Ca2+ signalling underlying pancreatitis.

In spite of significant scientific progress in recent years, acute pancreatitis (AP) is still a dangerous and in up to 5% of cases deadly disease with no specific cure. It is self-resolved in the majority of cases, but could result in chronic pancreatitis (CP) and increased risk of pancreatic cancer (PC). One of the early events in AP is premature activation of digestive pro-enzymes, including trypsinogen, inside pancreatic acinar cells (PACs) due to an excessive rise in the cytosolic Ca2+ concentration, which is the result of Ca2+ release from internal stores followed by Ca2+ entry through the store operated Ca2+ channels in the plasma membrane. The leading causes of AP are high alcohol intake and biliary disease with gallstones obstruction leading to bile reflux into the pancreatic duct. Recently attention in this area of research turned to another cause of AP - Asparaginase based drugs - which have been used quite successfully in treatments of childhood acute lymphoblastic leukaemia (ALL). Unfortunately, Asparaginase is implicated in triggering AP in 5-10% of cases as a side effect of the anti-cancer therapy. The main features of Asparaginase-elicited AP (AAP) were found to be remarkably similar to AP induced by alcohol metabolites and bile acids. Several potential therapeutic avenues in counteracting AAP have been suggested and could also be useful for dealing with AP induced by other causes. Another interesting development in this field includes recent research related to pancreatic stellate cells (PSCs) that are much less studied in their natural environment but nevertheless critically involved in AP, CP and PC. This review will attempt to evaluate developments, approaches and potential therapies for AP and discuss links to other relevant diseases.

Aberrant Ca(2+) signalling through acidic calcium stores in pancreatic acinar cells.

Pancreatic acinar cells possess a very large Ca(2+) store in the endoplasmic reticulum, but also have extensive acidic Ca(2+) stores. Whereas the endoplasmic reticulum is principally located in the baso-lateral part of the cells, although with extensions into the granular area, the acidic stores are exclusively present in the apical part. The two types of stores can be differentiated pharmacologically because the endoplasmic reticulum accumulates Ca(2+) via SERCA pumps, whereas the acidic pools require functional vacuolar H(+) pumps in order to maintain a high intra-organellar Ca(2+) concentration. The human disease acute pancreatitis is initiated by trypsinogen activation in the apical pole and this is mostly due to either complications arising from gall bladder stones or excessive alcohol consumption. Attention has therefore been focussed on assessing the acute effects of bile acids as well as alcohol metabolites. The evidence accumulated so far indicates that bile acids and fatty acid ethyl esters - the non-oxidative products of alcohol and fatty acids - exert their pathological effects primarily by excessive Ca(2+) release from the acidic stores. This occurs by opening of the very same release channels that are also responsible for normal stimulus-secretion coupling, namely inositol trisphosphate and ryanodine receptors. The inositol trisphosphate receptors are of particular importance and the results of gene deletion experiments indicate that the fatty acid ethyl esters mainly utilize sub-types 2 and 3.

Regulation of early response genes in pancreatic acinar cells: external calcium and nuclear calcium signalling aspects.

Nuclear calcium signalling has been an important topic of investigation for many years and some aspects have been the subject of debate. Our data from isolated nuclei suggest that the nuclear pore complexes (NPCs) are open even after depletion of the Ca(2+) store in the nuclear envelope (NE). The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P(3) receptors [Ins(1,4,5)P(3)Rs], most likely on both sides of the NE and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P(3)Rs by Ins(1,4,5)P(3). We have also investigated the possible consequences of nuclear calcium signals: the role of Ca(2+) in the regulation of immediate early genes (IEG): c-fos, c-myc and c-jun in pancreatic acinar cells. Stimulation with Ca(2+)-mobilizing agonists induced significant increases in levels of expression. Cholecystokinin (CCK) (10 nm) evoked a substantial rise in the expression levels, highly dependent on external Ca(2+): the IEG expression level was lowest in Ca(2+)-free solution, increased at the physiological level of 1 mm [Ca(2+)](o) and was maximal at 10 mm [Ca(2+)](o), i.e.: 102 +/- 22% and 163 +/- 15% for c-fos; c-myc -73 +/- 13% and 106 +/- 24%; c-jun -49 +/- 8% and 59 +/- 9% at 1 and 10 mm of extracellular Ca(2+) respectively. A low CCK concentration (10 pm) induced a small increase in expression. We conclude that extracellular Ca(2+) together with nuclear Ca(2+) signals induced by CCK play important roles in the induction of IEG expression.

