Levels of the Autophagy-Related 5 Protein Affect Progression and Metastasis of Pancreatic Tumors in Mice.

BACKGROUND AND AIMS: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. METHODS: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/-;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. RESULTS: A5+/-;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/-;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/-;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/-;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. CONCLUSIONS: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.

Intracellular rupture, exocytosis and actin interaction of endocytic vacuoles in pancreatic acinar cells: initiating events in acute pancreatitis.

KEY POINTS: Giant trypsin-containing endocytic vacuoles are formed in pancreatic acinar cells stimulated with inducers of acute pancreatitis. F-actin envelops endocytic vacuoles and regulates their properties. Endocytic vacuoles can rupture and release their content into the cytosol of acinar cells. Endocytic vacuoles can fuse with the plasma membrane of acinar cells and exocytose their content. ABSTRACT: Intrapancreatic activation of trypsinogen is an early event in and hallmark of the development of acute pancreatitis. Endocytic vacuoles, which form by disconnection and transport of large post-exocytic structures, are the only resolvable sites of the trypsin activity in live pancreatic acinar cells. In the present study, we characterized the dynamics of endocytic vacuole formation induced by physiological and pathophysiological stimuli and visualized a prominent actin coat that completely or partially surrounded endocytic vacuoles. An inducer of acute pancreatitis taurolithocholic acid 3-sulphate and supramaximal concentrations of cholecystokinin triggered the formation of giant (more than 2.5 µm in diameter) endocytic vacuoles. We discovered and characterized the intracellular rupture of endocytic vacuoles and the fusion of endocytic vacuoles with basal and apical regions of the plasma membrane. Experiments with specific protease inhibitors suggest that the rupture of endocytic vacuoles is probably not induced by trypsin or cathepsin B. Perivacuolar filamentous actin (observed on the surface of ∼30% of endocytic vacuoles) may play a stabilizing role by preventing rupture of the vacuoles and fusion of the vacuoles with the plasma membrane. The rupture and fusion of endocytic vacuoles allow trypsin to escape the confinement of a membrane-limited organelle, gain access to intracellular and extracellular targets, and initiate autodigestion of the pancreas, comprising a crucial pathophysiological event.

Systemic histone release disrupts plasmalemma and contributes to necrosis in acute pancreatitis.

BACKGROUND: Clinical and experimental acute pancreatitis feature histone release within the pancreas from innate immune cells and acinar cell necrosis. In this study, we aimed to detail the source of circulating histones and assess their role in the pathogenesis of acute pancreatitis. METHODS: Circulating nucleosomes were measured in patient plasma, taken within 24 and 48 h of onset of acute pancreatitis and correlated with clinical outcomes. Using caerulein hyperstimulation, circulating histones were measured in portal, systemic venous and systemic arterial circulation in mice, and the effects of systemic administration of histones in this model were assessed. The sites of actions of circulating histones were assessed by administration of FITC-labelled histones. The effects of histones on isolated pancreatic acinar cells were further assessed by measuring acinar cell death and calcium permeability in vitro. RESULTS: Cell-free histones were confirmed to be abundant in human acute pancreatitis and found to derive from pancreatitis-associated liver injury in a rodent model of the disease. Fluorescein isothianate-labelled histones administered systemically targeted the pancreas and exacerbated injury in experimental acute pancreatitis. Histones induce charge- and concentration-dependent plasmalemma leakage and necrosis in isolated pancreatic acinar cells, independent of extracellular calcium. CONCLUSION: We conclude that histones released systemically in acute pancreatitis concentrate within the inflamed pancreas and exacerbate injury. Circulating histones may provide meaningful biomarkers and targets for therapy in clinical acute pancreatitis.

Epithelial-mesenchymal transition, IP3 receptors and ER-PM junctions: translocation of Ca2+ signalling complexes and regulation of migration.

