Intracellular free ionized calcium in the pathogenesis of acute pancreatitis

Intracellular free ionized calcium in the pathogenesis of acute pancreatitis

BEST Baillière’s Clinical Gastroenterology Vol. 13, No. 2, pp 241–251, 1999 B A I L L I È R E ’ S 3 PRACTICE & RESEARCH Intracellular free ionize...

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Baillière’s Clinical Gastroenterology Vol. 13, No. 2, pp 241–251, 1999

B A I L L I È R E ’ S



Intracellular free ionized calcium in the pathogenesis of acute pancreatitis Michael G. T. Raraty*


Clinical Research Fellow Departments of Surgery and Physiology

Ole H. Petersen


Professor of Physiology Department of Physiology

Robert Sutton


Senior Lecturer in Surgery Department of Surgery

John P. Neoptolemos


Professor of Surgery Department of Surgery University of Liverpool, Liverpool, UK

Acute pancreatitis is a common, often severe disease with multiple causes. Many of the aetiological factors responsible for triggering acute pancreatitis have been identified but the pathophysiological mechanism by which they do so is still poorly understood. Free calcium ions within the cytosol of the acinar cell ([Ca2+]i) act as a key intracellular second messenger in the processes of stimulus–secretion coupling and may be crucial in the pathogenesis of acute pancreatitis. [Ca2+]i signals have been shown to be disrupted early in experimental pancreatitis, and it is known that an abnormal rise in [Ca2+]i is toxic by a variety of mechanisms. It has been demonstrated that abnormal, prolonged elevations in [Ca2+]i result from caerulein hyperstimulation and ethanol treatment, and it is likely that all the known causes of acute pancreatitis can cause similar disruptions. Elevations in [Ca2+]i have also been shown to be associated with both acinar cell vacuolization and intracellular enzyme activation, both of which are key steps in the pathogenesis of acute pancreatitis. A disturbance of intracellular Ca2+ signalling and the generation of an abnormal elevation in [Ca2+]i appears to be the common factor linking all the known triggers for acute pancreatitis and initiating the further sequence of pathological events leading to clinical disease. Key words: pancreatitis aetiology; pancreatitis pathophysiology; calcium signalling.

* Address correspondence to: Mr. M. G. T. Raraty, FRCS, Research Fellow, University of Liverpool Department of Surgery, 5th Floor UCD Block, Duncan Building, Daulby Street, Liverpool, L69 3GA, UK. 1521–6918/99/020241 + 11 $12.00/00

© 1999, Baillière Tindall


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Acute pancreatitis is a disease which is increasing in incidence (Jaakkola and Nordback, 1993) and which carries a significant mortality (Mann et al, 1994), but for which there is still no definitive treatment (Banks, 1993). This lack of any specific therapies may largely be ascribed to our poor understanding of the early pathological mechanisms involved, although the later progression of the disease to a systemic inflammatory process is becoming much better understood (Wilson et al, 1998). The commonest causes of acute pancreatitis remain gallstone disease and excess ethanol consumption, which, together, account for 80–90% of cases; however, a multitude of other trigger factors have been identified as causing pancreatitis, and these may be grouped into five broad categories as shown in Table 1. Given the diverse nature of these aetiological factors it is notable that they all give rise to a similar disease entity. Once the processes of acute pancreatitis have been initiated, the underlying cause cannot be determined from the course of the pancreatitis alone, but only by examining other features of the likely causes. It is, therefore, apparent that there must be some common pathway linking all the various aetiological factors together and then progressing to the clinical entity that we know as acute pancreatitis.

Table 1. Known aetiological factors responsible for acute pancreatitis. Mechanical causes (obstruction)

Cholelithiasis Gastric/biliary surgery Pancreas divisum Trauma ERCP Malignancy Ascaris infestation Duodenal obstruction

Metabolic causes

Alcohol Hyperlipidaemia Hypercalcaemia Drugs, for example thiazides, oestrogens, azathioprine Trinidadian scorpion venom

Infective causes

Mumps Coxsackie B Cytomegalovirus Cryptococcus HIV

Vascular causes (ischaemia)

