateral region, that also releases calcium into the cytosol. (6) Depletion of endoplasmic reticulum calcium ion concentrations stimulates opening of store-operated calcium channels (SOC) in the plasma membrane, allowing calcium into the cell to restore endoplasmic reticulum calcium concentrations. (7) Calcium released into the apical region of the cell initiates mitochondrial stimulus-metabolism and exocytosis. (8) Calcium released less frequently in the basolateral region stimulates perinuclear and sub-plasmalemmal mitochondrial stimulus-metabolism, calcium pumps and transcription. (9) Elevated cytosolic calcium levels prompt uptake into by the sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps. (10) Elevated calcium levels are also cleared by the action of plasma membrane calcium ATPase (PMCA) pumps out of the cell.

Under resting conditions the endoplasmic reticulum continually leaks calcium into the cytosol, which is normally counterbalanced exactly by SERCA pump uptake [11, 39]. Should SERCA pump function be inhibited, as from thapsigargin administration, the calcium leak causes elevation of cytosolic calcium concentrations, which if sustained results in typical premature digestive Criddle /McLaughlin /Murphy /Petersen / Sutton

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enzyme activation, vacuolisation and cell injury [29]. Under conditions of agonist stimulation, the enzyme phosphotidylinositol 3-kinase (PI3K) mediates prolonged elevations of cytosolic calcium concentrations via an inhibitory action on SERCA [40]. This effect may explain why pharmacological inhibition or knockout of PI3K is protective in experimental acute pancreatitis [41–43]. The effect of ethanol metabolites on cytosolic calcium concentrations is more pronounced, since an effect on the SERCA pump is combined with an effect on the PMCA pump [35, 36]. Ethanol itself has little effect on calcium signalling within the pancreatic acinar cell, and acetaldehyde, the principal oxidative metabolite, has no demonstrable effect [35]. Non-oxidative fatty acid ethyl ester metabolites, however, in addition to an effect on IP3R calcium release, accumulate in mitochondria and undergo hydrolysis by esterases [44] to release toxic concentrations of fatty acids [36]. The fatty acids inhibit mitochondrial respiration and ATP production, and thus impair SERCA and PMCA pump function, leading to sustained elevations in cytosolic calcium concentrations. These effects of fatty acid ethyl esters are paralleled by the development of pancreatitis in vivo [45, 46]. The mechanism of entry of calcium into the pancreatic acinar cell has yet to be fully elucidated [11]. It is likely that store-operated channels, including transient receptor potential channels (TRPCs) found as TRPC1, 3 and 6 in the pancreatic acinar cell [47], sensitive to the

calcium concentration within the endoplasmic reticulum, play a major role [11, 48] (shown in figure 1). Such signalling is mediated via STIM (stromal interaction molecule) protein family members, that are activated via calcium store depletion in Drosophila S2 cells and human T lymphocytes [49]. It is notable that sustained, global rises of cytosolic calcium and consequent cell injury are prevented by the removal of extracellular calcium prior to exposure of pancreatic acinar cells to the precipitants described above [29, 33, 35, 36]. The elucidation of acinar cell calcium entry mechanisms is thus an important area of investigation, as a number of potential pharmacological targets could be envisaged, including blockage of the entry channel itself, or inhibition of its activity.

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Premature Digestive Enzyme Activation

The central role of trypsinogen as a one-way master switch for normal digestive enzyme activation carries a vulnerability, namely that premature activation may initiate an activation cascade of toxic intracellular digestive enzymes [29, 31, 38, 41]. Mutations of the gene for cationic trypsinogen (PRSS1, the predominant human trypsinogen), which result in more immediate activation and/ or resistance to normal degradation, cause hereditary pancreatitis [50, 51]. Nonsense and splicing mutations of this gene appear protective in the development of pancre439

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atitis [52]. A rapidly autocatalytic variant of anionic trypsinogen (PRSS2, the second most frequent human trypsinogen) has been found to confer protection against pancreatitis [53]. Trypsin exerts local protective effects via trypsin-mediated stimulation of the protease-activated receptor (PAR-2) [54, 55], although PAR-2s also mediate deleterious systemic effects [54]. Nevertheless, the view that premature trypsinogen activation, a hallmark of pancreatitis [29, 31, 38, 41], is deleterious has been confirmed by the finding that mutations of the serine protease inhibitor Kazal type 1 (SPINK 1) confer an increased risk of pancreatitis [56], whilst overexpression of SPINK 1 ameliorated the severity of experimental acute pancreatitis [57].