Disconnection of a cell from its epithelial neighbours and the formation of a mesenchymal phenotype are associated with profound changes in the distribution of cellular components and the formation of new cellular polarity. We observed a dramatic redistribution of inositol trisphosphate receptors (IP3Rs) and stromal interaction molecule 1 (STIM1)-competent endoplasmic reticulum-plasma membrane junctions (ER-PM junctions) when pancreatic ductal adenocarcinoma (PDAC) cells disconnect from their neighbours and undergo individual migration. In cellular monolayers IP3Rs are juxtaposed with tight junctions. When individual cells migrate away from their neighbours IP3Rs preferentially accumulate at the leading edge where they surround focal adhesions. Uncaging of inositol trisphosphate (IP3) resulted in prominent accumulation of paxillin in focal adhesions, highlighting important functional implications of the observed novel structural relationships. ER-PM junctions and STIM1 proteins also migrate to the leading edge and position closely behind the IP3Rs, creating a stratified distribution of Ca(2+) signalling complexes in this region. Importantly, migration of PDAC cells was strongly suppressed by selective inhibition of IP3Rs and store-operated Ca(2+) entry (SOCE), indicating that these mechanisms are functionally required for migration.

Endoplasmic reticulum-plasma membrane junctions: structure, function and dynamics.

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are contact sites between the ER and the PM; the distance between the two organelles in the junctions is below 40 nm and the membranes are connected by protein tethers. A number of molecular tools and technical approaches have been recently developed to visualise, modify and characterise properties of ER-PM junctions. The junctions serve as the platforms for lipid exchange between the organelles and for cell signalling, notably Ca(2+) and cAMP signalling. Vice versa, signalling events regulate the development and properties of the junctions. Two Ca(2+) -dependent mechanisms of de novo formation of ER-PM junctions have been recently described and characterised. The junction-forming proteins and lipids are currently the focus of vigorous investigation. Junctions can be relatively short-lived and simple structures, forming and dissolving on the time scale of a few minutes. However, complex, sophisticated and multifunctional ER-PM junctions, capable of attracting numerous protein residents and other cellular organelles, have been described in some cell types. The road from simplicity to complexity, i.e. the transformation from simple 'nascent' ER-PM junctions to advanced stable multiorganellar complexes, is likely to become an attractive research avenue for current and future junctologists. Another area of considerable research interest is the downstream cellular processes that can be activated by specific local signalling events in the ER-PM junctions. Studies of the cell physiology and indeed pathophysiology of ER-PM junctions have already produced some surprising discoveries, likely to expand with advances in our understanding of these remarkable organellar contact sites.

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models.

BACKGROUND & AIMS: Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release-activated calcium modulator ORAI1 is the most abundant Ca(2+) entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. METHODS: Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. RESULTS: GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. CONCLUSIONS: Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

The role of Ca2+ influx in endocytic vacuole formation in pancreatic acinar cells.

The inducers of acute pancreatitis trigger a prolonged increase in the cytosolic Ca(2+) concentration ([Ca(2+)]c), which is responsible for the damage to and eventual death of pancreatic acinar cells. Vacuolization is an important indicator of pancreatic acinar cell damage. Furthermore, activation of trypsinogen occurs in the endocytic vacuoles; therefore the vacuoles can be considered as 'initiating' organelles in the development of the cell injury. In the present study, we investigated the relationship between the formation of endocytic vacuoles and Ca(2+) influx developed in response to the inducers of acute pancreatitis [bile acid taurolithocholic acid 3-sulfate (TLC-S) and supramaximal concentration of cholecystokinin-8 (CCK)]. We found that the inhibitor of STIM (stromal interaction molecule)/Orai channels, GSK-7975A, effectively suppressed both the Ca(2+) influx (stimulated by inducers of pancreatitis) and the formation of endocytic vacuoles. Cell death induced by TLC-S or CCK was also inhibited by GSK-7975A. We documented the formation of endocytic vacuoles in response to store-operated Ca(2+) entry (SOCE) induced by thapsigargin [TG; inhibitor of sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) pumps] and observed strong inhibition of TG-induced vacuole formation by GSK-7975A. Finally, we found that structurally-unrelated inhibitors of calpain suppress formation of endocytic vacuoles, suggesting that this Ca2+-dependent protease is a mediator between Ca(2+) elevation and endocytic vacuole formation.