Cardio-pulmonary bypass Peri-arteritis nodosa Embolism

Genetic causes

Hereditary pancreatitis

All the earliest events identified in the pathogenetic pathway of acute pancreatitis take place within the pancreatic acinar cells themselves, and similar features have been described in all experimental models of acute pancreatitis (Steer and Saluja, 1993). These features include loss of secretory polarity, the appearance of intracellular vacuoles, colocalization of zymogens with lysosomal enzymes, premature enzyme activation, and disordered exocytosis of zymogens. The common factor among all of these disturbances is that they are all related to the process of enzyme secretion, suggesting that the common mechanism in the pathogenesis of acute pancreatitis may

Intracellular free ionized calcium in the pathogenesis of acute pancreatitis


be a disruption to the normal pathway of stimulus–secretion coupling, enzyme processing and zymogen secretion for which the acinar cell is responsible. Intracellular calcium ions are a crucial intracellular messenger involved in these processes within the acinar cell and, therefore, may be an important factor in the pathogenesis of acute pancreatitis and may be the common factor linking all the disparate causes to a single pathogenetic pathway for acute pancreatitis (Ward et al, 1995). CALCIUM SIGNALLING IN NORMAL PANCREATIC ACINAR CELLS Calcium is an ubiquitous intracellular second messenger with a variety of physiological effects throughout the animal kingdom. These actions include the control of cell growth, initiation of muscle contraction, platelet activation, control of secretion and the triggering of apoptosis. Stimulation of many types of cell has been shown to evoke an increase in the concentration of free calcium ions within the cytosol ([Ca2+]i) and in pancreatic acinar cells, a rise in [Ca2+]i has been observed both after stimulation with acetylcholine and with cholecystokinin (Muallem, 1989). This is one of the most clearly observed early events within the acinar cell prior to a secretory response (Petersen, 1992). Due to their polarized nature, isolated pancreatic acinar cells have been one of the most valuable models in the study of intracellular calcium signals (Thorn et al, 1993). Changes in [Ca2+]i may be observed directly by the use of calcium-sensitive fluorescent indicators such as fura-2 (Grynkiewicz et al, 1985), or indirectly by the examination of other Ca2+-dependent events such as electrophysiological studies of Ca2+-dependent Cl– channels (Petersen and Maruyama, 1989). The following account of the stimulus–secretion coupling cascade has been elucidated by a combination of such studies. Under normal ‘resting’ conditions the concentration of calcium within the cytosol of the acinar cell ([Ca2+]i) is of the order of 10–7 M, compared with levels of around 10–3 M in the extracellular fluid and 10–4 M in the internal stores (Petersen et al, 1998), the most important of which, within the acinar cell and from the point of view of the amount of Ca2+ stored, is the endoplasmic reticulum which is anatomically continuous with the nuclear envelope (Petersen et al, 1998). The differences in concentration between cytosol and both external fluid and intracellular membrane-bound stores creates a concentration gradient favouring Ca2+ entry. This concentration gradient is maintained by the actions of a plasma membrane Mg2+-dependent Ca2+-ATPase (Kribben et al, 1983) and an endoplasmic reticulum Ca2+-ATPase (Bayerdorffer et al, 1984). A Na+-Ca2+ co-transporter has also been described but it is doubtful whether this is of any significance in the pancreatic acinar cell (Bayerdorffer et al, 1985). There are also a number of specific Ca2+ entry channels which are controlled by agonists (receptor-operated Ca2+ channels) and by changes in the filling status of the intracellular stores (store-operated Ca2+ channels). The maintenance of a low cytoplasmic calcium concentration enables small local increases in [Ca2+]i in different regions of the cell to be utilized as a signal to control intracellular events, and is also important in that high levels of [Ca2+]i are known to be toxic to many types of cell (Nicotera et al, 1992). Some of the complex mechanisms involved in the maintenance of a low resting [Ca2+]i are summarized in Figure 1. Pancreatic secretagogues, such as acetylcholine (ACh) and cholecystokinin (CCK), bind to and activate G-protein-linked transmembrane receptors on the basolateral plasma membrane of the acinar cell (Rosenzweig et al, 1983). G-protein activation then leads to the activation of phospholipase C-β, which cleaves membrane-bound


M. G. T. Raraty et al




SOC Ca2+ Ca2+ ADP + Pi

H+ Ca2+ + Na Mitochondrion



ADP + Pi


Gap junction Ca2+


ATP Ca2+-binding Ca2+ proteins


Ca2+ Endoplasmic reticulum



Ca2+ADP + Pi ATP

Ca2+ IP3

Zymogen granule


Figure 1. Some of the many mechanisms involved in the maintenance of a low [Ca2+]i within the acinar cell. SOC = store-operated Ca2+ channel; ROC = receptor-operated Ca2+ channel; ATP = adenosine triphosphate; ADP + Pi = adenosine diphosphate + inorganic phosphate, IP3 = inositol trisphosphate. Ca2+ is pumped out of the cell and into intracellular stores against a concentration gradient by specific ATPases. Influx into the cell is controlled by several plasma membrane receptors and release from internal stores by IP3 and ryanodine receptors (not shown).

phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol and free inositol 1,4,5trisphosphate (IP3). A second pathway generates cyclic adenosine diphosphate ribose (cADPr) from nicotinamide adenine diphosphate (NAD+) (Berridge, 1993). IP3 and cADPr are highly soluble molecules which rapidly diffuse throughout the cytosol and bind to IP3 and ryanodine (cADPr) receptors located in the endoplasmic reticulum (ER) membrane and on the zymogen granules (Gerasimenko et al, 1996) to induce release of Ca2+. Recent evidence suggests that CCK can induce release of Ca2+ via either pathway, but that changes in intracellular glucose concentration may act as a switch between the two messengers (Cancela et al, 1998). These IP3 and cADPr receptors appear to be concentrated in the parts of the endoplasmic reticulum closely associated with the zymogen granules at the apical, secretory pole of the cell (Lee et al, 1997) from where Ca2+ release occurs (Mogami et al, 1997). Positive feedback on these channels by Ca2+ ions magnifies the response and triggers a rapid local elevation in [Ca2+]i. Released Ca2+ is then rapidly removed once again by binding to cytosolic proteins such as calmodulin, by re-uptake into the ER store and by extrusion from the cell in order to return [Ca2+]i to its normal resting level. Continued stimulation of the cell with low doses of agonists results in the generation of repetitive spikes or oscillations in [Ca2+]i which, in the case of ACh, are restricted to the secretory pole of the cell (Thorn et al, 1993), but in the case of CCK are also associated with occasional longer-lasting transients throughout the cell. These transients may be involved in the integration of signals into the nucleus and the regulation of gene transcription and protein processing. The precise mechanism by which such an

Intracellular free ionized calcium in the pathogenesis of acute pancreatitis


oscillatory response is generated is not yet fully understood; however, it is known that each oscillation is associated with a burst of exocytotic activity and release of zymogens into the duct lumen (Maruyama and Petersen, 1994). The sequence of steps involved in the generation of a [Ca2+]i signal are illustrated in Figure 2. Most of the IP3 generated is rapidly de-activated by dephosphorylation to terminate the signal but some is further phosphorylated to 1,3,4,5-tetrakisphosphate (IP4) which stimulates refilling of the intracellular calcium stores. Depletion of the intracellular stores also triggers refilling via store-operated Ca2+ channels in the plasma membrane (also sometimes referred to as capacitative calcium entry).






DAG IP3 Ca2+

ADP + Pi


ADP + Pi Ca2+ ATP


Figure 2. Stimulus–secretion coupling within the acinar cell. CCK = cholecystokinin; R = receptor, PLCβ = phospholipase C-β; PIP2 = phosphatidyl inositol bisphosphate; IP3 = inositol trisphosphate; DAG = diacyl glycerol; ATP = adenosine triphosphate; ADP + Pi = adenosine diphosphate + inorganic phosphate. See text for full explanation. The graph illustrates oscillations in [Ca2+]i in response to stimulation with 50 pM CCK.

Supramaximal stimulation of acinar cells, in contrast, induces a completely different pattern of [Ca2+]i signal. Instead of an oscillatory response, there is a larger, more prolonged increase in [Ca2+]i which is initiated at the secretory pole but which rapidly spreads to involve the whole of the cytosol (Toescu et al, 1992). Figure 3 shows an example of such a rise due to hyperstimulation with CCK. Such a global rise in [Ca2+]i is known to be toxic to many types of cell by a variety of different mechanisms, including mitochondrial dysfunction, disruption of the cytoskeleton and activation of catabolic enzymes (Nicotera et al, 1992). Abnormally high levels of [Ca2+]i have been shown to be involved in the damage produced by abusive stimulation of cell-surface receptors (Thorn et al, 1993), by ischaemia-reperfusion injury and oxygen free radicals (Arnould et al, 1992), by numerous toxic chemicals (Canga et al, 1988; Pauwels et al,


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Figure 3. The typical [Ca2+]i response of an acinar cell exposed to 10 nM of cholecystokinin. This dose is higher than the dose required to induce maximal secretion (approximately 1 nM).