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Some two decades ago, electron microscopic studies of experimental acute pancreatitis induced by hyperstimulation led to the suggestion that co-localisation of zymogen granules and lysosomes initiates premature activation of zymogens through the action of lysosomal hydrolases, particularly cathepsin B [58, 59]. Subsequent secretagogue hyperstimulation experiments with cathepsin B knockout mice demonstrated that this lysosomal enzyme makes a major contribution to trypsinogen activation and pancreatic necrosis, but the systemic inflammatory response appeared unchanged, as assessed by lung injury [60]. Cathepsin B has since been found in the human pancreatic secretory pathway, co-localised with digestive pro-enzymes within zymogen granules and present in pancreatic juice [61]. In vitro, cathepsin B only Criddle /McLaughlin /Murphy /Petersen / Sutton

activates trypsinogen below pH 5.2, while the interior pH of zymogen granules typically falls no lower than 5.5 [62], although during the initial stages of acute pancreatitis further acidification may occur as a result of increased activity by vacuolar ATPase [63]. Nevertheless, a fall to a pH below 5 would seem unlikely, unless or until fusion of zymogen granules with the more acid lysosomes occurs. An alternative explanation for the role of cathepsin B is derived from work demonstrating this enzyme to have a proapoptotic role, including that in bile duct-ligated cathepsin B knockout mice [64]. Cathepsin B contributes to lysosomal permeabilisation during cytotoxicity, releasing lysosomal proteases into the cytosol which cause mitochondrial dysfunction [65]. Pharmacological inhibition of cathepsin B blocks apoptosis induced by p53 and cytotoxic agents [66]. After bile duct ligation in normal mice, hepatic inflammation as measured by transcription of CXC chemokines and neutrophil infiltration, and

fibrogenesis as measured by stellate cell activation and collagen deposition, both increase [64]. The comparative extent of these changes in cathepsin B knockout mice is reduced, as is hepatocyte apoptosis, mitochondrial cytochrome c release and elevation in alanine aminotransferase [64]. In this case, it would appear that extensive apoptosis has a proinflammatory role, perhaps because hepatocyte necrosis is accelerated as a result. It may be that the reduced pancreatic injury observed in hyperstimulated cathepsin B knockout mice [60] may be due to a reduction in apoptosis, which might otherwise augment inflammation and tissue injury because of its extent and conversion of apoptosis to necrosis. In keeping with this interpretation, the percentage of pancreatic cells undergoing apoptosis was significantly lower at 1, 3 and 8 h after hyperstimulation in the cathepsin B knockout compared to wild-type animals, after which necrosis became preponderant, more so in the wild-type animals [60]. Nevertheless, this interpretation must be squared with data indicating that apoptosis has a protective role in pancreatitis [67–69]. Whatever the mechanism by which cathepsin B contributes to pancreatic injury, an alternative explanation is required for the initiation of premature digestive enzyme activation. Since trypsinogen activation does not occur in hyperstimulated acinar cells pretreated with BAPTA, a calcium chelator that prevents prolonged, abnormal elevations in cytosolic calcium concentrations [29, 30], such elevations may interfere with zymogen granules directly. High intragranular concentrations of calcium are required for the stability of the zymogen contents [70], most of which are tightly condensed in the granular matrix. The second messengers IP3 and cADPR have been shown to induce calcium release from zymogen granules, attributed to activation of IP3Rs and RyRs on granular membranes [71]. Although this has been contested and attributed to release from another apical store [72], it is a feature common to other types of secretory granules [70, 73]. Increasing data indicate that such release comes from both zymogen granules and lysosomes [74]. A local perigranular rise of calcium induced by these second messengers activates calcium-dependent potassium channels present in the granular membrane, permitting uptake of potassium into the granule [70]. Since the matrix behaves as an ion exchanger [70, 73], calcium ions are replaced by potassium ions, causing disaggregation of the matrix, likely to facilitate trypsinogen activation [75]. The complexity of ionic interaction is increased, however, since there are effectively three compartments, namely the perigranular cytosol, intra-

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Fig. 4. Schema showing effect of non-oxidative ethanol metabo-

lites on calcium within the pancreatic acinar cell: (1) Fatty acid ethyl esters (FAEEs) are formed by synthase enzymes abundant in the pancreatic acinar cell, and induce release of calcium from via ER release channels (IP3R and RyR), and bind within mitochondria. (2) Emptying of ER calcium stores induces further calcium entry via store-operated calcium channels (SOC). (3) Hydrolase enzymes induce excessive local release of fatty acids (FA) from FAEEs within mitochondria, which impair ␤-oxidation and uncouple oxidative phosphorylation, leading to decreased ATP production. (4) SERCA and PMCA calcium pumps are inhibited by an inadequate supply of ATP, leading to cytosolic calcium overload that induces necrosis.