The infrared absorption spectrum of radioactive water isotopologue H215O.

A room temperature line list for the H215O radioactive isotopologue of the water molecule is computed using the variational nuclear-motion DVR3D program suite and an empirical high-precision potential energy function. The line list consists of rotation-vibrational energies and Einstein-A coefficients, covering a wide spectral range from 0 to 25000 cm-1 and the total angular momenta J up to 30. Estimates of air-broadening coefficients are provided. Experimentally derived energies of H216O, H217O and H218O from the literature are used to provide improved energies for important states with uncertainty estimates for the H215O. A number of the wmost promising spectroscopic ranges for the detection of H215O are proposed. The calculated absorption spectrum should be useful for the study gaseous radioactive water at IR region, determining concentration, etc.

Cellular geography of IP3 receptors, STIM and Orai: a lesson from secretory epithelial cells.

Pancreatic acinar cells exhibit a remarkable polarization of Ca2+ release and Ca2+ influx mechanisms. In the present brief review, we discuss the localization of channels responsible for Ca2+ release [mainly IP3 (inositol 1,4,5-trisphosphate) receptors] and proteins responsible for SOCE (store-operated Ca2+ entry). We also place these Ca2+-transporting mechanisms on the map of cellular organelles in pancreatic acinar cells, and discuss the physiological implications of the cellular geography of Ca2+ signalling. Finally, we highlight some unresolved questions stemming from recent observations of co-localization and co-immunoprecipitation of IP3 receptors with Orai channels in the apical (secretory) region of pancreatic acinar cells.

Autophagy, Acute Pancreatitis and the Metamorphoses of a Trypsinogen-Activating Organelle.

Recent studies have highlighted the importance of autophagy and particularly non-canonical autophagy in the development and progression of acute pancreatitis (a frequent disease with considerable morbidity and significant mortality). An important early event in the development of acute pancreatitis is the intrapancreatic activation of trypsinogen, (i.e., formation of trypsin) leading to the autodigestion of the organ. Another prominent phenomenon associated with the initiation of this disease is vacuolisation and specifically the formation of giant endocytic vacuoles in pancreatic acinar cells. These organelles develop in acinar cells exposed to several inducers of acute pancreatitis (including taurolithocholic acid and high concentrations of secretagogues cholecystokinin and acetylcholine). Notably, early trypsinogen activation occurs in the endocytic vacuoles. These trypsinogen-activating organelles undergo activation, long-distance trafficking, and non-canonical autophagy. In this review, we will discuss the role of autophagy in acute pancreatitis and particularly focus on the recently discovered LAP-like non-canonical autophagy (LNCA) of endocytic vacuoles.

Dynamic changes in cytosolic and mitochondrial ATP levels in pancreatic acinar cells.

