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Acute pancreatitis (AP) is a life-threatening inflammatory disease with no specific therapy. Excessive cytoplasmic Ca2+ elevation and intracellular ATP depletion are responsible for the initiation of AP. Inhibition of Ca2+ release-activated Ca2+ (CRAC) channels has been proposed as a potential treatment, and currently, a novel selective CRAC channel inhibitor CM4620 (Auxora, CalciMedica) is in Phase 2b human trials. While CM4620 is on track to become the first effective treatment for AP, it does not produce complete protection in animal models. Recently, an alternative approach has suggested reducing ATP depletion with a natural carbohydrate galactose. Here, we have investigated the possibility of using the smallest effective concentration of CM4620 in combination with galactose. Protective effects of CM4620, in the range of 1-100 n m, have been studied against necrosis induced by bile acids, palmitoleic acid, or l-asparaginase. CM4620 markedly protected against necrosis induced by bile acids or asparaginase starting from 50 n m and palmitoleic acid starting from 1 n m. Combining CM4620 and galactose (1 m m) significantly reduced the extent of necrosis to near-control levels. In the palmitoleic acid-alcohol-induced experimental mouse model of AP, CM4620 at a concentration of 0.1 mg/kg alone significantly reduced edema, necrosis, inflammation, and the total histopathological score. A combination of 0.1 mg/kg CM4620 with galactose (100 m m) significantly reduced further necrosis, inflammation, and histopathological score. Our data show that CM4620 can be used at much lower concentrations than reported previously, reducing potential side effects. The novel combination of CM4620 with galactose synergistically targets complementary pathological mechanisms of AP., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Physiological Society.)

Mutations in RYR1 encoding the ryanodine receptor (RyR) skeletal muscle isoform (RyR1) are a common cause of inherited neuromuscular disorders. Despite its expression in a wide range of tissues, non-skeletal muscle manifestations associated with RYR1 mutations have only been rarely reported. Here, we report three patients with a diagnosis of Central Core Disease (CCD), King-Denborough Syndrome (KDS) and Malignant Hyperthermia Susceptibility (MHS), respectively, who in addition to their (putative) RYR1-related disorder also developed symptoms and signs of acute pancreatitis. In two patients, episodes were recurrent, with severe multisystem involvement and sequelae. RyR1-mediated calcium signalling plays an important role in normal pancreatic function but has also been critically implicated in the pathophysiology of acute pancreatitis, particularly in bile acid- and ethanol-induced forms. Findings from relevant animal models indicate that pancreatic damage in these conditions may be ameliorated through administration of the specific RyR1 antagonist dantrolene and other compounds modifying pancreatic metabolism including calcium signalling. These observations suggest that patients with RYR1 gain-of-function variants may be at increased risk of developing acute pancreatitis, a condition which should therefore be considered in the health surveillance of such individuals., Competing Interests: Declaration of Competing Interest The authors do not have any conflicts of interest to declare in relation to this submission., (Copyright © 2023. Published by Elsevier B.V.)

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 Ca 2+ concentration, which is the result of Ca 2+ release from internal stores followed by Ca 2+ entry through the store operated Ca 2+ 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., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

There is growing evidence that intracellular calcium plays a primary role in the pathophysiology of the pancreas in addition to its crucial importance in major physiological functions. Pancreatic acinar cells have a remarkably large amount of Ca(2+) stored in both the endoplasmic reticulum (ER) and the acidic stores. The vast majority of the classical ER Ca(2+) store is located in the basal part of the acinar cells with extensions protruding into the apical area, however, the acidic stores are exclusively located in the secretory granular area of the cells. Both types of Ca(2+) store respond to all three intracellular Ca(2+) messengers - inositol trisphosphate (InsP3), cyclic-ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). The two stores interact with each other via calcium-induced calcium release; however, they can be separated using pharmacological tools. The ER relies on sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) that can be blocked by the specific inhibitor thapsigargin. The acidic store requires a low pH that can be modified by blocking vacuolar H(+)-ATPase. The acidic store is particularly important for pathological processes in the pancreas. Acute pancreatitis is initiated as a result of calcium overload in the apical pole, which leads to trypsinogen activation; two major causes are gall bladder stones and excessive alcohol consumption. Excessive Ca(2+) release from the acidic stores plays a major role in both scenarios; however NAADP-induced calcium release from acidic stores is particularly important for bile-induced pancreatitis. Cell-permeable calmodulin (CaM) activators such as CALP3 boost the natural protective effect of CaM by inhibiting excessive calcium release from the internal stores through inositol trisphosphate (InsP3R) and ryanodine receptors (RyR). Alternatively calcium overload can be dramatically reduced by inhibiting Ca(2+)-release-activated Ca(2+) (CRAC) currents that are required to reload the internal stores and therefore provide effective protection against the major triggers of acute pancreatitis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (2011 Elsevier Ltd. All rights reserved.)