1989; Boobis et al, 1990) and by NK cell-induced apoptosis and necrosis (Berke, 1995). It should also be noted that supramaximal stimulation of acinar cells in vivo leads to cell injury and acute pancreatitis and is the basis of perhaps the most widely used experimental model of pancreatitis: caerulein hyperstimulation (Lampel and Kern, 1977). Indeed, it is possible to hypothesize mechanisms whereby all the common triggers for acute pancreatitis could induce a rise in [Ca2+]i and, thereby, initiate cell damage and acute pancreatitis (Ward et al, 1995). DISRUPTION OF INTRACELLULAR CALCIUM SIGNALS IN EXPERIMENTAL ACUTE PANCREATITIS Pancreatic acinar cell [Ca2+]i signals have been examined during the early stages of experimentally induced pancreatitis (Ward et al, 1996). Pancreatitis was induced by intraperitoneal injections of caerulein to mice at hourly intervals at a dose of 50 µg/kg per injection. This dose is sufficient to induce a mild form of oedematous pancreatitis. Intraperitoneal injections of saline solution to a second batch of mice were used as a control. Pancreatic tissue was removed after 0, 1, 3, 5 and 7 injections and a suspension of isloated acini prepared by collagenase digestion. Cytosolic Ca2+ signalling was then examined by measuring fura-2 fluorescence. This compound demonstrates differential fluorescence in that it is excited maximally at either 340 nm or 380 nm, depending on whether or not it is Ca2+-bound; it is, therefore, possible to calculate [Ca2+]i by measuring fluorescence at both of these wavelengths and ratioing the two readings (Grynkiewicz et al, 1985). Physiological concentrations of ACh induced a normal oscillatory [Ca2+]i response in 80–90% of control cells, and this response was maintained in cells from mice injected with saline. The proportion of cells demonstrating this response from mice treated with ACh, however, progressively diminished with increasing numbers of injections, such that after five injections only 20% of the cells showed a normal

Intracellular free ionized calcium in the pathogenesis of acute pancreatitis


response (see Table 2). Instead, a greater proportion of cells showed a single, abnormal [Ca2+]i peak, or no response at all. A similar pattern of response was observed on treating the cells with physiological doses of CCK. These studies demonstrated progressive disruption to the normal pattern of acinar cell [Ca2+]i signalling in vitro early after the induction of experimental pancreatitis in vivo. Responses to treatment with supramaximal doses of ACh (500 nM) appeared to be much better preserved; however, even in this instance there was a loss of the normal spatial differentiation of the response. In the normal acinar cell the [Ca2+]i rise is first seen at the secretory pole and then spreads to involve the rest of the cell (Kasai and Augustine, 1990; Toescu et al, 1992). After induction of pancreatitis, however, initiation of the signal at the secretory pole was lost and, instead, the cells demonstrated a diffuse rise in [Ca2+]i throughout the cell. This loss of functional polarity is mirrored by the loss of structural polarity which has been described by other investigators (Adler et al, 1982; Lerch et al, 1992) and may be responsible for the abnormal discharge of digestive enzymes from the lateral plasma membrane which has been seen in some studies on both experimental and human disease (Adler et al, 1982; Kloppel et al, 1986). The mechanism by which continued stimulation leads to a disruption of [Ca2+]i signals is not yet known. In theory, the pathway could be interrupted at any point from binding of secretagogue to the discharge of Ca2+ from the cell to terminate a response but it seems that alteration of the sensitivity of the IP3 receptors on the intracellular stores is the most likely site of disruption (Matozaki et al, 1990; Xu et al, 1996). Table 2.* The response of acinar cells to ACh in experimental pancreatitis. Injection 0 1 3 5 7

Control (saline) Number of cells (%) 19 35 30 43 35

(79) (81) (80) (90) (97)

Experimental (caerulein) Number of cells (%) 34 21 5 2

(74) (78) (20) (6)

* The number (%) of cells maintaining a normal oscillatory [Ca2+]i response to 100 nM ACh after repeated injections of caerulein or saline. There was a significant difference between control and experimental cells after both five and seven injections (χ2Y, P < 0.001), and a significant linear trend in experimental results (χ2trend = 46.72, P < 0.001).