BACKGROUND & AIMS: Previous studies of pancreatic acinar cells characterized the effects of Ca(2+)-releasing secretagogues and substances, inducing acute pancreatitis on mitochondrial Ca(2+), transmembrane potential, and NAD(P)H, but dynamic measurements of the crucial intracellular adenosine triphosphate (ATP) levels have not been reported. Here we characterized the effects of these agents on ATP levels in the cytosol and mitochondria. METHODS: ATP levels were monitored using cytosolic- or mitochondrial-targeted luciferases. RESULTS: Inhibition of oxidative phosphorylation produced a substantial decrease in cytosolic ATP comparable to that induced by inhibition of glycolysis. Cholecystokinin-8 (CCK) increased cytosolic ATP in spite of accelerating ATP consumption. Acetylcholine, caerulein, and bombesin had similar effect. A bile acid, taurolithocholic acid 3-sulfate (TLC-S); a fatty acid, palmitoleic acid (POA); and palmitoleic acid ethyl ester (POAEE) reduced cytosolic ATP. The ATP decrease in response to these substances was observed in cells with intact or inhibited oxidative phosphorylation. TLC-S, POA, and POAEE reduced mitochondrial ATP, whereas physiological CCK increased mitochondrial ATP. Supramaximal CCK produced a biphasic response composed of a small initial decline followed by a stronger increase. CONCLUSIONS: Both glycolysis and oxidative phosphorylation make substantial contributions to ATP production in acinar cells. Ca(2+)-releasing secretagogues increased ATP level in the cytosol and mitochondria of intact isolated cells. TLC-S, POA, and POAEE reduced cytosolic and mitochondrial ATP. When cells rely on nonoxidative ATP production, secretagogues as well as TLC-S, POA, and POAEE all diminish cytosolic ATP levels.

Influence of the Filler Particles' Surface Morphology on the Polyurethane Matrix's Structure Formation in the Composite.

This article presents the surface morphology effect of silicon carbide (SiC) particles on the polyurethane binder's structure formation in a dispersed-filled composite. The difference in the morphology and surface relief of filler particles was ensured by the implementation of plasma chemical modification. As a result of this modification, the filler consisted of core-shell particles characterized by a SiC core and a carbon shell (SiC@C), as well as a carbon shell decorated with silicon nanoparticles (SiC@C/SiNP) or nanos (SiC@C/SiNW). The study of the relaxation properties of polyurethane composites has shown that the strongest limiting effect on the molecular mobility of boundary layer's chain segments is exerted by a highly developed surface with a complex relief of SiC@C/SiNP and SiC@C/SiNW particles. An empirical method was proposed to find the polymer fractions spent on the formation of the boundary, transition and bulk layers of the polymer matrix in the composite. It was shown that the morphology of the filler particles' surface does not affect the dependence of the boundary layer thickness on the filler's volume fraction. However, with an increase in the degree of surface development, the boundary layer thickness decreases.

Features of Functionalization of the Surface of Alumina Nanofibers by Hydrolysis of Organosilanes on Surface Hydroxyl Groups.

Small additions of nanofiber materials make it possible to change the properties of polymers. However, the uniformity of the additive distribution and the strength of its bond with the polymer matrix are determined by the surface of the nanofibers. Silanes, in particular, allow you to customize the surface for better interaction with the matrix. The aim of our work is to study an approach to silanization of nanofibers of aluminum oxide to obtain a perfect interface between the additive and the matrix. The presence of target silanes on the surface of nanofibers was shown by XPS methods. The presence of functional groups on the surface of nanofibers was also shown by the methods of simultaneous thermal analysis, and the stoichiometry of functional groups with respect to the initial hydroxyl groups was studied. The number of functional groups precipitated from silanes is close to the number of the initial hydroxyl groups, which indicates a high uniformity of the coating in the proposed method of silanization. The presented technology for silanizing alumina nanofibers is an important approach to the subsequent use of this additive in various polymer matrices.

Modulation of calcium signalling by mitochondria.

In this review we will attempt to summarise the complex and sometimes contradictory effects that mitochondria have on different forms of calcium signalling. Mitochondria can influence Ca(2+) signalling indirectly by changing the concentration of ATP, NAD(P)H, pyruvate and reactive oxygen species - which in turn modulate components of the Ca(2+) signalling machinery i.e. buffering, release from internal stores, influx from the extracellular solution, uptake into cellular organelles and extrusion by plasma membrane Ca(2+) pumps. Mitochondria can directly influence the calcium concentration in the cytosol of the cell by importing Ca(2+) via the mitochondrial Ca(2+) uniporter or transporting Ca(2+) from the interior of the organelle into the cytosol by means of Na+/Ca(2+) or H+/Ca(2+) exchangers. Considerable progress in understanding the relationship between Ca(2+) signalling cascades and mitochondrial physiology has been accumulated over the last few years due to the development of more advanced optical techniques and electrophysiological approaches.