Calcium signalling and pancreatic cell death: apoptosis or necrosis?

Secretagogues, such as cholecystokinin and acetylcholine, utilise a variety of second messengers (inositol trisphosphate, cADPR and nicotinic acid adenine dinucleotide phosphate) to induce specific oscillatory patterns of calcium (Ca(2+)) signals in pancreatic acinar cells. These are tightly controlled in a spatiotemporal manner, and are coupled to mitochondrial metabolism necessary to fuel secretion. When Ca(2+) homeostasis is disrupted by known precipitants of acute pancreatitis, for example, hyperstimulation or non-oxidative ethanol metabolites, Ca(2+) stores (endoplasmic reticulum and acidic pool) become depleted and sustained cytosolic [Ca(2+)] elevations replace transient signals, leading to severe consequences. Sustained mitochondrial depolarisation, possibly via opening of the mitochondrial permeability transition pore (MPTP), elicits cellular ATP depletion that paralyses energy-dependent Ca(2+) pumps causing cytosolic Ca(2+) overload, while digestive enzymes are activated prematurely within the cell; Ca(2+)-dependent cellular necrosis ensues. However, when stress to the acinar cell is milder, for example, by application of the oxidant menadione, release of Ca(2+) from stores leads to oscillatory global waves, associated with partial mitochondrial depolarisation and transient MPTP opening; apoptotic cell death is promoted via the intrinsic pathway, when associated with generation of reactive oxygen species. Apoptosis, induced by menadione or bile acids, is potentiated by inhibition of an endogenous detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), suggesting its importance as a defence mechanism that may influence cell fate.

The distribution of the endoplasmic reticulum in living pancreatic acinar cells.

Studies on pancreatic acinar cells provided the original evidence for the Ca(2+) releasing action of inositol 1,4,5-trisphosphate (IP(3)). Ironically, this system has presented problems for the general theory that IP(3) acts primarily on the endoplasmic reticulum (ER), because the IP(3)-elicited Ca(2+) release occurs in the apical pole, which is dominated by zymogen granules (ZGs) and apparently contains very little ER. Using confocal and two-photon microscopy and a number of different ER-specific fluorescent probes, we have now investigated in detail the distribution of the ER in living pancreatic acinar cells. It turns out that although the bulk of the ER, as expected, is clearly located in the baso-lateral part of the cell, there is significant invasion of ER into the granular pole and each ZG is in fact surrounded by strands of ER. This structural evidence from living cells, in conjunction with recent functional studies demonstrating the high Ca(2+) mobility in the ER lumen, provides the framework for a coherent and internally consistent theory for cytosolic Ca(2+) signal generation in the apical secretory pole, in which the primary Ca(2+) release occurs from ER extensions in the granular pole supplied with Ca(2+) from the main store at the base of the cell by the tunnel function of the ER.

Membrane repair: Ca(2+)-elicited lysosomal exocytosis.

Cells in exposed positions are subject to injury and therefore need membrane repair mechanisms. Ca(2+) entry inevitably follows membrane rupture and recent studies indicate that this elicits repair via Ca(2+)-activated exocytosis of lysosomes, regulated by lysosomal synaptotagmin VII.

Two different but converging messenger pathways to intracellular Ca(2+) release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate.

Hormones and neurotransmitters mobilize Ca(2+) from the endoplasmic reticulum via inositol trisphosphate (IP(3)) receptors, but how a single target cell encodes different extracellular signals to generate specific cytosolic Ca(2+) responses is unknown. In pancreatic acinar cells, acetylcholine evokes local Ca(2+) spiking in the apical granular pole, whereas cholecystokinin elicits a mixture of local and global cytosolic Ca(2+) signals. We show that IP(3), cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) evoke cytosolic Ca(2+) spiking by activating common oscillator units composed of IP(3) and ryanodine receptors. Acetylcholine activation of these common oscillator units is triggered via IP(3) receptors, whereas cholecystokinin responses are triggered via a different but converging pathway with NAADP and cyclic ADP-ribose receptors. Cholecystokinin potentiates the response to acetylcholine, making it global rather than local, an effect mediated specifically by cyclic ADP-ribose receptors. In the apical pole there is a common early activation site for Ca(2+) release, indicating that the three types of Ca(2+) release channels are clustered together and that the appropriate receptors are selected at the earliest step of signal generation.