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granular solution and intragranular matrix. Also, the exact role of hydrogen ions is unclear, since acidification by vacuolar ATPase is implicated in premature digestive enzyme activation [63], yet trypsinogen is activated more readily in an alkaline environment [76].

Mitochondrial Dysfunction

Pancreatic acinar cell mitochondria are distributed in perigranular, perinuclear and sub-plasmalemmal regions [21]. Perigranular acinar cell mitochondria comprise a buffer barrier that limits the spread of physiological calcium signals from the apical pole of the acinar cell [20], where such signals initiate secretion and requisite mitochondrial ATP production [11, 23–25]. Dysfunction of the perigranular mitochondrial buffer zone permits excessive globalisation of calcium signals [11, 20], and disrupts secretion. For example, cholecystokinin hyperstimulation (nM) of isolated normal mouse pancreatic acinar cells depolarises the mitochondrial membrane [77], whilst normal apical to basal progression of calcium signals is increasingly lost upon quasi-physiological stimulation (pM) of acinar cells isolated from mice with increasingly severe hyperstimulation-induced acute pancreatitis [78], suggesting destruction of the buffer barrier. Other recognised causes of acute pancreatitis, notably bile acids [33, 34] and non-oxidative ethanol metabolites [35, 36, 45, 46], the latter via their intracellular conversion to free fatty acids, promote sustained elevations in acinar cell cytosolic calcium concentrations with complete mitochondrial depolarisation [36]. In the case of non-oxidative ethanol metabolites this inhibitory action on mitochondria is associated with a concomitant fall of cellular ATP levels in pancreatic acinar cells, resulting in cellular necrosis [35, 36]. Such mitochondrial targeting, displayed in figure 4 with its effects on pancreatic acinar cell calcium concentrations, is consistent with previous evidence in myocardial tissue which showed that over 70% of intracellularly synthesised fatty acid ethyl esters are bound to this organelle and undergo hydrolysis to free fatty acids [79]. Dramatic falls of ATP levels have been reported in several in vivo animal models of acute pancreatitis, including cerulein hyperstimulation and oleic acid infusion [80, 81], indicating that cellular energy production is compromised as pancreatitis develops. This reduction may constitute both a trigger and exacerbating factor, depending on the pathogenetic mechanism. One consequence is an upregulation of mitochondrial ATP synthase, observed 442

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after both cerulein hyperstimulation [82] and chronic alcohol exposure [83]. This upregulation may compensate for decreased ATP production, but is insufficient when toxic effects are pronounced. A major consequence of cellular ATP depletion is that any increase in cytosolic calcium cannot be removed by the cell, since the ATP-dependent SERCA and PMCA pumps are rundown [36], as shown in figure 4. Under these conditions calcium entry into the acinar cell triggered by endoplasmic reticulum store depletion continues unabated [11, 30, 48]. This is of particular significance since the Na+/Ca2+ exchanger is not present in the pancreatic acinar cell and cannot contribute to the control of these calcium changes [11, 84, 85]. The importance of intracellular ATP levels in cytosolic calcium clearance has been highlighted in experiments where supplementary ATP was administered directly into the acinar cell interior via patch pipette [36]. Prolonged, global elevations of cytosolic calcium concentrations induced by non-oxidative ethanol metabolites were prevented, indicating that SERCA and PMCA rundown contribute to such sustained elevations [36, 85].

Energetics and Cell Fate

Prolonged elevations of cytosolic calcium concentrations cause mitochondrial calcium overload, leading to disruption of normal organellar function, inhibition of ATP production [86], enhanced generation of reactive oxygen species [87], mitochondrial membrane depolarisation [36] and opening of the mitochondrial permeability transition pore (MPTP) [88]. If the MPTP remains open, ATP production is lost and necrosis occurs. Since mitochondria are located close to the endoplasmic reticulum [89], they may be especially vulnerable to cytosolic calcium overload from endoplasmic reticulum calcium release. While transient opening of the MPTP is likely to release cytochrome c [90], cytochrome c has been shown to bind to endoplasmic reticulum IP3Rs, preventing closure of the channel by high cytosolic calcium concentrations, thereby potentiating calcium release from the endoplasmic reticulum [91] into the cytosol. Apoptosis removes cells largely without inflammation through programmed cell death that may occur via intrinsic or extrinsic pathways [27, 90, 92]. The intrinsic pathway depends on mitochondrial release of cytochrome c, which may occur when calcium overload contributes to transient induction of the MPTP [88, 90], although cytochrome c release does not typically depend on inducCriddle /McLaughlin /Murphy /Petersen / Sutton