ELEVATED [Ca2+]i AS A TRIGGER FOR ACUTE PANCREATITIS The studies described above provide evidence that the normal processes of Ca2+ signalling are disrupted early in the course of acute pancreatitis, although they do not prove any causative link between these two events. Further studies, however, have demonstrated evidence of such a link (Zhou et al, 1996). Caerulein hyperstimulation was used to induce pancreatitis in rats. In normal rats a dose of caerulein of 50 µg/kg/hour was required to induce pancreatitis, but in rats also given calcium gluconate in order to raise serum [Ca2+], acute pancreatitis could be induced by doses of caerulein as low as 5 µg/kg/hour. Infusion of calcium alone has also been shown to cause acinar cell injury by blocking the normal exocytosis of zymogen granules, thus leading to accumulation of zymogens within the acinar cell and the formation of autophagic vacuoles (Frick et al, 1995). Further studies have shown that the administration of calcium channel blockers such as verapamil and diltiazem to experimental


M. G. T. Raraty et al

animals can ameliorate the pancreatitis induced by caerulein hyperstimulation (Wang et al, 1995; Zhou et al, 1996), by a choline-deficient, ethionine-supplemented (CDE) diet (Lake-Bakaar and Lyubsky, 1995) and by taurocholate infusion to the pancreatic duct (Leahy et al, 1993). Although verapamil is known to be an antagonist at voltagegated Ca2+ channels, recent evidence indicates that there is a degree of sequence homology between the transmembrane regions of these and of ligand-gated Ca2+ channels (Petersen, 1996). If Ca2+ channel blockers do have some activity on the ligandgated channels in the acinar cell membrane, they may have exerted their effects in these experiments by preventing excessive Ca2+ entry and prolonged [Ca2+]i elevation. A number of studies have also shown evidence of premature, intracellular enzyme activation induced by elevated [Ca2+]i (Mithöfer et al, 1995a,b). In these studies, infusion of CaCl2 to rats either alone (Mithöfer et al, 1995a), or in combination with the induction of systemic hypotension (Mithöfer et al, 1995b) increased serum amylase activity, pancreatic and serum levels of trypsinogen activation peptide (TAP), and pancreatic wet weight together with histological evidence of acinar necrosis. Further studies on isolated pancreatic acini in vitro have confirmed that elevated [Ca2+]i, in combination with caerulein stimulation, leads to an increase in intracellular TAP production (Frick et al, 1997). IN VITRO STUDIES In order to address the fundamental question of whether a rise in [Ca2+]i is directly responsible for triggering the cascade of changes seen in acute pancreatitis, we have performed studies on isolated acini in vitro as a model for acute pancreatitis. Normal acinar cells were isolated from mice and stimulated with high-dose CCK to mimic the effects of caerulein hyperstimulation in vivo. This treatment induces a large, abnormal rise in [Ca2+]i with a prolonged plateau phase in which [Ca2+]i remains elevated above baseline levels (see Figure 3). After 60 minutes of stimulation there was extensive evidence of cell damage consistent with pancreatitis as measured by the appearance of intracellular vacuoles. These changes could be completely abolished by pre-treating the cells with the Ca2+-chelating compound 1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′tetraacetic acid (BAPTA). In order to confirm that the observed damage was due to the changes in [Ca2+]i, and not to some other pathway triggered by CCK, we performed similar experiments using the tumour promoter thapsigargin. This compound is a specific inhibitor of the ER Ca2+-ATPase (Thastrup et al, 1990) and when applied to acinar cells generates a rise in [Ca2+]i by allowing leakage from the ER stores down the concentration gradient. The pattern of this rise is very similar to that seen after CCK hyperstimulation, although it is generated by a completely separate mechanism. Thapsigargin was found to induce similar acinar cell vacuolization to CCK and, again, pre-treatment with BAPTA greatly ameliorated the damage seen (Raraty et al, 1998b). These findings provide direct evidence that a rise in [Ca2+]i per se can induce morphological changes in acinar cells consistent with acute pancreatitis. Studies are continuing to look at intracellular enzyme activation in response to a rise in [Ca2+]i in this model. GENERATION OF AN ABNORMAL [Ca2+]i RISE IN VIVO If an abnormal, prolonged elevation in [Ca2+]i is, indeed, the common pathogenetic mechanism by which the various disparate triggers of acute pancreatitis act, then it