LAP-like non-canonical autophagy and evolution of endocytic vacuoles in pancreatic acinar cells.

Activation of trypsinogen (formation of trypsin) inside the pancreas is an early pathological event in the development of acute pancreatitis. In our previous studies we identified the activation of trypsinogen within endocytic vacuoles (EVs), cellular organelles that appear in pancreatic acinar cells treated with the inducers of acute pancreatitis. EVs are formed as a result of aberrant compound exocytosis and subsequent internalization of post-exocytic structures. These organelles can be up to 12 µm in diameter and can be actinated (i.e. coated with F-actin). Notably, EVs can undergo intracellular rupture and fusion with the plasma membrane, providing trypsin with access to cytoplasmic and extracellular targets. Unraveling the mechanisms involved in cellular processing of EVs is an interesting cell biological challenge with potential benefits for understanding acute pancreatitis. In this study we have investigated autophagy of EVs and discovered that it involves a non-canonical LC3-conjugation mechanism, reminiscent in its properties to LC3-associated phagocytosis (LAP); in both processes LC3 was recruited to single, outer organellar membranes. Trypsinogen activation peptide was observed in approximately 55% of LC3-coated EVs indicating the relevance of the described process to the early cellular events of acute pancreatitis. We also investigated relationships between actination and non-canonical autophagy of EVs and concluded that these processes represent sequential steps in the evolution of EVs. Our study expands the known roles of LAP and indicates that, in addition to its well-established functions in phagocytosis and macropinocytosis, LAP is also involved in the processing of post-exocytic organelles in exocrine secretory cells. ABBREVIATIONS: AP: acute pancreatitis; CCK: cholecystokinin; CLEM: correlative light and electron microscopy; DPI: diphenyleneiodonium; EV: endocytic vacuole; LAP: LC3-associate phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PACs: pancreatic acinar cells; PFA: paraformaldehyde; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; Res: resveratrol; TAP: trypsinogen activation peptide; TEM: transmission electron microscopy; TLC-S: taurolithocholic acid 3-sulfate; TRD: Dextran Texas Red 3000 MW Neutral; ZGs: zymogen granules.

Knockout of the Mitochondrial Calcium Uniporter Strongly Suppresses Stimulus-Metabolism Coupling in Pancreatic Acinar Cells but Does Not Reduce Severity of Experimental Acute Pancreatitis.

Acute pancreatitis is a frequent disease that lacks specific drug treatment. Unravelling the molecular mechanisms of acute pancreatitis is essential for the development of new therapeutics. Several inducers of acute pancreatitis trigger sustained Ca2+ increases in the cytosol and mitochondria of pancreatic acinar cells. The mitochondrial calcium uniporter (MCU) mediates mitochondrial Ca2+ uptake that regulates bioenergetics and plays an important role in cell survival, damage and death. Aberrant Ca2+ signaling and mitochondrial damage in pancreatic acinar cells have been implicated in the initiation of acute pancreatitis. The primary aim of this study was to assess the involvement of the MCU in experimental acute pancreatitis. We found that pancreatic acinar cells from MCU-/- mice display dramatically reduced mitochondrial Ca2+ uptake. This is consistent with the drastic changes of stimulus-metabolism coupling, manifested by the reduction of mitochondrial NADH/FAD+ responses to cholecystokinin and in the decrease of cholecystokinin-stimulated oxygen consumption. However, in three experimental models of acute pancreatitis (induced by caerulein, taurolithocholic acid 3-sulfate or palmitoleic acid plus ethanol), MCU knockout failed to reduce the biochemical and histological changes characterizing the severity of local and systemic damage. A possible explanation of this surprising finding is the redundancy of damaging mechanisms activated by the inducers of acute pancreatitis.