Active mitochondria surrounding the pancreatic acinar granule region prevent spreading of inositol trisphosphate-evoked local cytosolic Ca(2+) signals.

Agonist-evoked cytosolic Ca(2+) spikes in mouse pancreatic acinar cells are specifically initiated in the apical secretory pole and are mostly confined to this region. The role played by mitochondria in this process has been investigated. Using the mitochondria-specific fluorescent dyes MitoTracker Green and Rhodamine 123, these organelles appeared as a bright belt concentrated mainly around the secretory granule area. We tested the effects of two different types of mitochondrial inhibitor on the cytosolic Ca(2+) concentration using simultaneous imaging of Ca(2+)-sensitive fluorescence (Fura 2) and electrophysiology. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied in the presence of the Ca(2+)-releasing messenger inositol 1,4, 5-trisphosphate (IP(3)), the local repetitive Ca(2+) responses in the granule area were transformed into a global rise in the cellular Ca(2+) concentration. In the absence of IP(3), CCCP had no effect on the cytosolic Ca(2+) levels. Antimycin and antimycin + oligomycin had the same effect as CCCP. Active mitochondria, strategically placed around the secretory pole, block Ca(2+) diffusion from the primary Ca(2+) release sites in the granule-rich area in the apical pole to the basal part of the cell containing the nucleus. When mitochondrial function is inhibited, this barrier disappears and the Ca(2+) signals spread all over the cytosol.

Calcium uptake via endocytosis with rapid release from acidifying endosomes.

A number of specific cellular Ca2+ uptake pathways have been described in many different cell types [1] [2] [3]. The possibility that substantial quantities of Ca2+ could be imported via endocytosis has essentially been ignored, although it has been recognized that endosomes can store Ca2+ [4] [5]. Exocrine cells can release significant amounts of Ca2+ via exocytosis [6], so we have investigated the fate of Ca2+ taken up via endocytosis into endosomes. Ca2+-sensitive and H+-sensitive fluorescent probes were placed in the extracellular solution and subsequently taken up into fibroblasts by endocytosis. Confocal microscopy was used to assess the distribution of fluorescence intensity. Ca2+ taken up by endocytosis was lost from the endosomes within a few minutes, over the same period as endosomal acidification took place. The acidification was inhibited by reducing the extracellular Ca2+ concentration, and Ca2+ loss from the endosomes was blocked by bafilomycin (100 nM), a specific inhibitor of the vacuolar proton ATPase. Quantitative evaluation indicated that endocytosis causes substantial import of Ca2+ because of rapid loss from early endosomes.

The calcium store in the nuclear envelope.

The nuclear envelope has a relatively small volume, but is connected up to the vastly larger endoplasmic reticulum. The Ca2+ concentration in the lumen of the interconnected nuclear envelope and endoplasmic reticulum network is in the resting state maintained at a level of more than 100 microM. There are specific Ca2+ release channels present in the inner nuclear membrane that can be activated by inositol trisphosphate or cADP ribose. The system, therefore, allows selective release of Ca2+ into the nucleoplasm which could be important for the control of specific types of gene expression.

Short pulses of acetylcholine stimulation induce cytosolic Ca2+ signals that are excluded from the nuclear region in pancreatic acinar cells.