Intracellular free ionized calcium in the pathogenesis of acute pancreatitis


should be possible to demonstrate that all the causes listed in Table 1 can cause such a disruption. Unfortunately, few of the in vivo models of acute pancreatitis lend themselves very easily to measurements of [Ca2+]i. The best in vitro model, CCK hyperstimulation, which is similar to caerulein hyperstimulation in vivo, can be shown to be associated with elevations in [Ca2+]i (see above) but there is no in vitro equivalent for a pancreatic duct ligation or bile salt infusion model of pancreatitis. Ethanol remains one of the commonest causes of acute pancreatitis and this can be studied in vitro. In such studies we have shown that both ethanol and its non-oxidative metabolites, the fatty acid ethyl esters, do indeed cause elevations in [Ca2+]i very similar to those observed after CCK hyperstimulation (Raraty et al, 1998a). Of the other aetiological factors of acute pancreatitis listed in Table 1, mechanisms by which each may cause a rise in [Ca2+]i have been described (Ward et al, 1995). Ductal obstruction and hypertension may create a secretory block, thus leading to accumulation of zymogens within the acinar cell and premature intracellular activation such as has been described in hypercalcaemia itself (Frick et al, 1995). Both low-density and high-density lipoproteins have been shown to raise [Ca2+]i directly (Bochkov et al, 1992) as have a number of drugs (Morley et al, 1992; Frick et al, 1993). Some viruses have been shown to affect [Ca2+]i in cell cultures (Michelangeli et al, 1991) and ischaemia has been shown to induce a rise in [Ca2+]i in several types of cell (Arnould et al, 1992). Thus, although most of these studies are on cells other than pancreatic acinar cells, it is likely that all of the known triggers for acute pancreatitis could act by inducing changes in [Ca2+]i. FROM [Ca2+]i RISE TO PANCREATITIS It is clear that intracellular calcium ions are a key messenger in stimulus–secretion coupling and, therefore, that disruption of normal [Ca2+]i signals will, inevitably, interfere with the normal processes of signal transduction and, hence, zymogen release. It has also been shown that abnormal [Ca2+]i signals can induce morphological changes similar to those seen in acute pancreatitis, and evidence is accumulating that this is also associated with intracellular activation of digestive enzymes. All of the known triggers for acute pancreatitis may be linked by their ability to induce an abnormal elevation in [Ca2+]i which, in turn, may then go on to trigger intracellular enzyme activation and, hence, the other local and systemic manifestations of acute pancreatitis. Further work is still needed to prove a direct connection between a rise in [Ca2+]i and the intracellular activation of enzymes and release of inflammatory mediators. It is also still unclear by what mechanism a rise in [Ca2+]i could induce such changes, although it seems increasingly clear that it does. If, indeed Ca2+ is shown to be central to the pathogenesis of acute pancreatitis then more research into specific Ca2+ channel blockers may open up the possibility of intervention much earlier in the course of the disease than has hitherto been possible. REFERENCES Adler G, Rohr G & Kern HF (1982) Alteration of membrane fusion as a cause of acute pancreatitis in the rat. Digestive Diseases and Sciences 27: 993–1002. Arnould T, Michiels C, Alexandre I & Remacle J (1992) Effect of hypoxia on intracellular calcium concentration of human endothelial cells. Journal of Cell Physiology 152: 215–221. Banks PA (1993) Medical management of acute pancreatitis and complications. In Go VLW, DiMagno EP, Gardner JD et al (eds) The Pancreas: Biology, Pathobiology and Disease, pp 593–611. New York: Raven Press.