F1F0-ATP Synthase Inhibitory Factor 1 in the Normal Pancreas and in Pancreatic Ductal Adenocarcinoma: Effects on Bioenergetics, Invasion and Proliferation.

F1F0-ATP synthase inhibitory factor 1 (IF1) inhibits the reverse mode of F1F0-ATP synthase, and therefore protects cellular ATP content at the expense of accelerated loss of mitochondrial membrane potential (ΔΨm). There is considerable variability in IF1 expression and its influence on bioenergetics between different cell types. High levels of IF1 in a number of cancers have been linked to increased glycolysis, resistance to cell death, increased migration and proliferation. However, neither the expression nor role of IF1 in the normal pancreas or in pancreatic cancer has been characterized. In this study, we found that pancreatic ductal adenocarcinoma (PDAC) patients express higher levels of IF1 in cancerous cells than in pancreatic acinar cells (PACs). PDAC cell lines have a higher IF1 content and IF1/ATP synthase ratio than PACs. The observed differences are consistent with the ability of the respective cell types to maintain ΔΨm and ATP levels in conditions of chemical hypoxia. Acinar cells and PDAC cells preferentially express different IF1 isoforms. Both knockdown and knockout of IF1 in the PANC-1 pancreatic cancer cell line modified cellular bioenergetics and decreased migration, invasion and proliferation suggesting the putative importance of IF1 for PDAC growth and metastasis.

An efficient method is required to transfect non-dividing cells with genetically encoded optical probes for molecular imaging.

Genetically encoded fluorescent and bioluminescent reporters are now widely used for imaging and understanding of intracellular signaling in response to extracellular stimuli in real time in single living cells. Primary cultures are a valuable tool, and are often preferred over transformed or immortalized cell lines, since they are biologically more relevant and important in biomedical research and therapeutic development. To incorporate genetically encoded reporters into the primary culture of non-dividing cells, such as mouse or human pancreatic acinar cells, is not an easy task. This short review discusses the different methods available to transfect cell lines and primary cultures while especially focusing on pancreatic acinar cells with genetically encoded optical reporters to advance knowledge of the pathophysiology of pancreatitis.

How to win ATP and influence Ca(2+) signaling.

This brief review discusses recent advances in studies of mitochondrial Ca(2+) signaling and considers how the relationships between mitochondria and Ca(2+) responses are shaped in secretory epithelial cells. Perhaps the more precise title of this review could have been "How to win ATP and influence Ca(2+) signaling in secretory epithelium with emphasis on exocrine secretory cells and specific focus on pancreatic acinar cells". But "brevity is a virtue" and the authors hope that many of the mechanisms discussed are general and applicable to other tissues and cell types. Among these mechanisms are mitochondrial regulation of Ca(2+) entry and the role of mitochondria in the formation of localized Ca(2+) responses. The roles of Ca(2+) signaling in the physiological adjustment of bioenergetics and in mitochondrial damage are also briefly discussed.

Mitochondrial calcium in the life and death of exocrine secretory cells.

The remarkable recent discoveries of the proteins mediating mitochondrial Ca(2+) transport (reviewed in this issue) provide an exciting opportunity to utilise this new knowledge to improve our fundamental understanding of relationships between Ca(2+) signalling and bioenergetics and, importantly, to improve the understanding of diseases in which Ca(2+) toxicity and mitochondrial malfunction play a crucial role. Ca(2+) is an important activator of exocrine secretion, a regulator of the bioenergetics of exocrine cells and a contributor to exocrine cell damage. Exocrine secretory cells, exocrine tissues and diseases affecting exocrine glands (like Sjögren's syndrome and acute pancreatitis) will, therefore, provide worthy research areas for the application of this new knowledge of the Ca(2+) transport mechanisms in mitochondria.