Calcium signaling of pancreatic acinar cells in the pathogenesis of pancreatitis

Abstract

Pancreatitis is an increasingly common and sometimes severe disease that lacks a specific therapy. The pathogenesis of pancreatitis is still not well understood. Calcium (Ca²⁺) is a versatile carrier of signals regulating many aspects of cellular activity and plays a central role in controlling digestive enzyme secretion in pancreatic acinar cells. Ca²⁺ overload is a key early event and is crucial in the pathogenesis of many diseases. In pancreatic acinar cells, pathological Ca²⁺ signaling (stimulated by bile, alcohol metabolites and other causes) is a key contributor to the initiation of cell injury due to prolonged and global Ca²⁺ elevation that results in trypsin activation, vacuolization and necrosis, all of which are crucial in the development of pancreatitis. Increased release of Ca²⁺ from stores in the intracellular endoplasmic reticulum and/or increased Ca²⁺ entry through the plasma membrane are causes of such cell damage. Failed mitochondrial adenosine triphosphate (ATP) production reduces re-uptake and extrusion of Ca²⁺ by the sarco/endoplasmic reticulum Ca²⁺-activated ATPase and plasma membrane Ca²⁺-ATPase pumps, which contribute to Ca²⁺ overload. Current findings have provided further insight into the roles and mechanisms of abnormal pancreatic acinar Ca²⁺ signals in pancreatitis. The lack of available specific treatments is therefore an objective of ongoing research. Research is currently underway to establish the mechanisms and interactions of Ca²⁺ signals in the pathogenesis of pancreatitis.

Core tip: Calcium (Ca²⁺) overload is crucial in the pathogenesis of pancreatitis, which results in trypsin activation, vacuolization and necrosis. Such cell injury results from increased Ca²⁺ released from intracellular endoplasmic reticulum Ca²⁺ stores, increased Ca²⁺ entry through the plasma membrane and Ca²⁺ pump defects. Current findings have provided further insight into the roles and mechanisms of Ca²⁺ overload in pancreatitis. The lack of specific treatments is a stimulus for ongoing research. This review summarizes recent advances in our understanding of Ca²⁺ signaling in the pathogenesis of pancreatitis, and discusses how research has guided our search for potential therapeutic targets.

INTRODUCTION

Pancreatitis remains a disease with significant morbidity and lethality, and is typically caused by alcohol abuse or complications arising from biliary disease[1]. The pathogenesis of pancreatitis is multi-factorial and has not yet been clarified[2-5]. In recent years, several pancreatic mechanisms have been proposed, such as trypsinogen activation[6], pancreatic microcirculation malfunction[7], calcium (Ca²⁺) overload[8-10] and inflammatory pathways[11-13]. Among these various theories, Ca²⁺ overload is receiving increasing attention and is being extensively investigated in the pathogenesis of pancreatitis[14]. Recent advances in our understanding of Ca²⁺ signaling of pancreatic acinar cells in the pathogenesis of pancreatitis are reviewed in this article, including a discussion on how research has guided our search for potential therapeutic targets.

PHYSIOLOGICAL AND PATHOLOGICAL CA²⁺ SIGNALS IN PANCREATIC ACINAR CELLS

As the most universal carrier of biological signals, intracellular Ca²⁺ is involved in the modulation of virtually all cellular functions, from its origin at fertilization to its end in the apoptotic process[15]. Intracellular Ca²⁺ acts both as a first and a second messenger to control cellular functions via regulating free-Ca²⁺ concentrations in the cytoplasm, for example, controlling the contraction and relaxation of muscles, and regulating secretion from exocrine glands[16]. Ca²⁺ signals elicited by physiological stimulation are transient and mostly localized in the granule-containing apical pole, whereas sustained global elevation of cytosolic Ca²⁺ concentrations can be fatal[17-19]. The digestive enzymes produced by pancreatic acinar cells are packaged in zymogen granules in the apical pole[20]. Physiological stimulation elicits proenzyme exocytosis exclusively through the apical membrane[21]. Ca²⁺ overload causes inappropriate intracellular trypsin activation, vacuolization and necrosis[20,22-26], which contribute to subsequent cell injury and are often fatal in human acute pancreatitis[27]. Pretreatment with pharmacological Ca²⁺ chelators or blockers was found to prevent premature digestive enzyme activation, vacuolization, skeletal disruption and acinar cell necrosis induced by Ca²⁺ overload[28].