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Bayerdorffer E, Haase W & Schulz I (1985) Na+/Ca2+ countertransport in plasma membrane of rat pancreatic acinar cells. Journal of Membrane Biology 87: 107–119. Bayerdorffer E, Streb H, Eckhardt L et al (1984) Characterization of calcium uptake into rough endoplasmic reticulum of rat pancreas. Journal of Membrane Biology 81: 69–82. Berke G (1995) The CTL’s kiss of death. Cell 81: 9–12. Berridge MJ (1993) A tale of two messengers. Nature 365: 388–389. Bochkov V, Tkachuk V, Buhler F & Resink T (1992) Phosphoinositide and calcium signalling responses in smooth muscle cells: comparison between lipoproteins, Ang II and PDGF. Biochemical and Biophysical Research Communications 188: 1295–1304. Boobis AR, Seddon CE, Nasseri-Sina P & Davies DS (1990) Evidence for a direct role of intracellular calcium in paracetamol toxicity. Biochemical Pharmacology 39: 1277–1281. Cancela JM, Mogami H, Tepikin AV & Petersen OH (1998) Intracellular glucose switches between cyclic ADP-ribose and inositol triphosphate triggering of cytosolic Ca2+ spiking. Current Biology 8: 865–868. Canga L, Levi R & Rifkind AB (1988) Heart as a target organ in 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity: decreased beta-adrenergic responsiveness and evidence of increased intracellular calcium. Proceedings of the National Academy of Sciences of the USA 85: 905–909. Frick TW, Speiser DE, Bimmler D & Largiader F (1993) Drug-induced acute pancreatitis—further criticism. Digestive Diseases 11: 113–132. *Frick TW, Fernandez del Castillo C, Bimmler D & Warshaw AL (1997) Elevated calcium and activation of trypsinogen in rat pancreatic acini. Gut 41: 339–343. Frick TW, Mithöfer K, Fernandez del Castillo C et al (1995) Hypercalcemia causes acute-pancreatitis by pancreatic secretory block, intracellular zymogen accumulation, and acinar cell injury. American Journal of Surgery 169: 167–172. Gerasimenko OV, Gerasimenko JV, Belan PV & Peterson OH (1996) Inositol trisphosphate and cyclic ADPribose-mediated release of Ca2+ from single isolated pancreatic zymogen granules. Cell 84: 473–480. Grynkiewicz G, Poenie M & Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260: 3440–3450. Jaakkola M & Nordback I (1993) Pancreatitis in Finland between 1970 and 1989. Gut 34: 1255–1260. Kasai H and Augustine GJ (1990) Cytosolic Ca2+ gradients triggering unidirectional fluid secretion from exocrine pancreas. Nature 348: 735–738. Kloppel G, Dreyer T, Willemer S et al (1986) Human acute pancreatitis: its pathogenesis in the light of immunocytochemical and ultrastructural findings in acinar cells. Virchows Archiv A (Pathology Anatomy and Histology) 409: 791–803. Kribben A, Tyrakowski T & Schulz I (1983) Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes. American Journal of Physiology 244: G480–G490. Lake-Bakaar G & Lyubsky S (1995) Dose-dependent effect of continuous subcutaneous verapamil infusion on experimental acute-pancretitis in mice. Digestive Diseases and Sciences 40: 2349–2355. *Lampel M & Kern HF (1977) Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchows Archiv A (Pathology Anatomy and Histology) 373: 97–117. Leahy AL, Darzi A, Grace P et al (1993) Verapamil is beneficial in a model of post-ERCP pancreatitis. European Journal of Gastroenterology and Hepatology 5: 467–469. Lee MG, Xu X, Zeng WZ et al (1997) Polatized expression of Ca2+ channels in pancreatic and salivary gland cells—correlation with initiation and propagation of [Ca2+]i waves. Journal of Biological Chemistry 272: 15 765–15 770. Lerch MM, Saluja AK, Dawra R et al (1992) Acute necrotizing pancreatitis in the opossum: earliest morphological changes involve acinar cells. Gastroenterology 103: 205–213. Mann DV, Hershman MJ, Hittinger R & Glazer G (1994) Multicenter audit of death from acute-pancreatitis. British Journal of Surgery 81: 890–893. Maruyama Y & Petersen OH (1994) Delay in granular fusion evoked by repetitive cytosolic Ca2+ spikes in mouse pancreatic acinar cells. Cell Calcium 16: 419–430. Matozaki T, Goke B, Tsunoda Y et al (1990) Two functionally distinct cholecystokinin receptors show different modes of action on Ca2+ mobilization and phospholipid hydrolysis in isolated rat pancreatic acini. Studies using a new cholecystokinin analog, JMV-180. Journal of Biological Chemistry 265: 6247–6254. Michelangeli F, Ruiz MC, del-Castillo JR et al (1991) Effect of rotavirus infection on intracellular calcium homeostasis in cultured cells. Virology 181: 520–527. *Mithöfer K, Fernandez del Castillo C, Frick TW et al (1995a) Acute hypercalcemia causes acute-pancreatitis and ectopic trypsinogen activation in the rat. Gastroenterology 109: 239–246. Mithöfer K, Warshaw AL, Frick TW et al (1995b) Calcium administration augments pancreatic injury and ectopic trypsinogen activation after temporary systemic hypotension in rats. Anesthesiology 83: 1266–1273.