Physiological stimulation of pancreatic acinar cells by cholecystokinin and acetylcholine activate a spatial-temporal pattern of cytosolic [Ca(+2)] changes that are regulated by a coordinated response of inositol 1,4,5-trisphosphate receptors (IP(3)Rs), ryanodine receptors (RyRs) and calcium-induced calcium release (CICR). For the present study, we designed experiments to determine the potential role of Bcl-2 proteins in these patterns of cytosolic [Ca(+2)] responses. We used small molecule inhibitors that disrupt the interactions between prosurvival Bcl-2 proteins (i.e. Bcl-2 and Bcl-xl) and proapoptotic Bcl-2 proteins (i.e. Bax) and fluorescence microfluorimetry techniques to measure both cytosolic [Ca(+2)] and endoplasmic reticulum [Ca(+2)]. We found that the inhibitors of Bcl-2 protein interactions caused a slow and complete release of intracellular agonist-sensitive stores of calcium. The release was attenuated by inhibitors of IP(3)Rs and RyRs and substantially reduced by strong [Ca(2+)] buffering. Inhibition of IP(3)Rs and RyRs also dramatically reduced activation of apoptosis by BH3I-2'. CICR induced by different doses of BH3I-2' in Bcl-2 overexpressing cells was markedly decreased compared with control. The results suggest that Bcl-2 proteins regulate calcium release from the intracellular stores and suggest that the spatial-temporal patterns of agonist-stimulated cytosolic [Ca(+2)] changes are regulated by differential cellular distribution of interacting pairs of prosurvival and proapoptotic Bcl-2 proteins.

Inositol trisphosphate and cyclic ADP-ribose, main intracellular Ca(2+) messengers, induce release from the intracellular Ca(2+) stores via inositol trisphosphate and ryanodine receptors, respectively. Recently, studies using novel messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releasing Ca(2+) from calcium stores in organelles other than endoplasmic reticulum (ER) have been conducted. However, technical difficulties of Ca(2+) measurements in relatively small Ca(2+) stores prompted us to develop a new, more sensitive, and less damaging two-photon permeabilization technique. Applied to pancreatic acinar cells, this technique allowed us to show that all three messengers - IP(3), cADPR, and NAADP - release Ca(2+) from two intracellular stores: the endoplasmic reticulum and an acidic store in the granular region. This chapter describes a detailed procedure of using this technique with pancreatic acinar cells.

Nuclear calcium signalling has been a controversial battlefield for many years and the question of how permeable the nuclear pore complexes (NPCs) are to Ca2+ has been the subject of a particularly hot dispute. Recent data from isolated nuclei suggest that the NPCs are open even after depletion of the Ca2+ store in the nuclear envelope. Other research has suggested that a new Ca2+ -releasing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), can liberate Ca2+ only from acidic organelles, probably lysosomes, rather than from the traditional Ca2+ store in the endoplasmic reticulum (ER). Recent work indicates that NAADP can release Ca2+ from the nuclear envelope (NE), which has a thapsigargin-sensitive, ER-type Ca2+ store. NAADP acts in a manner similar to inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] or cyclic ADP-ribose (cADPR): all three messengers are equally able to reduce the Ca2+ concentration inside the NE and this is associated with a transient rise in the nucleoplasmic Ca2+ concentration. The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P3 receptors [Ins(1,4,5)P3Rs], and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P3Rs by Ins(1,4,5)P3.

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.

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.

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.

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.

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 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.

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.

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.

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.

Experiments were performed on six volunteers, in order to investigate the effect of the activity of the m. biceps branchii on the median and ulnar nerves. The reflex of the m. biceps brachii was evoked by a sudden stretch and the EMG responses of the m. biceps brachii, m. abductor dig. V. and thenar muscles were recorded. Hand muscles produced early EMG potentials, the latency between them, as well as the onset of the electrical response recorded from the m. biceps brachii, being about the same as the conduction time of nerve excitation from biceps to the hand muscles. The possible mechanisms of the origin of the early EMG activity recorded from the hand muscles are discussed.