RELEASE OF CA²⁺ FROM THE ENDOPLASMIC RETICULUM

There are two types of G protein-coupled receptors localized on the plasma membrane, namely, acetylcholine (ACh) and cholecystokinin (CCK) receptors[8]. ACh is a secretagogue that activates phospholipase C (PLC) through ACh receptor ligand binding, which in turn cleaves phosphatidylinositol 4,5-bisphosphate into the classic Ca²⁺-releasing messengers inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol to mobilize Ca²⁺ and activate protein kinase C respectively[29]. The other principal secretagogue in acinar cells is the hormone CCK, which exists in multiple molecular forms, such as CCK8 and CCK58. CCK interacts with its receptor and activates adenosine diphosphate-ribosyl cyclase to produce the novel Ca²⁺-releasing agent nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate-ribose (cADPR).

There are two types of regulated Ca²⁺-release channels localized on the endoplasmic reticulum (ER) membrane, namely, the IP₃ receptors (IP₃R) and ryanodine receptors (RyR). IP₃R are concentrated in the apical part of the acinar cell and binding of IP₃ activates gated Ca²⁺ channels to release intracellular stored Ca²⁺ from the ER, which participates in the apical cytosolic Ca²⁺-spiking response to stimulation with physiological concentrations of ACh[10,19,30,31]. RyR in the basal region of acinar cells are activated by NAADP and cADPR, and oligomers form gated Ca²⁺ channels to release intracellular Ca²⁺ from ER stores[32] in response to stimulation with physiological concentrations of CCK[33-35]. Intriguingly, the Ca²⁺ response mediated by RyR was observed in the apical pole in mouse acinar cells and required functional IP₃R, which could be interpreted as co-localization and coordination of RyR and IP₃R[36].

Hyperstimulation with agents (in contrast to physiological stimulation) can induce acinar cell injury by IP₃R-induced release of Ca²⁺ from the ER. The Ca²⁺ increase spreads from the apical pole to the basolateral part of the acinar cell, and a sustained global Ca²⁺ elevation causes pancreatitis-like cellular changes, such as abnormal intracellular enzyme activation, vacuolization and necrosis[20]. Treatment with IP₃R inhibitors, such as caffeine and 2-aminoethoxydiphenyl borate, can reduce abnormal Ca²⁺ signals and the probability of ethanol-induced pancreatitis, but the low affinity and multiple actions restrict its therapeutic potential[37,38]. Hyperstimulation by CCK8 is specifically dependent on functional RyR, and induces toxic pancreatitis-like changes as a result of sustained global elevation of Ca²⁺ released from the ER. These aberrant Ca²⁺ signals and acinar cell injuries can be blocked in vitro and in vivo by pretreating with RyR inhibitors[8,39]. Hyperstimulation by CCK also activates PLC, which generates IP₃ and elicits Ca²⁺ overload[20].

Although the ER is a large Ca²⁺ store in the basolateral part of pancreatic acinar cells, there are also extensive acidic Ca²⁺ stores present in the apical part, which similarly release Ca²⁺ into the cytoplasm through IP₃, cADPR and NAADP signaling. Hyperstimulation from bile acids and alcohol metabolites can elicit pathological Ca²⁺ release from both the ER and acidic stores[40,41].

STORE-OPERATED CA²⁺ (SOC) INFLUX

Another abnormal Ca²⁺ signal in the pathogenesis of pancreatitis is extracellular Ca²⁺ entry, which is regulated at the plasma membrane of acinar cells by SOC channels[42]. Under physiological conditions, CCK and ACh induce the release of Ca²⁺ from the ER, followed by Ca²⁺ extrusion from the cell, suggesting that SOC entry is required to elevate intracellular Ca²⁺. The molecular mechanism underlying these pancreatic Ca²⁺-entry channels is ill-defined. Current research suggests that Ca²⁺-entry channels belong to the transient receptor potential family, including Ca²⁺ release-activated Ca²⁺ channel protein 1 (Orai1), transient receptor potential channel 1, and stromal interaction molecule (STIM) 1[43,44]. Recent studies indicate that STIM proteins serve as sensors, are concentrated in the ER membrane, and monitor the Ca²⁺ concentration in the ER lumen. When the luminal concentration is reduced in response to secretagogue stimulation, STIM proteins sense the changes, accumulate and translocate to the plasma membrane where they co-localize with and activate Orai1 channels[45-48].