Intracellular free ionized calcium in the pathogenesis of acute pancreatitis


Mogami H, Nakano K, Tepikin AV & Petersen OH (1997) Ca2+ flow via tunnels in polarised cells: recharging of apical Ca2+ stores by focal Ca2+ entry through basal membrane patch. Cell 88: 49–55. Morley P, Whitfield JF, Vanderhyden BC et al (1992) A new, non-genomic estrogen action: the rapid release of intracellular calcium. Endocrinology 131: 1305–1312. Muallem S (1989) Calcium transport pathways of pancreatic acinar cells. Annual Review of Physiology 51: 83–105. *Nicotera P, Bellomo G & Orrenius S (1992) Calcium-mediated mechanisms in chemically induced cell death. Annual Review of Pharmacology and Toxicology 32: 449–470. Pauwels PJ, van Assouw HP, Leysen JE & Janssen PAJ (1989) Ca2+-mediated neuronal death in rat brain neuronal cultures by veratridine: protection by flunarizine. Molecular Pharmacology 36: 525–531. Petersen CCH (1996) Store operated calcium entry. Seminars in the Neurosciences 8: 293–300. *Petersen OH (1992) Stimulus–secretion coupling: cytoplasmic calcium signals and the control of ion channels in exocrine acinar cells. Journal of Physiology 448: 1–51. Petersen OH & Maruyama Y (1989) Electrophysiology of salivary and pancreatic acinar cells. In Forte JG, Schultz SG (eds) Handbook of Physiology, pp 25–50. Petersen OH, Gerasimenko OV, Gerasimenko JV et al (1998) The calcium store in the nuclear envelope. Cell Calcium 23: 87–90. Raraty MGT, Lloyd Mills C, Ward JB et al (1998a) Ethanol and fatty acid ethyl esters, but not acetaldehyde, induce elevations in cytosolic calcium in mouse pancreatic acinar cells typical of acute pancreatitis. Digestion 59: 249 (abstract). Raraty MGT, Ward JB, Vaillant C et al (1998b) Attenuation of cytosolic calcium rise from hyperstimulation or store depletion prevents vacuolization in mouse pancreatic acinar cells. Digestion 59: 201 (abstract). Rosenzweig SA, Miller LJ & Jamieson JD (1983) Identification and localization of cholecystokinin binding sites on rat pancreatic plasma membranes and acinar cells: a biochemical and autoradiographic study. Journal of Cell Biology 96: 1288–1297. Steer ML & Saluja AK (1993) Experimental acute pancreatitis: studies of the early events that lead to cell injury. In Go VLW, DiMagno EP, Gardner JD et al (eds) The Pancreas: Biology, Pathobiology and Disease, pp 489–500. New York: Raven Press. Thastrup O, Cullen PJ, Drobak BK et al (1990) Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proceedings of the National Academy of Sciences of the USA 87: 2466–2470. *Thorn P, Lawrie AM, Smith PM et al (1993) Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate. Cell 74: 661–668. Toescu EC, Lawrie AM, Petersen OH & Gallacher DV (1992) Spatial and temporal distribution of agonistevoked cytoplasmic Ca2+ signals in exocrine acinar cells analysed by digital image microscopy. EMBO Journal 11: 1623–1629. Wang XD, Deng XM, Haraldsen P et al (1995) Antioxidant and calcium-channel blockers counteract endothelial barrier injury-induced by acute-pancreatitis in rats. Scandinavian Journal of Gastroenterology 30: 1129–1136. *Ward JB, Petersen OH, Jenkins SA & Sutton R (1995) Is an elevated concentration of acinar cytosolic-free ionized calcium the trigger for acute-pancreatitis. Lancet 346: 1016–1019. *Ward JB, Sutton R, Jenkins SA & Petersen OH (1996) Progressive disruption of acinar cell calcium signaling is an early feature of cerulein-induced pancreatitis in mice. Gastroenterology 111: 481–491. Wilson PG, Manji M & Neoptolemos JP (1998) Acute pancreatitis as a model of sepsis. Journal of Antimicrobial Chemotherapy 41: 51–63. Xu X, Zeng W & Muallem S (1996) Regulation of the IP3 activated Ca2+ channel by activation of G proteins. Journal of Biological Chemistry 271: 11 737–11 744. *Zhou W, Shen F, Miller J E et al (1996) Evidence for altered cellular calcium in the pathogenetic mechanism of acute pancreatitis in rats. Journal of Surgical Research 60: 147–155.


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