tion of the MPTP [88]. Thus the oxidant menadione, at a dose of 30 ␮M, induces only partial depolarisation of mitochondria and apoptosis of pancreatic acinar cells [90], resulting from activation of the intrinsic pathway [90], although some lysosomal, cathepsin-dependent activation also occurs [93]. While the extrinsic pathway depends on cell surface death-receptor-mediated signalling, both pathways may be accelerated by cathepsin activity [64–66, 92], and both pathways require calciumactivated cysteine proteases (caspases) for orderly progression [27, 88, 92]. In contrast, necrosis is disruptive; inflammation is induced and tissue or organ function jeopardised [28]. It has been suggested, therefore, that while acinar cell necrosis is a key contributor to pancreatitis, acinar cell apoptosis protects against the disease [67–69]. Thus, prior induction of pancreatic acinar cell apoptosis protected mice against cerulein-induced pancreatitis [67], whereas inhibition of caspase activity was found to induce necrotizing pancreatitis [69]. Nevertheless, it is possible that when extensive apoptosis is induced, if necrosis is superimposed, the resultant organ injury may be made worse by the prior induction of apoptosis [64–66]. As a critical fall in ATP levels, with or without other stresses, can define the transition between apoptosis and necrosis [94, 95], it is notable that various forms of experimental pancreatitis are associated with decreased cellular ATP content [80, 81]. The importance of such a decline in the ATP:ADP ratio to the fate of pancreatic acinar cells is suggested from experiments in which the chronic administration of ethanol sensitised rats to necrosis over apoptosis in response to lipopolysaccharide injection [96]. Thus, ethanol and lipopolysaccharide resulted in a significant decline in the ATP:ADP ratio, more necrosis and more inflammation, compared to lipopolysaccharide alone [96]. Toxins such as non-oxidative ethanol metabolites [35, 36, 45, 46] and bile acids [33, 34, 77], which inhibit mitochondrial function and decrease cellular ATP, are likely to induce cellular necrosis by compromise of energy production, exacerbating the injurious effects of sustained cytosolic calcium elevations induced by these agents.

(SIRS) that may lead to multiple organ failure [97]. Excessive acinar cell cytosolic calcium concentrations trigger activation of the transcription factor NF-␬B [32, 98]. This factor promotes expression and release of inflammatory cytokines, including tumour necrosis factor-␣ [99, 100], which in turn instigate the recruitment and activation of inflammatory cells [100, 101]. Also trypsinogen itself, which is activated by elevated cytosolic calcium concentrations [29, 31], upregulates the intercellular adhesion molecule ICAM-1 that targets leukocytes into areas of tissue injury [101]. Amplification and propagation of the acute inflammatory response may underlie multiple organ failure [97, 102]. Other triggers include calcium-dependent and calcium-independent isoforms of protein kinase C (PKC-␦ and PKC-␧), which contribute to cholecystokinin hyperstimulation-induced increases of NF␬B in pancreatic acinar cells [103], and may contribute to ethanol toxicity [104].

Where Do the Signals Point?

Recent developments in our understanding of signalling pathways that contribute to the pathogenesis of acute pancreatitis permit the design of rational pharmacological interventions that could prevent or treat this debilitating condition [85]. Abnormal patterns of calcium signalling appear central to acinar cell injury. Physiological control is compromised, and sustained elevations of cytosolic calcium lead to premature enzyme activation and cell damage [29–38]. Inhibition of calcium release from the endoplasmic reticulum provides one line of attack for which there are already supportive data [85]. An alternative approach would be inhibition of calcium entry pathways [30]. Specific measures to protect mitochondrial function or boost energy production could constitute an effective approach to limit necrosis and the ultimate extent of pancreatic and systemic damage. Thus in alcoholic or hyperlipidaemic pancreatitis, inhibition of fatty acid ethyl ester synthases, or blockade of their metabolism to free fatty acids via hydrolase enzymes [36, 44, 46, 85], could prove protective.

Signals to Inflammation

Acute pancreatitis resolves promptly in 80% of cases, whereas in the remaining 20% severe pancreatic injury, often with extensive pancreatic necrosis, induces an overwhelming systemic inflammatory response syndrome Signals to Pancreatitis

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