Orai1 channels are localized not only in the apical part of acinar cells, but also in the basal and lateral membranes, which cover about 95% of the pancreatic acinar cell surface[43]. Following ER Ca²⁺ store depletion, Orail1 interacts with STIM and activates SOC channels. The wide distribution of Orai1 channels enables sustained Ca²⁺ entry under physiological conditions, without the need for local Ca²⁺ concentrations, and refilling of the ER Ca²⁺ stores after agonist-elicited depletion[8]. However, Orai1 activity can result in an abnormal sustained global Ca²⁺ elevation following pathological stimulation, such as by high, toxic concentrations of CCK8, alcohol and bile acid, which all elicit intracellular Ca²⁺ overload that is mostly dependent on external Ca²⁺ influx[20,23,25]. Therefore, SOC entry may be crucial for the development of acute pancreatitis. Without external sustained global Ca²⁺ entry, cellular injury does not occur[20,22-25,49]. Removal of external Ca²⁺ or abrogation of elevated Ca²⁺ with a Ca²⁺ chelator can protect acinar cells against abnormal changes, such as trypsinogen activation and vacuolization[20,25,49]. SOC channel blockers might therefore be a possible therapeutic approach for the treatment of acute pancreatitis[7].

CA²⁺ PUMP DEFECTS

Sarco/endoplasmic reticulum Ca²⁺-activated adenosine triphosphate (ATP)ase (SERCA) is an ER Ca²⁺ pump which actively re-uptakes Ca²⁺ into the ER lumen to compensate for resting leakage into the cytosol[8,50]. Under normal physiological conditions, the elevation of intracellular Ca²⁺ can activate the SERCA pump[19,27,51], and Ca²⁺ release elicited by stimulation is followed by Ca²⁺ re-uptake. The rate of uptake decreases as luminal Ca²⁺ concentration increases until the uptake rate equals the resting leak rate[8]. Pathological stimulation by bile acids or fatty acids can elicit Ca²⁺ overload by inhibiting the SERCA pump and depolarizing the inner mitochondrial membrane, resulting in reduced ATP production, which in turn lessens the ability of the SERCA pump to restore ER Ca²⁺ stores[25]. Prolonged and uncompensated Ca²⁺ overload released from ER stores can cause thapsigargin activation and vacuolization in pancreatic acinar cells, which can be visualized directly[20].

All eukaryotic cells export Ca²⁺ through two pathways, the plasma membrane Ca²⁺-ATPase (PMCA; commonly called the Ca²⁺ pump) and the Na⁺-Ca²⁺ exchanger (NCE), to prevent Ca²⁺ overload and for the maintenance of intracellular Ca²⁺ at the appropriately low level[52,53]. The PMCA has high Ca²⁺ affinity but low transport capacity and is ATP-dependent. Any elevation of cytosolic Ca²⁺ can activate the PMCA to rapidly extrude Ca²⁺ in physiological conditions. Whereas ER-released Ca²⁺ is localized in the apical part and Ca²⁺ entry occurs across the basolateral surface, PMCA Ca²⁺ extrusion is confined to a small apical region only, which restricts the PMCA function as a fine-tuner of cell cytosolic Ca²⁺. Pathological stimulation can depolarize mitochondria and cause a deficiency in ATP production, which inhibits Ca²⁺ extrusion and aggravates the cytosolic Ca²⁺ overload[24,54].

The NCE has a low Ca²⁺ affinity and is a high-capacity transmembrane protein of the plasma membrane involved in Ca²⁺ homeostasis, and is especially important in excitable cells. Because of its high capacity, the NCE can extrude Ca²⁺ at a much higher rate than the PMCA, serving as the fast Ca²⁺ transporting system. For example, activation of the NCE prevents Ca²⁺ overload induced by pathological stimulation and cell death in neurons. Inactivation of the NCE can cause neuronal death, which can be visualized directly[55]. In pancreatic acinar cells, the NCE is of little quantitative importance, which explains why Ca²⁺ overloading is particularly dangerous in pancreatic acinar cells[19,27].