We have investigated the spreading of cytosolic Ca2+ signals generated by acetylcholine stimulation (using either microionophoresis or pressure application) of isolated pancreatic acinar cells (or small cell clusters) using confocal microscopy of Ca2+-sensitive fluorescence (fura red). We have been particularly interested in the effects of short vigorous pulses of acetylcholine (ACh) stimulation since, in the pancreas, ACh secreted from nerve endings is quickly eliminated by the action of ACh esterase. We focused on three regions: the secretory pole (secretory granule area), the nucleus and the basal area outside the nucleus. The nuclei were visualized by using the specific nuclear stain Hoechst 33342. With ionophoretic application, a long-lasting stimulation with ACh (10 s and longer) induces large Ca2+ transients of similar amplitude in all the three selected regions of the cell. Short applications (about 3 s) of ACh result in a Ca2+ rise in the secretory pole, whereas no changes in cytoplasmic Ca2+ were detected in the basal, nonnuclear region or in the nucleus. We found that at the peak of such localised Ca2+ responses, evoked either by ACh ionophoresis or pressure application, significant Ca2+ concentration gradients (up to 400 nM/microm) can be established along the line connecting the secretory pole with the nucleus. In some experiments slightly longer applications (about 5 s) of ACh produce Ca2+ transients in both the secretory region and in the basal, nonnuclear regions of the cells, whereas the nuclear [Ca2+] remained largely unaffected. Estimation of the ACh concentration in the vicinity of the cell under investigation indicated that values of about 1 microM were attained in the pressure application experiments. These results show directly that the nucleus of pancreatic acinar cells can be effectively protected from relatively large Ca2+ transients generated in the secretory pole of pancreatic acinar cells by short pulses of near-maximal ACh concentrations. This indicates that calcium-dependent secretion (both fluid and digestive enzymes) can occur without changes of the intranuclear [Ca2+] and consequently without activation of numerous calcium dependent nuclear processes.

Calcium transport pathways in the nucleus.

Due to the availability of new biophysical and biochemical techniques, there has recently been considerable progress in our understanding of Ca2+ transport inside, as well as into and out of, the nucleus. A number of Ca2+ transport pathways have been localized specifically in the outer or inner nuclear membrane and the Ca2+ permeability through the nuclear pore complex has been assessed. The nuclear envelope has characteristics similar to those of a leaky epithelium. The leak is through the nuclear pore complex. The outer nuclear membrane contains the Ca2+ ATPase whereas the functionally important inositol trisphosphate (IP3)-activated Ca2+ release channels are specifically localized in the inner nuclear membrane.

Inositol trisphosphate and cyclic ADP-ribose-mediated release of Ca2+ from single isolated pancreatic zymogen granules.

In pancreatic acinar cells low (physiological) agonist concentrations evoke cytosolic Ca2+ spikes specifically in the apical secretory pole that contains a high density of secretory (zymogen) granules (ZGs). Inositol 1,4,5-trisphosphate (IP3) is believed to release Ca2+ from the endoplasmic reticulum, but we have now tested whether the Ca(2+)-releasing messengers IP3 and cyclic ADP-ribose (cADPr) can liberate Ca2+ from AGs. In experiments on single isolated ZGs, we show using confocal microscopy that IP3 and cADPr evoke a marked decrease in the free intragranular Ca2+ concentration. Using a novel high resolution method, we have measured changes in the Ca2+ concentration in the vicinity of an isolated AG and show that IP3 and cADPr cause rapid Ca2+ release from the granule, explaining the agonist-evoked cytosolic Ca2+ rise in the secretory pole.

ATP-dependent accumulation and inositol trisphosphate- or cyclic ADP-ribose-mediated release of Ca2+ from the nuclear envelope.

Uptake and release of Ca2+ from isolated liver nuclei were studied with fluorescent probes. We show with the help of digital imaging and confocal microscopy that the Ca(2+)-sensitive fluorescent probe Fura 2 is concentrated in or around the nuclear envelope and that the distribution of Fura 2 fluorescence is similar to that of an endoplasmic reticulum marker. The previously demonstrated ATP-dependent uptake of Ca2+ into isolated nuclei and release of the accumulated Ca2+ by inositol 1,4,5-trisphosphate (IP3) are therefore due to transport of Ca2+ into and out of the nuclear envelope and not the nucleoplasm. Dextrans labeled with fluorescent Ca2+ indicators (calcium-Green 1 and Fura 2) are distributed uniformly in the nucleoplasm and can be used to show that changes in the external Ca2+ concentration produce rapid changes in the nucleoplasmic Ca2+ concentration. Nevertheless, IP3 and cyclic ADP-ribose evoke transient intranuclear Ca2+ elevations. The release from the Ca2+ stores in or around the nuclear envelope appears to be directed into the nucleoplasm from where it can diffuse out through the permeable nuclear pore complexes.