As another Ca²⁺ store, mitochondria also participate in maintaining cytosolic Ca²⁺ homeostasis in pancreatic acinar cells. Mitochondria surround the apical pole in a perigranular belt, separating zymogen granules from the basolateral part of the acinar cell, and are also positioned just beneath the plasma membrane and surrounding the nucleus[19,27,55-58]. The membrane potential across the inner mitochondrial membrane is the driving force behind mitochondrial uptake of Ca²⁺ into the matrix through the Ca²⁺ uniporter, a Ca²⁺-selective ion channel[59,60]. Mitochondria in pancreatic acinar cells play an important role in maintaining cytosolic Ca²⁺ homeostasis[56-58]. When cytosolic Ca²⁺ is elevated by physiological stimulation, mitochondria sense the Ca²⁺ in the environment and take up Ca²⁺ via the Ca²⁺ uniporter[60]. Ca²⁺ spikes released from the ER occurring in the apical region can cause immediate Ca²⁺ uptake into the mitochondrial matrix, preventing further spread of the Ca²⁺ signal into the basolateral part of the acinar cell, which contains the nucleus. Perigranular mitochondria function as a Ca²⁺ buffer barrier[8], causing Ca²⁺ uptake termination and Ca²⁺ removal via the mitochondrial NCE[61,62]. Mitochondrial Ca²⁺ uptake activates Krebs cycle enzymes and drives ATP production, supplying ATP for SERCA-mediated Ca²⁺ re-uptake into the ER and PMCA-mediated Ca²⁺ extrusion[27,63]. Pathological stimulation that can induce experimental pancreatitis, such as with bile salts, fatty acids and CCK or its analog, can depolarize the inner mitochondrial membrane, inducing further collapse of the mitochondrial membrane potential and impairment of ATP production[64]. This situation prevents perigranular mitochondrial Ca²⁺ re-uptake and mitochondria cannot buffer the apical Ca²⁺ elevation, causing the local Ca²⁺ signal to spread to the whole of the acinar cell[30]. Failure of ATP production reduces the ability of the SERCA and PMCA pumps to take Ca²⁺ back into the ER and for extrusion, which contributes to Ca²⁺ overload. This is the most likely explanation for why pretreatment with Ca²⁺ chelators can limit the global and sustained elevation of Ca²⁺.

TARGETS FOR POTENTIAL THERAPY

To date, there is no specific treatment for either acute or chronic pancreatitis[39,65-67]. The current therapy for pancreatitis is limited to the inhibition of proteolytic enzymes. Protease inhibitors have a modest preventative role in experimental animal models, however, they fail to show therapeutic value in clinical treatment[68,69]. An aberrant increase in cytosolic Ca²⁺ is a key molecular event in the pathogenesis of pancreatitis. Intracellular Ca²⁺ overload is a major reason for pancreatic acinar cell injury from toxin stimulation that induces pancreatitis[7,20,22-25,49]. Abnormal, prolonged, global Ca²⁺ signals lead to premature enzyme activation, vacuole formation and acinar cell damage. Thus, it is clinically relevant to identify the targets of the aberrant Ca²⁺ signals[70]. New avenues are required based on current findings in our understanding of Ca²⁺ signaling in the pathogenesis of pancreatitis (Figure 1). Possible interventions include: (1) inhibition of Ca²⁺ entry pathways; (2) enhancement of Ca²⁺ extrusion; and (3) inhibition of the primary Ca²⁺ release from the ER; and iv) protection of mitochondrial function, which can serve as potential therapeutic targets (Table 1). Recent progress in our understanding of Ca²⁺ signals of pancreatic acinar cells in the pathogenesis of pancreatitis now provides opportunities for the developments of better therapeutic approaches.

Figure 1.

Diagram showing possible interventions for therapeutic targets of pancreatitis. Blockage of Ca²⁺ entry will probably depend on inhibition of the store-operated Ca²⁺ (SOC) channel in the pancreatic acinar cell. Activation of plasma membrane Ca²⁺-adenosine triphosphate (ATP)ase (PMCA) and sarco/endoplasmic reticulum Ca²⁺-activated ATPase (SERCA) would enhance Ca²⁺ extrusion from the cell and endoplasmic reticulum (ER). Intracellular Ca²⁺ release from the ER can be prevented through inhibition of the inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR). Preconditioning strategies could protect mitochondrial function to ensure adequate ATP production extrusion by Ca²⁺ pumps and for pancreatic acinar cells to survive intact.

Table 1.

Potential therapeutic targets

Potential therapeutic targets
Store-operated Ca2+ channel
IP3 receptor
Ryanodine receptor
SERCA
PMCA
Mitochondria

IP₃: 1,4,5-trisphosphate; PMCA: Plasma membrane Ca²⁺-adenosine triphosphate (ATP)ase; SERCA: Sarco/endoplasmic reticulum Ca²⁺-activated ATPase.